Caldesmon,an actin-linked regulatory protein,comes across glucocorticoids

The glucocorticoids (GCs), the most downstream effectors of the hypothalamic-pituitary-adrenal (HPA) axis, are the main mediators of stress response. Stress-triggered GCs as well as acute and chronic GC treatment can impair the structural plasticity and function of the brain. The exposure of perinatal animals and humans to excess stress or GCs can affect the brain development, resulting in altered behaviors in the adult offspring of animals and an increased risk of psychiatric disorders in humans. Despite the numerous studies documenting these effects, the underlying mechanism remains unclear. In this commentary we will focus on the effect of excess GCs on cortical development. We have recently showed that excess-GC-dependent retardation of the radial migration of neural progenitor cells (NPCs) is caused by the dysregulation of actin-myosin interaction via upregulation of caldesmon (CaD), an actin-linked regulatory protein. The elucidation of the molecular mechanisms that underlie the detrimental action of GCs on cortical development will expand our understanding of how stress/GCs alter the formation of neural networks and affect behaviors later in life.Key words: neuronal migration, actin-myosin interaction, cortical development, stress, psychiatric disorderIt has been well documented that the elevation of GCs in response to stress impairs the development and function of the brain.^1–3 Prenatal stress is associated with an increase in abnormal behaviors of adult offspring.² In humans, maternal stress increases the risk of psychiatric disorders, including anxiety, depression and schizophrenia, in the offspring during adolescence and adulthood.^3–6 The treatment of neonatal rats with GC transiently retards their brain development,^3,5,7 and giving GC to pregnant sheep retards fetal brain growth.⁸ Antenatal exposure to synthetic GC in humans results in a reduced cortex convolution index and surface area in infants.⁹ Thus, excessive stress or GC exposure during the perinatal stages affects the brain development and subsequently causes abnormal behaviors in experimental animals and an increased incidence of psychiatric disorders in humans. Despite the numerous studies documenting these detrimental effects of GCs, the underlying mechanism remains unclear.Cortical development progresses by two types of neuronal migration: radial and tangential.^10,11 Postmitotic NPCs in the ventricular zone (VZ) migrate radially along radial glial fibers toward the surface of the neocortex, until they reach their final destination within the cortical plate (CP). About 80–90% of all cortical neurons arise from NPCs by radial migration. The remaining cells, which include the majority of GABAergic interneurons, migrate tangentially from the ganglionic eminence to the neocortex. Several human disorders that arise from defective neuronal migration have been identified. These disorders include periventricular nodular heterotopia and lissencephaly, which are caused by mutations in the genes involved in radial migration.¹² More commonly than gene mutations, however, environmental factors such as stress-triggered GCs as described above are implicated in inducing abnormalities in brain development. Most recently, we have found that excess GCs cause a change in radial migration during cortical development via the dysregulation of actomyosin system by GC-induced upregulation of CaD.¹³     

Guiding Neuronal Cell Migrations

Neuronal migration is, along with axon guidance, one of the fundamental mechanisms underlying the wiring of the brain. As other organs, the nervous system has acquired the ability to grow both in size and complexity by using migration as a strategy to position cell types from different origins into specific coordinates, allowing for the generation of brain circuitries. Guidance of migrating neurons shares many features with axon guidance, from the use of substrates to the specific cues regulating chemotaxis. There are, however, important differences in the cell biology of these two processes. The most evident case is nucleokinesis, which is an essential component of migration that needs to be integrated within the guidance of the cell. Perhaps more surprisingly, the cellular mechanisms underlying the response of the leading process of migrating cells to guidance cues might be different to those involved in growth cone steering, at least for some neuronal populations.The migration of newly born neurons is a precisely regulated process that is critical for the development of brain architecture. Neurons arise from the proliferative epithelium that covers the ventricular space throughout the neural tube, an area named the ventricular zone (VZ). From there, newly born neurons adopt two main strategies to disperse throughout the central nervous system (CNS), designated as radial and tangential migration (Hatten 1999; Marín and Rubenstein 2003). During radial migration, neurons follow a trajectory that is perpendicular to the ventricular surface, moving alongside radial glial fibers expanding the thickness of the neural tube. In contrast, tangentially migrating neurons move in trajectories that are parallel to the ventricular surface and orthogonal to the radial glia palisade (Fig. 1). Besides their relative orientation, some of the basic mechanisms underlying the movement of cells using each of these two modes of migration are also different. For example, radially migrating neurons often use radial glial fibers as substrate, whereas tangentially migrating neurons do not seem to require their support to migrate. Even so, neurons may alternate from radial to tangential movement and vice versa during the course of their migration. This suggests that both types of migrations share common principles, in particular those directly related to the cell biology of movement (Marín et al. 2006).Open in a separate windowFigure 1.Representative migrations in the developing CNS. Multiple migrations coexist during embryonic development at different areas of the central nervous system. This schema summarizes some of these migrations during the second week of the embryonic period in the mouse. Neurons use tangential and radial migration to reach their final destination; both strategies are used by the same neurons at different stages of development (i.e., cortical interneurons in the forebrain and precerebellar neurons in the hindbrain). (IML) intermediolateral region of the spinal cord; (IO) inferior olive nucleus; (LGE) lateral ganglionic eminence; (LRN) lateral reticular nucleus; (MGE) medial ganglionic eminence; (NCx) neocortex; (OB) olfactory bulb.One of the structures that better illustrates how both types of migrations are integrated during brain development is the cerebral cortex, and so we will primarily refer to studies performed on cortical neurons for this review. The adult cerebral cortex contains two main classes of neurons: glutamatergic cortical projection neurons (also known as pyramidal cells) and GABAergic interneurons. Pyramidal cells are generated in the ventricular zone (VZ) of the embryonic pallium—the roof of the telencephalon—and reach their final position by radial migration (Rakic 2007). In contrast, cortical interneurons are born in the subpallium—the base of telencephalon—and reach the cerebral cortex through a long tangential migration (Corbin et al. 2001; Marín and Rubenstein 2001).The earliest cortical neurons form a transient structure known as the preplate, around embryonic day 10 (E10) of gestation age in the mouse. This primordial layer consists of Cajal-Retzius cells and the first cohort of pyramidal neurons, which will eventually populate the subplate. Cajal-Retzius cells, which play important roles during neuronal migration, arise from discrete pallial sources and colonize the entire surface of the cortex through tangential migration (Bielle et al. 2005; Takiguchi-Hayashi et al. 2004; Yoshida et al. 2006). The next cohort of pyramidal cells forms the cortical plate (CP) by intercalating in the preplate and splitting this primitive structure in a superficial layer, the marginal zone (MZ or layer I), and a deep layer, the subplate. The development of the neocortex progresses with new waves of neurons that occupy progressively more superficial positions within the CP (Gupta et al. 2002; Marín and Rubenstein 2003). Birth dating studies have shown that layers II–VI of the cerebral cortex are generated in an “inside-out” sequence. Neurons generated earlier reside in deeper layers, whereas later-born neurons migrate past existing layers to form superficial layers (Angevine and Sidman 1961; Rakic 1974). In parallel to this process, GABAergic interneurons migrate to the cortex, where they disperse tangentially via highly stereotyped routes in the MZ, SP, and lower intermediate zone/subventricular zone (IZ/SVZ) (Lavdas et al. 1999). Interneurons then switch from tangential to radial migration to adopt their final laminar position in the cerebral cortex (Ang et al. 2003; Polleux et al. 2002; Tanaka et al. 2003).     

The essential role of LIS1, NDEL1 and Aurora-A in polarity formation and microtubule organization during neurogensis

