Role of hyaluronan in glioma invasion

Gliomas are the most common primary intracranial tumors. Their distinct ability to infiltrate into the extracellular matrix (ECM) of the brain makes it impossible to treat these tumors using surgery and radiation therapy. A number of different studies have suggested that hyaluronan (HA), the principal glycosaminoglycan (GAG) in the ECM of the brain, is the critical factor for glioma invasion. HA-induced glioma invasion was driven by two important molecular events: matrix metalloproteinase (MMP) secretion and upregulation of cell migration. MMP secretion was triggered by HA-induced focal adhesion kinase (FAK) activation, which transmits its signal through ERK activation and nuclear factor kappa B (NFκB) translocation. Another important molecular event is osteopontin (OPN) expression. OPN expression by AKT activation triggers cell migration. These results suggest that HA-induced glioma invasion is tightly regulated by signaling mechanisms, and a detailed understanding of this molecular mechanism will provide important clues for glioma treatment.Key words: hyaluronan, matrix metalloproteinase, osteopontin, emodin, invasion, gliomaMalignant gliomas are highly invasive and infiltrative tumors that have a poor prognosis with a median survival of only one year.^1,2 A major barrier to effective malignant glioma treatment is the invasion of these cells into brain parenchyma. Because of this fact, local therapies such as surgery or radiation therapy are not effective.³ Glioma cells invade through the ECM of the brain by activating a number of coordinated cellular programs, which include those necessary for migration and invasion.³ Therefore, a detailed understanding of the mechanisms underlying this invasive behavior is essential for the development of novel effective therapies.During glioma invasion, tumor cells closely interact with the ECM. Although brain tumor cells may share some of the invasive characteristics with tumors that arise outside of the central nerve system (CNS), the particular structure and composition of the brain ECM suggest the existence of unique invasive mechanisms for brain tumors.⁴Brain ECM is composed of typical ECM proteins and a HA scaffold with associated glycoproteins and proteoglycans.⁵ Typical ECM proteins such as laminin, type-IV collagen and fibronectin have been implicated in the invasion of other tumors by regulating cell adhesion and migration.⁶ However HA, which is associated with proteoglycans and GAGs, is especially abundant in the brain parenchyma compared to other tissues.⁷ Furthermore, malignant gliomas contain higher amounts of HA than normal brain tissue.⁷ These facts raise the possibility that HA might play an important role in glioma invasion, a process that is distinct from other non-CNS derived tumors.     

Cell adhesion molecules in context: CAM function depends on the neighborhood

Cell adhesion molecules (CAMs) are now known to mediate much more than adhesion between cells and between cells and the extracellular matrix. Work by many researchers has illuminated their roles in modulating activation of molecules such as receptor tyrosine kinases, with subsequent effects on cell survival, migration and process extension. CAMs are also known to serve as substrates for proteases that can create diffusible fragments capable of signaling independently from the CAM. The diversity of interactions is further modulated by membrane rafts, which can co-localize or separate potential signaling partners to affect the likelihood of a given signaling pathway being activated. Given the ever-growing number of known CAMs and the fact that their heterophilic binding in cis or in trans can affect their interactions with other molecules, including membrane-bound receptors, one would predict a wide range of effects attributable to a particular CAM in a particular cell at a particular stage of development. The function(s) of a given CAM must therefore be considered in the context of the history of the cell expressing it and the repertoire of molecules expressed both by that cell and its neighbors.Key words: cell adhesion molecule, fibroblast growth factor receptor, epidermal growth factor receptor, L1, NCAM, Neuroglian, fasciclin, membrane raft, ankyrin, doublecortin, ezrin, radixin, moesinCell migration, axon extension and dendrite arborization are all essential processes in creating the complex neural architectures of the developing brain. A number of CAMs, including those of the immunoglobulin superfamily (IgCAMs), integrins and cadherins, are known to mediate signaling between cells and between cells and the extracellular matrix (ECM). Because the greatest amount of IgCAM research has focused on L1-CAM and NCAM and their invertebrate homologs Neuroglian and Fasciclin II, these molecules will be the primary focus of this Commentary & View. IgCAMs are so named because their extracellular domains contain immunoglobulin repeats (usually 5–6). The Ig repeats are usually followed by fibronectin type-3 (Fn III) domains (2–5) and either transmembrane plus cytoplasmic domains or a glycosyl-phosphatidylinositol (GPI) linkage (reviewed in refs. ^1–3). IgCAMs can bind homophilically and heterophilically via their Ig and/or Fn III domains to achieve cell-cell and cell-ECM adhesion, which can simply stabilize the architecture of neural tissue, but can also transmit information to the cell interior.⁴ For example, a number of IgCAMs are known to bind to the cytoskeleton via linker molecules including Ankyrin, Doublecortin and members of the Ezrin-Radixin-Moesin family (ERMs).^5–12 Some of these linking interactions are thought to allow engagement or disengagement of a molecular “clutch module” (reviewed in ref. ¹³) similar to coupling of integrins to F-actin flow via focal adhesion proteins and are believed to be important in growth cone function and synaptogenesis.^14,15 Work from these groups suggests these linker molecules are expressed in different developmental windows, so that ERMs and Doublecortin are important in L1-mediated neurite outgrowth and suppression of neurite branching, while subsequent Ankyrin expression and binding to L1 blocks outgrowth and fosters axon stabilization and synaptogenesis.^6–12 Another important example of outside-in signaling by IgCAMs is their ability, via Ankyrin''s multivalent binding sites, to cluster and position many receptors and channels at specific cellular locations such as axon initial segments and nodes of Ranvier (reviewed in ref. ¹² and ¹⁶). IgCAMs have also been shown to be substrates for matrix metalloproteases (MMPs), which can cleave extracellular domains, allowing the fragments to act as diffusable signaling molecules and changing the signaling effects of the remaining membrane-bound moieties.^17,18     

The roles of cell adhesion molecules in tumor suppression and cell migration: A new paradox

In addition to mediating cell adhesion, many cell adhesion molecules act as tumor suppressors. These proteins are capable of restricting cell growth mainly through contact inhibition. Alterations of these cell adhesion molecules are a common event in cancer. The resulting loss of cell-cell and/or cell-extracellular matrix adhesion promotes cell growth as well as tumor dissemination. Therefore, it is conventionally accepted that cell adhesion molecules that function as tumor suppressors are also involved in limiting tumor cell migration. Paradoxically, in 2005, we identified an immunoglobulin superfamily cell adhesion molecule hepaCAM that is able to suppress cancer cell growth and yet induce migration. Almost concurrently, CEACAM1 was verified to co-function as a tumor suppressor and invasion promoter. To date, the reason and mechanism responsible for this exceptional phenomenon remain unclear. Nevertheless, the emergence of these intriguing cell adhesion molecules with conflicting roles may open a new chapter to the biological significance of cell adhesion molecules.Key words: hepaCAM, cell adhesion molecules, tumor suppressor, migration, E-cadherin, CADM1, integrin α7, CEACAM1It is well known that many cell adhesion molecules function as tumor suppressors (reviewed in ref. ¹). These molecules exert their tumor suppressive effect mainly through cell-adhesion-mediated contact inhibition. Cell adhesion molecules allow cells to communicate with one another or to the extracellular environment by mediating cell-cell or cell-extracellular matrix (ECM) interactions (reviewed in refs. ² and ³). Broadly, these proteins can be classified into five families including immunoglobulin superfamily, integrins, cadherins, selectins and CD44. Apart from participating in the development and maintenance of tissue architecture, cell adhesion molecules serve as cell surface receptors critical for capturing, integrating and transmitting signals from the extracellular milieu to the cell interior (reviewed in refs. ² and ³). These signaling events are vital for the regulation of a wide variety of cellular functions including embryogenesis, immune and inflammatory responses, tissue repair, cell migration, differentiation, proliferation and apoptosis. Alterations of these cell adhesion molecules are a common event in cancer (reviewed in refs. ^{1, 2, 4} and ⁵). The disrupted cell-cell or cell-ECM adhesion significantly contributes to uncontrolled cell proliferation and progressive distortion of normal tissue architecture. More importantly, changes in cell adhesion molecules play a causal role in tumor dissemination. Loss of cell adhesion contacts allows malignant cells to detach and to escape from the primary mass. Gaining a more motile and invasive phenotype, these cells break down the ECM and eventually invade and metastasize to distal organs.Based on the above understanding, it is conventionally accepted that cell adhesion molecules with tumor suppressor activity, when expressed in cancer cells, are able to exert inhibitory effect on cell motility. The ability of cells in migration/motility is a prerequisite for cancer invasion and metastasis (reviewed in refs. ¹ and ⁵). Indeed, a number of cell adhesion molecule-tumor suppressors have been reported to be capable of reducing cell migration. The most classical example is E-cadherin, a calcium-dependent cell adhesion molecule. E-cadherin is expressed exclusively in epithelial cells and its expression is commonly suppressed in tumors of epithelial origins. The cytoplasmic domain of E-cadherin interacts with catenins to establish an intracellular linkage with the actin cytoskeleton (reviewed in ref. ⁶). The assembly of E-cadherin with the cytoskeleton via catenins at the sites of adherens junctions is important for the stabilization of cell-cell adhesions. Disruption of E-cadherin-mediated cell-cell adhesion, due to loss of expression or function of E-cadherin and/or catenins, is assocated with tumor development and progression (reviewed in ref. ⁷). Forced expression of E-cadherin in several cancer cell lines not only slows down cell growth^8,9 but also significantly reduces the invasiveness of the cells.^10,11 On the other hand, inhibition of E-cadherin by function-blocking antibodies and antisense RNA restores the invasiveness in non-invasive transformed cells.¹¹ Furthermore, using a transgenic mouse model of pancreatic beta-cell carcinogenesis, it has been demonstrated that E-cadherin-mediated cell adhesion is important in preventing the transition from well differentiated adenoma to invasive carcinoma.¹²Cell adhesion molecule 1 (CADM1), another example, has also been implicated in cancer progression. CADM1 is a member of the immunoglobulin superfamily and mediates cell-cell adhesion.¹³ The molecule associates with the actin cytoskeleton via the differentially expressed in adenocarcinoma of the lung (DAL1) protein; and the formation of CADM1-DAL1 complex is dependent on the integrity of actin cytoskeleton.¹⁴ Inactivation of the CADM1 and/or DAL1 gene usually through methylation has been reported in diverse human cancers.^15,16 A paper by Ito et al. showed that restoration of CADM1 expression in esophageal squamous cell carcinoma cells not only suppresses cell growth, but also retards cell motility and invasion.¹⁶In contrast to E-cadherin and CADM1, integrin α7 is a cell-ECM adhesion molecule which also possesses tumor suppressor activity. Ren et al. showed that integrin α7 gene is mutated in several human malignances; and the mutations are associated with an increase in cancer recurrence.¹⁷ Forced expression of integrin α7 in integrin α7-deficient leiomyosarcoma cells results in decreased colony formation and slower cell motility. Conversely, knockdown of integrin α7 in lung cancer cells expressing wild-type integrin α7 increases the colony number and cell motility rate. In addition, the researchers revealed that mice bearing xenograft tumors overexpressing integrin α7 have reduced tumor size with no obvious metastasis.In 2005, we first reported the identification of a cell adhesion molecule belonging to the immunoglobulin superfamily, designated as hepaCAM.¹⁸ To date, we have shown that the gene is frequently downregulated in a variety of human cancers.^18,19 Re-expression of hepaCAM in the hepatocellular carcinoma HepG2 cells¹⁸ and breast cancer MCF7 cells¹⁹ inhibits colony formation and retards cell proliferation. In addition, expression of hepaCAM in MCF7 cells results in cell cycle arrest at the G₂/M phase and cellular senescence. Concurrently, the expression of several senescence-associated proteins including p53, p21 and p27 is enhanced. Moreover, downregulation of p53 by p53-specific small interfering RNA in cells expressing hepaCAM clearly reduces p21 without changing p27 and alleviates senescence, indicating that hepaCAM induces senescence through a p53/p21-dependent pathway.¹⁹ Together, the data suggest that hepaCAM is a tumor suppressor. Interestingly, the expression of hepaCAM in both HepG2 and MCF7 cells stimulates both cell-ECM adhesion and cell migration.^18,20,21 The function of hepaCAM as a tumor suppressor in cell migration is contradictory to other cell adhesion molecule-tumor suppressors. Noteworthily, hepaCAM-mediated cell motility is evidenced by its direct interaction with the actin cytoskeleton.²¹Evidences are currently emerging to support the contradictory roles of cell adhesion molecules that both inhibit cell growth and promote cell motility when restored in cancer cells. In addition to hepaCAM, the immunoglobulin superfamily carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is implicated to function as a tumor suppressor and a metastasis promoter. The characteristics and functions of CEACAM1 have been demonstrated in individual reports. CEACAM1 is frequently downregulated or dysregulated in multiple human tumors,^22–25 and is capable of suppressing cell growth and inducing apoptosis.^26–28 Ebrahimnejad et al. demonstrated that exogenous expression of CEACAM1 enhances melanoma cell invasion and migration; and this enhanced motility can be reverted by anti-CEACAM antibodies.²⁹ The ability of CEACAM to co-stimulate tumor suppression and invasion was finally established by Liu et al. in restricting thyroid cancer growth but promoting invasiveness.³⁰ Introduction of CEACAM1 into CEACAM1-deficient thyroid cancer cells results in G₁/S phase cell cycle arrest accompanied by elevated p21 expression and diminished Rb phosphorylation. Overexpression of CEACAM1 also increases cell-ECM adhesion and promotes cell migration and tumor invasiveness. In xenografted mice, CEACAM1 expression results in reduced tumor growth but increased tumor invasiveness. Conversely, silencing of endogenous CEACAM1 accelerates tumor growth and suppresses invasiveness.³⁰It is an exciting issue to address why a cell adhesion molecule is able to suppress tumor growth yet promote tumor progression. Could there be a molecular switch that controls the functions of the gene between a tumor suppressor and a migration promoter in cancer or are the functions executed simultaneously? The expression level, the extracellular cues as well as the interacting partners of the cell adhesion molecules may likely play a critical role in regulating its functions. The question is under what circumstances these factors come into play. To answer all these questions, and maybe more, on the intriguing findings of these proteins, more extensive and intensive experimentation is required. Nevertheless, it is obvious that the emergence of these cell adhesion molecules that function in a contradictory manner opens a new chapter to the biological significance of cell adhesion molecules.     

Chemokine arrest signals to leukocyte integrins trigger bi-directional-occupancy of individual heterodimers by extracellular and cytoplasmic ligands

