Integrins and cell signaling in chondrocytes

Integrins are adhesion receptor heterodimers that transmit information from the extracellular matrix (ECM) to the cell through activation of cell signaling pathways. Chondrocytes express several members of the integrin family including alpha5beta1 which is the primary chondrocyte receptor for fibronectin. Cell signaling mediated through integrins regulates several chondrocyte functions including differentiation, matrix remodeling, responses to mechanical stimulation and cell survival. Integrin-mediated activation of members of the mitogen-activated protein kinase family likely plays a key role in transmitting signals regulating chondrocyte gene expression. Upstream mediators of mitogen-activated protein kinase (MAP kinase) activation include focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (pyk2) which are both expressed by chondrocytes. A better understanding of chondrocyte integrin signaling is needed to define the mechanisms by which the ECM regulates chondrocyte function.     

Regulation of integrin activity by MIA

MIA (melanoma inhibitory activity) has been identified as a small protein secreted from malignant melanoma cells, which interacts with extracellular matrix proteins including fibronectin. Here, we show that MIA negatively regulates the activity of the mitogen-activated protein kinase pathway in malignant melanoma. Using far Western blotting and co-immunoprecipitation we searched for MIA-binding cell surface proteins. We found that MIA interacts with integrin alpha4beta1 and alpha5beta1, leading to down-regulation of integrin activity and reduction of mitogen-activated protein kinase signaling. These findings also suggest that MIA may play a role in tumor progression and the spread of malignant melanomas via mediating detachment of cells from extracellular matrix molecules by modulating integrin activity. Inhibiting MIA functions in vivo may therefore provide a novel therapeutic strategy for metastatic melanoma disease.     

R-Ras signals through specific integrin alpha cytoplasmic domains to promote migration and invasion of breast epithelial cells.

Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the alpha2, but not the alpha5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin beta cytoplasmic domains was observed for the alpha2 cytoplasmic domain; alpha2beta1-mediated migration was inhibited by the expression of excess alpha2 but not alpha5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin alpha subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin alpha cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion.     

Enterocytic differentiation of the human Caco-2 cell line correlates with alterations in integrin signaling

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with down-regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco-2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen-activated protein (MAP) kinase. The differentiation of Caco-2 cells was associated with: 1) down-regulation of beta1 integrin expression at the mRNA and protein levels; 2) increased FAK expression together with decreased FAK autophosphorylation; 3) decreased FAK's ability to associate with PI3-kinase and pp60c-src; and 4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco-2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by beta1 integrins in Caco-2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line.     

The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.     

TNFalpha-induced apoptosis and integrin switching in human extravillous trophoblast cell line

Differentiation of extravillous trophoblast cells (EVT) to an invasive phenotype plays an essential role in establishing and maintaining feto-placental organization during human pregnancy. A switch in integrin expression occurs during this differentiation and is accompanied by changes in the extracellular matrix (ECM). Alteration of EVT behavior is also modulated by cytokines. To investigate the molecular interactions involved in the EVT differentiation, we examined the effects of cytokines and ECM on the human EVT cell line, TCL1 cells. We found that tumor necrosis factor alpha (TNFalpha) induced apoptosis in TCL1 cells but not in JEG3 cells derived from choriocarcinoma while the addition of interleukin-1beta, leukemia inhibitory factor, or transforming growth factor had no effect on TCL1 cells. This apoptosis was suppressed when TCL1 cells were seeded on fibronectin (Fn), collagen type I (C1), collagen type IV (C4), or laminin (Ln). Wortmannin, a specific PI3 kinase inhibitor, inhibited this suppression. Spreading assays and adhesion blocking assays indicated that TCL1 cells express integrin-alpha5 and -alpha6 and beta1 and beta4 subunits. Adhesion on Fn is mediated by alpha5beta1, and adhesion on C1, C4, or Ln is mediated by alpha6beta1 integrins. TNFalpha suppressed alpha6 integrin expression and enhanced alpha1 integrin expression in a dose-dependent manner. In addition, aggregation of beta1 subunits on C4 was detected after addition of TNFalpha. Taken together, these results suggest that TNFalpha and ECM, through activation of PI3 kinase mediated by beta1 integrin signaling, might collaboratively regulate differentiation of trophoblast cells through integrin signaling in establishing and maintaining successful pregnancy.     

Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal

We previously demonstrated contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169-184). Ectopic expression of the integrin alpha5 or alpha6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin alpha subunit ratios regulate this decision. Ectopic expression of truncated alpha5 or alpha6A indicate that the alpha5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of alpha6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha5 and alpha6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta1 signaling. Ectopically expressed IL2R-alpha5 or IL2R-alpha6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta1A integrin subunit or IL2R-beta1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha5 or alpha6A ratios also significantly affects activation of beta1 integrin signaling pathways. Ectopic alpha5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta1A subunit and is modulated by the alpha subunit cytoplasmic domains; (b) perturbing alpha subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.     