Lissencephaly is a devastating neurological disorder caused by to defective neuronal migration. LIS1 (or PAFAH1B1), the gene mutated in lissencephaly patients and its binding protein NDEL1 were found to regulate cytoplasmic dynein function and localization. LIS1 and NDEL1 also play a pivotal role on a microtubule regulation and determination of cell polarity. For example, LIS1 is required for the precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. On the other hand, NDEL1 is essential for mitotic entry as an effector molecule of Aurora-A kinase. In addition, an atypical protein kinase C (aPKC)-Aurora-A-NDEL1 pathway is critical for the regulation of microtubule organization during neurite extension. These findings suggest that physiological functions of LIS1 and NDEL1 in neurons have been ascribed for proteins fundamentally required for cell cycle progression and control. In turn, cell cycle regulators may exert other functions during neurogenesis in a direct or an indirect fashion. Thus far, only a handful of cell cycle regulators have been shown to play physiological cell cycle-independent roles in neurons. Further identification of such proteins and elucidation of their underlying mechanisms of action will likely reveal novel concepts and/or patterns that provide a clear link between their seemingly distinct cell cycle and neuronal functions.Key words: microtubule, mitotic kinase, neurite, cell polarity, migrationDuring the development of the mammalian central nervous system, the self-renewal of neural stem cells can occur either by symmetric cell divisions, which generate two daughter cells with the same fate, or by asymmetric cell divisions, which generate one daughter cell that is identical to the mother cell and a second, different non-stem-cell progenitor (reviewed in refs. ¹ and ²). Neural non-stem-cell progenitors typically undergo symmetric, differentiating divisions, each of which generates two neurons, which are terminally differentiated, post-mitotic cells. These post-mitotic neural progenitors migrate from their birth place at the ventricular zone to their final destinations in cortical plate (reviewed in ref. ³). Coinciding with the proper positioning of post-mitotic neurons, neurons project neurite and dendrites to targets with the assistant of molecular guidance cues in the local environment. Proper navigations of neurite and dendrite processes ensure synapse formations, which are the basis of brain function. In the series of developmental steps, the determination of neuronal polarity is critically important (reviewed in refs. ⁴ and ⁵). A polarity complex of Par3, Par6, and atypical protein kinase C (aPKC) functions in various cell-polarization events including axon formation.^6,7 GTPases that regulate actin cytoskeletal dynamics have been implicated in cell polarization. Recent findings provide insights into polarization mechanisms and show intriguing crosstalk between small GTPases and members of polarity complexes in regulating cell polarization (reviewed in ref. ⁸). Thus, determination of neuronal polarity and regulation of cytoskeletal organization are intimately related.LIS1 was identified as the first gene mutated in isolated lisssencephaly sequence (ILS), a human neuronal migration defect.^9,10 LIS1 and its binding protein, NDEL1 regulate the function and localization of cytoplasmic dynein^11,13 as part of an evolutionarily conserved pathway.^14,15 Genetic analysis of fungi displaying defective nuclei migration led to the identification of a number of genes and their protein products involved in this process. For example, mutations of nudA (coding for cytoplasmic dynein heavy chain) and genes coding for other subunits of the dynein complex inhibit nuclear migration, including nudC (mammalian NudC, mNudC), nudE (Ndel1 and Nde1) and nudF (Lis1). We recently demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules in vitro (Suppl. movies 1 and 2), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein.¹⁶ We demonstrated anterograde co-migration of cytoplasmic dynein and LIS1 (Suppl. movies 3 and 4). When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Lis1 KO cells exhibited biased distribution around the centrosome and aberrant distribution of organelles. Our favorite model is that LIS1 fixes cytoplasmic dynein on soluble microtubules in an “idling” state, thereby creating a microtubule-LIS1-dynein complex, which could be transported by kinesin to the plus-end of microtubules.Lis1 is also essential for the precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells.¹⁷ Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. We believe the role of LIS1 in promoting the anterograde transport of cytoplasmic dynein on kinesin as part of a microtubule-LIS1-dynein complex, as described in the previous paragraph, is responsible for controlling spindle orientation, since when LIS1 is reduced, cortical dynein fixed on the surface of the cell is also reduced. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells.¹⁷ Thus, Lis1 is intimiately involved in the determination of cell polarity as an effector molecule, which regulates dynein localization and/or function as well as microtubule organization.Interestingly, more than half of LIS1 protein is degraded at the cell cortex after transport to the plus-end of MTs via calpain-dependent proteolysis. We recently demonstrated that inhibition or knockdown of calpain protects LIS1 from proteolysis resulting in the augmentation of LIS1 levels in Lis1^+/− mouse embryonic fibroblast (MEF) cells, which leads to rescue of the aberrant distribution of cytoplasmic dynein and intracellular components including mitochondria and β-COP positive vesicles.¹⁸ We also showed that presence of calpain inhibitors improves neuronal migration of Lis1^+/− cerebellar granular neurons.¹⁸ This study demonstrates that stabilization of proteins in disorders caused by haploinsufficiency is a potential therapeutic strategy and provides a proof-of-principle for this notion.NDEL1, a binding partner of LIS1 is also essential for the regulation of cytoplasmic dynein and microtubule organization.^12,13 In particular, NDEL1 is phosphorylated by cyclin dependent kinase1 (CDK1) in mitotic cells, or CDK5 in post-mitotic neurons, and this phosphorylation is essential for proper targeting of NDEL1 binding proteins to the centrosome.¹⁹ NDEL1 is also a substrate of the mitotic kinase Aurora-A, by which NDEL1 connects Aurora-A to other target molecules for the regulation of microtubule organization.²⁰ Interestingly, NDEL1 is differentially phosphorylated by Aurora-A and CDK1. It is possible that distinct pools of NDEL1 may be targeted by each kinase, or conversely the affects of each kinase may counteract each other within the same pool of NDEL1.Aurora-A is a one of representative mitotic kinase, whose homologues have been reported in various organisms including yeast, nematodes, fruit flies and vertebrates (reviewed in ref. ²¹). The three human homologues of Aurora kinases (A, B and C) are essential for proper execution of various mitotic events and are important for maintaining genomic integrity. Aurora-A is mainly localized at spindle poles and the mitotic spindle during mitosis, where it regulates the functions of centrosomes, spindles and kinetochores required for proper mitotic progression. In particular, Aurora-A plays a pivotal role on microtubule reorganization during remodeling from interphase microtubules to mitotic microtubules, i.e., the mitotic spindle. We recently reported molecular and cell biological data that support a unique role of aPKC-Aurora-A-NDEL1 pathway on microtubule dynamics at the neurite hillock during neurite extension.²² PKCζ phosphorylates Aurora-A at T287 and activates it, which augments interaction with TPX2 and facilitates activation of Aurora-A at the neurite hillock, followed by S251 phosphorylation of NDEL1 and recruitment. Inhibition of PKCζ/λ, depletion of Aurora-A and disruption of Ndel1 severely affected neurite extension and microtubule dynamics, suggesting that the aPKC-Aurora-A-NDEL1 pathway is an important regulatory system of microtubule oranization within neurite processes (Fig. 1A).Open in a separate windowFigure 1Models of microtubule remodeling. (A) Neurite extension: an unknown upstream cue polarity activates aPKC followed by T287 phosphorylation of Aurora-A. T287 phosphorylation of Aurora-A facilitates binding of the Aurora-A activator, TPX2 resulting in activation of Aurora-A at the neurite hillock, which leads to phosphorylation of NDEL1, one of effector molecules of Aurora-A. Finally, phosphorylation of NDEL1 triggers remodeling microtubules during neurite extension. (B) Spindle formation: NDEL1 is differentially phosphorylated at T219 and Ser251 by CDK1 and Aurora-A, respectively at the beginning of mitotic entry. NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. (C) Neuronal migration: during neuronal migration, NDEL1 may be differentially phosphorylated at T219 and Ser251 by CDK5 and Aurora-A, respectively. 14-3-3ɛ might negatively regulate Aurora-A kinase.Our preliminary data suggest that Aurora-A is also activated by neurons during migration, and may further link signaling components already implicated in neuronal migration. Mice deficient in Ywhae that encondes 14-3-3ɛ have defects in brain development and neuronal migration, similar to defects observed in mice heterozygous with respect to Lis1.²³ Mice heterozygous with respect to both genes have more severe migration defects than single heterozygotes. Heterozygous deletions of 17p13.3 in human result in the human neuronal migration disorders isolated lissencephaly sequence (ILS) and the more severe Miller-Dieker syndrome (MDS). Mice carrying double heterozygous mutations of Ywhae and Lis1 are therefore thought to be a mouse model of MDS. Intriguingly, 14-3-3ɛ binds to NDEL1 after phosphorylation by CDK1/CDK5, protecting phospho-NDEL1 from phosphatase attack.14-3-3 proteins mediate multiple cellular events, including scaffolding of signaling molecules, regulation of enzyme catalysis, and subcellular targeting. In the C. elegans, 14-3-3 homolog, Par5 is required for correct anterior-posterior zygote polarization.²⁴ In addition, phosphorylation-dependent interactions between 14-3-3ɛ, and the tight junction-associated protein Par3 had been reported.²⁵ Intriguingly, 14-3-3ɛ is a centrosomal protein,²⁶ suggesting that 14-3-3ɛ, Aurora-A and NDEL1 might create a complex at the centrosome, which may then be involved in the determination of polarity and neuronal migration. These findings might be the result of the known role of Aurora-A as a regulator of microtubule network. Microtubules are emanated from MTOC, and are extended into the chromosome, nucleus or the cell periphery (Fig. 1). These microtubule flows associated with the dynamic remodeling will provide enough force to maintain a neurite process, a spindle body or a leading process.Post-mitotic neurons, however, lose their mitotic competence permanently. Intuitively, once a neural progenitor differentiates into a neuron, the post-mitotic neurons have severed all ties with the cell cycle, in which the expression of cell cycle proteins are assumed to be not expressed. Emerging evidence reveals that this holds true for a handful of core cell cycle regulators, which facilitate the differentiation and maturation of neurons, suggesting that “core“ cell cycle regulators serve diverse postmitotic functions that span various developmental stages of a neuron, including neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity (reviewed in ref. ²⁷). Among the essential kinases that function in mitosis are Aurora kinases, evolutionarily conserved serine-threonine kinases that maintain genomic stability and are required for mitotic progression. Although they share conserved regions, each member (Aurora A, B and C) contributes distinctly to cell cycle progression. Aurora-A is essential for mitotic entry, centrosome maturation during late G2 and prophase, centrosome separation during bipolar spindle assembly and mitotic spindle organization (reviewed in refs. ²¹ and ²⁸). During mitotic progression, Aurora-A loss of function prevents centrosomal separation prior to mitotic spindle formation and results in monopolar spindles.²⁹ We reported an essential role of Aurora-A during neurite extension. Wirtz-Peitz et al. reported that Aurora-A phosphorylates Par-6.³⁰ This phosphorylation cascade triggered by the activation of Aurora-A is responsible for the asymmetric localization of Numb in mitosis, which provides further evidence for crosstalk of PAR proteins and Aurora-A.³⁰ Apart from neurons, the interactions between the prometastatic scaffolding protein HEF1/Cas-L/NEDD9 and the oncogenic Aurora-A kinase at the basal body of cilia had been reported.³¹ This pathway is both necessary and sufficient for ciliary resorption and constitutes an unexpected non-mitotic activity of Aurora-A in vertebrates.Aurora-A kinase, Plk1 or CDK1 has been recognized as a mitotic kinase, which regulates mitotic entry. Cells in which these genes are mutated display defective mitotic entry. Individual proteins however, have multiple functions within specific cellular context. For example, Aurora-A may participate remodeling microtubule in mitotic spindle formation and in remodeling of microtubule organization during neurite extension or neuronal migration. Apart from existing concept, elucidation of multiple functions of cell cycle regulators will provide us with a better understanding of the extent to which they exert physiological cell cycle-independent neuronal functions.     

