Tensin is potentially involved in extracellular matrix production in mesangial cells

Tensin, a focal adhesion protein, is expressed in renal tubular epithelial cells (TECs). Tensin-null mice develop multiple large cysts in the renal proximal tubules. However, the role of tensin in human glomeruli remains unclear. In this study, we assessed tensin localization in human kidney and interaction between tensin and other adhesion components. In human mesangial cells (MCs) and TECs, we confirmed mRNA and protein expressions of tensin by RT-PCR and immunoprecipitation. In normal kidney, immunohistochemistry revealed that tensin was localized in MCs and parietal epithelial cells as well as TECs. In biopsy specimens, the expression of tensin was significantly increased in areas of mesangial expansion in patients with IgA nephropathy and diabetic nephropathy. These results suggest that the expression of tensin is associated with extracellular matrix (ECM) production. In vitro, immunocytochemistry revealed that MCs express tensin mainly at the ends of actin stress fibers and apparently in the focal adhesion areas. Integrin 5, but not 1 and 3, colocalized with tensin. Vinculin and focal adhesion kinase (FAK) were coprecipitated by tensin, suggesting that tensin can mediate signal transduction between cell and ECM through these molecules. Tensin may play important roles in mesangial ECM production through an adhesion complex with integrin 5, FAK, and vinculin.     

Amplification and oscillations in the FAK/Src kinase system during integrin signaling

Integrin signaling is a major pathway of cell adhesion to extracellular matrices that regulates many physiological cell behaviors such as cell proliferation, migration or differentiation and is implied in pathologies such as tumor invasion. In this paper, we focused on the molecular system formed by the two kinases FAK (focal adhesion kinase) and Src, which undergo auto- and co-activation during early steps of integrin signaling. The system is modelled using classical kinetic equations and yields a set of three nonlinear ordinary differential equations describing the dynamics of the different phosphorylation forms of FAK. Analytical and numerical analysis of these equations show that this system may in certain cases amplify incoming signals from the integrins. A quantitative condition is obtained, which indicates that the total FAK charge in the system acts as a critical mass that must be exceeded for amplification to be effective. Furthermore, we show that when FAK activity is lower than Src activity, spontaneous oscillations of FAK phosphorylation forms may appear. The oscillatory behavior is studied using bifurcation and stability diagrams. We finally discuss the significance of this behavior with respect to recent experimental results evidencing FAK dynamics.     

The paxillin LD motifs

Adapter/scaffold proteins, through their multidomain structure, perform a fundamental role in facilitating signal transduction within cells. Paxillin is a focal adhesion adapter protein implicated in growth factor- as well as integrin-mediated signaling pathways. The amino-terminus of paxillin contains five leucine-rich sequences termed LD motifs. These paxillin LD motifs are highly conserved between species as well as within the paxillin superfamily. They mediate interactions with several structural and regulatory proteins important for coordinating changes in the actin cytoskeleton associated with cell motility and cell adhesion as well as in the regulation of gene expression.     

Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

Syndecans are transmembrane proteoglycans that support integrin-mediated adhesion. Well documented is the contribution of syndecan-4 that interacts through its heparan sulphate chains to promote focal adhesion formation in response to fibronectin domains. This process has requirements for integrin and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in any epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were unable to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence for an indirect interaction of beta1 integrin with both syndecan ectodomains was obtained, all of which suggests a distinct mechanism of integrin-mediated adhesion.     

α2,6-Sialyltransferase Gene Transfection into a Human Glioma Cell Line (U373 MG) Results in Decreased Invasivity

Abstract: Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP-NeuAc:Galβ1,4GlcNAc α2,6-sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated α3β1 integrin. Adhesion-mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125^fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion-mediated signal transduction and block glioma cell invasivity in vitro.     

Inhibition of integrin-mediated adhesion and signaling disrupts retinal development

Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling play essential roles in neural development. In this study, we have used echistatin, an RGD-containing short monomeric disintegrin, to investigate the role of integrin-mediated adhesion and signaling during retinal development in Xenopus. Application of echistatin to Xenopus retinal-derived XR1 glial cells inhibited the three stages of integrin-mediated adhesion: cell attachment, cell spreading, and formation of focal adhesions and stress fibers. XR1 cell attachment and spreading increased tyrosine phosphorylation of paxillin, a focal adhesion associated protein, while echistatin significantly decreased phosphorylation levels of paxillin. Application of echistatin or beta(1) integrin function blocking antibody to the embryonic Xenopus retina disrupted retinal lamination and produced rosette structures with ectopic photoreceptors in the outer retina. These results indicate that integrin-mediated cell-ECM interactions play a critical role in cell adhesion, migration, and morphogenesis during vertebrate retinal development.     

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.     

PINCH2 is a new five LIM domain protein, homologous to PINCHand localized to focal adhesions

PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2. Here we describe a new isoform of PINCH, which we call PINCH2. Therefore, we rename PINCH to PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, we observed differential expression pattern of the mRNAs. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. Finally, we demonstrate that PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes.     

Intracellular modifiers of integrin alpha 6p production in aggressive prostate and breast cancer cell lines

Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent “inside-out” signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent “inside-out” signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype.     