Integrin heterodimers acquire high affinity to endothelial ligands by extensive conformational changes in both their α and β subunits. These heterodimers are maintained in an inactive state by inter-subunit constraints. Changes in the cytoplasmic interface of the integrin heterodimer (referred to as inside-out integrin activation) can only partially remove these constraints. Full integrin activation is achieved when both inter-subunit constraints and proper rearrangements of the integrin headpiece by its extracellular ligand (outside-in activation) are temporally coupled. A universal regulator of these integrin rearrangements is talin1, a key integrin-actin adaptor that regulates integrin conformation and anchors ligand-occupied integrins to the cortical cytoskeleton. The arrest of rolling leukocytes at target vascular sites depends on rapid activation of their α4 and β2 integrins at endothelial contacts by chemokines displayed on the endothelial surface. These chemotactic cytokines can signal within milliseconds through specialized Gi-protein coupled receptors (GPCRs) and Gi-triggered GTPases on the responding leukocytes. Some chemokine signals can alter integrin conformation by releasing constraints on integrin extension, while other chemokines activate integrins to undergo conformational activation mainly after ligand binding. Both of these modalities involve talin1 activation. In this opinion article, I propose that distinct chemokine signals induce variable strengths of associations between talin1 and different target integrins. Weak interactions of the integrin cytoplasmic tail with talin1 (the cytoplasmic integrin ligand) dissociate unless the extracellular ligand can simultaneously occupy the integrin headpiece and transmit, within milliseconds, proper allosteric changes across the integrin heterodimer back to the tail-talin1 complex. The fate of this bi-directional occupancy of integrins by both their extracellular and intracellular ligands is likely to benefit from immobilization of both ligands to cortical cytoskeletal elements. To properly anchor talin1 onto the integrin tail, a second integrin partner, Kindlin-3 may be also required, although an evidence that both partners can simultaneously bind the same integrin heterodimer is still missing. Once linked to the cortical actin cytoskeleton, the multi-occupied integrin complex can load weak forces, which deliver additional allosteric changes to the integrin headpiece resulting in further bond strengthening. Surface immobilized chemokines are superior to their soluble counterparts in driving this bi-directional occupancy process, presumably due to their ability to facilitate local co-occupancy of individual integrin heterodimers with talin1, Kindlin-3 and surface-bound extracellular ligands.Key words: adhesion, migration, endothelium, cytoskeleton, shear stress, immunityFirm adhesion of leukocytes to blood vessels is tightly regulated by integrins and their cognate ligands.^1,2 These include the α₄ integrins, VLA-4 (α₄β₁) and α₄β₇, and the β₂ integrins, LFA-1 (α_Lβ₂) and Mac-1 (α_Mβ₂). Accumulated data suggest that these counter-receptors are structurally adapted to operate under disruptive blood-derived shear forces.³ A remarkable feature of leukocyte integrins is that their affinity state and microclustering can be regulated within fractions of seconds.^4,5 The most robust signals for leukocyte integrins are transduced by chemoattractants, mostly chemokines displayed on the vessel wall.⁶ A growing body of evidence suggests that the Gi protein coupled receptors of these endothelial chemokines elicit diverse signaling pathways in distinct leukocyte subtypes,^2,22 which use two common downstream elements to coactive all leukocyte integrins: talin1 and Kindlin-3.⁷ In this review, I will describe a model explaining how chemokine signals to these elements regulate the conformation of all leukocyte integrins by facilitating a coupled bi-directional occupancy and activation via both their cytoplasmic and headpiece domains.Recent structural and biophysical studies suggest that leukocyte integrins can alternate between inactive bent conformers, which are clasped heterodimers, and variable unclasped heterodimers with extended ectodomains exhibiting intermediate and high affinity to ligand.⁵ Most leukocyte integrins are maintained in an inactive resting state,² whereas in situ chemokine-stimulated integrins unfold and extend 10–25 nm above the cell surface, allowing their headpiece to readily recognize immobilized ligand on a counter surface.⁸ These extended integrins must undergo extensive rearrangements of their headpiece I-domains induced by external endothelial-displayed ligands in order to arrest rolling leukocytes on blood vessel walls. In leukocytes, these two canonical switches are very short-lived, implying the necessity for a stabilization. It is therefore likely that any type of robust integrin activation must involve bi-directional occupancy of the integrin by both its extracellular ligand and one or more cytoplasmic partners.⁹The main cytoplasmic integrin-activating adaptor in leukocytes and platelets is talin1.^10,11 Talin knock down in multiple cell types results in nearly total loss of integrin activation.^12,13 This actin binding adaptor binds different integrin β subunit tails with low affinity,¹⁴ which can be locally increased by in situ generated PI(4,5)P2 (PIP2). This phosphoinositide presumably binds to the FERM domain within the talin head and thereby enhances talin binding to a membrane proximal NPXY motif on the β integrin tail, a key event in integrin heterodimer unclasping.^15,16 Recent studies suggest, however, that mere talin association may be insufficient to unclasp and activate the integrin heterodimer. Thus, the beta subunit tail may need to get co-occupied by the integrin co-activator, Kindlin, in order to optimize talin association with this integrin subunit.^17,18 In leukocytes, Talin1 and the Kindlin family member, Kindlin 3 co-activate both VLA-4 and LFA-1 and this co-activation is dramatically enhanced by multiple chemokine triggered effectors, the nature of which has begun to unfold¹⁹ (Fig. 1). I would like to propose that talin1-Kindlin-3 co-binding to the β tails of these and other leukocyte integrins is insufficient to switch these integrins to a conformation able to bind their soluble extracellular ligands due to fast dissocia-tion of PIP2-activated talin1 from the integrin cytoplasmic tail complex. This short lived talin-integrin complex may, on the other hand, get stabilized, if the integrin headpiece can simultaneously bind an immobilized extracellular ligand and undergo immediate outside-in activation, before the talin1 has dissociated from the integrin beta tail (Fig. 1). Such full confor-mational switch can result in additional allosteric changes in the integrin-bound talin which may expose vinculin binding sites and further increase talin-actin associations that reinforce this bi-directional allosteric integrin activation.²⁰Open in a separate windowFigure 1Bi-directional integrin activation requires simultaneous co-occupancy of the integrin heterodimer by extracellular and cytoplasmic ligands. A proposed scheme for chemokine-triggered integrin activation on leukocytes. Integrin conformation is allosterically modulated in a bidirectional manner by at least two sets of ligands, extracellular and cytoplasmic. The degree of activation is dictated primarily by unclasping of the integrin heterodimer, a process dependent on the binding of the activated talin FERM domain to a specific site on the integrin β tail. (1) Inactive integrin. (2–5) Four postulated integrin conformations triggered by distinct chemokine signals. (2) Talin FERM domain activation close to the target integrin is a rate limiting step in integrin activation. This activation is triggered by PIP2 locally generated by talin-associated PIP5Kγ (purple rectangle) stimulated by a nearby Gi-coupled chemokine receptor. (3) Kindlin-3 binding to the integrin β tail stabilizes the otherwise weak talin1-integrin tail complex. The activated integrin can bind a soluble extracellular ligand with a low affinity due to a high k_off of the soluble ligand from the integrin headpiece. (4) In the absence of Kindlin-3, chemokine triggered, PIP2-activated talin1 binds only transiently the integrin tail (High k_off). The semiactivated integrin, even if occupied by an immobilized extracellular ligand, cannot undergo full bi-directional activation. (5) When both the extracellular ligand and talin are properly anchored, their escape from the integrin is dramatically reduced, lowering the k_off. Low k_off increases the probability of simultaneous bi-directional occupancy of both the integrin headpiece by the extracellular ligand and of the integrin tail by talin1 and Kindlin-3. This results in optimal bi-directional integrin activation and unclasping of the heterodimer. Stable linkages also allow this bi-directionally occupied integrin to undergo extensive mechanical strengthening by low forces applied on the headpiece; this further activates the headpiece I domains, further separates the β and α subunits from each other, and maximally stabilizes the unclasped integrin. Force application through the high affinity-talin complex can stretch the talin rod domain and expose vinculin binding sites (VBS). Since integrin ligands are generally multivalent, rapid integrin dimerization can take place to further stabilize the focal adhesion (not shown). Additional cytoplasmic partners of specific leukocyte integrins like a-actinin, L-plastin and RAPL may further strengthen subsets of focal adhesions. These and other cytosolic partners bind different integrin targets with different affinities. Therefore the effect of each of these partners on both the kinetics and stability of the talin1-integrin tail complex may vary with the cell type, the integrin type, the strength of the chemokine signal and the proximity between the integrin and its stimulatory GPCR.How can such postulated simultaneous bi-directional occupancy of a leukocyte integrin can be so rapidly triggered by a chemokine signal encountered during leukocyte rolling on blood vessels? An attractive mechanism for in situ facilitation of talin1 binding to the integrin β tail by chemokine signals involves chemokine triggered Gi stimulated RhoA and Rac1 GTPases and their downstream target, the PIP2 generating enzyme PIP5Kγ in the vicinity of the in situ activated integrin¹⁹ (Fig. 1). Additional talin1 molecules may also be recruited to the vicinity of this initially stimulated integrin by RIAM,²¹ an effector that associates with activated Rap-1, one of the key chemokine stimulated GTPases involved in rapid integrin mediated activation in both leukocytes and platelets.^22,23 To bidirectionally bind and activate their integrin targets, both the cytoplasmic integrin ligands, Talin1 and Kindlin-3 and the extracellular integrin ligand may need to achieve low dissociation rates from the integrin tail and headpiece, respectively. Why would an immobilized extracellular ligand be superior to soluble extracellular ligand in capacity to bi-directionally bind and activate a leukocyte integrin? The probability that a given surface-bound ligand, rather than a soluble integrin ligand would escape from its cognate integrin receptor following its dissociation is very small, since reactants in viscous medium are more likely to recombine than to diffuse apart.²⁴ Thus, surface-immobilized single integrin ligands may rebind the integrins they recenty dissociated from much more frequently than their soluble counterparts. Similarly, the cytoplasmic ligands talin1 and Kindlin may need to remain immobile once occupying their target integrin tail. Such immobilization of talin1 can be optimized by talin anchoring to the cortical cytoskeleton.²⁵ Talin may be also restricted from immediate dissociation from the integrin tail by Kindlin-3. An optimal integrin activating chemokine signal would therefore not only need to recruit and induce talin1 association with the β subunit of the target integrin and opening of the integrin clasp, but also need to keep the talin in an immobile state, and thereby maintain its low dissociation rate from its integrin tail sites.As both the integrin headpiece and the integrin subunits are predicted to undergo faster opening and separation in the presence of applied forces,^26,27 another tradeoff of this postulated immobilization of both the intracellular and extracellular integrin ligands is optimal force sensing of the integrin heterodimer. Application of force on the bidirectionally occupied integrin and its cognate ligands would be possible only if the integrin, its extracellular ligand, and talin1 are all properly anchored.^3,28,29 Force transduction through the integrin-talin1 complex can transmit additional conformational changes on the integrin-occupied talin by exposing vinculin binding sites on the talin rod.³⁰ Additional chemokine signals may induce talin rod phosphorylation and other changes in actin-talin associations (Fig. 1) that may further facilitate talin anchorage to the cortical cytoskeleton and subsequent microclustering of adjacent ligand-occupied integrins. It is well recognized that ligand occupancy anchors integrins to the cortical cytoskeleton.³¹ Thus, the anchorage states of both the extracellular and the cytoplasmic ligands of a given integrin may facilitate bidirectional integrin occupancy and optimize force driven bi-directional activation of the integrin-ligand complex and subsequent dimerization of ligand-occupied integrins. The ability of different integrin-ligand complexes to undergo diverse mechanochemical rearrangements provides a broad spectrum of integrin-ligand bond strengths, accounting for the unique capacity of chemokine stimulated leukocyte integrins to support both firm and reversible adhesions of leukocytes to their endothelial ligands.     

Genetic Analyses of Integrin Signaling

The development of multicellular organisms, as well as maintenance of organ architecture and function, requires robust regulation of cell fates. This is in part achieved by conserved signaling pathways through which cells process extracellular information and translate this information into changes in proliferation, differentiation, migration, and cell shape. Gene deletion studies in higher eukaryotes have assigned critical roles for components of the extracellular matrix (ECM) and their cellular receptors in a vast number of developmental processes, indicating that a large proportion of this signaling is regulated by cell-ECM interactions. In addition, genetic alterations in components of this signaling axis play causative roles in several human diseases. This review will discuss what genetic analyses in mice and lower organisms have taught us about adhesion signaling in development and disease.Almost all cells in multicellular organisms are surrounded by a three-dimensional organized meshwork of macromolecules that constitute the extracellular matrix (ECM). The ECM is a dynamic structure that is generated and constantly remodeled by cells that secrete and manipulate its components into a precise configuration. It functions as a structural framework that provides cells with positional and environmental information, but also forms specialized structures such as cartilage, tendons, basement membranes (BM), bone, and teeth. In addition to its structural properties, the ECM acts as a signaling platform that regulates a large number of cellular functions. It is capable of binding growth factors, chemokines, and cytokines thereby modulating their bioavailability and activity. On the other hand, the ECM is recognized by multiple cell surface receptors that transmit information from the extracellular environment by propagating intracellular signals (for a review, see Hynes 2009).The major cell surface receptors that recognize and assemble the ECM are integrins. Integrins are heterodimeric transmembrane proteins composed of α and β subunits. Eighteen α subunits and eight β subunits can assemble in 24 different combinations with overlapping substrate specificity and cell-type-specific expression patterns (Hynes 2002; Humphries et al. 2006). This, together with the ability of different heterodimers to assemble specific intracellular signaling complexes, provides multiple layers of signaling specificity to these receptors. Conversely, the integrin expression profile of a given cell type determines which ECM components it can bind. Signals arising from integrins regulate virtually all aspects of cell behavior, including cell migration, survival, cell cycle progression, and differentiation.Genetics has proven to be a powerful tool to dissect the functions of ECM–cell interactions in complex organisms. To date, all of the integrin subunits and their major ligands have been deleted in mice. Given the large variety of cellular processes regulated by adhesion signaling, it is not surprising that a significant subset of these proteins has proven to be essential for embryonic development and/or tissue maintenance. However, in addition to underlining the importance of cell-ECM interactions in development, genetic studies also revealed critical roles for tissue- and cell-type-specific modes of adhesion signaling and provided important insights into human disease.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

Matrix metalloproteinase (MMP) and TGFβ1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGFβ) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGFβ-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.Key words: wound healing, migration, matrix metalloproteinase, transforming growth factor, skin, corneaWound healing is a well-ordered but complex process involving many cellular activities including inflammation, growth factor or cytokine secretion, cell migration and proliferation. Migration of skin keratinocytes and corneal epithelial cells requires the coordinated expression of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), small GTPases, and macrophage stimulating protein (reviewed in refs. ¹ and ²). The epithelial cells in turn regulate the expression of matrix metalloproteinases (MMPs), extracellular matrix (ECM) proteins and integrins during cell migration.^1,3,4 TGF-β is a well-known cytokine involved in processes such as cell growth inhibition, embryogenesis, morphogenesis, tumorigenesis, differentiation, wound healing, senescence and apoptosis (reviewed in refs. ⁵ and ⁶). It is also one of the most important cytokines responsible for promoting the migration of skin keratinocytes and corneal epithelial cells.^3,6,7TGFβ has two quite different effects on skin keratinocytes: it suppresses their multiplication and promotes their migration. The TGFβ-induced cell growth inhibition is usually mediated by Smad signaling, which upregulates expression of the cell cycle inhibitor p21^WAF1/Cip1 or p12^CDK2-AP1 in HaCaT skin keratinocyte cells and human primary foreskin keratinocytes.^8,9 Keratinocyte migration in wounded skin is associated with strong expression of TGFβ and MMPs,¹ and TGFβ stimulates the migration of manually scratched wounded HaCaT cells.¹⁰ TGFβ also induces cell migration and inhibits proliferation of injured corneal epithelial cells, whereas it stimulates proliferation of normal corneal epithelial cells via effects on the MAPK family and Smad signaling.^2,7 Indeed, skin keratinocytes and corneal epithelial cells display the same two physiological responses to TGFβ during wound healing; cell migration and growth inhibition. However as mentioned above, TGFβ has a different effect on normal cells. For example, it induces the epithelial to mesenchymal transition (EMT) of normal mammary cells and lens epithelial cells.^11,12 It also promotes the differentiation of corneal epithelial cells, and induces the fibrosis of various tissues.^2,6The MMPs are a family of structurally related zinc-dependent endopeptidases that are secreted into the extracellular environment.¹³ Members of the MMP family have been classified into gelatinases, stromelysins, collagenases and membrane type-MMPs (MT-MMPs) depending on their substrate specificity and structural properties. Like TGFβ, MMPs influence normal physiological processes including wound healing, tissue remodeling, angiogenesis and embryonic development, as well as pathological conditions such as rheumatoid arthritis, atherosclerosis and tumor invasion.^13,14The expression patterns of MMPs during skin and cornea wound healing are well studied. In rats, MMP-2, -3, -9, -11, -13 and -14 are expressed,¹⁵ and in mice, MMP-1, -2, -3, -9, -10 and -14 are expressed during skin wound healing.¹ MMP-1, -3, -7 and -12 are increased in corneal epithelial cells during Wnt 7a-induced rat cornea wound healing.¹⁶ Wound repair after excimer laser keratectomy is characterized by increased expression of MMP-1, -2, -3 and -9 in the rabbit cornea, and MMP-2, -9 in the rat cornea.^17,18 The expression of MMP-2 and -9 during skin keratinocyte and corneal epithelial cell migration has been the most thoroughly investigated, and it has been shown that their expression generally depends on the activity of MMP-14. MMP-14 (MT1-MMP) is constitutively anchored to the cell membrane; it activates other MMPs such as MMP-2, and also cleaves various types of ECM molecules including collagens, laminins, fibronectin as well as its ligands, the integrins.¹³ The latent forms of some cytokines are also cleaved and activated by MMP-14.¹⁹ Overexpression of MMP-14 protein was found to stimulate HT1080 human fibrosarcoma cell migration.²⁰ In contrast, the attenuation of MMP-14 expression using siRNA method decreased fibroblast invasiveness,²¹ angiogenesis of human microvascular endothelial cells,²² and human skin keratinocyte migration.¹⁰ The latter effect was shown to result from lowering MMP-9 expression. Other studies have shown that EGF has a critical role in MMP-9 expression during keratinocyte tumorigenesis and migration.^23,24 On the other hand, TGFβ modulates MMP-9 production through the Ras/MAPK pathway in transformed mouse keratinocytes and NFκB induces cell migration by binding to the MMP-9 promoter in human skin primary cultures.^25,26 Enhanced levels of pro-MMP-9 and active MMP-9 have also been noted in scratched corneal epithelia of diabetic rats.²⁷There is evidence that MMP-14 activates a number of intracellular signaling pathways including the MAPK family pathway, focal adhesion kinase (FAK), Src family, Rac and CD44, during cell migration and tumor invasion.^19,20,28 In COS-7 cells, ERK activation is stimulated by overexpression of MMP-14 and is essential for cell migration.²⁹ These observations all indicate that MMP-14 plays an important role in cell migration, not only by regulating the activity or expression of downstream MMPs but also by processing and activating migration-associated molecules such as integrins, ECMs and a variety of intracellular signaling pathays.³⁰Cell migration during wound healing is a remarkably complex phenomenon. TGFβ is just one small component of the overall process of wound healing and yet it triggers a multitude of reactions needed for cell migration. It is important to know what kinds of molecules are expressed when cell migration is initiated, but it is equally important to investigate the roles of these molecules and how their expression is regulated. Despite the availability of some information about how MMPs and signaling molecules can influence each other, much remains to be discovered in this area. It will be especially important to clarify how MMP-14 influences other signaling pathways since its role in cell migration is not restricted to digesting ECM molecules but also includes direct or indirect activation of cellular signaling pathways.     