L1 cell adhesion molecules as regulators of tumor cell invasiveness

Fast growing malignant cancers represent a major therapeutic challenge. Basic cancer research has concentrated efforts to determine the mechanisms underlying cancer initiation and progression and reveal candidate targets for future therapeutic treatment of cancer patients. With known roles in fundamental processes required for proper development and function of the nervous system, L1-CAMs have been recently identified as key players in cancer biology. In particular L1 has been implicated in cancer invasiveness and metastasis, and has been pursued as a powerful prognostic factor, indicating poor outcome for patients. Interestingly, L1 has been shown to be important for the survival of cancer stem cells, which are thought to be the source of cancer recurrence. The newly recognized roles for L1CAMs in cancer prompt a search for alternative therapeutic approaches. Despite the promising advances in cancer basic research, a better understanding of the molecular mechanisms dictating L1-mediated signaling is needed for the development of effective therapeutic treatment for cancer patients.Key words: L1CAMs, cancer, metastasis, axon guidance, cancer stem cell, migration, invasionA major obstacle in oncology is the early diagnosis and curative therapeutic intervention of locally invasive cancers that rapidly disseminate from the primary tumor to form metastases. The standard treatment for malignant tumors consists of surgical removal of the tumor mass followed by chemo- and radiotherapy in order to eradicate the remaining cancer cells. Despite such aggressive intervention, a population of resistant cancer cells often remains intact and is thought to be the source of cancer recurrence.During the past decades, cancer basic research has focused on determining the molecular mechanisms underlying cancer initiation and progression that can provide a basis for the development of new and effective therapeutic treatments for cancer patients. An important finding was the discovery that cancer onset and development are often associated with alterations in the expression of cell adhesion molecules, which are likely to stimulate tumor cell invasiveness by signaling mechanisms that enhance cell migration.¹ The L1 family of neural cell adhesion molecules (L1-CAMs), which is comprised of four structurally related transmembrane proteins L1, CHL1, NrCAM and neurofascin (Fig. 1), is now in the spotlight of cancer research due to their upregulation in certain human tumors. L1-CAMs are transmembrane molecules of the immunoglobulin superfamily, characterized by an extracellular region of six immunoglobulin-like domains and four to five fibronectin type III repeats, followed by a highly conserved cytoplasmic domain, which is reversibly linked to the cell cytoskeleton through binding to ankyrin and ERM proteins (ezrin-radixin-moesin).² Its multi-domain structure allows complex heterophilic interactions with diverse cell receptors, although homophilic interactions also have a crucial role in L1-CAMs mediated signaling.Open in a separate windowFigure 1L1-CAMs: All have 6 Ig domains and 4–5 FN domains. The 186 kD Neurofascin isoform has a mucin-like Pro/Ala/Thr-rich (PAT) domain, while the 155 kD has only the 4 FN domains. RGD and DGEA motifs interact with integrins, while the FigQ/AY motif binds to ankyrin. ERM binding sites are indicated. The RSLE motif in L1 recruits AP2/clathrin adaptor for endocytosis.A wealth of studies has revealed L1-CAMs as pivotal components for proper development of the nervous system through regulation of cell-cell interactions. L1-CAMs have critical roles in neuronal migration and survival, axon outgrowth and fasciculation, synaptic plasticity and regeneration after trauma.² Neither CHL1 nor L1 is present on mature astrocytes, oligodendroglia or endothelial blood vessel cells in the brain, but CHL1 is upregulated in astrocytes upon injury³ and is present on oligodendroglial precursors.^4,5 During neural development, L1 plays an important role in the migration of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.⁶ In the cerebellum, L1 is required for the inward migration of granule neurons from the external granular layer and cooperates with NrCAM in regulating neuronal positioning.² Similarly, CHL1 controls area-specific migration and positioning of deep layer cortical neurons in the neocortex.⁷ In addition to its role in neuronal precursor positioning, L1 plays a crucial role in axon guidance, which is governed by repellent and attractive response mechanisms directed by Ephrins and Semaphorins and their receptors (Ephs, Neuropilins, Plexins).² The importance of L1-CAMs in the development and function of the nervous system is exemplified by developmental neuropsychiatric disorders that are associated with mutation or genetic polymorphisms in genes encoding L1 (X-linked mental retardation) and CHL1 (low IQ, speech and motor delay). Polymorphisms in L1 and CHL1 genes are also associated with schizophrenia, and NrCAM gene polymorphisms are linked to autism in some populations.²Recent studies have described upregulation of L1 in a variety of tumor types. Overexpression of L1 correlates with tumor progression and metastasis in certain human gliomas,⁸ melanoma,⁹ ovarian¹⁰ and colon carcinomas.^11–13 Interestingly, L1 was found to be present only in cells at the invasive front of colon cancers but not in the tumor mass.¹² L1 is also associated with micrometastasis to both lymph nodes and bone marrow in patients bearing other cancers, suggesting a potential role in early metastatic spread.¹¹ L1 has now been pursued as both a biomarker and a powerful prognostic factor, indicative of poor outcome for patients as observed for epithelial ovarian carcinoma¹⁰ and colorectal cancer.¹¹ More recently, L1 has been shown to be overexpressed in a small fraction of glioma cells, termed glioma stem cells, which are capable of self-renewal and generate the diverse cells that comprise the tumor.¹⁴ First characterized in acute myeloid leukemia,¹⁵ cancer stem cells have been recently described in a variety of solid tumors, including breast cancer, lung cancer and gastrointestinal tumors.¹⁶ In gliomas, L1 expression was shown to be required for maintaining the growth and survival of glioma stem cells.¹⁴ These findings suggest that L1 may be implicated not only in cancer invasiveness but also in cancer survival. It will be important to determine if L1 is also upregulated in other cancer stem cells as well as to define the role of L1-mediated signaling in other cancers. Although not extensively investigated, NrCAM has also been shown to be overexpressed in glioblastoma cell lines and several cases of high grade astrocytoma¹⁷ and ependymomas.¹⁸ Studies are needed to address whether CHL1 and neurofascin play analogous roles in cancer onset and progression.The molecular mechanisms of L1-mediated signaling that govern the migration of neuronal precursors and guidance of axons during the development of the nervous system may also be used by cancer cells to facilitate invasion and cancer progression. Integrins are well-characterized cooperative partners for L1-CAMs, and signal transduction pathways activated by this complex are known to promote cell adhesion and directional motility. L1/integrin-mediated signaling may converge with growth factor signaling networks to promote motility. Like L1, CHL1 cooperates with integrins to stimulate migration. All L1-CAMs reversibly engage the actin cytoskeleton through a conserved motif FigQ/AY in the cytoplasmic domain that contains a crucial tyrosine residue required for binding the spectrin adaptor ankyrin. Phosphorylation of the FigQY tyrosine decreases ankyrin binding, whereas dephosphorylation promotes L1-ankyrin interaction. Dynamic adhesive interactions controlled by phosphorylation/dephosphorylation of the ankyrin motif in L1 family members may enable a cell to cyclically attach and detach from the ECM substrate or from neighboring cells, thus facilitating migration.¹ Another way L1 promotes cell migration is by stimulating endocytosis of integrins, reducing cell adhesion to the extracellular matrix.¹⁹ Thus, it is reasonable to speculate that upregulation of L1 in cancer may result in increased L1-mediated signaling and, consequently, increased cell migration.L1-CAMs are cleaved by metalloproteases, releasing functionally active ectodomain fragments that are laid down as “tracks” on the extracellular matrix (ECM). These fragments can cause autocrine activation of signal transduction pathways, promoting cell migration through heterophilic binding to integrins.²⁰ Specifically, L1 is cleaved constitutively or inducibly by the ADAM family metalloproteases (a disintegrin and metalloprotease) ADAM10 and ADAM17, which stimulates cell migration and neurite outgrowth during brain development.^20,21 In colon cancer, L1 colocalizes with ADAM 10 at the invasive front of the tumor tissue, suggesting that L1 shedding may play a role in cancer invasiveness.¹² Similarly, CHL1 is shed by ADAM8, which was reported to promote cell migration and invasive activity of glioma cells in vitro and is highly expressed in human brain tumors including glioblastoma multiforme, correlating with invasiveness in vivo.²² Furthermore, NrCAM, found in pancreatic, renal and colon cancers, is subject to ectodomain shedding,²³ but its function in regulating cell migration or invasion has not yet been studied.Given the newly recognized roles of L1 in tumor progression, a growing body of experimental studies has explored novel therapeutic approaches targeting L1-CAMs. Antibody-based therapeutic strategies are being pursued to functionally inhibit homophilic and heterophilic interactions of cell adhesion molecules to suppress tumor invasive motility. L1 monoclonal antibodies reduce in vivo growth of human ovarian and colon carcinoma cells in mouse xenograft models.^13,24,25 L1 targeting using lentiviral-mediated short hairpin RNA (shRNA) interference decreases growth and survival of glioma stem cells in vitro, suppresses tumor growth, and increases survival of tumor-bearing animals.