Focal adhesion and actin organization by a cross-talk of TM4SF5 with integrin alpha2 are regulated by serum treatment

The biological functions of transmembrane 4 L6 family member 5 (TM4SF5) homologues to a tumor-associated antigen L6 are unknown, although it is over-expressed in certain forms of cancer. In the present study, the ectopic expression of TM4SF5 in Cos7 cells reduced integrin signaling under serum-containing conditions, but increased integrin signaling upon serum-free replating on substrates. TM4SF5 regulated actin organization and focal contact dynamics via the serum treatment-dependent differential regulation of FAK Tyr925 and paxillin Tyr118 phosphorylations and their localizations on peripheral cell boundaries. Y925F FAK mutation abolished the TM4SF5 effects. TM4SF5 associated with integrin alpha2 subunit, and this association was abolished by serum treatment. Furthermore, functional blocking anti-integrin alpha2 antibody abolished TM4SF5-enhanced signaling activity and caused membrane blebbing with abnormal actin organization. TM4SF5 increased chemotactic but decreased haptotactic migration. Altogether, this study reveals the functions of TM4SF5 collaborative with integrin signaling to alter focal contact dynamics, actin reorganization, and migration. Furthermore, this study suggests a mechanism of cross-talk between TM4SF5 and integrin which is further regulated by growth factor signaling.     

Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in extracellular matrix-induced cell migration and the activation of extracellular signal-regulated kinase (ERK). We showed here that transfection of the MT1-MMP gene into HeLa cells promoted fibronectin-induced cell migration, which was accompanied by fibronectin degradation and reduction of stable focal adhesions, which function as anchors for actin-stress fibers. MT1-MMP expression attenuated integrin clustering that was induced by adhesion of cells to fibronectin. The attenuation of integrin clustering was abrogated by MT1-MMP inhibition with a synthetic MMP inhibitor, BB94. When cultured on fibronectin, HT1080 cells, which endogenously express MT1-MMP, showed so-called motile morphology with well-organized focal adhesion formation, well-oriented actin-stress fiber formation, and the lysis of fibronectin through trails of cell migration. Inhibition of endogenous MT1-MMP by BB94 treatment or expression of the MT1-MMP carboxyl-terminal domain, which negatively regulates MT1-MMP activity, resulted in the suppression of fibronectin lysis and cell migration. BB94 treatment promoted stable focal adhesion formation concomitant with enhanced phosphorylation of tyrosine 397 of focal adhesion kinase (FAK) and reduced ERK activation. These results suggest that lysis of the extracellular matrix by MT1-MMP promotes focal adhesion turnover and subsequent ERK activation, which in turn stimulates cell migration.     

Macrocyclic chelator assembled RGD multimers for tumor targeting

Macrocyclic chelators have been extensively used for complexation of metal ions. A widely used chelator, DOTA, has been explored as a molecular platform to assemble multiple bioactive peptides in this paper. The multivalent DOTA-peptide bioconjugates demonstrate promising tumor targeting ability.     

Chelate oxorhenium to assemble new integrin antagonists

Assembly of independent chemical modules through oxorhenium coordination by a NS₂ + S chelation motif was applied to the synthesis of RGD (Arg-Gly-Asp) analogs. Modules were assembled through oxorhenium chelation to give a series of 18 metal complexes in good yields and satisfactory purities. Screening of these oxorhenium coordinates as antagonists of integrins αVβ3, αIIbβ3 and αVβ5 led to the identification of 3 bioactive compounds that exhibit submicromolar affinities for the 3 integrins. Preliminary studies showed that the corresponding oxotechnetium complexes are stable in mice plasma and therefore could be proposed for the molecular imaging of pathologies that overexpress integrins αVβ3 and αVβ5.     

Presentation of functional foreign peptides on the surface of SV40 virus-like particles

Viral capsids of simian virus 40 (SV40) are highly efficient gene delivery vehicles that infect a broad range of cells and tissues. To develop a controlled, cell type-specific delivery system, we sought to display foreign peptides on the capsid surface by genetically manipulating the major capsid protein Vp1. Here we report the identification of two sites within the surface loops of Vp1 that can accommodate foreign peptides in such a way that the foreign peptides are displayed on the surface of the virus-like particles (VLPs) without interfering with VLP assembly or the packaging of viral DNA. Insertion of Flag-tags but not RGD integrin-binding motifs at these sites strongly inhibited cell attachment of VLPs, which normally associate with host cells through cell surface molecules such as major histocompatibility complex (MHC) class I and ganglioside GM1. Instead, VLPs carrying the RGD motifs bound to integrin in vitro and to the cell surface in an RGD-dependent manner. Thus, insertion of foreign sequences into the surface loops of Vp1 can reduce natural virus-cell interactions and even confer an ability to bind to a new target receptor. This study demonstrates the potential usefulness of this strategy for the development of novel delivery vehicles with different cell tropisms.     

RGD-IL-24, A Novel Tumor-Targeted Fusion Cytokine: Expression,Purification and Functional Evaluation

Targeting drugs to tumor cells is a central challenge for improving existing cancer therapies. ACDCRGDCFCG peptide (RGD-4C) binds to αvβ3 integrin, which is selectively expressed in tumor blood vessels and on the surface of some tumor cells. Interleukin 24 (IL-24) is a novel cancer growth-suppressing and apoptosis-inducing cytokine. To enhance the antitumor effect, we coupled RGD-4C to the N-terminus of IL-24 and expressed RGD-IL-24 in Escherichia coli. Cell proliferation and adhesion experiments revealed that RGD-IL-24 specifically binds to MCF-7 cancer cells, and induces apoptosis of MCF-7 cancer cells. These studies support the use of the RGD-IL-24 protein in tumor-targeting therapy.