Integrin switching modulates adhesion dynamics and cell migration

When cells are stimulated to move, for instance during development, wound healing or angiogenesis, they undergo changes in the turnover of their cell-matrix adhesions. This is often accompanied by alterations in the expression profile of integrins—the extracellular matrix receptors that mediate anchorage within these adhesions. Here, we discuss how a shift in expression between two different types of integrins that bind fibronectin can have dramatic consequences for cell-matrix adhesion dynamics and cell motility.Key words: integrin, fibronectin, migration, cytoskeleton, dynamicsCells attach to the extracellular matrix (ECM) that surrounds them in specialized structures termed “cell-matrix adhesions.” These come in different flavors including “focal complexes” (small adhesions found in membrane protrusions of spreading and migrating cells), “focal adhesions” (larger adhesions connected by F-actin stress fibers that are derived from focal complexes in response to tension), “fibrillar adhesions” (elongated adhesions associated with fibronectin matrix assembly), and proteolytically active adhesions termed “podosomes” or “invadopodia” found in osteoclasts, macrophages and certain cancer cells. Common to all these structures is the local connection between ECM proteins outside- and the actin cytoskeleton within the cell through integrin transmembrane receptors. The intracellular linkage to filamentous actin is indirect through proteins that concentrate in cell-matrix adhesions such as talin, vinculin, tensin, parvins and others.¹Cell migration is essential for embryonic development and a number of processes in the adult, including immune cell homing, wound healing, angiogenesis and cancer metastasis. In moving cells, cell-matrix adhesion turnover is spatiotemporally controlled.² New adhesions are made in the front and disassembled in the rear of cells that move along a gradient of motogenic factors or ECM proteins. This balance between formation and breakdown of cell-matrix adhesions is important for optimal cell migration. Several mechanisms regulate the turnover of cell-matrix adhesions. Proteolytic cleavage of talin has been identified as an important step in cell-matrix adhesion disassembly³ and FAK and Src family kinases are required for cell-matrix adhesion turnover and efficient cell migration.^4,5 Besides regulating phospho-tyrosine-mediated protein-protein interactions within cell-matrix adhesions, the FAK/Src complex mediates signaling downstream of integrins to Rho GTPases, thus controlling cytoskeletal organization.^6,7 The transition from a stationary to a motile state could involve (local) activation of such mechanisms.Interestingly, conditions of increased cell migration (development, wound healing, angiogenesis, cancer metastasis) are accompanied by shifts in integrin expression with certain integrins being lost and others gained. Most ECM proteins can be recognized by various different integrins. For instance, the ECM protein, fibronectin (Fn) can be recognized by nine different types of integrins and most of these bind to the Arg-Gly-Asp (RGD) motif in the central cell-binding domain. Thus, cell-matrix adhesions formed on Fn contain a mixture of different integrins and shifts in expression from one class of Fn-binding integrins to another will alter the receptor composition of such adhesions. This may provide an alternative means to shift from stationary to motile.Indeed, we have found that the type of integrins used for binding to Fn strongly affects cell migration. We made use of cells deficient in certain Fn-binding integrins and either restored their expression or compensated for their absence by overexpression of alternative Fn-binding integrins. This allowed us to compare in a single cellular background cell-matrix adhesions containing α5β1 to those containing αvβ3. Despite the fact that these integrins support similar levels of adhesion to Fn, only α5β1 was found to promote a contractile, fibroblastic morphology with centripetal orientation of cell-matrix adhesions⁸ (Fig. 1). Moreover, RhoA activity is high in the presence of α5β1 and these cells move in a random fashion with a speed of around 25 mm/h. By contrast, in cells using αvβ3 instead, adhesions distribute across the ventral surface, RhoA activity is low, and these cells move with similar speed but in a highly persistent fashion.^8,9 Finally, photobleaching experiments using GFP-vinculin and GFP-paxillin demonstrated that cell-matrix adhesions containing α5β1 are highly dynamic whereas adhesions containing αvβ3 are more static.⁹Open in a separate windowFigure 1Immunofluorescence images. GE11 cells, epithelial β1 knockout cells derived from mouse embryos chimeric for the integrin β1 subunit endogenously express various av integrins, including low levels of αvβ3 and αvβ5. Ectopic expression of β1 leads to expression of α5β1 and induced α5β1-mediated adhesion to Fn (left image) whereas ectopic expression of β3 (in the β1 null background) leads to strong expression of αvβ3 and induced αvβ3-mediated adhesion to Fn (right image). Adhesions containing either α5β1 or αvβ3 show distinct distribution and dynamics (paxillin; green) and cause different F-actin organization (phalloidin; red). Cartoons: Differences in cell-matrix adhesion dynamics may be explained by differential binding of soluble Fn molecules (blue) or different molecular determinants of the interaction with immobilized Fn (red). See text for details.It has been observed that α5β1 and αvβ3 use different recycling routes. Interfering with Rab4-mediated recycling of αvβ3 causes increased Rab11-mediated recycling of α5β1 to the cell surface. In agreement with our findings, the shift to α5β1 leads to increased Rho-ROCK activity and reduced persistence of migration.¹⁰ One possible explanation for the different types of migration promoted by these two Fn-binding integrins might involve different signaling and/or adaptor proteins interacting with specific amino acids in their cytoplasmic tails. However, this appears not to be the case: α5β1 in which the cytoplasmic tails of α5 or β1 are replaced by those of αv or β3, respectively, behaves identical to wild type α5β1: it promotes a fibroblast-like morphology with centripetal orientation of cell-matrix adhesions and it drives a non-persistent mode of migration.^8,11 Together, these findings point to differences between α5β1 and αvβ3 integrins in the mechanics of their interaction with Fn, which apparently modulates intracellular signaling pathways in control of cell-matrix adhesion dynamics and cell migration.How might this work? It turns out that although α5β1 and αvβ3 similarly support cell adhesion to immobilized (stretched) Fn, only α5β1 efficiently binds soluble, folded (“inactive”) Fn.¹¹ We have proposed that such interactions with soluble Fn molecules (possibly secreted by the cell itself) may weaken the interaction with the immobilized ligand thereby causing enhanced cell-matrix adhesion dynamics in the presence of α5β1,¹¹ (Fig. 1). Preferential binding of soluble Fn by α5β1 could be explained by differences in accessibility of the RGD binding pocket between α5β1 (more exposed) and αvβ3 (more hidden) as suggested by others.¹² If this is the case, immobilization (“stretching”) of Fn apparently leads to reorientation of the RGD motif in such a way that it is easily accessed by both integrins.The issue is considerably complicated by the fact that other recognition motifs are present in the Fn central cell-binding domain. In addition to the RGD sequence in the tenth Fn type 3 repeat (IIIFn10), binding of α5β1, but not αvβ3, also depends on the PHSRN “synergy” sequence in IIIFn9.^13–15 The relative contribution of these motifs is controversial and there is structural data pointing either towards a model in which IIIFn9 interacts with α5β1 or towards a model in which IIIFn9 exerts long-range electrostatic steering resulting in a higher affinity interaction without contacting the integrin.^16,17 Cell adhesion studies have suggested that an interaction of α5β1 with the synergy region stabilizes the binding to RGD.^14,18 Such a two-step interaction may facilitate binding to full length, folded Fn for instance by altering the tilt angle between IIIFn9 and IIIFn10 leading to optimal exposure of the RGD loop, perhaps explaining why αvβ3 (which may not interact with the synergy site) poorly binds soluble Fn.Others have shown that the RGD motif alone is sufficient for mechanical coupling of αvβ3 to Fn whereas the synergy region is required to provide mechanical strength to the α5β1-Fn bond.¹⁹ It appears that the interaction of α5β1 with Fn is particularly dynamic with various conformations of α5β1 interacting with different Fn binding surfaces, including the RGD and synergy sequences as well as other regions in IIIFn9. Thus, besides the above model based on differential binding to soluble Fn molecules, differences in the complexity and dynamics of interactions with immobilized Fn that determine functional binding strength could also underlie the different dynamics of cell-matrix adhesions containing either α5β1 or αvβ3 (Fig. 1).Precisely how mechanical differences in receptor-ligand interactions result in such remarkably distinct cellular responses is poorly understood. In addition to effects on cell-matrix adhesion dynamics and cytoskeletal organization it is also associated with different activities of Rho GTPases, indicating that mechanical differences between these two integrins must translate into differential activation of intracellular signaling pathways.^8,9,11 Possibly, different adhesion dynamics due to distinct mechanisms of receptor-ligand interaction result in different patterns of F-actin organization, which, in turn, affects the formation of signaling platforms. It is also possible that differences in the extent of integrin clustering have an impact on the conformation of one or more cytoplasmic components of the cell-matrix adhesions containing either α5β1 or αvβ3. This could lead to hiding or exposing binding sites for signaling molecules (e.g., upstream regulators of Rho GTPases) or substrates. Whatever the mechanism involved, altering the integrin composition of cell-matrix adhesions through shifts in integrin expression as observed during development, angiogenesis, wound healing and cancer progression may be a driving force in the enhanced cell migration that characterizes those processes.     

Rap GTPase-mediated adhesion and migration: A target for limiting the dissemination of B-cell lymphomas?