¹⁴ These findings raise the possibility that L1 represents a cancer stem cell-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors. Cancer stem cells represent a potential target for future treatment of different cancer as these cells are believed to be responsible for cancer recurrence.²⁶ Promoting cancer stem cell differentiation by drug treatment could potentially reduce stem cells properties of self-renewal and proliferation, leading to inhibition of tumor growth.Inhibitors of metalloproteases that block L1-CAM shedding represent a potentially novel approach to curtailing tumor invasiveness. Chemical inhibitors of ADAMS are appealing for glioma therapy due to their diffusability, which circumvents blood-brain barrier limitations. Another novel approach involves the secreted axon repellent protein, Semaphorin 3A (Sema3A). L1-CAMs serve as co-receptors for Sema3A by cis binding in the plasma membrane to Neuropilin-1, important for repellent axon guidance.² Interestingly, Sema3A inhibits invasiveness of prostate cancer cells²⁷ and migration and spreading of breast cancer cells in in vitro assays,²⁸ and thus may also be mediated by L1-CAMs. Such an approach could be potentially useful in mitigating invasion of cancer cells in gliomas and other tumors that are known to express L1 and Neuropilins. However, effective strategies for some types of cancer can promote cancer progression in other types. For example, Sema3A has been shown to contribute to the progression of pancreatic cancer²⁹ and colon cancer.³⁰ Thus, it is imperative that the molecular mechanisms underlying L1-mediated signaling are understood in a tissue specific manner. Despite the promising advances in cancer basic research, much more research is needed to better design strategies for cancer therapy.     

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.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

Netrin-1 signaling for sensory axons: Involvement in sensory axonal development and regeneration

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a “waiting period” of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.Key words: netrin-1, dorsal root ganglion, axon guidance, chemorepellent, Unc5, spinal cord, axon regenerationDeveloping axons navigate to their targets by responding to attractive and repulsive guidance cues working in a contact-dependent or diffusible fashion in their environment (reviewed in ref. ¹). During early development of the primary sensory system, centrally projecting sensory axons from dorsal root ganglion (DRG) neurons extend toward the dorsolateral region of the spinal cord (Fig. 1A and C), where they enter the spinal cord exclusively through the dorsal root entry zone (DREZ), and never orient themselves toward the notochord or the ventral spinal cord (Fig. 1A; reviewed in ref. ²). We previously showed that the notochord but not the ventral spinal cord secretes semaphorin 3A (Sema3A), which is known to be a chemorepellent for DRG axons at early developmental stages (Fig. 1A).³ This is the reason why DRG axons never project toward the notochord. Along the same line, it is highly possible that the ventral spinal cord may secrete some chemorepulsive cue other than Sema3A for DRG axons.Open in a separate windowFigure 1Netrin-1 plays a critical role in sensory axonal guidance as an axon chemorepellent. (A) A schematic diagram of a thoracic transverse section of an E10 mouse embryo, summarizing the possible mechanism of netrin-1 action in early DRG axonal guidance. When DRG axons project toward the DREZ in the dorsal spinal cord (dSC), ventrally derived netrin-1 chemorepels DRG axons to prevent them from orienting aberrantly toward the ventral spinal cord (vSC) (upper). NC; notochord. In netrin-1-deficient embryos, some DRG axons misorient themselves toward the ventral spinal cord, because of the absence of netrin-1 proteins in the ventral spinal cord (lower). (B) At E12.5 when DRG axons grow to the marginal zone of the spinal cord longitudinally (arrows) to form the dorsal funiculus (DF), netrin-1 proteins are transiently expressed in a subpopulation of dorsal spinal cord cells adjacent to the dorsal funiculus (upper). In netrin-1-deficient embryos, the dorsal funiculus is disorganized because DRG axons are no longer waiting for invading the dorsal mantle layer (lower). (C) Gain-of-function experiments by electroporation confirm the repulsive activity of netrin-1 toward DRG axons. When netrin-1 is misexpressed in the dorsal spinal cord, the number of DRG axons that enter the DREZ is significantly reduced compared with the control, because some DRG axons fail to project toward the DREZ and turn in the wrong direction.After entering the spinal cord, DRG axons grow to the marginal zone of the spinal cord longitudinally to form the dorsal funiculus without projecting to the dorsal mantle layer for a few days (this delay of the axonal projection to the mantle layer is referred to as the ‘waiting period;’ Fig. 1B). A few days later, proprioceptive afferents of DRGs begin to send collaterals into the dorsal layers, and cutaneous afferents project ventrally through the dorsal layers.⁴ This evidence raises the possibility that some repulsive cues transiently prevent the collaterals of DRGs from penetrating the dorsal spinal cord during this waiting period.Netrins are a family of secreted proteins that play a key role in axonal guidance, cell migration, morphogenesis and angiogenesis.⁵ Netrin-1 is a bifunctional axonal guidance cue, attracting some axons including commissural axons via the Deleted in Colorectal Cancer (DCC) receptor and repelling others via Unc5 receptors (reviewed in ref. ⁶). However, it has not been clear whether netrin-1 plays a role in sensory axonal guidance during development.Several observations strongly suggest a role for netrin-1 in DRG axonal guidance as a repulsive guidance cue during development.^7,8 First, in the mouse embryo at embryonic day (E) 10–11.⁵ when many DRG axons orient themselves to reach the DREZ, netrin-1 is strongly expressed in the floor plate of the ventral spinal cord but not in the dorsal spinal cord (Fig. 1A). Second, at E12.5 when DRG neurons extend their axons longitudinally along the dorsolateral margin of the spinal cord, netrin-1 is expressed in the dorsolateral region adjacent to the DREZ (Fig. 1B), but its expression is down-regulated in the dorsal spinal cord at E13.5 when many collaterals have entered the mantle layer. Third, repulsive netrin-1 receptor Unc5c is expressed in the DRG neurons during development.These observations motivated us to explore whether netrin-1/Unc5c signaling contributes to DRG axonal guidance. We used cell and tissue cultures combined with tissues from netrin-1-deficient mice. We clearly showed that netrin-1 exerts a chemorepulsive activity toward developing DRG axons and that the ventral spinal cord-derived repulsive activity depends on netrin-1 in vitro.⁸ Additional evidence for a chemorepulsive role of netrin-1 came from the observation of DRG axonal trajectories in netrin-1-deficient mice.^7,8 In netrin-1-deficient embryos at E10, we showed that some DRG axons became misoriented toward the ventral spinal cord, probably because of the absence of netrin-1 proteins in the ventral spinal cord (Fig. 1A). In addition, at E12.5 when DRG axons grow to the marginal zone of the spinal cord longitudinally to form the dorsal funiculus, the dorsal funiculus is disorganized in netrin-1-deficient embryos, because in the absence of netrin-1 DRG axons are not waiting for invading the dorsal mantle layer adjacent to the dorsal funiculus (Fig. 1B). Gain-of-function experiments further confirmed the repulsive activity of netrin-1 toward DRG axons (Fig. 1C). These lines of evidence lead us to the conclusion that dorsally derived netrin-1 plays an important role in providing the ‘waiting period’ for extension of collaterals from sensory afferents and that ventrally derived netrin-1 prevents sensory axons from misorienting themselves toward the ventral spinal cord.At later developmental stages (E13.5), DRG axons still possess a weak responsiveness to the chemorepulsive activity of netrin-1 in vitro.⁸ In addition, both postnatal and adult DRG neurons respond to netrin-1-induced axon inhibition.⁹ Consistent with these results, DRG neurons at not only later developmental stages (E13.5) but also postnatal stages express the repulsion-mediating netrin-1 receptor Unc5c.^8,9Generally, lesioning of the dorsal column projection of sensory axons results in a complete lack of regeneration. The possible explanation for the complete lack of regeneration is that the environment, the lesion site itself and/or oligodendrocytes adjacent to the lesion, may be non-permissive for regenerating axons.¹⁰ Sema3A and chondroitin sulfate proteoglycans (CSPGs) are candidates as major inhibitors of sensory axonal regeneration in the spinal cord, because they are expressed in the lesion site and can inhibit DRG axonal growth in vitro.^3,11–14 Recently, Kaneko et al. showed that a selective inhibitor of Sema3A also enhances axonal regeneration and functional recovery in a subpopulation of sensory neurons after lesioning of the dorsal column.¹² More recently, McMahon''s group clearly demonstrated that enzymatic degradation of CSPGs on the dorsal column lesion of the spinal cord promotes sensory axonal regeneration and functional recovery.^13,14 Although these treatments greatly improved functional recovery, complete sensory axonal growth and functional recovery have not been yet achieved after the spinal cord injury. To promote further recovery of sensory axonal regeneration in the CNS, we should focus on other candidate inhibitors of CNS injury sites.Following spinal cord injury, the expression of the attraction- mediating netrin-1 receptor DCC decreases, while the expression level of the repulsive receptor Unc5c returns to normal.¹⁵ Levels of netrin-1 expression also stay unchanged in neurons and oligodendrocytes adjacent to the lesion site. Together with the in vitro evidence described above, these data strongly suggest a possible role for netrin-1 as a novel inhibitor of CNS myelin for regenerating DRG axons in the dorsal column-lesioned spinal cord. Further studies will be required to show directly the functional recovery of sensory axons in the spinal cord by perturbation of netrin-1 in and around the lesion site after spinal cord injury.     