A protein kinase C/Ras/ERK signaling pathway activates myeloid fibronectin receptors by altering beta1 integrin sialylation

Here we report that myeloid cells differentiating along the monocyte/macrophage lineage down-regulate the ST6Gal-I sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence, the beta1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of alpha5beta1 fibronectin receptors. Pharmacologic inhibitors of protein kinase C, Ras, and MEK, but not phosphoinositide 3-kinase, block ST6Gal-I down-regulation, integrin hyposialylation, and fibronectin binding. In contrast, constitutively active MEK stimulates these same events, indicating that ERK is both a necessary and sufficient activator of hyposialylation-dependent integrin activation. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified alpha5beta1 receptors bind fibronectin more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of these integrins with recombinant ST6Gal-I. Finally, we have mapped the N-glycosylation sites on the beta1 integrin to better understand the potential effects of differential sialylation on integrin structure/function. Notably, there are three N-glycosylated sites within the beta1 I-like domain, a region that plays a crucial role in ligand binding. Our collective results suggest that variant sialylation, induced by a specific signaling cascade, mediates the sustained increase in cell adhesiveness associated with monocytic differentiation.     

Alpha2beta1 integrin regulates lineage commitment in multipotent human colorectal cancer cells

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the beta1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to beta1 integrin. Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin. Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor alpha2beta1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.     

Beta1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits.

Integrins are found in adhesion structures, which link the extracellular matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of beta1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that beta1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The alpha4beta1 integrin exhibits a distinct distribution from other beta1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: alpha5beta1 integrins colocalize with adaptor protein-2 in coated pits, while alpha4beta1 integrins do not. This parallels our earlier observation that of the two laminin receptors, alpha1beta1 and alpha6beta1, only alpha1beta1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of alpha1beta1 and alpha5beta1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.     

Urinary-type plasminogen activator (uPA) expression and uPA receptor localization are regulated by alpha 3beta 1 integrin in oral keratinocytes

Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.     

Alpha 4 integrin signaling activates phosphatidylinositol 3-kinase and stimulates T cell adhesion to intercellular adhesion molecule-1 to a similar extent as CD3, but induces a distinct rearrangement of the actin cytoskeleton.

Dynamic regulation of beta(2) integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity alpha(4)beta(1) integrins to VCAM-1 strengthens alpha(L)beta(2) integrin-mediated adhesion to ICAM-1. In this study, we compared beta(2) integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: alpha(4) integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either alpha(4) integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of beta(2) integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by alpha(4) integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect alpha(4) integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca(2+) ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following alpha(4) integrin or CD3 stimulation of human peripheral blood T cells.     

Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development

Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.     

A co-clustering model involving alpha5beta1 integrin for the biological effects of GPI-anchored human carcinoembryonic antigen (CEA)

CEA functions as an intercellular adhesion molecule and is up-regulated in a wide variety of human cancers, including colon, breast and lung. Its over-expression inhibits cellular differentiation, blocks cell polarization, distorts tissue architecture, and inhibits anoikis of many different cell types. Here we report results concerning the molecular mechanism involved in these biological effects, where relatively rapid molecular changes not requiring alterations in gene expression were emphasized. Confocal microscopy experiments showed that antibody-mediated clustering of a deletion mutant of CEA (DeltaNCEA), normally incapable of self binding and clustering, led to the co-localization of integrin alpha5beta1 with patches of DeltaNCEA on the cell surface. Activation of alpha5, as defined by an anti-alpha5 mAb-sensitive increase in cell adhesion to immobilized fibronectin, and an increased binding of soluble fibronectin to cells, was also observed. This was accompanied by the recruitment of integrin-linked kinase (ILK), protein kinase B (PKB/Akt), and the mitogen-activated protein kinase (MAPK) to membrane microdomains and the phosphorylation of Akt and MAPK. Inhibition of PI3-K and ILK, but not MAPK, prevented the alpha5beta1 integrin activation. Conversely, anti-alpha5 antibody inhibited the PI3-K-mediated activation of Akt, implying the involvement of outside-in and inside-out signaling in integrin activation. Therefore we propose that CEA-mediated signaling involves clustering of CEA and co-clustering and activation of the alpha5beta1 and associated specific signaling elements on the internal surfaces of membrane microdomains. These changes may represent a molecular mechanism for the biological effects of CEA.     

Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of alphabeta(1), alpha(v)beta(3), and alpha(v)beta(5) integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.     

The integrin alpha5beta1 regulates chondrocyte hypertrophic differentiation induced by GTP-bound transglutaminase 2.