B-cell lymphomas, which arise in lymphoid organs, can spread rapidly via the circulatory system and form solid tumors within multiple organs. Rate-limiting steps in this metastatic process may be the adhesion of lymphoma cells to vascular endothelial cells, their exit from the vasculature and their migration to tissue sites that will support tumor growth. Thus proteins that control B-cell adhesion and migration are likely to be key factors in lymphoma dissemination, and hence potential targets for therapeutic intervention. The Rap GTPases are master regulators of integrin activation, cell motility and the underlying cytoskeletal, adhesion and membrane dynamics. We have recently shown that Rap activation is critical for B-lymphoma cells to undergo transendothelial migration in vitro and in vivo. As a consequence, suppressing Rap activation impairs the ability of intravenously injected B-lymphoma cells to form solid tumors in the liver and other organs. We discuss this work in the context of targeting Rap, its downstream effectors, or other regulators of B-cell adhesion and migration as an approach for limiting the dissemination of B-lymphoma cells and the development of secondary tumors.Key words: B-cell lymphomas, Rap GTPases, extravasation, chemokines, integrins, metastasisB-cell lymphomas are frequently occurring malignancies that are often aggressive and difficult to treat. Abnormally proliferating B cells that acquire survival-promoting mutations originate within the bone marrow or the lymphoid organs but can traffic via the blood and lymphatic systems to other organs, where they can form solid tumors. A consequence of the genetic mechanisms that generate a large repertoire of antigen-detecting B-cell receptors (BCR) and antibodies is an increased frequency of chromosomal translocations and mutations that can lead to oncogenic transformation.¹ During B-cell development in the bone marrow, the vast diversity of the BCR repertoire within an individual is generated by the random rearrangement of the VDJ gene segments that encode the BCR. Subsequent to antigen binding, highly proliferating B cells within the germinal centers of secondary lymphoid organs undergo somatic hypermutation of the genes encoding the immunoglobulin portion of the BCR in order to generate antibodies of higher affinity (“affinity maturation”). These cells can also undergo a second DNA rearrangement event associated with immunoglobulin class switching. Aberrant DNA rearrangements or somatic hypermutation can lead to oncogenic transformation. As examples, translocation of the c-myc gene into the IgH locus is characteristic of Burkitt''s lymphoma whereas somatic hypermutation of genes that encode prosurvival proteins (e.g., pim-1) is associated with diffuse large B-cell lymphomas,² the most common type of non-Hodgkin lymphoma.The ability of B-cell lymphomas to spread to multiple organs reflects the migratory capacity of their normal counterparts. B cells circulate continuously throughout the body via the blood and lymphatic systems. The extravasation of B cells out of the blood and into tissues is a multi-step process that requires selectin-mediated rolling on the surface of vascular endothelial cells, intergin-mediated firm adhesion to the endothelial cells, and migration across the endothelial cell monolayer that makes up the vessel wall (Fig. 1).^3–6 These steps are orchestrated by chemokines and adhesion molecules that are displayed on the surface of the vascular endothelial cells. Chemokines initiate signaling within the B cell that results in integrin activation. The collaboration between chemokine receptor signaling and outside-in integrin signaling causes B cells to reorganize their cytoskeleton. This cytoskeletal reorganization allows B cells to spread on the surface of the vascular endothelial cells, migrate to sites suitable for extravasation (e.g., junctions between endothelial cells) and then deform themselves in order to move across the endothelial cell layer.⁷ The ability of B-cell lymphomas to follow these constitutive organ-homing cues allows them to spread to multiple organs throughout the body, making them difficult to combat. Diffuse large B-cell lymphomas are highly aggressive precisely for this reason and readily spread to the liver, kidneys and lungs.⁸ Thus, identifying key proteins that regulate the extravasation of B-cell lymphomas could suggest new therapeutic strategies for treating these malignancies.⁹Open in a separate windowFigure 1Rap activation is required for multiple steps in lymphoma dissemination. B-cell lymphomas exit the vasculature using the same mechanisms as normal B cells. Once B cells are tethered via selectin-mediated rolling, chemokines immobilized on the surface of vascular endothelial cells convert integrins to a high affinity state via a mechanism that involves activation of the Rap GTPases. This permits firm adhesion. Adhered B cells migrate across the endothelium and then send out actin-rich protrusions, which penetrate the endothelial barrier to reach the subendothelial matrix. The formation of these membrane processes, and the subsequent movement of the cells through the junctions, requires activation of the Rap, Rho and Rac GTPases. Once in the tissue, B-lymphoma cells assume a polarized morphology and can migrate towards optimal growth niches.The ubiquitously-expressed Rap GTPases are master regulators of cell adhesion, cell polarity, cytoskeletal dynamics and cell motility.¹⁰ Receptor-induced conversion of the Rap GTPases to their active GTP-bound state (Rap-GTP) allows them to bind multiple effector proteins and thereby orchestrate their localization and function. These downstream effectors of Rap-GTP control integrin activation, actin polymerization and dynamics and the formation of protrusive leading edges in migrating cells (see below and Fig. 2). In both normal B cells and B-lymphoma cell lines, signaling via chemoattractant receptors, the BCR and integrins all activate Rap.^11–13 Moreover, we have shown that chemokine-induced Rap activation is essential for the chemoattractants CXCL12 (SDF-1), CXCL13 and sphingosine-1-phosphate (S1P) receptors to stimulate B-cell migration and adhesion.^12,14 Rap activation is also important for receptor-induced actin polymerization, cell spreading and cytoskeletal reorganization in both primary B cells and B-lymphoma cells.¹⁵ These findings suggested that Rap activation might be essential for the in vivo metastatic spread of B-cell lymphomas.Open in a separate windowFigure 2The Rap GTPases are master regulators of actin dynamics, cell morphology, cell polarity and integrin-mediated adhesion. The Rap GTPases are activated subsequent to the binding of chemokines to their receptors or activated integrins to their ligands. The active GTP-bound form of Rap binds effector proteins that promote integrin activation, actin polymerization and membrane protrusion, as well as activation of the Pyk2 and FAK tyrosine kinases, which modulate cell spreading, adhesion and migration. Rap-GTP also plays a key role in establishing cell polarity and may direct membrane vesicles to the leading edge of the cell. See text for details. MTOC, microtubule-organizing center.To test this hypothesis, we suppressed Rap activation in A20 murine B-lymphoma cells, a cell line derived from an aggressive diffuse large B-cell lymphoma. We blocked Rap activation in these cells by expressing a Rap-specific GTPase-activating protein (GAP), RapGAPII, which enzymatically converts Rap1 and Rap2 proteins to their inactive GDP-bound states. Injecting stable A20/RapGAPII and A20/empty vector transfectants intravenously into mice showed that Rap activation was required for these cells to form solid lymphomas within organs such as the liver.¹⁶ Solid tumor formation was delayed and reduced when A20/RapGAPII cells were injected instead of A20/control cells. Strikingly, the lymphoma cells isolated from the tumors that developed in mice injected with A20/RapGAPII cells had downregulated RapGAPII expression and regained the ability to activate Rap. Thus tumor formation reflected a strong in vivo selection for lymphoma cells capable of activating Rap. This indicates that Rap-dependent signaling is critical for the metastatic spread of B-cell lymphomas.The ability of B-lymphoma cells to exit the vasculature and migrate into the underlying tissue is likely to be a rate-limiting step in the metastasis of B-cell lymphomas. We showed that this extravasation step is a Rap-dependent process for B-cell lymphomas. To do this, we performed competitive in vivo homing assays in which differentially-labeled A20/vector and A20/RapGAPII cells were co-injected into the tail veins of mice.¹⁶ Analyses performed 1–3 days after injecting the cells showed that A20/RapGAPII cells exhibited a greatly reduced ability to arrest and lodge in the liver, compared to control cells. The liver produces large amounts of the chemokine CXCL12 and is a major site of lymphoma homing and tumor formation. More detailed studies revealed that the control A20 cells that lodged in the liver had entered the liver parenchyma and had an elongated morphology, as expected for cells that are migrating within the tissue and interacting with resident cells. In contrast, a larger fraction of the A20/RapGAPII cells were round and appeared to still be within the vasculature. These findings suggest that Rap activation is required for efficient extravasation of lymphoma cells in vivo, as had previously been shown for in T cells in vitro.¹⁷Leukocyte extravasation is a multi-step process that requires initial adhesion to the vascular endothelium followed by crawling on the luminal surface of the endothelial cells until a suitable site for migration through the endothelial barrier is located. We found that Rap activation was required for the initial adhesion of A20 cells to vascular endothelial cells in vitro.¹⁶ Whether integrin-mediated adhesion is an absolute requirement for tumor cells to arrest within organ vasculature remains an open question as tumor cells can be physically trapped in small vessels in a manner that is independent of integrins or other adhesion molecules (Freeman SA, unpublished data). In contrast, the ability of lymphoma cells to generate polarized membrane protrusions that invade junctions between vascular endothelial cells and then move through the junctions is likely to have a strong dependence on Rap-mediated integrin activation and Rap-mediated cell polarization and cytoskeletal reorganization. Indeed, we found that Rap activation was required for A20 B-lymphoma cells to form membrane projections that penetrated endothelial junctions in vitro, and for the subsequent transendothelial migration of A20 cells.¹⁶In addition to this well-characterized paracellular mode of extravasation in which leukocytes crawl across endothelial cells until they arrive at cell-cell junctions and then migrate across the endothelial cell layer, leukocytes can also extravasate via a transcellular route.¹⁸ T cells can send invadopodia through endothelial cells, which upon contacting the subendothelial matrix pull the cell through and across the endothelial cell. The paracellular and transcellular routes of leukocyte extravasation may involve distinct modes of leukocyte motility and cytoskeletal reorganization. For example, activation of WASp and Src is required for transcellular extravasation of T cells, but dispensable for paracellular extravasation.¹⁸ Our data suggest that Rap activation is involved in the paracellular extravasation of B-cell lymphomas. It is not known if lymphoma cells, which are considerably larger than normal leukocytes, can undergo transcellular extravasation, and if so, whether Rap-dependent signaling is required. Determining the relative contributions of these two modes of extravasation, as well as their underlying molecular mechanisms, could facilitate the development of therapeutic approaches for reducing lymphoma cell extravasation and dissemination.Rap GTPases are ubiquitously expressed and are involved in critical processes such as the formation of tight junctions between vascular endothelial cells.¹⁹ Therefore, targeting downstream effectors of Rap that mediate specific aspects of adhesion and migration may be a more reasonable way to limit lymphoma dissemination than targeting Rap activation. As shown in Figure 2 and reviewed by Bos,¹⁰ the effector proteins that are regulated either directly or indirectly by Rap-GTP control several modules that are critical for cell adhesion and migration.Activated Rap is an essential component of the inside-out signaling pathway by which chemokine receptors activate integrins. Rap-GTP recruits the adaptor protein RapL as well as RIAM/talin complexes to the cytoplasmic domains of integrins.^20,21 This results in conformational changes in the integrin extracellular domains that increase their affinity for adhesion molecules, such as those present on the surface of vascular endothelial cells. Actin-dependent intracellular forces exerted by talin on the integrin cytoplasmic domains also increase integrin affinity²² and may be regulated by Rap-GTP, which promotes actin polymerization (see below).Effector proteins that bind Rap-GTP include upstream activators of Rac and Cdc42,^23,24 GTPases that promote dynamic actin polymerization at the leading edge of migrating cells and at the growing ends of membrane protrusions. Activated Rac and Cdc42 act via the WASp and WAVE proteins to induce branching actin polymerization that drives membrane protrusion and the formation of lamellipodia and filopodia. Other Rap effectors, the RIAM²⁵ and AF-6 adaptor proteins,²⁶ allow Rap-GTP to recruit Ena/Vasp and profilin, proteins that prime actin monomers for incorporation into actin filaments, a rate-limiting step in actin filament assembly.The Pyk2 and FAK tyrosine kinases are key regulators of cell adhesion, cell migration and cell morphology, and we have shown that they are also downstream targets of Rap-GTP signaling.²⁷ Rap-dependent actin dynamics is critical for the activation of Pyk2 and FAK in B-lymphoma cells. Moreover the kinase activities of Pyk2 and FAK are required for B cell spreading, a key aspect of cell adhesion and motility.²⁷ The importance of this Rap/Pyk2 signaling module is supported by the observation that B cells from Pyk2-deficient mice have a severe defect in chemokine-induced migration.²⁸Rap effectors also promote the establishment of cell polarity, another key aspect of cell motility. Rap-GTP binds the evolutionarily-conserved Par3/6 polarity complex²⁹ and promotes the microtubule-dependent transport of vesicles containing integrins to the leading edge of migrating cells and to cell-cell contact sites such as immune synapses.^30,31A key question is whether modulating the expression or activity of individual targets of Rap signaling can effectively limit the dissemination of B-cell lymphomas. An exciting recent paper supports the idea that targeting proteins involved in cell motility may be an effective way to limit the spread and growth of B-cell lymphomas.⁹ Using a library of short hairpin RNAs (shRNAs) directed against 1,000 genes thought to be involved in lymphoma progression, Meachem et al. found that two regulators of the actin cytoskeleton, Rac2 and twinfilin (Twf1), were key determinants of lymphoma motility, invasiveness and progression. shRNA-mediated knockdown of either Rac2 or Twf1 expression dramatically inhibited the growth of Eµ-myc B-cell lymphomas in mice, a model for the development of human Burkitt lymphomas. The decreased lymphoma tumorgenicity, as well as the decreased ability of the lymphoma cells to engraft in the spleen and bone marrow and then metastasize to secondary sites such as the liver was associated with the cells'' inability to migrate and crawl in vitro. This is consistent with our finding that inhibiting the in vitro migration and adhesion of B-lymphoma cells by suppressing Rap activation correlated with reduced extravasation and tumor formation in vivo.The involvement of both Rap and Rac2 in lymphoma motility and dissemination may reflect the fact that these two GTPases lie in the same pathway. Rap-GTP has been shown to bind the Rac activator Vav2 and promote Rac activation.²³ Conversely, Batista and colleagues showed that Rac2 acts upstream of Rap to promote Rap activation and modulate B-cell adhesion and immune synapse formation.³² Although the interrelationship of Rap and Rac2 in B-cell lymphomas remains to be clarified, the Rac2/Rap signaling module is a potential target for limiting the spread of B-cell lymphomas. Inhibiting this Rac2/Rap module that controls B-cell motility and adhesion may reduce both the extravasation of lymphoma cells into organs as well as the ability of B-lymphoma cells to crawl to sites within the organ where they can establish a suitable metastatic niche. Migration through the subendothelial stroma to find optimal growth niches is a rate-limiting step in the dissemination of many types of tumors.³³ Blocking Rap-dependent adhesion may also prevent B-lymphoma cells from forming critical adhesive interactions with tissue-resident stromal cells. In vitro, the survival of many B-cell lymphomas depends on integrin engagement^34,35 and the subsequent activation of pro-survival signaling pathways (e.g., the PI 3-kinase/Akt pathway) by integrin signaling.³⁶ It is not known whether Rap-dependent adhesion and the ensuing integrin-mediated survival signaling are required for B-cell lymphomas to form solid tumors at secondary sites in vivo.A series of recent papers has identified the hematopoietic lineage-restricted adaptor protein kindlin-3 as a key regulator of integrin activation in leukocytes. Kindlin-3 is required for leukocyte adhesion in vitro and for in vivo extravasation,^37–39 making it a potential target for limiting the spread of B-cell lymphomas. Kindlin-3 binds to the cytoplasmic domain of several integrin beta subunits but the mechanism by which it promotes integrin activation is not known. An interesting question is whether Rap-GTP, or the RapL/RIAM/talin complexes that are recruited to integrins by Rap-GTP, regulate the localization or function of kindlin-3. Whether or not Rap and kindlin-3 act in the same pathway, it would be interesting to test whether knocking down the expression of kindlin-3 reduces the dissemination of B-cell lymphomas in either the A20 cell model we have used or the Eµ-myc B-cell lymphoma model used by Meachem et al.⁹Although we have thus far referred to the Rap GTPases collectively as “Rap,” there are five Rap GTPases in humans and mice, Rap1a, Rap1b, Rap2a, Rap2b and Rap2c, each encoded by a separate gene. Several reports have suggested distinct functions for Rap1 versus Rap2,^14,40 but it is not known to what extent the functions of the five Rap proteins are redundant or unique. Although many studies have not assessed Rap2 activation, loss-of-function approaches such as overexpressing Rap-specific GAPs or expressing the dominant-negative Rap1N17 protein may suppress the activation of all Rap proteins. Nevertheless, the possibility that different Rap proteins have distinct functions, coupled with cell type-specific differences in the expression of the Rap proteins, may present additional opportunities for targeting Rap signaling in tumor cells. Rap1b is much more abundant than Rap1a in B cells and recent work has shown that Rap1b-deficient murine B cells exhibit impaired migration and adhesion in vitro, as well as impaired in vivo homing.^41,42 If B-lymphoma cells also express much more Rap1b than Rap1a, then Rap1b could be a target for limiting the spread of these malignant B cells. An important caveat is that Rap1b is also the most abundant Rap1 isoform in platelets and plays a critical role in platelet aggregation and clotting.^43,44As master regulators of cell adhesion and migration, the Rap GTPases and the signaling pathways they control are obvious therapeutic targets for limiting the spread of B-cell lymphomas. Other signaling pathways that impact B-cell migration and adhesion, perhaps independently of Rap, are also attractive targets. Our in vivo experiments and those of Meachem et al.⁹ provide direct evidence that interfering with key regulators of adhesion and migration can dramatically limit the dissemination of B-cell lymphomas and the development of secondary tumors in critical organs. Further studies are needed to determine if this approach would be a useful therapeutic strategy for patients with B-cell lymphoma.Finally, it will be of interest to determine whether gain-of-function mutations that increase Rap signaling, or activate other pathways that promote B cell migration and adhesion, contribute to the aggressiveness of certain types of B-cell lymphomas. Increased Rap activation is associated with enhanced invasiveness in several types of tumors.^45,46 If this were true for B-cell lymphomas, then Rap-GTP levels could be a useful prognostic marker for aggressive lymphomas, in addition to being a potential therapeutic target.     

Anchoring stem cells in the niche by cell adhesion molecules

Adult stem cells generally reside in supporting local micro environments or niches, and intimate stem cell and niche association is critical for their long-term maintenance and function. Recent studies in model organisms especially Drosophila have started to unveil the underlying mechanisms of stem anchorage in the niche at the molecular and cellular level. Two types of cell adhesion molecules are emerging as essential players: cadherin-mediated cell adhesion for keeping stem cells within stromal niches, whereas integrin-mediated cell adhesion for keeping stem cells within epidermal niches. Further understanding stem cell anchorage and release in coupling with environmental changes should provide further insights into homeostasis control in tissues that harbor stem cells.Key words: stem cell, niche, anchorage, cell adhesion, extracellular matrix, cadherin, integrinTissue-specific adult stem cells are characterized by their prolonged self-renewal ability and potentiality to differentiate into one or more types of mature cells. These unique properties make stem cells essential for maintaining tissue homeostasis throughout life. It is generally believed that all adult stem cells reside in specific microenvironments named niches, which provide physical support and produce critical signals to maintain stem cell identity and govern their behavior.^1–4 Consequently, intimate stem cell and niche association is a pre-requisite for stem cell''s long-term maintenance and function. How stem cells are kept within the niche is thus an important issue in stem cell biology. Characterization of a number of stem cell niches in model organisms has led to the classification of niches into two general types: stromal niches where stem cells have direct membrane contact with the niche cells and epidermal niches where stem cells are usually associated with the extracellular matrix (ECM), and do not directly contact any fixed stromal cells.¹ Studies in Drosophila have led to the cellular and functional verification of the stem cell niche theory^5,6 and not surprisingly, have also led to the discovery of the molecular mechanisms anchoring stem cells to the niche. Here I consider recent studies in Drosophila on types of cell adhesions used to anchor stem cells in the niches, and summarize cell adhesion molecules utilized in the most characterized niches in the mammalian tissues, and suggest that cadherin-mediated cell-to-cell adhesion and integrin-mediated cell-to-ECM adhesion are possibly two general mechanisms that function in respective stromal or epidermal niches for stem cell anchorage in diverse organisms.     

A novel role for the Ste20 kinase SLK in adhesion signaling and cell migration

With over 60 members, the Sterile 20 family of kinases has been implicated in numerous biological processes, including growth, survival, apoptosis and cell migration. Recently, we have shown that, in addition to cell death, the Ste20-like kinase SLK is required for efficient cell migration in fibroblasts. We have observed that SLK is involved in cell motility through its effect on actin reorganization and microtubule-induced focal adhesion turnover. Scratch wounding of confluent monolayers results in SLK activation. The induction of SLK kinase activity requires the scaffold FAK and a MAPK-dependent pathway. However, its recruitment to the leading edge of migrating fibroblasts requires the activity of the Src family kinases. Since SLK is microtubule-associated, it may represent one of the signals delivered to focal contacts that induces adhesions turnover. A speculative model is proposed to illustrate the mechanism of SLK activation and recruitment at the leading edge of migrating cells.Key words: cell migration, cell adhesion, SLK, microtubules, adhesion turnoverCell migration is involved in multiple biological processes such as development, tissue regeneration, immune surveillance and tumor metastasis. Numerous studies reported a multitude of cellular and molecular players that take part in the signaling networks that regulate cell migration.^1,2 Recently, we reported the participation of a new member, the Ste20 serine/threonine kinase SLK, in the regulation of cell migration. We have shown that SLK is a novel adhesion disassembly signal that is activated and recruited downstream of the FAK/Src complex following scratch wound-induced migration.³ Furthermore, SLK-dependent signals are required to mediate microtubule-dependent focal adhesion tunrnover.³ These findings provide new insights into the mechanisms of cell migration and adhesion dynamics.Since sterile 20 protein (Ste20p) acts as a MAP4K in yeast, it was suggested that mammalian homologues of Ste20p also function as MAP4K.⁴ Several members of the Ste20 family of kinases have been identified in mammals and implicated in various biological processes such as stress responses, cell death and cytoskeletal reorganization.⁵ We and others previously identified a novel Ste20-related kinase termed SLK, which is a part of a signaling pathway mediating c-Jun terminal kinase 1 (JNK1) activation and apoptosis in cultured fibroblasts.^6–8 In addition, recent reports showed that SLK is involved in C2C12 myoblast differentiation and plays a role in cell cycle progression.^9,10 SLK is ubiquitously expressed, but during embryogenesis it is highly enriched in muscle and neuronal tissues.¹¹ It has been shown that SLK is associated with the microtubule cytoskeleton and we have demonstrated that SLK-induced disassembly of actin stress fibers can be inhibited by dominant negative Rac1.^12–14Recently, SLK was identified as a member of a new signaling pathway that induce vasodilatation in response to angiotensin II type 2 receptor activation.¹⁵ It was reported that SLK negatively regulates RhoA-dependent functions by phosphorylation of RhoA at Ser188.¹⁵ These findings suggest that SLK represents a novel relaxation signal involved in cytoskeletal remodeling and cell migration.We have observed that SLK is recruited to the leading edge of migrating fibroblasts by a mechanism involving c-Src signaling.³ The molecular mechanism regulating SLK recruitment is still unclear but is likely to implicate the association of SLK with another protein. The translocation of SLK could involve a microtubule-dependent mechanism leading to its redistribution to peripheral adhesions, using actin stress fibers as tracks. The Rho GTPases have been shown to be important in the targeting of signaling components, such as c-Src, to specific adhesion sites.^16,17 Whether SLK recruitment to the leading edge requires the Rho GTPases remains to be investigated. The Rho-mDia pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and is responsible for delivering APC/Cdc42 and c-Src to their respective sites of action.¹⁸ One attractive possibility is that mDia facilitates SLK-microtubule translocation in a c-Src dependent manner.Integrin molecules which link the extracellular matrix to the intracellular machinery are key players in initiating polarized cell migration into the wound. We investigated SLK activity in a scratch-induced migration model and have been able to decipher various signaling components regulating SLK activation.³ Using knockdown and dominant negative approaches, we showed that SLK is required for microtubule-dependent focal adhesion turnover and cell migration downstream of the FAK/Src complex.³The molecular mechanisms by which microtubules contribute to cell migration have been intensively studied. Geiger''s group provided the first demonstration that cytoskeletal modulation, such as microtubule disruption, triggers integrin-dependent signaling in the absence of external growth factor stimulation.¹⁹ The authors suggested that the involvement of microtubules in adhesion dependent signaling is related to microtubule interaction with the contractile actin-myosin system.¹⁹ By using a nocodazole washout system, it was shown that FAK and the GTPase dynamin are required for microtubule-induced focal adhesion disassembly.²⁰Adhesion turnover involves a number of adapters and signaling molecules, most of which are engaged in FAK signaling pathways.²¹ FAK stimulates adhesion disassembly through a signaling pathway that includes extracellular signal-regulated kinase (ERK) and myosin light chain kinase (MLCK).²² Our data have shown that SLK is activated downstream of FAK/Src/MAPK signaling, suggesting that SLK may be a new target of this pathway that leads to adhesion disassembly. Furthermore, if RhoA is a bona fide substrate for SLK in fibroblasts, then by phosphorylating and inhibiting RhoA, SLK could tilt the Rho/Rac antagonistic interplay toward relaxation and adhesion disassembly. Downstream targets of FAK and Src kinase activity often regulate the recruitment of adapter and structural protein complexes to adhesions.²² The integration of molecules such as zyxin, α-actinin or paxillin into focal contacts can lead to their stabilization and maturation into focal adhesions.²² Interestingly, depending on their phosphorylation state, these components can promote adhesion destabilization and turnover. Therefore, it is tempting to speculate that activated SLK at the leading edge may phosphorylate key signaling components to induce adhesion turnover.A recent study has shown that the frequency of microtubule catastrophes is higher at focal adhesion sites and this event leads to a local release of microtubule regulatory proteins, such as GEF-H1 and APC.²³ Signaling molecules that are released from the microtubules at adhesions could directly associate with molecular factors concentrated at the adhesion plaques, such as Src, PAK and Arp2/3. Furthermore, it was speculated that microtubule catastrophe could be associated with phosphorylated paxillin-dependent protein complexes.²³ One possibility is that through the microtubule, SLK is delivered to focal contacts or adhesions where it serves as a scaffold for disassembling signals. Alternatively, SLK may be phsophorylating key signaling molecules, which ultimately leads to adhesion destabilization and turnover.Overall, our recent findings suggest that SLK is novel regulator of focal adhesion turnover and cell migration (Fig. 1). The molecular mechanisms regulating SLK activity and SLK-dependent adhesion turnover remain to be uncovered and await the identification of SLK substrates.Open in a separate windowFigure 1Model for SLK activation and recruitment at the leading edge. A proportion of SLK is microtubule-associated, likely through a microtubule-binding protein (X). Following activation of the FAK/c-Src complex, signaling through the MAPK pathway can activate and recruit the microtubule-SLK complex, inducing adhesion turnover by destabilization of the actin network or focal contacts/adhesions through an unknown mechanism. (C) denotes a cargo protein coupling the microtubule to polymerized actin. Nocodazole treatment fails to recruit SLK resulting in stable adhesions.     

