A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.     

Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.     

Cas-L is required for beta 1 integrin-mediated costimulation in human Tcells.

Beta 1 integrins provide a costimulus for TCR/CD3-driven T cell activation and IL-2 production in human peripheral T cells. However, this beta 1 integrin-mediated costimulation is impaired in a human T lymphoblastic line, Jurkat. We studied the molecular basis of this impaired costimulation and found that Cas-L, a 105-kDa docking protein, is marginally expressed in Jurkat T cells, whereas Cas-L is well expressed in peripheral T cells. Cas-L is a binding protein and a substrate for focal adhesion kinase and is tyrosine phosphorylated by beta 1 integrin stimulation. We here show that the transfection of wild-type Cas-L in Jurkat T cells restores beta 1 integrin-mediated costimulation. However, Cas-L transfection had no effect on CD28-mediated costimulation, indicating that Cas-L is specifically involved in the beta 1 integrin-mediated signaling pathway. Furthermore, transfection of the Cas-L Delta SH3 mutant failed to restore beta 1 integrin-mediated costimulation in Jurkat cells. Cas-L Delta SH3 mutant lacks the binding site for focal adhesion kinase and is not tyrosine phosphorylated after beta 1 integrin stimulation. These findings strongly suggest that the tyrosine phosphorylation of Cas-L plays a key role in the signal transduction in the beta 1 integrin-mediated T cell costimulation.     

EGFR and beta1 integrins utilize different signaling pathways to activate Akt

Akt, also called PKB, is a serine/threonine kinase that plays a major role in cell survival. It can be activated by several cellular receptors, including integrins and growth factor receptors, in PI3K-dependent manners. In this study, we analyzed the two current models for Akt activation upon beta1 integrin-mediated adhesion: via focal adhesion kinase and via transactivation of the EGF receptor. Distinct differences in the pathways leading to phosphorylation and activation of Akt from stimulated beta1 integrins and EGF receptor were observed, including opposing sensitivity to the tyrosine kinase inhibitors PP2 and Gefitinib. Using knockout cells and integrin mutant cells, we show that beta1 integrins can induce phosphorylation of Akt at Ser473 and Thr308 and Akt kinase activity independently of the EGF receptor activity, focal adhesion kinase, and the Src family members. In contrast to stimulation with EGF, beta1 integrin-mediated adhesion did not induce Akt tyrosine phosphorylation. Moreover, tyrosine phosphorylation of Akt was found not to be required for its catalytic activity. The results identify a previously unrecognized mechanism by which beta1 integrins activate the PI3K/Akt pathway.     

Control of TCR-mediated activation of beta 1 integrins by the ZAP-70 tyrosine kinase interdomain B region and the linker for activation of T cells adapter protein

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.     

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.     

The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility

Integrin affinity is modulated by intracellular signaling cascades, in a process known as "inside-out" signaling, leading to changes in cell adhesion and motility. Protein kinase C (PKC) plays a critical role in integrin-mediated events; however, the mechanism that links PKC to integrins remains unclear. Here, we report that PKCepsilon positively regulates integrin-dependent adhesion, spreading, and motility of human glioma cells. PKCepsilon activation was associated with increased focal adhesion and lamellipodia formation as well as clustering of select integrins, and it is required for phorbol 12-myristate 13-acetate-induced adhesion and motility. We provide novel evidence that the scaffolding protein RACK1 mediates the interaction between integrin beta chain and activated PKCepsilon. Both depletion of RACK1 by antisense strategy and overexpression of a truncated form of RACK1 which lacks the integrin binding region resulted in decreased PKCepsilon-induced adhesion and migration, suggesting that RACK1 links PKCepsilon to integrin beta chains. Altogether, these results provide a novel mechanistic link between PKC activation and integrin-mediated adhesion and motility.     

A novel function for the Tec family tyrosine kinase Itk in activation of beta 1 integrins by the T-cell receptor

Stimulation of T cells via the CD3--T-cell receptor (TCR) complex results in rapid increases in beta 1 integrin-mediated adhesion via poorly defined intracellular signaling events. We demonstrate that TCR-mediated activation of beta 1 integrins requires activation of the Tec family tyrosine kinase Itk and phosphatidylinositol 3-kinase (PI 3-K)-dependent recruitment of Itk to detergent-insoluble glycosphingolipid-enriched microdomains (DIGs) via binding of the pleckstrin homology domain of Itk to the PI 3-K product PI(3,4,5)-P(3). Activation of PI 3-K and the src family kinase Lck, via stimulation of the CD4 co-receptor, can initiate beta 1 integrin activation that is dependent on Itk function. Targeting of Itk specifically to DIGs, coupled with CD4 stimulation, can also activate beta 1 integrin function independently of TCR stimulation. Changes in beta 1 integrin function mediated by TCR activation of Itk are also accompanied by Itk-dependent modulation of the actin cytoskeleton. Thus, TCR-mediated activation of beta 1 integrins involves membrane relocalization and activation of Itk via coordinate action of PI 3-K and a src family tyrosine kinase.     