Fluid flow and guidance of collective cell migration

Collective cell migration is emerging as a significant component of many biological processes including metazoan development, tissue maintenance and repair and tumor progression. Different contexts dictate different mechanisms by which migration is guided and maintained. In vascular endothelia subjected to significant shear stress, fluid flow is utilized to properly orient a migrating group of cells. Recently, we discovered that the developing zebrafish pronephric epithelium undergoes a similar response to luminal fluid flow, which guides pronephric epithelial migration towards the glomerulus. Intratubular migration leads to significant changes in kidney morphology. This novel process provides a powerful in vivo model for further exploration of the mechanisms underlying mechanotransduction and collective migration.Key words: collective migration, fluid flow, mechanotransduction, development, kidney, zebrafishThe term “collective cell migration” (collective motion) was first introduced to describe the behavior of starved Dictyostelium discoideum.¹ The term has rapidly gained general acceptance as encompassing a wide variety of coordinated cell migratory behaviors. A number of definitions have been proposed to unify the various collective migratory behaviors. Friedl et al.² defined it as “the movement of cell groups, sheets or strands consisting of multiple cells that are mobile yet simultaneously connected by cell-cell junctions.” This definition implies a number of features setting collective migration apart from other migratory behaviors. First, it points to the spatial restrictions on the individual cells within the migrating groups. The cells cannot leave the group and continue on their own. Therefore, they must respect the behavior of their neighbors and the overall migration occurs through the integration of individual cell activities across the collective. Second, it implies that different cells within the migrating group may play different roles. Some of them may not be migratory at all and simply “ride” the rest of the group, as indeed seen in border cell migration.³ Other cells within the group may further specialize into leaders and followers as can be seen in most current models of collective migration.⁴A variety of biological processes satisfy this definition. They include, among others, closure of wounded epithelial sheaths,³ physiological maintenance of intestinal epithelium,⁵ cancer invasion,^2,4,6 developmental processes of branching morphogenesis,^7,8 vascular sprouting,⁹ gastrulation,¹⁰ dorsal embryo closure,¹¹ as well as movements of some basal metazoan organisms such as sponges.¹² Over the years, a number of models emerged to study the process of collective migration.When starved, thousands of single cells of Dictyostelium discoideum aggregate and form a “slug” that migrates to the soil surface to form a fruiting body. This process has two general stages: the stage of aggregation, where individual migrating cells respond to cAMP concentration to form a multicellular aggregate¹³ and the stage of collective migration. In the latter stage, the leading (pre-stalk) cells of the slug secrete cAMP. In addition, they produce slime sheath that provides traction support for the aggregate. The slime sheath allows outermost cells of the aggregate to develop necessary traction for the entire slug to propel itself towards guidance cues. A number of molecular and cellular components have been recently identified to be important in this process, including integrin-, paxillin-like molecules and dynamic focal adhesion formation.¹⁴ Thus, Dictyostelium serves as a useful model for understanding the dynamic mechanisms of force formation in a migrating collective.Another well-established model of collective cell migration is the migration of border cells during ovary development in Drosophila. There, a small group of cells consisting of a central pair of polar cells surrounded by migratory outer border cells delaminate from the epithelium and migrate as a free group between nurse cells. Because of the tight nature of the migrating group, non-motile polar cells as well as mutant outer migratory cells can be carried within the group by their migratory companions.³ The migrating cluster uses nurse cells to generate the necessary traction to continue along the migratory path and rely on E-cadherin to accomplish this task.¹⁵ It has been proposed that the migrating border cell cluster is guided collectively wherein each outer border cell is inherently polarized, having an outer aspect and the inner aspect, so that the net migration of the cluster is simply the net of all the forces generated by the outer collective.¹⁶ It has been shown recently that both individual cell guidance and the collective cell guidance are at play in border cell migration.¹⁷Perhaps the best-studied examples of collective cell migration are found in the wound closure of epithelial sheets. Both kidney and gastric epithelial cell lines have been extensively studied in the wound closure assay to reveal important details of the collective migration that is a central process in wound repair. Recent studies have demonstrated the role of integrins, Rac, ERK, MAPK, Src and Pi3K among others as important molecular components of this processes.^18–21 A recent siRNA screen using breast epithelial cells identified a number of molecules that either inhibit or augment epithelial migration.²² This study revealed 42 genes previously unknown to be involved in migration. Many genes clustered within β-catenin, β1-integrin and actin networks in secondary analysis.While in vitro epithelial wound assays continue to provide insights into potential mechanisms of collective cell migration, the most developed in vivo vertebrate model comes from the studies of the zebrafish lateral line formation. In this process, the lateral line primordium cells move as a group in the anterior-posterior direction.^4,23 The migration is dependent on the interaction of stromal factor Cxcl12 along the guidance path and its receptor Cxcr4b.²⁴ The direction of migration is defined by the interplay between Fgf and Wnt signaling (rear and front of the migrating group, respectively). Wnt signaling in the front of the migrating lateral line inhibits Cxcr7b expression and promotes Cxcr4b expression. It also results in the secretion of Fgf ligands. Expression of sef at the front (also under control of Wnt) prevents Fgf from acting in this front domain. Fgf ligants interact with their receptors in the trailing end of the migrating group. As a result, the cells at the trailing end express dkk1 (to limit Wnt signaling) and Cxcr7b while downregulating Cxcr4b.^4,23,25,26 Thus, Cxcl12-Cxcr4b interaction is limited to the migration front. Cxcr7b expressed in the back of the migrating collective is believed to further interfere with Cxcl12-Cxcr4b interaction by sequestering Cxcl12. The net result of the differential signaling is the establishment of a distinct migratory front at the posterior aspect of the precursor population. At the same time, groups of cells at the back stop migrating and give rise to individual lateral line organs.The existence of a distinct migratory front is a unifying feature of all the models of collective migration described above. The migratory front defines the interface between the migratory collective and the tissues into which the migratory group advances. The front may be maintained by a stable pattern of signaling within the migrating group, as seen in lateral line migration where Wnt signaling at the front and FGF signaling at the back are maintained through mutual exclusion. Alternatively, the front may be maintained through spatial differences in concentrations of chemoattractants rendering the front of the group more migratory, as seen in the Drosophila border cell migration.³ In other systems, the migratory front may be maintained through cell-to-cell direct signaling, such as Notch signaling in determining the tips of vascular sprouts.⁹ Furthermore, migrating epithelial cultures in wound assays are inherently polarized by the presence of a free margin. Interestingly, the presence of the margin, which becomes the migratory front, is sufficient even in the absence of the wound to initiate a directed migration.²⁷ However, several new studies revealed that the existence of a distinct migratory front is not a universal or required feature of collective migration.^28–31Recently we discovered a novel form of collective migration that guides the morphogenesis and maturation of pronephric kidney.²⁸ The zebrafish pronephros is a simple bilaterally symmetrical structure consisting of two fused glomeruli, each connected to a pronephric tubule that runs posteriorly, eventually exiting at the level of the cloaca. The pronephros begins to function shortly after 1 dpf.²⁸ After the onset of its function, a signifi- cant maturation of the pronephros takes place, manifested at the structural level by the development of proximal convolution and re-positioning of nephron segment boundaries (Fig. 1). We demonstrated that both of these structural changes are a direct consequence of the collective epithelial migration that starts at about 30 hpf and lasts for the next three days. This proximal migration is governed by the onset of luminal fluid flow. The cells of the pronephric epithelium move enmasse towards the glomerulus and against the flow of urine. As a result, the proximal segment becomes compressed, shortened and convoluted. In contrast, the distal segment straightens and becomes longer (Fig. 1 and Suppl. Movie 1). This lengthening of the distal kidney is accompanied by cell proliferation that compensates for the proximal shift of kidney segments and allows for the en-masse migration to continue for three days.²⁸Open in a separate windowFigure 1Effect of pronephric migration on tubule architecture. (A) Schematic representation of zebrafish showing the pronephric kidney. Arrowhead points to the glomerulus. Arrow points to the pronephric tubule. (B) Pronephric architecture at 1 dpf before the onset of tubule flow. Dark shading indicates a distal segment. (C) Pronephric architecture at 3 dpf showing a markedly shortened and folded (convoluted) proximal segment. (D) Pronephric architecture at 3 dpf in the embryo with eliminated glomerular filtration. The position of the tubule convolution is now at the interface of the proximal and the distal tubules (see also Suppl. movie files 1 and 2).As mentioned above, this novel developmental process differs from most models of collective cell migration in at least one aspect; it lacks a distinct migratory front. In the absence of such front, the polarity of the migrating pronephric epithelium is established by using fluid flow as the guiding cue. When directed fluid flow is eliminated by obstructing the pronephros, the proximal migration is disrupted. Instead, the cells of the pronephric epithelium can often be seen migrating circumferentially, around the tube perimeter. This circumferential pseudo-migration correlates with the presence of local vortex currents in obstructed pronephroi due to the presence of beating cilia. Indeed, we failed to observe similar circumferential pseudomigratory behavior in paralyzed cilia mutants (unpublished data). In addition, we were able to engineer an ectopic convolution (about 500 µm distal to its normal location next to the glomerulus) by selectively eliminating proximal, but not distal sources of fluid flow (Fig. 1 and Suppl. Movie 2). This finding further supports the conclusion that luminal fluid flow guides the epithelial migration. It is still possible that different cells within the pronephric epithelium have distinct roles in orchestrating the migration. For instance, a small subset of cells could act as functional leaders and organize the migration process. Alternatively, luminal flow could directly interact with each migrating cell. Further studies should determine which scenario is present in the pronephros.There are at least two other systems where cell migration is governed by the mechanical forces generated by luminal fluid flow. Vascular endothelial cells respond to fluid shear stress, orient in the direction of the flow and migrate in the direction of shear force. This behavior is thought to be important in vascular remodeling.²⁹ A related model was developed in macaque placental trophoblast cells which demonstrate a similar behavior.³⁰ It is notable that in a wound assay, endothelial cells respond in a way similar to that in other in vitro wound models described above.³² Thus, more than one mode of guidance may be present in a given tissue.Significant advances have been made in our understanding of the cellular responses to shear stress in vascular endothelium. Endothelial cells sense and respond to fluid shear by utilizing a system of adhesion molecules including PECAM and VE-cadherin, integrin activation, activation of VEGFR, calcium influx, and modulation of the cytoskeleton by Rho family GTPases.^29,31 Recent evidence also suggests that sensory cilia play a role in the endothelial response to shear stress.^33,34 Fluid shear first induces lamellipodial cell extensions, followed by basal protrusions and new focal adhesion formation in the direction of the flow. Subsequent migration requires remodeling of adhesions and release of cell substratum attachments at the rear of the migrating cell.Migration of pronephric epithelial cells is likely to involve similar basic mechanisms. For instance, we have observed a strong correlation between the presence of directed lamellipodial extensions of epithelial cells on the tubule basement membranes and the basal phosphoFAK staining, suggesting that pronephric epithelial cells actively remodel their matrix attachments as they migrate. The similarities and differences between these two systems are likely to prove useful in determining how mechanical forces establish self-perpetuating cell movement. A notable difference between the pronephric cell migration and endothelial cell migration is that pronephric cells migrate against the flow as opposed to in the direction of the flow, suggesting that the exact nature of the process linking flow to migration may also be different.Several mechanisms may be at play in transducing the directional flow into directed migration of pronephric epithelia. First, ciliary function has been implicated in sensing fluid flow.³⁵ Thus, it is possible that bending the luminal cilia is key to the translation of luminal flow into collective epithelial cell migration. However, this potential mechanism was not supported by our observations. In particular, we tested the role of polycystins that are thought to be central to the flow sensing mechanism in the cilia.^33,36 We observed that polycystin morphants did not show an arrest or misorientation of migration until pronephric cycts were formed. This finding indicates that polycystins affect migration secondarily, due to pronephric obstruction and perturbation of flow, rather than by directly influencing epithelial migration. While polycystins do not appear to mediate mechanotransduction in pronephric epithelial migration, other members of the TRP ion channel family may be involved. Many TRP channels, such as TRPV, TRPC as well as other mechanosensitive ion channels, are thought to mediate transduction of mechanical stimuli into the intracellular signals.^37,38 It is possible that one or more such mechanisms are present in the pronephros.Alternatively, as discussed above, shear stress may be transduced at focal adhesions through integrin coupled intracellular signals with multiple potential intracellular targets, including Src, FAK, ILK, paxillin and p130Cas.³⁹ It has been shown, for example, that in cultured intestinal epithelial cells, a mechanical deformation of the substrate stimulates migration in FAK dependent manner.⁴⁰ Cell-cell junctions may also serve as a major site of mechanotransduction as was shown in vascular endothelia.³¹Other potential components of mechanotransduction include G-protein coupled receptors, which were shown to localize to the sites of focal adhesion and are known to be activated by shear stress and cyclic stretching.³⁸ Here, mechanical displacement may lead to the conformational change in the receptor molecule and the activation of downstream targets. In addition, Wnt and receptor tyrosine kinase signaling have been linked to mechanotransduction.^38,41 It remains to be determined which of these processes mediate the relation between pronephric flow and epithelial migration.It is possible, however, that multiple components (focal adhesion complexes, cell junctions, sensory cilia, etc.) interact with each other, and these interactions are integrated by the cell to generate a response to a mechanical stimulus. There is evidence showing that various components are indeed linked together by cytoskeleton.^34,42 The apical ciliary response to shear stress by cultured kidney cells (as measured by the cytoplasmic calcium increase) can be prevented by altering the integrity and the tensile properties of the cytoskeleton. The same result can be achieved by blocking the integrin interaction with extracellular matrix at the basal surface.⁴² Conversely, disrupting ciliary function in vascular endothelial cells significantly attenuates the overall response of the cell to fluid shear, the result that can also be achieved by disrupting cytoplasmic microtubule polymerization.³⁴These findings suggest a model in which multiple identified components of the mechanotransduction response are linked by cytoskeletal elements, that allow events at each specific location to influence the state of a different remote cell component directly.⁴³ For example, bending the cilium would have opposite mechanical effects on cell-cell junctions located in the direction of the bending, compared to those located on the opposite side.³⁴ Importantly, this mechanical communication is inherently bidirectional and would allow the cell to instantly integrate signals originating at different locations and initiate a robust and coordinated response to external mechanical cues.     