v-Crk regulates membrane dynamics and Rac activation

Cell migration is an integrated process that involves cell adhesion, protrusion and contraction. We recently used CAS (Crk-associated substrate, 130CAS)-deficient mouse embryo fibroblasts (MEFs) to examined contribution made to v-Crk to that process via its interaction with Rac1. v-Crk, the oncogene product of avian sarcoma virus CT10, directly affects membrane ruffle formation and is associated with Rac1 activation, even in the absence of CAS, a major substrate for Crk. In CAS-deficient MEFs, cell spreading and lamellipodium dynamics are delayed; moreover, Rac activation is significantly reduced and it is no longer targeted to the membrane. However, expression of v-Crk by CAS-deficient MEFs increased cell spreading and active lamellipodium protrusion and retraction. v-Crk expression appears to induce Rac1 activation and its targeting to the membrane, which directly affects membrane dynamics and, in turn, cell migration. It thus appears that v-Crk/Rac1 signaling contributes to the regulation of membrane dynamics and cell migration, and that v-Crk is an effector molecule for Rac1 activation that regulates cell motility.Key words: v-Crk, Rac, lamellipodia dynamics, cell migration, p130CASCell migration is a central event in a wide array of biological and pathological processes, including embryonic development, inflammatory responses, angiogenesis, tissue repair and regeneration, cancer invasion and metastasis, osteoporosis and immune responses.^1,2 Although the molecular basis of cell migration has been studied extensively, the underlying mechanisms are still not fully understood. It is known that cell migration is an integrated process that involves formation of cell adhesions and/or cell polarization, membrane protrusion in the direction of migration (e.g., filopodium formation and lamellipodium extension), cell body contraction and tail detachment.^1–3 Formation of cell adhesions, including focal adhesions, fibrillar adhesions and podosomes are the first step in cell migration. Cell adhesions are stabilized by attachment to the extracellular matrix (ECM) mediated by integrin transmembrane receptors, which are also linked to various cytoplasmic proteins and the actin cytoskeleton, which provide the mechanical force necessary for migration.^2,4 The next steps in the process of cell migration are filopodium formation and lamellipodium extension. These are accompanied by actin polymerization and microtubule dynamics, which also contribute to the control of cell adhesion and migration.⁵Focal adhesions are highly dynamic structures that form at sites of membrane contact with the ECM and involve the activities of several cellular proteins, including vinculin, focal adhesion kinase (FAK), Src family kinase, paxillin, CAS (Crk-associated substrate, p130CAS) and Crk.⁶ A deficiency in focal adhesion protein is associated with the severe defects in cell motility and results in embryonic death. For example, FAK deficiency disrupts mesoderm development in mice and delays cell migration in vitro,⁷ which reflects impaired assembly and disassembly the focal adhesions.⁸ In addition, mouse embryonic fibroblasts (MEFs) lacking Src kinase showed a reduced rate of cell spreading that resulted in embryonic death.⁹ Taken together, these findings strongly support the idea that cell adhesion complexes play crucial roles in cell migration.CAS is a hyperphosphorylated protein known to be a major component of focal adhesion complexes and to be involved in the transformation of cells expressing v-Src or v-Crk.¹⁰ CAS-deficient mouse embryos die in utero and show marked systematic congestion and growth retardation,⁴ while MEFs lacking CAS show severely impaired formation and bundling of actin stress fibers and delayed cell motility.^4,11,12 Conversely, transient expression of CAS in COS7 cells increases cell migration.¹¹ Crk-null mice also exhibit lethal defects in embryonic development,¹³ which is consistent with the fact that CAS is a major substrate for v-Crk, and both CAS and v-Crk are necessary for induction of cell migration.¹⁴ v-Crk consists of a viral gag sequence fused to cellular Crk sequences, which contain Src homology 2 (SH2) and SH3 domains but no kinase domain, and both CAS and paxillin bind to SH2 domains.^12,15,16 Despite the absence of a kinase domain, cell expressing v-Crk show upregulation of tyrosine phosphorylation of CAS, FAK and paxillin, which is consistent with v-Crk functioning as an adaptor protein.¹⁷ Moreover, this upregulation of tyrosine phosphorylation correlates well with the transforming activity of v-Crk.¹⁷ By contrast, tyrosine phosphorylation of FAK and CAS is diminished in Src kinase-deficient cells expressing v-Crk, and they are not targeted to the membrane, suggesting v-Crk signaling is Src kinase-dependent. After formation of the CAS/v-Crk complex, v-Crk likely transduces cellular signaling to Src kinase and FAK.¹² Notably, tyrosine phosphorylation of FAK and cell migration and spreading are all enhanced when v-Crk is introduced into CAS-deficient MEFs.¹² We therefore suggest that v-Crk activity, but not cellular Crk activity, during cell migration and spreading is CAS-independent.Membrane dynamics such as lamellipodium protrusion and membrane ruffling reportedly involve Rac1,¹⁸ α4β1 integrin,¹⁹ Arp2/3,⁶ and N-WASP,²⁰ and are enhanced in v-Crk-expressing CAS-deficient MEFs.²¹ Moreover, expression in those cells of N17Rac1, a dominant defective Rac1 mutant, abolished membrane dynamics at early times and delayed cell migration.²¹ v-Crk-expressing, CAS-deficient MEFs transfected with N17Rac1 did not begin spreading until one hour after being plated on fibronectin, and blocking Rac activity suppressed both membrane dynamics and cell migration. We therefore suggest that v-Crk is involved in cell attachment and spreading, and that this process is mediated by Rac1 activation. In addition, v-Crk expression apparently restores lamellipodium formation and ruffle retraction in CAS-deficient MEFs. Thus v-Crk appears to participate in a variety cellular signaling pathways leading to cell spreading, Rac1 activation, membrane ruffling and cell migration, even in the absence of CAS, its major substrate protein.In fibroblasts, the Rho family of small GTP-binding proteins (e.g., Cdc42, Rac and Rho) functions to control actin cytoskeleton turnover, including filopodium extension, lamellipodium formation and generation of actin stress fibers and focal adhesions.²² These GTPases function in a cascade, such that activation of Cdc42 leads to activation of Rac1, which in turn activates Rho.²² Once activated, Rho controls cell migration. Cell adhesion to ECM leads to the translocation of Rac1 and Cdc42 from the cytosol to the plasma membrane,²³ where they regulate actin polymerization at the leading edge.^19,24 Dominant negative Rac and Cdc42 mutants inhibit the signaling to cell spreading initiated by the interaction of integrin with ECM.²⁴ The fact that cellular levels of activated Rac are higher in cells adhering to ECM than in suspended cells further suggests that activation of Rac and Cdc42 is a critical step leading to membrane protrusion and ruffle formation. It is noteworthy in this regard that v-Crk is able to induce Rac activation and its translocation to plasma membrane.²¹Overall, the findings summarized in this article demonstrate that v-Crk participates in several steps leading to cell adhesion and spreading (Fig. 1), and the targeting of v-Crk to focal adhesion sites appears to be a prerequisite for regulation of cell migration and spreading via Rac activation. To fully understand its function, however, it will be necessary to clarify the role of v-Crk in Rac1 and Cdc42 activation initiated by integrin-ECM interactions.Open in a separate windowFigure 1Schematic diagram of v-Crk signaling in MEFs. Cell adhesion signaling initiated by the integrin-ECM interaction triggers v-Crk signaling mediated by Src kinase, after which focal adhesion proteins are tyrosine phosphorylated. These events lead to translocation of Rac from the cytosol to the membrane, where it promotes membrane protrusion and ruffle formation. Under basal conditions, Rac is bound with GDP and is inactive. Upon stimulation, Rac activation is mediated by guanine nucleotide exchange factors (GEFs) that stimulate the release of bound GDP and the binding of GTP. Activation of Rac is transient, however, as it is inactivated by GTPase activating protein (GAP).     

Is,indeed, the prion protein a Harlequin servant of “many” masters?