Integrin signaling cascades are operational in adult hippocampal synapses and modulate NMDA receptor physiology

Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin-associated tyrosine kinases, and if integrin signaling targets include NMDA-class glutamate neurotransmitter receptors. The integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, proline-rich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to beta1 integrin, and not obtained with control peptides, indicating that kinase activation was integrin-mediated. Both GRGDSP and fibronectin caused rapid Src kinase-dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and beta1 integrin-dependent increase in NMDA receptor-mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.     

Beta1 integrin/focal adhesion kinase-mediated signaling induces intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation

We have assessed characteristics of primary human osteoblasts, shedding light on signaling mediated by beta1 integrin. beta1 integrins are major receptors for these matrix glycoproteins. 1) Integrins beta1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphav were highly expressed on primary osteoblasts. 2) Engagement of beta1 integrins on osteoblasts by cross-linking with specific antibody or ligand matrices, such as fibronectin or collagen, augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on the surface. 3) Up-regulation of ICAM-1 and RANKL on osteoblasts by beta1 stimulation was completely abrogated by pretreatment with herbimycin A and genistein, tyrosine kinase inhibitors, or transfection of dominant negative truncations of focal adhesion kinase (FAK). 4) Engagement of beta1 integrins on osteoblasts induced tartrate-resistant acid phosphatase-positive multinuclear cell formation in the coculture system of osteoblasts and peripheral monocytes. 5) Up-regulation of tartrate-resistant acid phosphatase-positive multinuclear cell formation by beta1 stimulation was completely abrogated by transfection of dominant negative truncations of FAK. Our results indicate that beta1 integrin-dependent adhesion of osteoblasts to bone matrices induces ICAM-1 and RANKL expression and osteoclast formation via tyrosine kinase, especially FAK. We here propose that beta1 integrin/FAK-mediated signaling on osteoblasts could be involved in ICAM-1- and RANKL-dependent osteoclast maturation.     

Negative regulation of EGFR signalling through integrin-alpha1beta1-mediated activation of protein tyrosine phosphatase TCPTP

Integrin-mediated cell adhesion regulates a multitude of cellular responses, including proliferation, survival and cross-talk between different cellular signalling pathways. So far, integrins have been mainly shown to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signalling. Here we show that a collagen-binding integrin alpha(1)beta(1) functions as a negative regulator of epidermal growth factor receptor (EGFR) signalling through the activation of a protein tyrosine phosphatase. The cytoplasmic tail of alpha(1) integrin selectively interacts with a ubiquitously expressed protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) and activates it after cell adhesion to collagen. The activation results in reduced EGFR phosphorylation after EGF stimulation. Introduction of the alpha(1) cytoplasmic domain peptide into cells induces phosphatase activation and inhibits EGF-induced cell proliferation and anchorage-independent growth of malignant cells. These data are the first demonstration of the regulation of TCPTP activity in vivo and represent a new molecular paradigm of integrin-mediated negative regulation of receptor tyrosine kinase signalling.     

Adhesion, actin cytoskeleton organisation and the spreading of colon adenocarcinoma cells induced by EGF are mediated by α2β1 integrin low clustering through focal adhesion kinase

Both epidermal growth factor (EGF) and the extracellular matrix components have been implicated in the pathobiology of adenocarcinomas by somewhat poorly understood mechanisms. We have addressed this problem using an in vitro model comprising the colon adenocarcinoma cell line HT29-D4, wherein the role of EGF and type IV collagen on cell adhesion was examined. We demonstrated that the effect of EGF on HT29-D4 cell adhesion was regulated by type IV collagen in a time- and dose-dependent manner. The incorporation of a panel of monoclonal antibodies to integrins alpha1beta1, alpha2beta1 and alpha3beta1 in adhesion medium revealed that EGF-mediated increase in the cell adhesion was mediated essentially by alpha2beta1, and the use of flow cytometry led us to conclude that this EGF effect was mediated by an increase in alpha2beta1 activation and not by an increase in cell surface expression of integrin. An indirect immunofluorescence technique was employed to demonstrate that focal adhesion kinase (FAK) and alpha2beta1 integrin were present in focal complexes in large EGF-induced lamellipodia whereas actin cytoskeleton was organised in small tips that colocalised with FAK. This pattern was observed at early time points (15 min) with a strong FAK tyrosine phosphorylation and with an increase in mitogen-activated protein kinase activity (5-15 min) as measured by immunoprecipitation and immunoblotting. We conclude that at early time points of cell adhesion and spreading, EGF exerted an inside-out regulation of alpha2beta1 integrin in HT29-D4 cells. This regulation seemed to be mediated by EGF-dependent FAK phosphorylation entailing an increase in integrin activation and their recruitment in numerous focal complexes. Furthermore after activation, FAK induced aggregation of actin-associated proteins (paxillin, vinculin and other tyrosine phosphorylated proteins) in focal complexes, leading to organisation of actin cytoskeleton that is involved in lamellipodia formation. Finally, activated alpha2beta1 integrins intervened in all these processes clustered in small focal complexes but not in focal adhesions.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