Sweet cues: How heparan sulfate modification of fibronectin enables growth factor guided migration of embryonic cells

Growth factors regulate a diverse array of cellular functions including proliferation, survival and movement, and the ability to do this often involves interactions with the extracellular matrix (ECM) and particularly heparan sulfate proteoglycans (HSPGs). HSPGs have been shown to sequester growth factors and to act as growth factor co-receptors or receptors themselves. Recent studies, however, have revealed a new role for HSPGs in mediating the interactions of growth factors with the ECM. Specifically, heparan sulfate has been shown to modulate fibronectin structure to reveal previously masked growth factor binding sites. In vivo, this mechanism appears to control the guidance of migrating cells during embryonic development as HSPG-modification of fibronectin enables direct platelet derived growth factor-fibronectin interactions necessary for this process. A model based on this observation is discussed here as well as the possibility that other growth factors/morphogens utilize similar mechanisms involving fibronectin or additional ECM proteins.Key words: heparin, heparan sulfate, fibronectin, cell movement, cell motility, mesoderm, mesendoderm, embryonic development, embryo, XenopusThe ability of cells to move is critical to normal processes, such as embryonic development and wound healing, and is also a feature of pathological conditions such as cancer metastasis. In normal circumstances, cell movement is tightly regulated and leads to precise cell reorganizations. This is particularly evident during embryonic development where cells need to be in the right place at the right time to give and receive signals, to form tissues and organs and ultimately, to become a functional organism. To achieve this, cells must be able to move in a directed way. This requires that cells sense and move in response to extracellular signals, while coordinating changes in shape with the creation of traction and force and balancing attachment and detachment to the extracellular matrix (ECM) and neighboring cells.Evidence from a number of systems suggests that growth factors play a significant role in the spatial and temporal control of cell movements. In particular, members of the platelet derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) family act as guidance cues for cells in a variety of processes including the development of both invertebrate and vertebrate embryos (reviewed in ref. ¹). How such cues are laid down is an area of intensive investigation; however, a new study from our group suggests that the microenvironment within the ECM, principally the composition of heparan sulfate, modulates direct interactions between PDGF and fibronectin necessary for directed migration of embryonic cells during Xenopus development.²During the development of all vertebrates, cell movements begin at gastrulation, when changes in the shape, adhesion and motility of cells drive the complex series of tissue rearrangements necessary to establish the basic body plan of the embryo. In Xenopus embryos, some of the first cells to move during gastrulation are the anterior mesendoderm cells. These cells migrate in a characteristic pattern that provides a tractable model to identify the molecular control of directed cell movement. Anterior mesendoderm cells move away from the blastopore lip and towards the animal pole across a fibrillar, fibronectin-rich ECM that covers the ectoderm (see Fig. 1A; reviewed in ref. ³). They migrate as a sheet and are polarized with lamelliform protrusions extending in the direction of movement and the trailing edges underlapping one another like shingles on a roof (reviewed in ref. ³). This mesendoderm movement is guided by PDGF-A signaling.⁴ Immediately prior to and during Xenopus gastrulation, PDGF-A is expressed in the ectoderm while its receptor, PDGFRα, is expressed in the migrating mesendoderm cells (reviewed in ref. ⁵). Disruption of PDGF-A signaling by interference with either the receptor or ligand in vivo or in an ex vivo assay disorients the cells and the direction of migration is randomized, although the cells are still able to physically move.⁴Open in a separate windowFigure 1Heparan sulfate modification of fibronectin enables PDGF-AA-fibronectin interactions necessary for directed cell migration during Xenopus gastrulation. (A) A schematic of a Xenopus embryo immediately after the onset of gastrulation (stage 10+). The prospective mesoderm (red), ectoderm (blue) and endoderm (yellow) are indicated. The black circle indicates migrating anterior mesendoderm cells and the region of tissue shown in (B and C). (B) In vivo the inner surface of the ectoderm (blue blocks) is lined with a fibronectin-rich ECM. This fibronectin is proposed to be in its open conformation (blue dumbbells) due to the presence of heparan sulfate proteoglycans on the surface of these cells (indicated in red). PDGF (green triangle) secreted by the ectoderm cells binds to the C-terminus of the fibronectin. This PDGF is then available for PDGFRα (green fork) binding and activation. The PDGFRα is expressed on the migrating mesendoderm cells. This ligand receptor interaction guides the directed movement of these cells. (C) Heparinase III treatment of these tissues during matrix deposition digests the heparan sulfate side chains leaving the proteoglycan core (red line), which does not alter fibronectin structure leaving it in the closed conformation. PDGF secreted by the ectoderm cells can then diffuse from the site of secretion leaving it available to bind PDGFRαs over the entire surface of the mesendoderm cells and thus, disrupting directed movement. (D and E) A schematic of a “higher magnification” view of one cell in (B and C). A key of the symbols used in (B–D) is shown.PDGF-A must be associated with the ECM for directed mesendoderm migration. In an ex vivo assay, overexpression of the long form of PDGF-A, which associates with the ECM via a positively charged carboxy-terminal retention motif (reviewed in ref. ⁶) in the ectoderm cells, disrupts the normal guidance cues causing randomization of mesendoderm movement.⁴ In contrast, overexpression of the short form of PDGF-A, which diffuses from its site of secretion,⁶ does not.⁴ Our recent work now suggests that this growth factor-ECM association represents a direct interaction between PDGF-A and fibronectin.² Importantly, this interaction requires that the fibronectin is pre-exposed to heparan sulfate proteoglycan (HSPG) before PDGF-A can bind.² A similar interaction between the related growth factor VEGF and fibronectin is also enhanced by pre-treatment of the fibronectin with heparin or heparan sulfate.^7–9 Heparin acts by modifying the structure of fibronectin,⁸ mediating the transition from the globular form to a stable, more extended conformation,^7,8,10–12 which reveals growth factor binding sites.^7,8 These data are significant because in an ex vivo directed migration assay, treatment of the native ECM with heparinase III, which degrades heparan sulfate chains, abolishes the guidance cues and results in randomized mesendoderm migration.² Here we propose a model in which prior to and during Xenopus gastrulation, fibronectin is modified to an extended conformation by endogenous HSPGs as it is being laid down (Fig. 1B and D). PDGF-AA secreted from the ectoderm cells binds to this modified fibronectin and is thus, retained in the ECM (Fig. 1B and D). PDGF-AA then guides the mesendoderm cells by local activation of their cell surface PDGFRαs (Fig. 1B and D). In the absence of HSPG, the fibronectin remains in a more globular form and PDGF-AA binding sites are shielded (Fig. 1C and E). In this case, it is proposed that PDGF-AA diffuses away from the site of secretion resulting in a less localized activation of PDGFRαs and a loss of persistent cell migration (Fig. 1C and E). This idea is supported by data showing that flooding the native ECM with exogenous PDGF-AA prior to performing the ex vivo migration assay, also causes a randomization of directed movement.⁴ In this case, it seems likely that any gradient or uneven distribution of PDGF-AA is overwhelmed and the guidance cues consequently lost.In cultured cells, PDGF can also associate with HSPGs via its retention motif.¹³ However, in the Xenopus ex vivo directed migration assay, heparinase treatment of the ectoderm ECM after its deposition does not alter the direction of mesendoderm migration.^2,14 This suggests that in this system, PDGF-AA interacts directly with fibronectin and not with heparan sulfate chains, since heparinase III would have degraded them and caused the release of HS-bound PDGF-AA.¹³The type of proteoglycan as well as the specific structural composition of their heparan sulfate chains likely contributes to the modulation of PDGF-fibronectin binding. Both chain length and the chemical composition of heparan sulfate play crucial roles in the binding of VEGF to fibronectin, in which long (>22 saccharides) heparin chains with sulfation on the 6-O and N positions of glucosamine units are required for activity.⁸ This suggests that a localized control of heparan sulfate biosynthesis through the regulation of the array of enzymes involved might be a critical component to directed cell migration. Interestingly, an analysis of heparan sulfate expression during mouse lung development noted dynamic and discrete localization within the mesenchyme at sites of FGF10-mediated epithelial budding,¹⁵ indicating that rapid alterations of heparan sulfate structure may provide a general mechanism for positional control of growth factor activity.Several HSPGs are expressed in tissue specific patterns during Xenopus gastrulation including Syndecans-1 and -2 (expressed in the ectoderm);¹⁶ and Syndecan-4, Glypican-4 and Biglycan (expressed in the ectoderm and mesoderm).^16–19 Although the role(s) of these HSPGs in mesendoderm migration are not known, they do affect embryological processes that involve cell rearrangements. For example, Syndecan-2 has recently been shown to regulate the migration of organ primordia and fibrillogenesis in zebrafish embryos.²⁰ During gastrulation, knock-down of either Syndecan-1 or -2 disrupts the native ECM for mesendoderm migration and results in gaps in fibril deposition.²¹ In addition, Syndecan-4 and Glypican-4 are both essential for convergent extension, another important form of cell movement during gastrulation that involves the coordinated rearrangement of cells.^17,18 Similarly, Syndecan-4 is necessary for the directional migration of neural crest cells.²² In these cases, Syndecan-4 and Glypican-4 regulate the planar cell polarity (PCP) pathway, which is also involved in polarized matrix deposition during convergent extension.²³ Compellingly, embryos develop with anterior defects when either Syndecan-4 or Glypican-4 is knocked-down, indicating that mesendoderm cell migration is also likely to be affected.^17,18The Slb/Wnt-11 mediated-PCP pathway has been shown in zebrafish embryos to regulate the polarity and directional movement of ingressing mesendoderm cells.²⁴ In Slb/Wnt-11 mutants, however, although these processes are disrupted they are not completely abolished and evidence suggests that PDGF signaling through phosphatidyl inositol 3-kinase (PI3K) is responsible for establishing cell polarity and controlling the velocity of these cells.²⁵ Rho GTPases act downstream in both PCP and PDGF signaling pathways. Recent evidence suggests that RhoA and Rac1 are important for the polarity and protrusive activity of migrating mesendoderm cells and play a role in the guidance of neural crest cells,^22,26 but the mechanisms by which these pathways are integrated with signals from other factors that also modulate these processes are still to be determined.How retention of PDGF-AA in the ECM translates into directional migration still needs to be determined. For example, PDGF-AA may be present in a gradient such that receptors at the front of each cell are exposed to a different level of PDGF than those at the rear. Alternatively, control might involve selective expression of molecules that locally release PDGF from matrix binding sites. Either process might provide a means for uneven activation of PDGFRs and a polarized distribution of downstream signaling components at the leading and lagging edges. Such signaling mechanisms have been identified in other cell types and organisms including leukocytes, fibroblasts and Dictyostelium in which the initial response to a chemoattractant creates cell polarity that is further amplified by feedback (reviewed in ref. ²⁷). For example, in Dictyostelium PI3K and PTEN (phosphatase and tensin homolog) are regulated reciprocally to control the spatial and temporal levels of phosphatidyl inositol-3,4,5-triphosphate (PI(3,4,5)P₃). PI(3,4,5)P₃ is localized to the leading edge of the cell in response to an extracellular signal, whereas PTEN is downregulated in these regions. Recruitment of proteins that bind preferentially to PI(3,4,5)P₃, many of which affect remodeling of the actin cytoskeleton, further enhances this polarity in the direction of the chemoattractant.²⁷ In Xenopus, PDGF signaling does appear to regulate cell polarity since overexpression of PDGF-A in the ectoderm disorients the protrusive activity and abolishes the normal shingle arrangement of the receptor expressing, migrating mesendoderm cells.⁴ Similarly, in the zebrafish mesendoderm, PDGF signaling induces cell protrusions and polarization.²⁵ In this case, upon PDGF treatment, protein kinase B (PKB)/Akt become localized to the leading edge of the cells in a PI3K dependent manner.²⁵ Such mechanisms require the activation of PDGFR at the leading edge of the cell. In mammalian cells, however, it has been demonstrated that activation of epidermal growth factor receptor (EGFR) can spread laterally in the cell membrane.²⁸ If the same is true for PDGFR, even cells exposed to a gradient or localized source of PDGF-AA may lose spatial information. Recent evidence in Drosophila, however, suggests an alternate mechanism involving receptor endocytosis that ensures that a graded extracellular signal is maintained intracellularly.²⁹The Drosophila member of the PDGF/VEGF family, PVF1, along with epidermal growth factor, directs the migration of border cells towards the oocyte during oogenesis.^30–32 The patterns of expression of ligand and receptor are similar to those during Xenopus gastrulation in that a stationary cell, the oocyte, expresses the ligand, PVF1, while the motile border cells express its receptor, PVR.³⁰ PVR activation is concentrated at the leading edge, and specifically maintained in a localized region of the cell by endocytosis.²⁹ PDGFRβ endocytosis also regulates PDGF-dependent migration of NIH3T3 cells in culture.³³ This process involves recruitment of a ternary complex of DOCK4-Grb2-Dynamin2 to the leading edge of the migrating cell, which results in rapid receptor endocytosis, where it is maintained in an active phosphorylation state and leads to polarized signaling. Disruption of the complex, for example by overexpression of DOCK4 ΔC, which cannot bind Grb2, blocks chemotaxis in response to PDGF. Whether a similar mechanism creates spatial signaling during Xenopus gastrulation requires further investigation, but this tantalizing evidence suggests that it is a possibility.     

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).     