Tens of putative interacting partners of the cellular prion protein (PrP^C) have been identified, yet the physiologic role of PrP^C remains unclear. For the first time, however, a recent paper has demonstrated that the absence of PrP^C produces a lethal phenotype. Starting from this evidence, here we discuss the validity of past and more recent literature supporting that, as part of protein platforms at the cell surface, PrP^C may bridge extracellular matrix molecules and/or membrane proteins to intracellular signaling pathways.Key words: prion protein, PrP^C, extracellular matrix, cell adhesion molecules, neuritogenesis, p59fyn, Ca²⁺Initially, the discovery that the prion protein was the major, if not the unique, component of the prion agent causing transmissible spongiform encephalopathies (TSE)¹ has placed the protein in an extremely unfavorable light. Thereafter, however, a wealth of evidence has supported the notion that the protein positively influences several aspects of the cell physiology, and that its duality—in harboring both lethal and beneficial potentials—could be rationalized in terms of a structural switch. Indeed, the protein exists in at least two conformational states: the cellular, α helix-rich isoform, PrP^C, and the prion-associated β sheet-rich isoform, PrP^Sc.² If it is now unquestionable that the presence of PrP^C in the cell is mandatory for prion replication and neurotoxicity to occur,^3,4 nonetheless its physiologic function is still debatable, despite the long lasting effort, and the numerous, frequently genetically advanced, animal and cell model systems dedicated to the issue. From these studies the picture of an extremely versatile protein has emerged, whereby PrP^C acts in the cell defense against oxidative and apoptotic challenges, but also in cell adhesion, proliferation and differentiation, and in synaptic plasticity.^5,6 In an effort to converge these multiple propositions in an unifying functional model, different murine lines devoid of PrP^C have been studied. These animals, however, displayed no obvious phenotype,^7–9 suggesting that either PrP^C is dispensable during development and adult life or that compensative mechanisms mask the loss of PrP^C function in these paradigms. Thus, identifying the exact role of PrP^C in the cell would not only resolve an important biological question, but would also help elucidate the cellular steps of prion pathogenesis necessary for designing early diagnostic tools and therapeutic strategies for TSE.As is often the case, the employment of a model system unprecedented in prion research has recently disclosed a most interesting scenario with regards to PrP^C physiology, having unravelled, for the first time, a lethal phenotype linked to the absence of the protein.¹⁰ The paradigm is the zebrafish, which expresses two PrP^C isoforms (PrP1 and PrP2). Similarly to mammalian PrP^C, they are glycosylated and attached to the external side of the plasma membrane through a glycolipid anchor. PrP1 and PrP2 are, however, expressed in distinct time frames of the zebrafish embryogenesis. Accordingly, the knockdown of the PrP1, or PrP2, gene very early in embryogenesis impaired development at different stages, bypassing putative compensatory mechanisms. By focusing on PrP1, Malaga-Trillo et al. showed that the protein was essential for cell adhesion, and that this event occurred through PrP1 homophilic trans-interactions and signaling. This comprised activation of the Src-related tyrosine (Tyr) kinase p59fyn, and, possibly, Ca²⁺ metabolism, leading to the regulation of the trafficking of E-cadherin, a member of surface-expressed cell adhesion molecules (CAMs) responsible for cell growth and differentiation.¹¹ It was also reported that overlapping PrP1 functions were performed by PrP^Cs from other species, while the murine PrP^C was capable to replace PrP1 in rescuing, at least in part, the knockdown developmental phenotype. Apart from providing the long-sought proof for a vital role of PrP^C, the demonstration that a mammalian isoform corrected the lethal zebrafish phenotype strongly reinforces previous results—mainly obtained in a variety of mammalian primary neurons and cell lines—pointing to a functional interplay of PrP^C with CAMs, or extra cellular matrix (ECM) proteins, and cell signaling, to promote neuritogenesis and neuronal survival. A revisit of these data is the main topic of the present minireview.As mentioned, the capacity of PrP^C to act as a cell adhesion, or recognition, molecule, and to entertain interactions with proteins implicated in growth and survival, has already been reported for the mammalian PrP^C. A case in point is the interaction, both in cis- and trans-configurations, with the neuronal adhesion protein N-CAM12 that led to neurite outgrowth.¹³ Like cadherins, N-CAM belongs to the CAM superfamily. Following homo- or heterophylic interactions, it can not only mediate adhesion of cells, or link ECM proteins to the cytoskeleton, but also act as a receptor to transduce signals ultimately resulting in modulating neurite outgrowth, neuronal survival and synaptic plasticity.¹¹ Another example is the binding of PrPC to laminin, an ECM heterotrimeric glycoprotein, which induced neuritogenesis together with neurite adhesion and maintenance,^14,15 but also learning and memory consolidation.¹⁶ Further, it has been described that PrPC interacted with the mature 67 kDa-receptor (67LR) (and its 37 kDa-precursor) for laminin, and with glycosamminoglycans (GAGs), each of which is involved in neuronal differentiation and axon growth.^17–21 More recently, Hajj et al.²² have reported that the direct interaction of PrP^C with another ECM protein, vitronectin, could accomplish the same process, and that the absence of PrP^C could be functionally compensated by the overexpression of integrin, another laminin receptor.²³ Incidentally, the latter finding may provide a plausible explanation for the absence of clear phenotypes in mammalian PrP-null paradigms. By exposing primary cultured neurons to recombinant PrPs, others have shown that trans-interactions of PrP^C are equally important for neuronal outgrowth,^24,25 including the formation of synaptic contacts.²⁵ Finally, it has been demonstrated that the binding of PrP^C with the secreted co-chaperone stress-inducible protein 1 (STI1) stimulated neuritogenesis.²⁶ This same interaction had also a pro-survival effect, as did the interaction of PrP^C with its recombinant form.²⁴ Notably, the involvement of PrP^C in cell protection has been heightened by experiments with whole animals. By applying transient or permanent focal cerebral ischemia to the animals, it was found that their reduced brain damage correlated with spontaneous or adenoviral-mediated, upregulation of PrP^C,^27–29 (reviewed in ref. ³⁰), and that PrP^C deficiency aggravated their ischemic brain injury.^30,31 Thus, now that data are available from phylogenetically distant paradigms (zebrafish and mammalian model systems), it acquires more solid grounds the advocated engagement of PrP^C in homo/heterophilic cis/trans interactions to trigger signaling events aiming at neuronal—or, in more general terms, cell—survival and neuritogenesis. The latter notion is consistent with the delayed maturation of different types of PrP^C-less neurons, observed both in vitro and in vivo.^32,33If one assumes that the interaction of PrP^C with multiple partners (45 for PrP^C and PrP^Sc, as reviewed in Aguzzi et al.,⁵ or 46 considering the homophylic interaction) are all functionally significant, the most immediate interpretation of this “sticky” behavior entails that PrP^C acts as a scaffolding protein in different membrane protein complexes.^5,6 Each complex could then activate a specific signaling pathway depending on the type and maturation of cells, the expression and glycosylation of PrP^C, and availability of extra- and intra-cellular signaling partners. At large, all these signals have been shown to be advantageous to the cell. However, because in a cell only a subtle line divides the “good” from the “bad,” instances can be envisioned in which a pro-life signal turns into a pro-death signal. A typical example of this possibility is glutamate excitotoxicity resulting in dangerous, glutamate receptor-linked, Ca²⁺ overload. Likewise, an excessive or over-stimulated signal elicited by PrP^C, or by the putative complex housing the protein could become noxious to the cell. This possibility may explain why the massive expression of PrP^C caused degeneration of the nervous system,³⁴ and of skeletal muscles,^34,35 in transgenic animals. More intriguing is the finding that—in a mouse line expressing anchorless PrP^C—PrP^Sc was capable to replicate without threatening the integrity of neurons.³⁶ This may suggest that native membrane-bound PrP^C acts as, or takes part into, a “receptor for PrP^Sc”, and that lasting PrP^Sc-PrP^C interactions distort the otherwise beneficial signal of the protein/complex and cause neurodegeneration.³⁷ Consistent with this hypothesis is the finding that the in vivo antibody-mediated ligation of PrP^C provoked apoptosis of the antibody-injected brain area.³⁸ Speculatively, the action of N-terminally, or N-proximally truncated PrPs whose expression in PrP-less transgenic mice induced extensive neurodegeneration,^39–41 may be traced back to the same hyper-activation of PrP^C signaling. Possibly, this may hold true also for the synaptic impairment that, recorded only in PrP^C-expressing neurons, was attributed to the binding of amyloid beta (Aβ) peptide oligomers implicated in Alzheimer disease, to PrP^C.^42,43But which is (are) the cellular signaling pathway(s) conveyed by the engagement of PrP^C in different signaling complexes? In line with its multifaceted behavior, several intracellular effectors have been proposed, including p59fyn, mitogen-activated kinases (MAPK) Erk1/2, PI3K/Akt and cAMP-PKA. p59fyn is the most reported downstream effector, suggesting that, in accordance with its behavior, p59fyn could serve as the sorting point for multiple incoming and outgoing signals also in the case of PrP^C. The initial evidence of the PrP^C-p59fyn connection came from cells subjected to antibody-mediated cross-linking of PrP^C.⁴⁴ Later, it was shown that the PrP^C-p59fyn signal converged to Erk1/2 through a pathway dependent on (but also independent of) reactive oxygen species generated by NADPH oxidase.⁴⁵ A PrP^C-dependent activation of p59fyn^13,25 and Erk1/2 (but also of PI3K and cAMP-PKA)²⁴ was evident in other neuronal cell paradigms and consistent with the almost ubiquitous expression of PrP^C, in non-neuronal cells such as Jurkat and T cells.⁴⁶ Not to forget that in zebrafish embryonic cells activated p59fyn was found in the same focal adhesion sites harboring PrP1.¹⁰ Regarding the activation of the ERK1/2 pathway promoted by the PrP^C-STI1 complex, and leading to neuritogenesis, the role of p59fyn was not investigated.²⁶ The same holds true for the transduction of a neuroprotective signal by the PrP^C-STI1 complex involving the cAMP-PKA pathway.²⁶ Interestingly, this is not the only example reporting that engagement of PrP^C activates simultaneously two independent pathways. In fact, possibly after transactivating the receptor for the epidermal growth factor, the antibody-mediated clustering of PrP^C was shown to impinge on both the Erk1/2 pathway, and on a protein (stathmin) involved in controlling microtubule dynamics.⁴⁷Yet, if p59fyn is implicated in mammalian PrP^C-activated signaling cascade, a protein linking extracellular PrP^C to p59fyn is needed, given the attachment of the enzyme to the inner leaflet of the plasma membrane through palmitoylated/myristoylated anchors. In this, the PrP^C partner N-CAM (isoform 140) seems ideal to fulfill the task, given that p59fyn is part of N-CAM-mediated signaling. Indeed, after recruitment of N-CAM to lipid rafts—which may also depend on PrP^C,¹³—together with the receptor protein Tyr phosphatase α (RPTPα), the Tyr-phosphate removing activity of RPTPα allows the subsequent activation of p59fyn through an autophosphorylation step.⁴⁸ This event recruits and activates the focal adhesion kinase (FAK),¹¹ another non-receptor Tyr kinase. Finally, formation of the FAK-p59fyn complex triggers neuritogenesis through both Erk1/2 and PI3K/Akt pathways.^49,50 Parenthetically, the FAK-p59fyn and PI3K/Akt connection would be suitable to explain why aggravation of ischemic brain injury in PrP-deficient brains was linked to a depressed Akt activation.³¹ FAK-p59fyn complex, however, may be also involved in the signal triggered by the still mysterious PrP^C partner, 67LR. This protein was reported not only to act as a laminin receptor but also to facilitate the interaction of laminin with integrins,⁵¹ thereby possibly activating (through integrins) FAK-p59fyn-regulated pathways.⁴⁹ Conversely, other data have supported the candidature of caveolin-1 for coordinating the signal that from PrP^C reaches Erk1/2 through p59fyn.^44,45,52 Further scrutiny of this route has shown that it comprised players such as laminin and integrins (upstream), FAK-p59fyn, paxillin and the Src-homology-2 domain containing adaptor protein (downstream), and that caveolin-1, a substrate of the FAK-p59fyn complex, facilitated the interaction of these signaling partners by recruiting them in caveolae-like membrane domains.⁵³For the relevance they bear, we need to acknowledge recent propositions supporting the commitment of PrP^C with proteins whose function is unrelated from the above-mentioned cell adhesion or ECM molecules; namely, the β-site amyloid precursor protein (APP) cleaving enzime (BACE1) and the N-methyl-D-aspartate (NMDA)-receptor. BACE1 is a proteolytic enzyme involved in Aβ production. It has been shown that overexpressed PrP^C restricted, while depletion of PrP^C increased the access of BACE1 to APP, possibly because PrP^C interacts with BACE1 via GAGs.⁵⁴ Thus, native PrP^C reduces the production of Aβ peptides. A beneficial effect of PrP^C was also highlighted by Khosravani et al.⁵⁵ showing that, by physically associating with the subunit 2D of the NMDA-receptor, PrP^C attenuated neuronal Ca²⁺ entry and its possible excitotoxic effect. This clear example for the control of PrP^C on Ca²⁺ metabolism is particularly intriguing in light of previous reports linking Ca²⁺ homeostasis to PrP^C pathophysiology (reviewed in ref. ⁵⁶). Also, it is important to mention that a few partners of PrP^C or downstream effectors may initiate signals that increase intracellular Ca²⁺, and that, in turn, local Ca²⁺ fluctuations regulate some of the afore-mentioned pathways.^11,49,57,58In conclusion, although still somehow speculative, the implication of Ca²⁺ in PrP^C-dependent pathways raises the possibility that the different input signals originating from the interaction of PrP^C with diverse partners may all converge to the universal, highly versatile Ca²⁺ signaling. Were indeed this the case, then clearly the acting of PrP^C as Harlequin, the famous character of the 18^th century Venetian playwright Carlo Goldoni, who struggles to fill the orders of two masters, would be merely circumstantial.     

Cdk5: A regulator of epithelial cell adhesion and migration

Cell adhesion is a fundamental property of epithelial cells required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of a variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.Key words: Cdk5, Src, Rho, stress fibers, epithelial cells, cell adhesion, cell migrationAnchoring of epithelial cells to their basement membrane is essential to maintain their morphology, normal physiological function and survival. Cells attach to extracellular matrix components by means of membrane-spanning integrins, which cluster and link to the actin cytoskeleton via components of focal adhesions. At focal adhesions, actin is bundled into stress fibers, multi-protein cellular contractile machines that strengthen attachment and provide traction during migration.¹ Stress fiber contraction is generated by myosin II, a hexamer containing one pair of each non-muscle heavy chains (NMHCs), essential light chains, and myosin regulatory light chains (MRLC). Myosin motor activity is regulated by phosphorylation of MRLC at Thr18/Ser19 and is required to generate tension on actin filaments and to maintain stress fibers.¹ Although a number of kinases have been identified which phosphorylate MRLC at Thr18/Ser19, the principal kinases in most cells are myosin light chain kinase (MLCK)² and Rho-kinase (ROCK),³ a downstream effector of the small GTPas, RhoA.Rho family small GTPases play a central role in regulating many aspects of cytoskeletal organization and contraction.⁴ These GTPases are subject to both positive regulation by guanine nucleotide exchange factors (GEFs), such as GEF-H1,^5,6 and negative regulation by GTPase-activating proteins (GAPs), such as p190RhoGAP.⁷ As cells spread, the Rho-family GTPase, Cdc42, is activated at the cell periphery, leading to the formation of numerous filapodia. Focal adhesion formation is first seen at the tips of these filapodia as focal adhesion proteins such as talin and focal adhesion kinase (FAK) bind to the intracellular domains of localized integrins.⁸ Src is recruited to activated FAK at the nascent focal adhesion and generates binding sites for additional focal adhesion proteins by phosphorylating FAK and paxillin.⁹ Src activity is essential for the further maturation of the focal adhesion and for activating the Rho-family GTPase, Rac, leading to Arp2/3-dependent actin polymerization, formation of a lamellipodium and extension of the cell boundary. Simultaneously, Src inhibits RhoA by phosphorylating and activating its upstream inhibitor, p190RhoGAP. As the focal adhesion matures, Src is deactivated, allowing the Rho activation necessary for mDia-dependent actin polymerization,¹⁰ myosin-dependent cytoskeletal contraction⁵ and tight adhesion to the extracellular matrix. Since new focal adhesions continually form at the distal boundary of the spreading cell, the most mature and highly contracted stress fibers are localized at the center of the cell.Cell adhesion is an essential component of cell migration: if adhesion is too weak, cells can not generate the traction necessary for migration; if it is too strong, they are unable to overcome the forces that anchor them in place. Thus, the relationship between adhesion force and migration rate is a bell-shaped curve.¹¹ Migration rate increases as adhesive strength increases until an optimum value is reached. Thereafter, increases in adhesive strength decrease migration rate. Since the strength of adhesion depends on extracellular matrix composition as well as the types of integrin expressed in the cell, a decrease in adhesive strength may result in either faster or slower cell migration.Several lines of evidence indicate that the proline-directed serine/threonine kinase cyclin dependent kinase 5 (Cdk5) plays an integral role in regulating cell adhesion and/or migration in epithelial cells.^12–17 Cdk5 is an atypical member of cyclin dependent kinase family, which is activated by the non-cyclin proteins, p35 or p39.¹⁸ Cdk5 is most abundant in neuronal cells where it also regulates migration and cytoskeletal dynamics.¹⁹ In neurons, Cdk5 exerts its effects on migration at least in part by phosphorylating FAK,¹⁹ and the LIS1 associated protein, NDEL1.²⁰ In contrast, recent findings have revealed two novel pathways involved in Cdk5-dependent regulation of migration in epithelial cells.^16,17One of these newly discovered mechanisms links Cdk5 activation to control of stress fiber contraction.¹⁶ We have found that Cdk5 and its activator, p35, co-localize with phosphorylated myosin regulatory light chain (MRLC) on centrally located stress fibers in spreading cells.¹⁶ Moreover, Cdk5 is strongly activated in spreading cells as central stress fiber contraction becomes pronounced.²¹ Since contraction of these central stress fibers is primarily responsible for tight attachment between the cell and the extracellular matrix,⁵ the above findings suggested that Cdk5 might regulate cell adhesion by regulating MRLC phosphorylation. To test this possibility we inhibited Cdk5 activity by several independent means and found that MRLC phosphorylation was likewise inhibited. In addition, we found that inhibiting Cdk5 either prevented the formation of central stress fibers or led to their dissolution. The concave cell boundaries characteristic of contracting cells were also lost.¹⁶ Since MRLC lacks a favorable site for phosphorylation by Cdk5, we asked whether Cdk5 might affect the upstream signaling pathways that regulate MRLC phosphorylation. Experiments with specific pathway inhibitors indicated that the MRLC phosphorylation involved in stress fiber contraction in lens epithelial cells was regulated largely by Rho-kinase (ROCK). Inhibiting Cdk5 activity not only significantly reduced ROCK activity, but also blocked activation of its upstream regulator, Rho. To explore the mechanism behind the Cdk5-dependent regulation of Rho, we turned our attention to p190RhoGAP, which appears to play a major role in regulating Rho-dependent stress fiber contraction.⁷ This RhoGAP must be phosphorylated by Src to be active; as a result, Rho activity is low in the early stages of cell spreading, when Src activity is high. At later times, Src activity falls, p190RhoGAP activity is lost, and Rho-GTP is formed, enabling Rho-dependent myosin phosphorylation and stress fiber contraction.^9,10 We have found that inhibiting Cdk5 activity during this later stage of cell spreading increases Src activity and Src-dependent phosphorylation of its substrate, p190RhoGAP. This in turn leads to decreased Rho activity accompanied by loss of Rho-dependent myosin phosphorylation, dissolution of central stress fibers, and loss of cell contraction (Fig. 1). Moreover, inhibiting Src protects cells from the loss of Rho activation and dissolution of central stress fibers produced by inhibiting Cdk5.¹⁶ Since the effects of Cdk5 on Rho-dependent cytoskeletal contraction appear to be mediated almost entirely through Cdk5-dependent regulation of Src, it will be particularly important to determine how Cdk5 limits Src activity.Open in a separate windowFigure 1Cdk5 inhibition reduces contraction of preformed stress fibers. (A) Cells were spread on fibronectin for 60 min to adhere, allowing them to form focal adhesion and stress fibers (pre-incubation) and then further incubated for 2 h in absence (control) or presence of Cdk5 inhibitor (olomoucine) and stained with phalloidin. The cells without olomoucine (control) had concave boundaries and well-formed stress fibers. Olomoucine treated cells showed loss of central stress fibers and failure to contract. Scale bar = 20 µ. (B) experimental conditions were same as shown in (A). Cdk5 inhibitor, olomoucine, was added after 1 h of spreading (indicated as t = 0) and cells were incubated for an additional 2h in absence or presence of olomoucine. Cell lysates were immunoblotted with antibodies for pMRLC (upper) and MRLC (middle). Tubulin was used as a loading control (lower). Lane 1: untreated (at 0 h); Lane 2: untreated (at 2 h); Lane 3: Cdk5 inhibitor (olomoucine) treated. (C) results of three independent experiments of the type shown in (B) were quantified by densitometry and normalized to determine the relative levels of pMRLC at each time. Statistical analysis demonstrated a significant (p < 0.05) decrease in pMRLC level in olomoucine treated cells compared to untreated cells.The central stress fibers regulated by Cdk5 play a central role in anchoring cells to the substratum, and their loss when Cdk5 is inhibited will reduce adhesion. As discussed above, reduction in cell adhesion may either increase or decrease the rate of cell migration, depending on the cell type and extracellular matrix composition. In lens and corneal epithelial cells, the reduction in adhesion produced by Cdk5 inhibition promotes cell migration.^13,15,16,22 Moreover, regulation of Rho/Rho-kinase signaling by Cdk5 seems to be a major factor in determining the migration rate, since inhibitors of Cdk5 and Rho-kinase increased lens epithelial cell migration rate equivalently and inhibiting both produced no additional effect.¹⁶Interestingly, an independent line of investigation has shown that this is not the only mechanism underlying Cdk5-dependent regulation of cell adhesion and migration. Cdk5 also localizes at focal contacts at the cell periphery and phosphorylates the focal adhesion protein talin.¹⁷ The talin phosphorylation site has been identified as S425, near the FERM domain in the talin head region. Upon focal adhesion disassembly, this region is separated from the talin rod domain by calpain-dependent cleavage.²³ Phosphorylation at S425 by Cdk5 blocks ubiquitylation and degradation of the talin head by inhibiting interaction with the E3 ligase, Smurf1. This leads, ultimately, to greater stability of lamellipodia and newly formed focal adhesions, thus strengthening adhesion to the substrate.¹⁷ Although the exact molecular events involved in this stabilization are not yet clear, it has been suggested that the talin head may “prime” integrins to bind full length talin.²⁴ One possible scenario describing how this might occur is shown in Figure 2. By permitting the isolated head region to escape degradation following calpain cleavage, Cdk5-dependent phosphorylation may stablize a pool of talin head domains to bind focal contacts within the lamellipodium. It is known that the isolated talin head region can bind and activate integrins during cell protrusion.²⁵ The resulting integrin activation would be expected to stabilize the lamellipodium by strengthening integrin-dependent adhesion. Since the head domain lacks sites for actin binding, which are located in the talin rod domain,²⁶ the bound head domain would have to be replaced by full length talin to enable focal adhesion attachment to the cytoskeleton.²⁵ The head domain might promote this replacement by recruiting the PIP-kinase needed to generate PI(4,5) P₂,²³ which facilitates binding of full length talin to integrin by exposing the auto-inhibited integrin binding sites.²⁷ The binding of full length talin and the resulting link between the integrins and the actin cytoskeleton would then further strengthen adhesion.²⁵ This model predicts that full length talin would bind poorly in the absence of Cdk5 activity, due to degradation of the talin head and the resulting limited availability of PI(4,5)P₂, and thus provides a possible explanation for the observed rapid turnover of peripheral focal adhesions.¹⁷ Clearly, other models may be proposed to explain the increase in adhesion produced by talin head phosphorylation, and deciding among them will be an active area for future investigation. Nonetheless, it is now certain that talin is a key substrate for Cdk5 at focal adhesions.Open in a separate windowFigure 2Mechanism of Cdk5-dependent regulation of cell adhesion and migration. Binding of p35 to Cdk5 forms the active Cdk5/p35 kinase, which regulates cell adhesion and migration in two distinct ways. Cdk5-dependent phosphorylation of the talin head domain at Ser425 prevents its ubiquitylation and degradation, allowing it to persist following calpain cleavage. The phosphorylated talin head may then bind to integrin at peripheral sites and recruit PIP-K, which converts PI(4)P to PI(4,5)P₂. PI(4,5)P₂ may promote replacement of the talin head by full length talin. Full length talin recruits other focal adhesion proteins to form the mature focal adhesion. The talin tail provides the site for the actin binding and polymerization. Polymerized actin is subsequently bundled into stress fibers. Cdk5/p35 also regulates the Rho-dependent myosin phosphorylation necessary for stress fiber stability and cytoskeletal contraction by limiting Src activity. This in turn decreases Src-dependent phosphorylation of p190RhoGAP, favoring Rho-GTP formation, Rho-dependent stress fiber polymerization, stabilization and contraction. Both pathways modulate cell migration by increasing adhesive strength.In summary, the presently available data indicate that Cdk5 has at least two distinct functions in cell adhesion (Fig. 2). On the one hand, it stabilizes peripheral focal adhesions and promotes their attachment to the cytoskeleton by phosphorylating the talin head. On the other hand, once the actin cytoskeleton has been organized into stress fibers, Cdk5 enhances the Rho activation essential for stability and contraction of central stress fibers by limiting Src activity. The discovery that Cdk5 is involved in two separate events required for efficient migration, suggests that it may coordinate multiple signaling pathways. The known involvement of Cdk5 and its activator, p35, in regulating microtubule stability suggests yet another mechanism by which Cdk5 activity may regulate cytoskeletal function. Microtubules are closely associated with stress fibers²⁸ and their depolymerization has been shown to release the Rho activating protein, GEF-H1, leading to Rho activation and Rho-dependent myosin contraction.⁶ Since cell adhesion and migration play an important role in the progression of many pathological conditions, Cdk5, its substrates and its downstream effectors involved in cell adhesion may provide novel targets for therapeutic intervention.^15,29     