Differential role of beta(1C) and beta(1A) integrin cytoplasmic variants in modulating focal adhesion kinase, protein kinase B/AKT, and Ras/Mitogen-activated protein kinase pathways

The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation; beta(1C) inhibits proliferation, whereas beta(1A) promotes it. We investigated the ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) did not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) showed significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induced activation of both proteins. We show that Ras activation was strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines was attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals.     

The EphA4 receptor regulates dendritic spine remodeling by affecting beta1-integrin signaling pathways

Remodeling of dendritic spines is believed to modulate the function of excitatory synapses. We previously reported that the EphA4 receptor tyrosine kinase regulates spine morphology in hippocampal pyramidal neurons, but the signaling pathways involved were not characterized (Murai, K.K., L.N. Nguyen, F. Irie, Y. Yamaguchi, and E.B. Pasquale. 2003. Nat. Neurosci. 6:153-160). In this study, we show that EphA4 activation by ephrin-A3 in hippocampal slices inhibits integrin downstream signaling pathways. EphA4 activation decreases tyrosine phosphorylation of the scaffolding protein Crk-associated substrate (Cas) and the tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) and also reduces the association of Cas with the Src family kinase Fyn and the adaptor Crk. Consistent with this, EphA4 inhibits beta1-integrin activity in neuronal cells. Supporting a functional role for beta1 integrin and Cas inactivation downstream of EphA4, the inhibition of integrin or Cas function induces spine morphological changes similar to those associated with EphA4 activation. Furthermore, preventing beta1-integrin inactivation blocks the effects of EphA4 on spines. Our results support a model in which EphA4 interferes with integrin signaling pathways that stabilize dendritic spines, thus modulating synaptic interactions with the extracellular environment.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

Processing of integrin alpha(v) subunit by membrane type 1 matrix metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of focal adhesion kinase.

Recently, we have shown that membrane type 1 matrix metalloproteinase (MT1-MMP) exhibits integrin convertase activity. Similar to furin-like proprotein convertases, MT1-MMP directly processes a single chain precursor of alpha(v) integrin subunit (pro-alpha(v)) into the heavy and light alpha-chains connected by a disulfide bridge. To evaluate functionality of MT1-MMP-processed integrins, we examined breast carcinoma MCF7 cells co-expressing alpha(v)beta(3) integrin with either the wild type or mutant MT1-MMP in a variety of migration and adhesion tests. Specific inhibitors of proprotein convertases and MMP were employed in our cell system to attenuate the individual pathways of pro-alpha(v) maturation. We present evidence that MT1-MMP cleavage of pro-alpha(v) in the cells did not affect RGD-ligand binding of the resulting alpha(v)beta(3) integrin but enhanced outside-in signal transduction through a focal adhesion kinase pathway. Enhanced tyrosine phosphorylation of focal adhesion kinase in cells co-expressing MT1-MMP and alpha(v)beta(3) integrin contributed to efficient adhesion and, especially, migration of cells on vitronectin, a ligand of alpha(v)beta(3) integrin. These mechanisms underscore the significance of a coordinated interplay between MT1-MMP and alpha(v)beta(3) integrin in tumor cells and identify downstream signaling pathways resulting from their interactions. Regulation of integrin maturation and functionality may be an important role of MT1-MMP in tumor cells.     

Beta 1 integrins signal lipid second messengers required during cell adhesion.

Clustering of integrin receptors during cell adhesion stimulates signal transduction across the cell membrane. Second messengers are generated, activating cytosolic proteins and causing cytoskeletal assembly and rearrangement. HeLa cell adhesion to a collagen substrate has been shown to initiate an arachidonic acid-mediated signaling pathway, leading to the activation of protein kinase C (PKC) and cell spreading. To determine the role of integrin receptors in triggering this signaling pathway, monoclonal antibodies to beta 1 integrins were used to either cluster integrins on the cell surface or to provide an integrin-dependent substrate for cell adhesion. Using this approach, we have defined a pathway required for cell spreading that can be initiated by the ligation of integrins and leads to the activation of PKC. Specifically, our results indicate that clustering beta 1 integrins results in the activation of phospholipase A2 leading to the production of arachidonic acid and the activation of PKC.