Soluble GTP-bound transglutaminase 2 (TG2) induces hypertrophic differentiation in chondrocyte cultures in a beta1 integrin-dependent fashion. beta1 integrin subfamily consists of 12 heterodimers with 12 different alpha subunits and a beta1 subunit. To identify the specific integrin heterodimer(s) responsible for this process, we specifically blocked individual beta1 integrins on the CH-8 immortalized human chondrocytes during hypertrophic differentiation. Blockade of alpha5beta1 inhibited matrix metalloproteinase 13 (MMP-13), type X collagen expression, alkaline phosphatase activity and matrix calcification by 30-50% associated with weak effects of anti-alpha3beta1 and -alpha4beta1. Anti-alpha1beta1, -alpha2beta1 and -alpha6beta1 had no effect. To examine whether the dominant effect of integrin alpha5beta1 was due to a direct interaction with TG2, we incubated the chondrocytic cells on plates coated with GTP-bound TG2. The immobilized GTP-bound TG2 induced hypertrophic differentiation to the same extent as the soluble GTP-bound TG2, which was also inhibited by anti-alpha5beta1. CH-8 cells grown on plates coated with GTP-bound TG2 demonstrated adherence associated with focal adhesion kinase phosphorylation. These properties were inhibited by anti-alpha5beta1. Furthermore, engagement of alpha5beta1 on CH-8 cells via anti-alpha5beta1 antibody did, in fact, induce differentiation. Although CH-8 cells adhered to GTP-free TG2 via integrin alpha5beta1, the cells failed to undergo hypertrophic differentiation. Thus, integrin alpha5beta1 is critical for the chondrocyte hypertrophic differentiation induced by GTP-bound TG2, and this induction is ligand dependent.     

Regulation of laminin 1-induced pancreatic beta-cell differentiation by alpha6 integrin and alpha-dystroglycan

BACKGROUND: The ability to manipulate the development of pancreatic insulin-producing beta cells has implications for the treatment of type 1 diabetes. Previously, we found that laminin-1, a basement membrane trimeric glycoprotein, promotes beta-cell differentiation. We have investigated the mechanism of this effect, using agents that block the receptors for laminin-1, alpha6 integrin, and alpha-dystroglycan (alpha-DG). MATERIALS AND METHODS: Dissociated cells from 13.5-day postcoitum (dpc) fetal mouse pancreas were cultured for 4 days with laminin-1, with and without monoclonal antibodies and other agents known to block integrins or alpha-DG. Fetuses fixed in Bouin's solution or fetal pancreas cells fixed in 4% paraformaldehyde were processed for routine histology and for immunohistology to detect hormone expression and bromodeoxyuridine (BrdU) uptake. RESULTS: Blocking the binding of laminin-1 to alpha6 integrin with a monoclonal antibody, GoH3, abolished cell proliferation (BrdU uptake) and doubled the number of beta cells. Inhibition of molecules involved in alpha6 integrin signaling (phosphotidylinositol 3-kinase, F-actin, or mitogen-activated protein kinase) had a similar effect. Nevertheless, beta cells appeared to develop normally in alpha6 integrin-deficient fetuses. Blocking the binding of laminin-1 to alpha-DG with a monoclonal antibody, IIH6, dramatically decreased the number of beta cells. Heparin, also known to inhibit laminin-1 binding to alpha-DG, had a similar effect. In the presence of heparin, the increase in beta cells in response to blocking alpha integrin with GoH3 was abolished. CONCLUSIONS: These findings reveal an interplay between alpha6 integrin and alpha-DG to regulate laminin-1-induced beta-cell development. Laminin-I had a dominant effect via alpha-DG to promote cell survival and beta-cell differentiation, which was modestly inhibited by alpha6 signaling.     

Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.     

beta 1 integrin regulates fibroblast viability during collagen matrix contraction through a phosphatidylinositol 3-kinase/Akt/protein kinase B signaling pathway

Integrins regulate cell viability through their interaction with the extracellular matrix. Integrins can sense mechanical forces arising from the matrix and convert these stimuli to chemical signals capable of modulating intracellular signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is a major regulator of cell survival. It is not known, however, whether integrins, acting as mechanoreceptors, regulate cell survival via the PI3K/Akt pathway. Here, we show that in response to a matrix-derived mechanical stimulus, beta1 integrin regulated cell viability by regulating Akt activity in a PI3K-dependent fashion. To accomplish this, we employed fibroblasts cultured in collagen gels. During contraction of collagen matrices, fibroblasts underwent apoptosis. We demonstrate that ligation of beta1 integrin with anti-beta1 integrin antibodies protected fibroblasts from apoptosis. The nature of the survival signal activated by beta1 integrin engagement with antibody was mediated by PI3K acting through Akt/protein kinase B. We show that Akt phosphorylation decreased during collagen contraction and that this decrease correlated precisely with the onset of fibroblast apoptosis. Fibroblasts transfected with constitutively active PI3K displayed increased Akt phosphorylation and were protected from anoikis and collagen gel contraction-induced apoptosis. Our data identify a novel role for beta1 integrin in regulating fibroblast viability through a PI3K/Akt/protein kinase B signaling pathway in response to a matrix-derived mechanical stimulus.