Regulation of cancer invasiveness by the physical extracellular matrix environment

Long-term clinical outcomes are dependent on whether carcinoma cells leave the primary tumor site and invade through adjacent tissue. Recent evidence links tissue rigidity to alterations in cancer cell phenotype and tumor progression. We found that rigid extracellular matrix (ECM) substrates promote invasiveness of tumor cells via increased activity of invadopodia, subcellular protrusions with associated ECM-degrading proteinases. Although the subcellular mechanism by which substrate rigidity promotes invadopodia function remains to be determined, force sensing does appear to occur through myosin-based contractility and the mechanosensing proteins FAK and p130^Cas. In addition to rigidity, a number of ECM characteristics may regulate the ability of cells to invade through tissues, including matrix density and crosslinking. 3-D biological hydrogels based on type I collagen and reconstituted basement membrane are commonly used to study invasive behavior; however, these models lack some of the tissue-specific properties found in vivo. Thus, new in vitro organotypic and synthetic polymer ECM substrate models will be useful to either mimic the properties of specific ECM microenvironments encountered by invading cancer cells or to manipulate ECM substrate properties and independently test the role of rigidity, integrin ligands, pore size and proteolytic activity in cancer invasion of various tissues.Key words: cancer, invasion, invadopodia, rigidity, mechanotransduction, microenvironmentIn multicellular organisms, cells must sense and respond to multiple cues for proper functioning within tissues. Although most experimental research has focused on the regulation of cellular processes by external chemical signals, there is increasing recognition that mechanical forces also regulate critical cellular functions. Indeed, rigidity of the extracellular environment has been shown to regulate such diverse processes as muscle cell differentiation, stem cell lineage fate, breast epithelial signaling and phenotype, and fibroblast motility.^1–5In breast cancer, accumulating evidence suggests a role for tissue rigidity in promoting both the formation and invasiveness of tumors. Mammographic density of breast tissue has been correlated with increased cancer risk and included in models to predict the likelihood of in situ and invasive breast cancers.⁶ Histologically, dense breast tissue has increased stromal collagen content and in vitro analyses have shown that cancerous breast tissue is much stiffer than normal tissue (as represented by values for the elastic or Young''s moduli).^3,7 In addition, experimentally increased expression of collagen fibrils in a mouse mammary model of spontaneous breast cancer was recently shown to promote tumor formation, invasion and metastasis.⁸ Therefore, both clinical and animal data suggest a correlation between tissue density and cancer aggressiveness, and mechanical factors appear likely to play a role in this process.⁹A well-established mechanism by which extracellular matrix (ECM) rigidity signals can drive phenotypic transformations is through mechano-signal transduction (mechanotransduction) pathways in which external forces are transmitted via integrin receptors at linear focal adhesion structures to cytoskeletal and signaling proteins inside the cell. Actomyosin contractility leads to stretching and activation of proteins such as talin, p130^Cas and potentially focal adhesion kinase (FAK).^10–12 For example, stem cell lineage was found to be dependent on formation of cellular focal adhesions and actomyosin contractility in response to substrate tensile properties.² Mammary epithelial cells grown on compliant matrices will differentiate and polarize to form lactating 3-D structures that resemble in vivo acini but fail to do so on stiff matrices due to increased cytoskeletal contractility.³ Activation of mechanotransduction molecules, such as FAK, Rho and ROCK, are required for the rigidity-induced phenotype changes.^3,5 Using polyacrylamide (PA) gel systems, Yu-li Wang''s group found that rigid substrates induce fibroblast and epithelial cells to migrate away from each other instead of aggregating to form tissue-like structures.¹³ This transformation in phenotype is characteristic of the epithelial to mesenchymal transition and thought to be crucial for tumor cell migration.¹⁴A critical feature of tumor aggressiveness is the ability to invade across tissue boundaries, through degradation of ECM. The subcellular structures responsible for this invasive activity are thought to be invadopodia: actin-rich, finger-like cellular protrusions that proteolytically degrade local ECM. These structures are characteristic of invasive cells and have been implicated in tumor cell metastasis due to their association with ECM degradation.¹⁵ Similar structures, podosomes, are formed in src-transformed cells, as well as normal cells such as osteoclasts and dendritic cells that need to degrade matrix and/or cross tissue boundaries.¹⁶ In addition to mediating ECM degradation, podosomes have been postulated to function as adhesion structures, since well-characterized adhesion proteins localize to podosomes and many podosome-expressing cells no longer express focal adhesions.¹⁷ Furthermore, podosomes have been shown to be essential for chemotactic motility and transendothelial migration, although not for chemokinetic motility.^18,19We recently found that ECM rigidity increases both the number and activity of invadopodia, and this effect was dependent on the cellular contractile machinery (Fig. 1A).²⁰ Consistent with a role for mechanotransduction in this process, we found localization of the active, phosphorylated forms of the mechanosensing proteins FAK and p130^Cas in actively degrading invadopodia and an increase in invadopodia-associated degradation in breast cancer cells overexpressing FAK and p130^Cas. These results suggest that in breast cancer, increases in tissue rigidity may directly lead to increased cellular invasiveness and tumor progression.Open in a separate windowFigure 1Potential rigidity sensing mechanisms by invadopodia. (A) Invadopodia are typically identified by colocalization of fluorescent antibodies for actin and cortactin at puncta that correspond to areas of ECM degradation visualized as dark regions in FITC-labeled fibronectin (Fn) overlaying gelatin. In this case, ECM was layered on top of either soft (storage modulus = 360 Pa) or hard (storage modulus = 3,300 Pa) polyacrylamide gels (PA) to determine if invadopodia activity was regulated by differences in mechanical properties. On hard PA, invasive MCF10ACA1d breast carcinoma cells produced more invadopodia and degraded more ECM than on soft PA. Yellow arrows indicate examples of invadopodia. (B) The localization of rings of the contractile protein myosin IIA (myoIIA) surrounding invadopodia (actin puncta) suggests a role for these structures in mechanosensing by potentially linking invadopodia with the contractile apparatus to detect differences in substrate rigidity. An example ring structure is indicated with a yellow arrow and shown in the zoomed portion of the myosin IIA image, and an example of no or weak localization of myosin IIA with an invadopodium is indicated with the red arrow. (C) Activated forms of FAK and p130^Cas localize to invadopodia and depend on cytoskeletal contractility.²⁰ Rings of myosin IIA also frequently surround invadopodia. These results suggest that invadopodia may act as mechanosensing organelles, either directly through localized mechanoresponsiveness at the invadopodia or through longer-range connections to neighboring or even distant focal adhesions. In either case, traction forces may be generated as a result of changes in cytoskeletal tension in response to ECM properties. Alternatively, invadopodia function could be regulated in the absence of local traction forces, secondary to distant intracellular signaling that leads to alterations in whole cell phenotypic changes. (A and B) are reprinted from Current Biology, Volume 18, Nelson R. Alexander, Kevin M. Branch, Aron Parekh, Emily S. Clark, Izuchukwu C. Iwueke, Scott A. Guelcher and Alissa M. Weaver, Extracellular Matrix Rigidity Promotes Invadopodia Activity, pp. 1295–9, 2008; with permission from Elsevier.The localization of phosphorylated FAK and p130^Cas at invadopodia and the requirement for actomyosin contractility in our study suggests that invadopodia have the potential to act as mechanosensing organelles. This concept is supported by our finding that ∼40% of breast cancer cells cultured on rigid substrates had rings of myosin IIA surrounding invadopodia (Fig. 1B)²⁰ and the recent finding that similar podosome structures can exert local traction forces.²¹ In addition, a few studies have implicated integrin activity in invadopodia function as well as localized β1 and β3 integrins to invadopodia.^22–25 However, whether invadopodia can serve as tension-generating adhesion structures is controversial, in part because of the presence of both focal adhesions and invadopodia in many cancer cells (Fig. 1C).Regulation of invadopodia and podosome function is also not straightforward. Although our data,²⁰ along with results from Collin et al.,²¹ suggests that mechanical tension promotes invadopodia and podosome activity, in some systems podosome formation is promoted by a loss rather than a gain of cytoskeletal tension. That is, local cytoskeletal relaxation has been shown to promote podosome formation coincident with focal adhesion dissolution in both vascular smooth muscle cells treated with phorbol ester²⁶ and neuroblastoma cells.²⁷ A yin-yang activity between focal adhesions and podosomes has been known for many years, whereby activation of src kinase leads to both disassembly of focal adhesions²⁸ and formation of podosomes.²⁹ However, the role of tension in this process is unclear, particularly since activation of src kinase occurs downstream of mechanical stimuli³⁰ and should promote podosome/invadopodia activity, yet loss of tension apparently induces biological activities dependent on src kinase (focal adhesion disassembly and podosome formation).^26,27 For invadopodia, the role of tension is even less clear. Basic characterization studies need to be performed to establish molecular and structural differences between invadopodia and focal adhesions and to measure force profiles at the two structures. Since invadopodia have much smaller diameters compared to podosomes (50–100 nm vs ∼1 µm, respectively),^15,16 the latter task of determining traction forces may be difficult due to resolution limitations in measuring potentially tiny substrate displacements. The standard identification of invadopodia, by association of actin-rich puncta with sites of degradation of fluorescent ECM, adds another technical limitation since the thickness and fluorescence of the ECM matrix used to identify proteolytic activity may hinder visualization of embedded fluorescent beads in the underlying PA gel (displacement of beads is typically used to calculate traction forces).³¹ Thus, an important future direction should be the development of new in vitro experimental systems that have manipulable substrate properties and allow simultaneous identification of subcellular forces and proteolytic activity.The cellular response to rigidity is often characterized using PA gels with tunable stiffness in the range spanning that of normal and cancerous breast tissue (elastic moduli = 100–10,000 Pa).^3,7 PA gels will likely continue to be invaluable tools for understanding cellular responses to rigidity. However, this system is inherently simple and cannot fully replicate cellular events occurring in a complex in vivo ECM microenvironment. Given that invading breast cancer cells are likely to experience different microenvironments as they cross through the basement membrane (BM) and into neighboring collagenous stromal tissue (Fig. 2), biological hydrogels such as reconstituted basement membrane (Matrigel) and type I collagen gels are often utilized to mimic these ECM substrates. However, both of these models lack many of the chemical, physical, and mechanical characteristics of tissues found in vivo and have been recently questioned as suitable models for studying cancer cell invasion.³² Type I collagen gels have a fibrillar architecture but a low density and high porosity³³ and frequently lack crosslinking sites.³⁴ Although Matrigel contains many of the biochemical components of the BM, it is tumor-derived³⁵ and the major component is laminin-1, which is only abundant in fetal tissues.³⁶ By contrast, the major component of normal BM is type IV collagen. In addition, Matrigel is a solubilized preparation that lacks crosslinks³⁷ and a fibrillar component.³⁸ Both sparse collagen gels and Matrigel are quite compliant with Young''s moduli of ∼1,000 and ∼200 Pa, respectively;³ therefore, without further manipulation these substrates lack the rigidity required to mimic tumor-associated ECM.Open in a separate windowFigure 2Navigation of basement membranes and stromal collagen by invading cancer cells. Invasive cancer cells are thought to navigate different tissue microenvironments in the process of invasion. In order for invasion to occur, tumor cells must first breach the basement membrane, a thin and highly crosslinked specialized ECM that requires proteolytic degradation for subsequent transmigration. Once past this barrier, cells must proceed through the neighboring stroma composed of collagenous connective tissue. The meshwork in the stroma is looser and may facilitate diverse migration modes dependent on local microenvironmental conditions and cellular cohesiveness. These modes of migration include a single cell, proteinase-independent amoeboidal phenotype (left) and single cell (middle) and collective (right) proteinase-dependent mesenchymal phenotypes that locally degrade matrix at enzymatically active invadopodia. Note the absence of collagen stroma surrounding and along the migration track of proteolytically active cells. New physiologically relevant models that mimic these interactions in vitro will be useful to elucidate mechanisms of cancer cell migration and invasion in various tissues.In order to invade neighboring stromal tissue, carcinoma cells must first breach the BM, a complex, interwoven meshwork composed of type IV collagen, laminin, nidogen/entactin, and various proteoglycans and glycoproteins.³² The highly ordered and crosslinked type IV collagen network is regarded as the limiting barrier to cancer cell invasion since it forms pores on the order of 100 nm that are too small for passage of cells without proteolytic degradation of the BM.³² In addition to degradation, decreased BM synthesis may contribute to the initial steps of cancer invasion by altering the balance between BM formation and remodeling.³⁹ Once cancer cells cross the BM, they encounter stromal collagen tissue. In tumors, this desmoplastic stroma is frequently fibrotic due to increased ECM deposition and crosslinking by carcinoma-associated fibroblasts.⁹ Although controversial, cancer cells are thought to use a nonproteolytic, amoeboid mode to traverse this connective tissue;⁴⁰ therefore, different modes of migration may be necessary to traverse BM or stromal collagenous matrices (Fig. 2). However, the amoeboid phenotype has been described using either sparse collagen gels without crosslinks⁴¹ or Matrigel.⁴² In vivo, the process of invading through tumor-associated stromal collagen is likely to depend on the pore size, the crosslinking status, and whether cells are migrating collectively or individually.^34,43In light of these concerns and many others, there has been a push for more physiologically relevant in vitro models that represent closer approximations of BM or stromal collagen tissue. Successful models, whether natural or synthetic, must be able to mimic the composition, architecture and mechanical properties of the in vivo environment as well as support cell culture in ex vivo conditions. Natural substrates can be produced by cultured cells, such as the epithelial basement membranes synthesized by MDCK cells.³⁷ Alternatively, organotypic models derived from biological specimens have recently been utilized to study invasion. These materials can be based on processed biological tissue, such as detergent-extracted mouse embryo sections,⁴⁴ homogenized involution matrix,³⁸ and decellularized human dermis,⁴⁵ or on native tissue such as chick chorioallantoic membrane⁴⁶ and explanted peritoneal or mammary tissue.^34,37 In addition, the field of tissue engineering has already provided novel hybrid scaffolds and advanced tissue culturing methods that can be utilized for cancer research.⁴⁷ Biological materials developed for clinical use in tissue reconstruction and regeneration, such as small intestinal submucosa and urinary bladder matrix, are attractive candidates as new in vitro models since they maintain their tissue-like properties and have been extensively characterized.^48,49 These tissue-derived scaffolds are composed of well-defined structural and functional proteins, originally produced by cells in vivo, and maintain their complex 3-D architecture. Thus, such materials can provide an environment that recapitulates the chemical, physical and mechanical properties found in vivo.⁴⁸ In addition, synthetic materials, such as poly(ethylene glycol)-based hydrogels, will likely play a large role in cancer research since they can be designed with defined chemistries to obtain appropriate physical and mechanical properties as well as specific spatial arrangements of biologically relevant moieties on relevant length scales.^33,50 Similarly, engineered adhesive microenvironments created with microfabrication techniques can also be utilized to probe molecular and cellular phenomena.⁵¹ Due to this flexibility in fabrication, these materials are good candidates for novel in vitro models to probe the effects of specific mechanical, topographical and chemical factors on cellular migration and invasion.In summary, the physical microenvironment is increasingly recognized as a major influence on cellular phenotype. Recent data emphasizes the importance of mechanical factors in tumor progression, including cellular invasiveness. Exciting future directions include understanding how stromal and BM environments affect cellular invasiveness at multiple scales, including subcellular and molecular regulation of ECM degradation in response to ECM rigidity and the role of proteinases in crossing diverse tissue barriers. The development of novel model systems with appropriate biological and physical properties will facilitate all of these goals.     