Directional cell migration in vivo: Wnt at the crest

Directional cell migration is essential for almost all organisms during embryonic development, in adult life and contributes to pathological conditions. This is particularly critical during embryogenesis where it is essential that cells end up in their correct, precise locations in order to build a normal embryo. Many cells have solved this problem by following a gradient of a chemoattractant usually secreted by their target tissues. Our recent research has found an alternative, complimentary, mechanism where intracellular signals are able to generate cell polarity and directional migration in absence of any external chemoattactant. We used neural crest cells to study cell migration in vivo, by performing live imagining of the neural crest cell migrating during embryo development. We show that the Planar Cell Polarity (PCP) or non-canonical Wnt signaling pathway interacts with the proteoglycan syndecan-4 to control the direction in which cell protrusions are generated, and in consequence, the direction of migration. By analyzing the activity of the small GTPases using in vivo FRET imaging we showed that PCP signaling activates RhoA, while syndecan-4 inhibits Rac, both at the back of the neural crest cell. Here we discuss a model where these signals are integrated to generate directional migration in vivo.Key words: directional migration, cell migration, syndecan-4, PCP, non-canonical Wnt, neural crest, RhoA, RacThe ability of cells to move in a directed manner is a fundamental requirement for life. In multi-cellular organisms, this requirement begins in the embryo, where morphogenetic processes are dependent on the correct movement of large numbers of cells. In the adult too, cell migration plays a vital role in many systems including the immune system and wound healing. Cell migration defects can contribute to the pathology of many diseases including vascular diseases such as atherosclerosis, and chronic inflammatory diseases like asthma and multiple sclerosis. Likewise, metastasis in cancer is characterized by mis-regulation of the normal cell migration machinery and results in cells that are normally static becoming aggressively motile and invasive.Cell migration requires cell polarization and the formation of protrusions at one end of the cell. Polarization results in a different molecular ensemble at the front of the cell compared to that at the back. Cell protrusion formation at the front of the cell requires reorganization of the actin and microtubule cytoskeleton to produce a protrusion either in the form of a broad sheet-like lamellipodium or spiky filopodium. Small GTPases are well known modulators of these processes (reviewed in ref. ¹).Several mechanism has been proposed as involved in directional migration during embryo development, such as chemotaxis (migration toward an soluble chemoattractant),² haptotaxis (migration toward a substrate-bound chemoattractant),³ population pressure (migration from a region of high towards a region of low cell density)⁴ and contact inhibition of locomotion (change in the direction of migration as a consequence of cell-cell contact),⁵ being chemotaxis the most widely accepted and studied.The correct orientation of the cell and its protrusion is the keystone of directional migration and, in the case of chemotaxis, it is supposed to be controlled by the action of external chemical cues (chemoattractants) that are produced by or near to the target tissue.⁶ One of the best examples for chemoattraction in vivo is the migration of the progenitor germ cells, which are attracted by the chemokine SDF-1.² It has been shown in vitro and in vivo, that upon receiving a chemotactic signal, the cell becomes polarized in the direction of migration. Nevertheless, it is known that cells cultured in vitro can became polarized and exhibit directional migration in absence of extrinsic chemoattractants.⁷ Pankov et al. showed that persistent directional migration in vitro can be achieved solely by modulating the activity of the small GTPase, Rac: high levels of Rac promotes the formation of peripheral lamella during random migration, while slightly lower levels of Rac suppress peripheral lamella and favour the formation of a polarized cell with lamella just at the leading edge.⁷ Is it possible that a similar mechanism of directional migration could occur in vivo?The migration of Neural Crest (NC) cells has been used as a model to study directional cell migration in vivo.^8–10 The neural crest is an embryonic population of cells that are specified at the border between the neural plate and the epidermis.¹¹ Upon induction neural crest cells undergo an epithelial to mesenchymal transition,¹² detach from the neural tube and migrate following defined pathways that eventually allow them to colonize almost the entire embryo.¹³ Finally, after reaching their destination NC cells differentiate to form many different cell types including neurons, glia, cartilage, skeleton and pigment cells.¹⁴ The migration of the NC cells is critical for the proper differentiation of their derivatives and there are several human syndromes associated with failures in this process.The migration of NC cells is a highly ordered process; individual NC cells migrate with high persistence towards the direction of their targets,⁸ but until now it was not known how this directionality is controlled. A number of molecules have been identified as key players in neural crest migration, such as Ephrins, Semaphorins, Slit/Robo, etc. (reviewed in ref. ¹³). However most of these molecules work as inhibitory signals, which are required to restrict the migration of NC cells from prohibited areas. Although chemoattraction has been one of the proposed mechanisms to explain this directional migration, no chemoattractant has thus far been found in the NC.It has been known for many years that NC cells can migrate in vitro with a high directionality even in the absence of external signals.¹⁵ Therefore, our work has been focused on understanding how NC directionality is controlled. Recently, we have unveiled some of the molecules that control this directional migration in vitro. More importantly, we have been able to show that the same molecular machinery controls directional migration in vivo.^9,10One of the key factors that controls directional migration of NC cells is the Planar Cell Polarity (PCP) or non-canonical Wnt signaling pathway.^9,10,16 PCP signaling was first described in Drosophila, where a number of mutations were identified that disrupt the formation of bristles and hairs on the adult cuticle.¹⁷ In the Drosophila wing, epithelial cells are highly polarized, with a single hair outgrowth forming at the distal end of each cell. Mutations in PCP genes cause loss in cell polarity in this tissue with hairs forming in a disorganized pattern.¹⁸ In vertebrates, PCP signaling also regulates cell polarity during a number of different developmental processes including neural tube closure, cochlear hair orientation and ciliogenesis.¹⁹We have shown that the PCP pathway is essential for correct neural crest migration in Xenopus. Injection of dominant negative forms of the intracellular PCP component Dishevelled (Dsh), which inhibit the PCP pathway but not canonical Wnt signaling, block the migration of cranial neural crest cells in vivo.⁹ Recently this role has also been extended to zebrafish where directional migration of neural crest is severely disrupted in the PCP mutant trilobite (strabismus) and in embryos injected with a dominant negative form of Dsh or a morpholino against wnt5a,¹⁰ with no effect in neural crest cell motility.^9,10 Two factors, pescadillo and syndecan-4 that have recently been proposed as modulators of the PCP signaling,^20,21 are also required for NC migration.^10,21 Taken together, these data point to an essential role for PCP signaling in neural crest migration.What is the cellular and molecular mechanism by which PCP signaling controls migration of NC cells? In order to investigate this question we analyzed the direction of neural crest migration and cell polarity in vitro and in vivo after interfering with two elements of the PCP signaling pathway: syndecan-4 and Dsh. One of the key finding of our work was that the inhibition of NC migration through syndecan-4 depletion does not affect the velocity of cell migration, but significantly reduces the directional migration of the cells in vivo (Fig. 1A and B). Consequently, when the orientation of cell protrusions was analyzed we found that syndecan-4 depletion does not affect the formation of cell protrusions, but the direction in which the cell protrusions are generated during migration. More precisely, normal cells extend their lamellipodia at the front of the cell (Fig. 1D), while cells where syndecan-4 is inhibited generate protrusion in all directions (Fig. 1E). A similar analysis was performed for embryos expressing a mutated form of Dsh that works as a dominant negative of PCP signaling and an equivalent effect on directional migration and the orientations of cell protrusions was observed (Fig. 1C and F).Open in a separate windowFigure 1Directional migration of neural crest cells. (A and B) Example of track of a single cell migrating in vivo. (A) Control cell showing persistent directional migration. (B) Cell in which the PCP signaling has been inhibited, showing absence of directional migration. (C) Cell in which syndecan-4 has been inhibited, showing no persistent migration. (D–F) Analysis of cell polarity and model of directional migration. Fn: fibronectin; Syn4: syndecan-4. (D) Control cell. Activation of Fn/Syn4 and PCP/RhoA lead to inhibition of Rac at the back of the cell, with the consequence polarization and directional migration. (E) Inhibition of PCP signaling leads to absence of RhoA activity, and in consequence an increase of Rac activity at the back of the cell. It seems that the inhibition of Rac activity by Syn4 is not sufficient to keep low levels of Rac at the back of the cells. High levels of Rac at the back produce a loss in cell polarity and in directional migration. (F) Inhibition of Syn4 generates high levels of Rac activity by a double mechanism: absence of direct inhibition of Rac and absence of RhoA which is dependent on PCP signaling. High levels of Rac at the back produce a loss of cell polarity and directional migration.As cell protrusions are known to be controlled by small GTPases and as PCP and syndecan-4 signaling regulates the activities of small GTPases,^18,22 we analyzed the activity of cdc42, RhoA and Rac after interfering with Dsh and syndecan-4. We choose to perform FRET analysis of these molecules as it is a technique that allows the visualization of their localized activity. More interestingly we succeeded in performing FRET analysis in cells migrating in vivo for the first time. Our results show that syndecan-4 inhibits Rac activity, while Dsh signaling promotes RhoA activity. In addition, we show that RhoA inhibits Rac in neural crest cells.¹⁰ The regulation of Rac by syndecan-4 is similar to that seen in other cells types in vitro.^23,24The model that emerges from these results to explain directional migration of NC cells in vivo is as follows (Fig. 1D). After delamination NC cells come into contact with fibronectin in the extracellular matrix, which is known to provide the main substrate for neural crest cells during their migration.^25,26 The interaction of fibronectin with syndecan-4 leads to two major changes in the cell: activation of PCP signaling and inhibition of Rac activity. The activated PCP signaling becomes localized at the back of the cell. From here, PCP contributes to the inhibition of Rac at the back of the cell, through the activation of RhoA. The coordinated activities of syndecan-4 and PCP signaling lead to polarised Rac activity across the cell, with Rac enriched at the leading edge, where it promotes the polymerization of actin and formation of lamellipodia, resulting in directional migration (Fig. 1D). Inhibition of PCP signaling produces high levels of Rac all over the cell as Rac, an inhibitor of RhoA in many cell types including neural crest cells, is absent (Fig. 1E). This generates cell protrusions in all directions with the consequent loss of cell polarity. If syndecan-4 is absent, the levels of Rac activity are also high all over the cell as the inhibition of Rac by syndecan-4 is absent (Fig. 1F), which also leads to a loss of cell polarity.Although detailed study of the localized activity of small GTPases has not been performed for other migratory cells in vivo, it is likely that the machinery will be similar to the one described here for NC cells. For example, it is well established in Xenopus, zebrafish and chick embryos that the migration of mesodermal cells during gastrulation requires PCP signaling.^27–29 It has also been shown that gastrulation in Xenopus²⁰ and in zebrafish (unpublished observations) requires the activity of syndecan-4. Thus, it is expected that cell polarity established during the migration of mesodermal cells will be dependent on small GTPases controlled by non-canonical Wnt signaling and syndecan-4.This novel integrated view of PCP, syndecan-4 and small GTPase activity during directional cell migration in vivo is an important advance in our knowledge of cell migration. Nevertheless, how the PCP signaling becomes activated only at the back of the cell, is a key question that needs to be answered. Future studies will be necessary to solve this and other crucial problems.     

The potential of statin and stromal cell-derived factor-1 to promote angiogenesis