Integrin linked kinase (ILK) expression and function in vascular smooth muscle cells

Vascular smooth muscle cell (SMC) migration and proliferation contribute to arterial wound repair and thickening of the intimal layer in atherosclerosis, restenosis and transplant vascular disease. These processes are influenced by cell adhesion to molecules present in the extracellular matrix, and regulated by the integrin family of cell-surface matrix receptors. An important signaling molecule acting downstream of integrin receptors is integrin-linked kinase (ILK), a serine/threonine kinase and scaffolding protein. ILK has been implicated in cancer cell growth and survival through modulation of downstream targets, notably Akt and glycogen synthase kinase-3β (GSK3β). Evidence also exists to establish ILK as a molecular adaptor protein linking integrins to the actin cytoskeleton and regulating actin polymerization, and this function may not necessarily depend upon the kinase activity of ILK. ILK has been implicated in anchorage-independent growth, cell cycle progression, epithelial-mesenchymal transition (EMT), invasion and migration. In addition, ILK has been shown to be involved in vascular development, tumor angiogenesis and cardiac hypertrophy. Despite the documented involvement of integrin signaling in vascular pathologies, the function of ILK has not been well characterized in the SMC response to vascular injury. This brief review summarizes and puts into context the current literature on ILK expression and function in the vascular smooth muscle cell.Key words: smooth muscle cell, migration, extracellular matrix, atherosclerosis, cytoskeletonA large body of research is dedicated to elucidating the mechanisms by which smooth muscle cells (SMCs) contribute to thickening of the arterial wall in pathologies such as atherosclerosis and restenosis. After arterial injury and during neointimal hyperplasia, SMCs undergo a phenotypic switch characterized by the transition from a quiescent to an active/synthetic phenotype, and they begin to synthesize an abundant extracellular matrix.¹ In turn, interactions between cells and the matrix govern the process of neointimal thickening.² Cell surface integrin receptors play important roles in signaling proliferative and migratory cellular responses during arterial wound repair. Integrin-linked kinase (ILK) is an important downstream mediator of integrin signaling, yet little is known of its function in the arterial response to injury.Integrin-linked kinase (ILK) was originally identified as a serine-threonine kinase binding to the cytoplasmic domain of β₁- and β₃-integrin subunits.³ ILK functions to activate Akt and inhibit glycogen synthase kinase-3β (GSK3β),^4–6 and has been implicated in cancer cell growth and survival through modulation of these downstream targets. Given its role in anchorage-independent growth, survival and cell cycle progression,⁷ epithelial-mesenchymal transition (EMT), and invasion and migration,^8,9 it is often suggested that ILK be targeted for cancer treatment.¹⁰ ILK is also involved in vascular development^11,12 and tumor angiogenesis.^13,14Concurrent studies in model organisms and cell cultures point to a role for ILK as a molecular scaffold linking integrins to the actin cytoskeleton and regulating actin polymerization.^15–17 Furthermore, this scaffolding function may be independent of the kinase activity of ILK. In C. elegans, genetic ablation of pat-4/ilk (ILK homologue) leads to severe adhesion defects, muscle detachment and embryonic lethality.¹⁵ However PAT-4/ILK does not phosphorylate GSK3β in C. elegans.¹⁵ Similarly, in Drosophila melanogaster, loss of function mutants for ILK resulted in severe embryonic muscle-attachment defects and detachment of F-actin from the cell membrane, and the muscle attachment defect was rescued by expressing a kinase-deficient ILK.^15,17 Finally, tissue-specific conditional knockout of ILK in mouse chondrocytes results in defects in the skeleton,^18,19 and inhibition of cell adhesion, spreading and cytoskeletal assembly in chondrocytes in culture.¹⁸ These deficiencies were not attributable to impaired Akt or GSK3β signaling. In fact, the importance of ILK kinase function appears to be cell type-dependent. Inhibition of ILK activity in transformed cells resulted in a decrease in Akt phosphorylation and apoptosis, but had no effect in non-transformed cell types including vascular SMCs, thus calling into question the importance of ILK as a kinase in non-cancerous cell types.²⁰We have studied the function of ILK in vascular smooth muscle cell wound repair and found that ILK acted as a scaffolding protein at focal adhesion sites.²¹ In our experiments, immunostaining of cultured SMCs revealed co-localization of ILK and paxillin at focal adhesions, a finding which is consistent with a previous study.²² Several proteins such as PINCH1, parvins and paxillin interact directly with ILK to facilitate its localization to focal adhesions and coordinate actin organization and cell spreading.^23–25 Overexpression of an ILK-binding-deficient PINCH protein in tracheal SMCs led to decreased recruitment of ILK and PINCH to focal adhesions, and decreased association between ILK, paxillin and vinculin.²⁶We hypothesized that ILK acting as a scaffolding protein might regulate the SMC response to vascular injury. To study this, we examined ILK using in vitro models mimicking vascular injury. Silencing ILK expression with siRNA decreased cell adhesion to fibronectin, and accelerated cell proliferation and wound closure.²¹ However, silencing ILK in wounded SMCs did not attenuate the increase in Akt and GSK3β phosphorylation observed after wounding.²¹ Nonetheless, we observed rearrangement of focal adhesions and stress fibers in ILK-silenced SMCs, which may have contributed to the reduced adhesion to fibronectin and enhanced cell migration and proliferation. Thus it seems that the scaffolding role of ILK may be more important for focal adhesion dynamics and remodeling in SMCs than the kinase function of ILK. These results were also surprising because they imply that ILK functions to inhibit cell growth and motility, unlike several studies which have suggested that ILK signals to increase these processes.^7,8,10To address in vivo arterial wound repair, we studied ILK expression after balloon catheter injury of the rat carotid artery. Following balloon injury, SMCs undergo a process of dedifferentiation which includes enhanced proliferation and migration from the media to the intima. We found that ILK protein expression was dramatically decreased in the media during the SMC proliferative and migratory responses.21 The rapid decrease in ILK protein expression is consistent with the effects of silencing ILK in cultured SMCs. We propose that the decrease in ILK following injury facilitates the rearrangement of focal adhesions, altering cell adhesion to facilitate SMC migration and proliferation. The decrease in ILK expression in SMCs following injury may be related to the transition of these cells to a de-differentiated state. A recent study has shown that increased ILK expression correlates with cell differentiation in the luminal layers of the epithelium in the esophagus, colon and intestines when compared to the basal layers.²⁷ ILK was also prominent in more differentiated areas of malignant tumors. In our studies, we noted an increase in ILK expression in the layers of the intima closest to the vascular lumen. This was consistent with findings in another recent study reporting increased ILK protein expression in the intima of balloon-injured rat carotid arteries in vivo and in the developing intima of human saphenous veins cultured ex vivo.²⁸ We suggest that ILK is upregulated here in coincidence with the re-establishment of SMC quiescence.In addition to maintaining stable cell adhesion to matrix, in the quiescent differentiated SMC, ILK may function to mediate contraction and aid the cell in exerting force on surrounding extracellular matrix fibers. In SMCs, ILK is localized to myofilaments, and promotes cell contraction by directly phosphorylating myosin light chain (MLC) or myosin light chain phosphatase (MLCP).^9,29,30 Alternatively, ILK may activate smooth-muscle contraction indirectly via phosphorylation and activation of MLCP inhibitors including CPI-17 and PHI-1.²⁹ Consistent with a role for ILK in mediating contraction, stimulation of tracheal SMCs with acetycholine recruits ILK and PINCH to the cell membrane, and overexpression of an ILK-binding-deficient mutant PINCH attenuated the localization of ILK at adhesion sites, and attenuated actin polymerization, the activation of the actin nucleation initiator N-WASP, and the development of tension.²⁶ ILK has also been identified as a key regulator of cardiac myocyte contractility.³¹ Likewise, ILK is required in the skeletal muscle of zebrafish for integrin-matrix adhesion to maintain the stability of muscle fibres.³² Mice with a skeletal muscle-specific deletion of ILK develop muscular dystrophy and detachment of muscle cells from basement membranes.³³ ILK mutants also showed displacement of several focal adhesion proteins and reorganization of the actin cytoskeleton.³⁴Our results after silencing ILK expression differ somewhat from a previous study of ILK in vascular SMCs. Overexpression of wild- type ILK in SMCs increased cell migration in response to stromal derived factor-1 or angiotensin II, while overexpression of a kinase-dead mutant of ILK (E359K) suppressed SMC migration in Boyden chamber assays.³⁵ In contrast to this study, we have shown the effects of inhibiting endogenous ILK by siRNA. ILK-induced quiescence of SMC may require tight regulation of intracellular ILK levels such that both its suppression and its upregulation promote cell motility.Taken together, these studies reveal that the functions of ILK are broader and more complex than originally thought. This molecule has the potential to function as an adapter protein regulating cytoskeletal assembly and signal transduction from focal adhesion sites, as a protein kinase activating several signaling axes, and as a regulator of the mitotic spindle.^36,37 The breadth of ILK function in regulating cell-matrix interactions, cytoskeletal organization and cell signaling is of great importance to normal development and disease progression. Functional studies using both kinase-deficient ILK variants and ILK siRNA will allow researchers to specifically attribute cellular behaviors to the proposed functions of ILK, and to determine their relative importance in different cells and pathologies. Based on our studies using injury models mimicking cellular events in occlusive vascular disease, we propose that ILK functions to maintain SMCs in a stationary, contractile phenotype in the normal artery. Following arterial injury, decreased ILK expression facilitates the reorganization of focal adhesions and the actin cytoskeleton, allowing for more efficient SMC migration and proliferation to establish a thickened neointima.     

Regulation of Platelet-derived Growth Factor Receptor Function by Integrin-associated Cell Surface Transglutaminase

A functional collaboration between growth factor receptors such as platelet derived growth factor receptor (PDGFR) and integrins is required for effective signal transduction in response to soluble growth factors. However, the mechanisms of synergistic PDGFR/integrin signaling remain poorly understood. Our previous work showed that cell surface tissue transglutaminase (tTG) induces clustering of integrins and amplifies integrin signaling by acting as an integrin binding adhesion co-receptor for fibronectin. Here we report that in fibroblasts tTG enhances PDGFR-integrin association by interacting with PDGFR and bridging the two receptors on the cell surface. The interaction between tTG and PDGFR reduces cellular levels of the receptor by accelerating its turnover. Moreover, the association of PDGFR with tTG causes receptor clustering, increases PDGF binding, promotes adhesion-mediated and growth factor-induced PDGFR activation, and up-regulates downstream signaling. Importantly, tTG is required for efficient PDGF-dependent proliferation and migration of fibroblasts. These results reveal a previously unrecognized role for cell surface tTG in the regulation of the joint PDGFR/integrin signaling and PDGFR-dependent cell responses.Adhesion of cells to the extracellular matrix (ECM)² regulates a wide range of cellular processes, including cell survival, growth, migration, and differentiation. A central paradigm in the field entails both physical association and functional collaboration between integrins and growth factor receptors (GFRs) in the regulation of cell responses to the ECM and soluble growth factors (1). In particular, the engagement of β1 and αvβ3 integrins with ECM ligands transiently activates platelet-derived growth factor (PDGF) receptor-tyrosine kinase even in the absence of its soluble ligands and promotes and sustains growth factor-initiated signaling by PDGFR (2). Despite a significance of this synergistic signaling, the molecular mechanisms underlying the cross-talk between the two receptor systems remain unknown. A direct or indirect association between these two types of signaling receptors may be enhanced by their co-sequestering in cholesterol-enriched membrane microdomains (3). Because integrins and receptor-tyrosine kinases share many downstream signaling targets, integrin-ECM interaction may also increase availability of signal relay enzymes and adapter proteins to receptor-tyrosine kinases by promoting their recruitment from cytosol to the plasma membrane (4).PDGF is a major survival factor, mitogen, and motogen for mesenchymal cells (5). This ligand-receptor pair is implicated in tumor-associated processes, including autocrine growth stimulation of tumor cells, tumor angiogenesis, and regulation of stromal fibroblasts (6). Atherosclerosis in the vessel wall and restenosis after angioplasty also involve hyperactivation of the PDGF-PDGFR signaling axis in vascular smooth muscle cells (7). Likewise, skin wound healing and liver, lung, and kidney fibrosis depend on PDGF-mediated signaling and cell responses (8). Importantly, ECM composition and cell-matrix interactions modulate cell responsiveness to PDGF (9).Upon binding a dimeric PDGF molecule, PDGFR undergoes dimerization and autophosphorylation of tyrosine residues in trans because of the juxtaposition of cytoplasmic tails of the receptor. Phosphorylation of the conserved tyrosine residue in the kinase domain (Tyr-849 of PDGFRα and Tyr-857 of PDGFRβ) increases catalytic activity of the kinases, whereas autophosphorylation of tyrosine residues outside the kinase domain creates docking sites for signal transduction proteins containing Src homology 2 domains. The latter include various enzymes such as phosphatidylinositol 3-kinase, phospholipase Cγ, the Src family tyrosine kinases, the tyrosine phosphatase Shp-2, and the GTPase activating protein for Ras, RasGAP. Other PDGFR binding partners including Grb2, Grb7, Nck, Shc, and Crk lacking enzymatic activity but serve adapter functions in the downstream signaling pathways (10).Previous studies revealed a transient PDGF-independent tyrosine phosphorylation of PDGFRβ in human fibroblasts during adhesion on fibronectin or collagen type I, whereas similar PDGFRβ activation response was reproduced by application of external strain to quiescent cells (2). Clustering of integrins with fibronectin-coated beads was shown to stimulate PDGFR phosphorylation in fibroblasts (11). Furthermore, fibronectin was found to promote PDGF-mediated signaling in fibroblasts by increasing association of phosphatase Shp-2 with PDGFR and limiting the time that the negative signaling regulator, RasGAP, interacts with the receptor (4). Whereas these results implicate cell-ECM interactions and integrin function in the regulation of PDGFR activity, many details of this functional cross-talk remain unknown.Tissue transglutaminase (tTG) is a multifunctional protein that possesses Ca²⁺-dependent transamidating and GTPase activities (12). On the surface of various cells, all the tTG forms stable non-covalent complexes with β1 and β3 integrins and functionally collaborates with these receptors by acting as a co-receptor for fibronectin (13). This adhesive function of tTG is involved in the assembly of fibronectin matrices and cell migration on fibronectin (14–16). tTG broadly affects integrin signaling by promoting their clustering and increasing activation of focal adhesion kinase and RhoA (13, 17). Thus, we set to examine whether signaling mediated by GFRs, which depends on the integrin function, is altered by tTG.Here we present a novel mechanistic insight into the cross-talk between integrin and PDGFR signaling pathways. We provide evidence that tTG interacts with PDGFR on the cell surface and mediates its physical association with integrins. In turn, the formation of stable integrin-tTG-PDGFR ternary complexes promotes PDGFR activation and downstream signaling, regulates the receptor turnover, and amplifies PDGFR-mediated cellular responses. These studies reveal a novel function of tTG in coupling the adhesion-mediated and growth factor-dependent signaling pathways. They suggest that this tTG activity might be involved in pro-inflammatory function of this protein in normal wound healing and tissue fibrosis (18), vascular remodeling (19), and tumor metastasis (20).