Angiogenesis requires the mobilization of progenitor cells from the bone marrow (BM) and homing of progenitor cells to ischemic tissue. The cholesterol lowering drug Statins can stimulate angiogenesis via mobilization of BM derived endothelial progenitor cells (EPCs), promoting EPC migration, and inhibiting EPC apoptosis. The chemokine stromal cell-derived factor-1 (SDF-1) augments EPC chemotaxis, facilitates EPC incorporation into the neovasculature. The combined use of a statin to mobilize EPCs and local overexpression of SDF-1 to augment EPC homing to ischemic muscle resulted in superior angiogenesis versus use of either agent alone. Their effects are through augmenting EPC mobilization, incorporation, proliferation, migration and tube formation while inhibiting EPC apoptosis. Statin and SDF-1 therefore display synergism in promoting neovascularization by improving reperfusion of ischemic muscle, increasing progenitor cell presentation and capillary density in ischemic muscle, and diminishing apoptosis. These results suggest that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.Key words: angiogenesis, endothelial progenitor cells, statin, SDF-1, migrationAngiogenesis is the process by which new vessels form in ischemic tissue. The cytokine Stromal Cell Derived Factor-1 (SDF-1) is released into the circulation in response to ischemia and is an initiating signal in the angiogenesis process. SDF-1 mobilizes bone marrow cells (BMC) by binding to the cell surface receptor CXCR4. BMCs then enter the circulation and migrate to the ischemic site following the SDF-1 gradient. On arrival, BMCs promote angiogenesis by providing cellular elements such as endothelial cells (EC) and perivascular cells and also by secreting signaling proteins that mature the angiogenesis process. BMC surface CXCR4 expression and the SDF-1/CXCR4 interaction are essential for BMC to home to the injured site.Cell-based strategies to improve neovascularization of ischemic tissue have been achieved by injecting mononuclear cells derived from either BM¹ or peripheral blood, directly into ischemic muscle,² or by mobilizing BM-MNC with cytokines³ or other drugs such as statins.^4–6Statins are 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors and are primarily used to lower circulating cholesterol levels. In addition to reducing cholesterol synthesis, inhibition of the mevalonate pathway prevents synthesis of isoprenoid intermediates including geranylgeranylpyrophosphate. Geranylgeranylation is important in the posttranslational modification of intracellular signaling proteins, including Rho GTPases. This mechanism underlies many of the pleiotropic effects including the ability of statins to stabilize endothelial nitric oxide synthase mRNA and increase nitric oxide biosynthesis. In fact, statins have been shown to protect against ischemic injury of the heart and stimulate angiogenesis in ischemic limbs of normocholesterolemic animals.^7,8 The mechanism of action of statins has been demonstrated via mobilization of BM endothelial progenitor cells (EPCs) and facilitation of EPC incorporation into the neovasculature through a phosphoinositide-3 (PI-3) kinase-dependent pathway.^4–6 Statins have also been reported to enhance EPC migration, augment EPC chemotaxis and inhibit EPC apoptosis both in vitro and in vivo.^4,9,10SDF-1, an 89-amino acid polypeptide, is a member of the chemokine CXC subfamily originally isolated from murine bone marrow stromal cells.¹¹ SDF-1 was initially identified as a potent chemoattractant for lymphocytes and monocytes, and as an enhancer of B cell proliferation. SDF-1 is considered to be a key regulator of hematopoietic stem cell trafficking between BM and the peripheral circulation. SDF-1 is highly expressed in ischemic tissues.^12,13 Elevation of SDF-1 levels in peripheral blood results in BMC mobilization to the peripheral circulation with a concurrent decrease within the BM.¹⁴ SDF-1 not only mobilizes progenitor cells in BM but also directs them to the ischemic site by promoting cell migration and proliferation.^3,15 SDF-1 may generate a gradient similar to developmental morphogens during ischemia that provides the cues and directions for progenitor cell mobilization into peripheral blood and homing to ischemic tissues.^16,17 Furthermore, SDF-1 also reduces EPC apoptosis and enhances survival of the progenitor cells.^3,18 SDF-1, either delivered locally in its protein form,^3,19,20 or generated in situ via plasmid and viral vector-mediated gene expression,^10,21,22 enhances neovascularization by augmenting EPC recruitment into ischemic tissues.SDF-1 binding to its receptor CXCR4 on the cell surface provides essential signals for mobilization and homing of EPCs to the injured site.^23–25 SDF-1 binding with CXCR4 triggers internalization of CXCR4. This SDF-1/CXCR4 interaction results in elevation of cytoplasmic Ca²⁺ levels²⁶ and phosphorylation of PI-3 kinase and other protein kinases, e.g. Akt,²¹ MEK/ERK^27,28 and Janus kinase (JAK)-2.²⁹ Activation of Akt protein kinase further upregulates the activity of eNOS by increasing both eNOS expression and phosphorylation, which in turn catalyzes the production of nitric oxide (NO), an important signal molecule for vascular protection and remodeling.^21,26 Disruption of SDF-1/CXCR4 interaction impaired incorporation of EPC into sites of ischemia, and disturbed ischemic limb neo-vascularization.³⁰To explore if the combined use of a statin to mobilize BM EPCs and local overexpression of SDF-1 to augment EPC homing to ischemic muscle will result in superior angiogenesis versus use of either agent alone, we used the murine hindlimb ischemia model to determine the effects of Fluvastatin and SDF-1 on angiogenesis.¹⁰ Fluvastatin (5 mg/kg) was injected intra-peritoneally into the mice daily for 7 days to mobilize progenitor cells prior to ischemia-inducing surgery. NIH 3T3 cells transduced with the retroviral vector carrying SDF-1 gene were injected I.M. into the ischemic limb after surgery to locally deliver SDF-1 to ischemic muscle.²² The number of circulating EPCs increased 9–18 fold seven days post statin/SDF-1 treatment.Our data of single treatment with Fluvastatin are consistent with the previous reports that statins not only augment mobilization of progenitor cells by increasing circulating EPC originated from BM,^4,31 but also modulate their differentiation. We further give a new insight view of the mechanism for statin induced EPC mobilization. We found that statin induced activation of matrix metalloproteinases (MMP)-2 and -9 in EPC. The increased MMP activity could result in degradation of extracellular matrix.¹⁷ Progenitor cells will be such mobilized into circulation when the cellular attachment is reduced within the bone marrow niches. We show that statin alone can enhance the phosphorylation of Akt, promote EPC proliferation, migration and inhibit cell apoptosis in vitro. The proangiogenic effects of statin are also illustrated in vivo using a murine hind-limb ischemia model. In this model, Fluvastatin treatment results in more EPC in circulation, more BM derived progenitor cells in ischemic muscle, more cell proliferation, enhanced capillary formation, and diminished cell apoptosis; these effects end up in improved reperfusion versus control. The beneficial effects of statin on angiogenesis are independent of cholesterol since the total serum cholesterol level is not changed by Fluvastatin treatment under these experimental conditions.To be noted, the effect of statins on EPCs was found to be concentration dependent. EPC proliferation, migration and the inhibition of apoptosis are enhanced at low statin concentrations (10 nM and 100 nM) but are significantly inhibited at a higher statin concentration (1,000 nM). The toxic effect of statin at high concentration cannot be compensated by addition of SDF-1, indicating that Statin causes apoptosis in a pathway different from the pathway that SDF-1 uses to prevent EPC apoptosis. Increased apoptosis at the higher statin concentration could explain the reversed effect of stain in angiogenesis. These findings are consistent with the reports in which statins were found to have proangiogenic effects at low therapeutic concentrations but angiostatic effects at high concentrations, the latter effect being reversible by geranylgeranyl pyrophosphate.^32,33Combined statin and SDF-1 treatment significantly enhanced angiogenesis versus treatment with either reagent alone. More cell proliferation and less apoptosis were observed both in vitro and in vivo, along with increased cell migration and tube formation in vitro, and enhanced progenitor cell incorporation and higher capillary density in ischemic tissue in vivo. It is interesting to note that neither statin nor SDF-1 alone promotes EPC tube formation, but combined treatment results in significant EPC tube formation. These results suggest that SDF-1 and statin have different mechanisms of action with regards to the promotion of neovascularization. It is possible that each drug affects a specific subset of progenitor cells.The facilitative effect of both statin and SDF-1 on EPC proliferation and migration is involved with Akt phosphorylation and endothelial nitric oxide synthase (eNOS) activation. The mechanism by which statins promote angiogenesis is through, at least partly, improved nitric oxide bioavailability. Statins have been reported to induce eNOS mRNA stability³⁴ and eNOS activity through a PI3k/Akt dependent pathway.^31,35–37 However, neither eNOS mRNA/protein expression nor EPCs are reported to be essential for the therapeutic effect of Fluvastatin on hypoxia-induced pulmonary hypertension; Fluvastatin improved eNOS phosphorylation by a mechanism independent of Akt activation.³⁸ Our data favor a mechanism involving Akt phosphorylation since phosphorylated Akt is increased when EPCs are cultured in the presence of statin, and statin-enhanced EPC proliferation and migration were inhibited by the PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.The angiogenic effects of SDF-1 also involve increased production of NO²⁶ as NO is essential for EC migration and angiogenesis. SDF-1α gene transfer has been shown to enhance eNOS activity.²¹ Our in vitro data confirmed the involvement of Akt and eNOS in SDF-1 mediated cell migration.¹⁰ Phosphorylated Akt is increased when EPCs are cultured in the presence of SDF-1. The facilitative effect of SDF-1 on EPC migration is blocked by both the Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the eNOS inhibitor L-NMMA. In contrast, L-NMMA does not reverse the inhibitory effect of SDF-1 on apoptosis, indicating that the inhibitory effect of SDF-1 on apoptosis is not mediated through NO.²²We also show that the expression of MMP-2 and MMP-9 was increased when EPCs were cultured in the presence of statin or SDF-1. MMPs are a family of proteolytic enzymes that degrade components of the extracellular matrix (ECM). Degradation of ECM is an essential step for cell mobilization and migration. Our data indicate that the novel effect of statin and SDF-1 on migration is through enhancement of MMP-2 and MMP-9 activity, resulting in ECM degradation, thus promoting progenitor cell mobilization and migration. Both Akt phosphorylation and expression of MMP-2 and MMP-9 in EPCs are further enhanced by combined treatment with statin and SDF-1. This result indicates that treatment of EPCs with either statin or SDF-1 as monotherapy results in a sub-maximal angiogenic response. The effects of statin partially overlap with that of SDF-1; and the combined use of two factors appears to have an optimal effect on progenitor cells (Fig. 1).Open in a separate windowFigure 1Effect of statins and SDF-1 on promoting angiogenesis. Statin enhances the phosphorylation of Akt with a yet undefined mechanism. SDF-1 binding with the G-protein coupled membrane receptor CXCR4 results in phosphorylation of protein kinases like PI3 kinase and Akt. Activation of Akt then upregulates the activities of MMPs and eNOS. NOS catalyze the synthesis of NO which is essential for the EPC migration. MMPs degrade extracellular matrix to initiate cell migration. Activation of Akt also prevents cell apoptosis. These reactions promote cell migration and proliferation and enhance EPC survival. EPCs from bone marrow are thus mobilized into circulation. The circulating EPC are homed into ischemia area in lure of SDF-1. EPCs contribute to neovascularisation, either directly by incorporation into endothelium and differentiation into endothelial cells or indirectly by differentiating into perivascular cells that provide physical support and secrete signaling proteins and structural enzymes enabling the angiogenesis process. The effects of statin partially overlap with that of SDF-1; and the combined use of two factors appears to have an additive/synergistic effect on progenitor cells.In summary, the combination of progenitor cell mobilization with statin and targeted recruitment into the ischemic bed by SDF-1 leads to improved blood flow in the ischemic limb versus treatment with either agent alone. Statin and SDF-1 therefore display synergism in promoting neovascularization. This result suggests that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia. However, the use of statins as a clinical modifier of angiogenesis is still unproven. A great number of patients have been treated with these drugs and if they were potently proangiogenic, one might expect to see an increased risk of tumors. However, there is no evidence that these drugs encourage tumor development. Likewise, there is no definitive evidence for an antiangiogenic, tumor-modulating action of statins. We await further studies with interest.     

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     

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.     

Free edges in epithelia as cues for motility

One of the primary functions of any epithelium is to act as a barrier. To maintain integrity, epithelia migrate rapidly to cover wounds and there is intense interest in understanding how wounds are detected. Numerous soluble factors are present in the wound environment and epithelia can sense the presence of adjacent denuded extracellular matrix. However, the presence of such cues is expected to be highly variable, and here we focus on the presence of edges in the epithelial sheets as a stimulus, since they are universally and continuously present in wounds. Using a novel tissue culture model, free edges in the absence of any other identifiable cues were found to trigger activation of the epidermal growth factor receptor and increase cell motility. Edges bordered by inert physical barriers do not activate the receptor, indicating that activation is related to mechanical factors rather than to specific cell-cell interactions.Key words: cell migration, wound, healing, mechanotransduction, epithelial, edges, chronic ulcers, contact inhibition, sheet movementThe fundamental role of epithelia is to provide barriers between different compartments of the organism and to the outside environment. During development and in adulthood, epithelial cells employ their inherent ability to migrate as a collective sheet to generate or restore barrier function. Collective migration is essential for processes such as organogenesis and wound healing, and similar migratory mechanisms can go awry and contribute to cancer metastasis. Therefore, a considerable amount of research has been directed at understanding the cellular signals that initiate and sustain epithelial migration.^1–3In numerous epithelia, the epidermal growth factor receptor (EGFR) is activated by wounding, and blocking the activity of the receptor pharmacologically or by genetic techniques inhibits healing. Conversely, experimental stimulation of the EGFR results in enhancement of wound healing in many instances, underscoring the central role of the EGFR in the healing process.^4–6 Wounding induces proteolytic release of ligands, such as heparin-binding EGF-like growth factor (HB-EGF), from precursors located in the cell membrane in a mechanism that resembles EGFR transactivation by G-protein coupled receptors.^7–9 In a mammalian model of epithelial morphogenesis, eyelid closure in mice, epithelial sheet movement is also dependent on the proteolytic release of HB-EGF, which activates the EGFR.¹⁰ Therefore, not only are the biomechanical processes that control epithelial movements during morphogenesis and wound healing similar, but the signals that induce this motility are similar as well.Given its importance, it is not surprising that many mechanisms have evolved to regulate epithelial wound healing. Starting immediately after wounding, the epithelium is inundated with a large number of growth factors and cytokines produced by bordering tissues and infiltrating inflammatory cells.^1,11,12 In addition, epithelial cells themselves possess mechanisms that detect the presence of wounds. Epithelial cells in a monolayer are not stationary, but appear to move around in a lively fashion, which could theoretically produce wound closure because the cells could simply fill up the space that is opened up after wounding. In support of this, computer modeling has shown that the behavior of individually randomly moving cells can approximate the observed collective migration as a sheet.¹³ However, human corneal limbal epithelial (HCLE) and other cells react to wounding by increasing their velocities near edges,¹⁴ so they respond to wounds by changes in behavior and must therefore contain appropriate detection mechanisms.