Beta8 integrin binds Rho GDP dissociation inhibitor-1 and activates Rac1 to inhibit mesangial cell myofibroblast differentiation

Alpha(v)beta8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted alpha(v) or beta8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney alpha(v)beta8 function. We find that kidney beta8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that beta8 regulates normal mesangial cell differentiation. To interrogate beta8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated beta8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective beta8 stimulation enhanced beta8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8-/- mice backcrossed to a genetic background that permitted survival, or gdi-/- mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and alpha-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced alpha-smooth muscle actin organization. We conclude that the beta8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased beta8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype.     

Vav1 and Rac control chemokine-promoted T lymphocyte adhesion mediated by the integrin alpha4beta1

The chemokine CXCL12 promotes T lymphocyte adhesion mediated by the integrin alpha4beta1. CXCL12 activates the GTPase Rac, as well as Vav1, a guanine-nucleotide exchange factor for Rac, concomitant with up-regulation of alpha4beta1-dependent adhesion. Inhibition of CXCL12-promoted Rac and Vav1 activation by transfection of dominant negative Rac or Vav1 forms, or by transfection of their siRNA, remarkably impaired the increase in T lymphocyte attachment to alpha4beta1 ligands in response to this chemokine. Importantly, inhibition of Vav1 expression by RNA interference resulted in a blockade of Rac activation in response to CXCL12. Adhesions in flow chambers and soluble binding assays using these transfectants indicated that initial ligand binding and adhesion strengthening mediated by alpha4beta1 were dependent on Vav1 and Rac activation by CXCL12. Finally, CXCL12-promoted T-cell transendothelial migration involving alpha4beta1-mediated adhesion was notably inhibited by expression of dominant negative Vav1 and Rac. These results indicate that activation of Vav1-Rac signaling pathway by CXCL12 represents an important inside-out event controlling efficient up-regulation of alpha4beta1-dependent T lymphocyte adhesion.     

Discoidin domain receptor 1 activation suppresses alpha2beta1 integrin-dependent cell spreading through inhibition of Cdc42 activity

Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation.     

Integrin alpha2beta1 modulates EGF stimulation of Rho GTPase-dependent morphological changes in adherent human rhabdomyosarcoma RD cells

The ability of cells to undergo shape changes is essential for diverse cellular functions including cell growth, differentiation, and movement. The present study examines how an integration of the function of alpha2beta1 integrin with that of the receptor for epidermal growth factor (EGFR) modulates EGF-stimulated morphological changes in human rhabdomyosarcoma RD transfectant cells. Upon EGF stimulation, RD transfectant cells that lacked alpha2beta1 integrin expression (RDpF) underwent contraction; in contrast, expression of alpha2beta1 on RD cells (RDX2C2) resulted in transient cell spreading. Integrin alpha2 cytoplasmic domain played a critical role in the observed alpha2beta1-mediated conversion from a cell rounding to a cell spreading phenotype. Thus, the expression of an alpha2 cytoplasmic domain deletion variant (X2C0) or a chimeric alpha2beta1 containing the cytoplasmic domain of alpha4 (X2C4) or alpha5 (X2C5), instead of alpha2, failed to mediate spreading upon EGF stimulation. Using dominant negative (DN) mutants of RhoGTPases, results revealed that RhoA activation was required for both EGF-stimulated responses of cell rounding and spreading, Cdc42 functioned in the re-spreading of cells after undergoing EGF-stimulated contraction, and Rac1 was required in alpha2beta1-mediated RD cell spreading. Therefore, alpha2beta1 integrin function can switch the Rho GTPase-dependent cell shape changes in RD cells from an EGF-stimulated cell contraction to a spreading morphology. Together, results show that integrin alpha2 cytoplasmic domain plays an indispensable role in the ability of integrin alpha2beta1 to modulate EGF stimulation of Rho-GTPase-dependent morphological changes in RD cells.     

Activated Rac1 selectively up-regulates the expression of integrin alpha6beta4 and induces cell adhesion and membrane ruffles of nonadherent colon cancer Colo201 cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin beta4 dependent since an antibody for integrin beta4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin alpha6beta4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of alpha6beta4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated alpha6beta4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin alpha6beta4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

Syndecan-1 signals independently of beta1 integrins during Raji cell spreading

Syndecan-1-expressing Raji lymphoid cells (Raji-S1 cells) bind and spread rapidly when attaching to matrix ligands that contain heparan sulfate-binding domains. However, these ligands also contain binding sites for integrins, which are widely known to signal, raising the question of whether the proteoglycan core protein participates in generation of the signal for spreading. To address this question, the spreading of the Raji-S1 cells is examined on ligands specific for either beta1 integrins, known to be present on the Raji cells, or the syndecan-1 core protein. The cells adhere and spread on invasin, a ligand that activates beta1 integrins, the IIICS fragment of fibronectin, which is a specific ligand for the alpha4beta1 integrin, or mAb281.2, an antibody specific for the syndecan-1 core protein. The signaling resulting from adhesion to the syndecan-specific antibody appears integrin independent as (i) the morphology of the cells spreading on the antibody is distinct from spreading initiated by the integrins alone; (ii) spreading on the syndecan or integrin ligands is affected differently by the kinase inhibitors tyrphostin 25, genistein, and staurosporine; and (iii) spreading on the syndecan-specific antibody is not disrupted by blocking beta1 integrin activation with mAb13, a beta1 inhibitory antibody. These data demonstrate that ligation of syndecan-1 initiates intracellular signaling and suggest that this signaling occurs when cells expressing syndecan-1 adhere to matrix ligands containing heparan sulfate-binding domains.     

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.     

The small GTPase Rap1 is required for Mn(2+)- and antibody-induced LFA-1- and VLA-4-mediated cell adhesion

In T-lymphocytes the Ras-like small GTPase Rap1 plays an essential role in stimulus-induced inside-out activation of integrin LFA-1 (alpha(L)beta(2)) and VLA-4 (alpha(4)beta(1)). Here we show that Rap1 is also involved in the direct activation of these integrins by divalent cations or activating antibodies. Inhibition of Rap1 either by Rap GTPase-activating protein (RapGAP) or the Rap1 binding domain of RalGDS abolished both Mn(2+)- and KIM185 (anti-LFA-1)-induced LFA-1-mediated cell adhesion to intercellular adhesion molecule 1. Mn(2+)- and TS2/16 (anti-VLA-4)-induced VLA-4-mediated adhesion were inhibited as well. Interestingly, both Mn(2+), KIM185 and TS2/16 failed to induce elevated levels of Rap1GTP. These findings indicate that available levels of GTP-bound Rap1 are required for the direct activation of LFA-1 and VLA-4. Pharmacological inhibition studies demonstrated that both Mn(2+)- and KIM185-induced adhesion as well as Rap1-induced adhesion require intracellular calcium but not signaling activity of the MEK-ERK pathway. Moreover, functional calmodulin signaling was shown to be a prerequisite for Rap1-induced adhesion. From these results we conclude that in addition to stimulus-induced inside-out activation of integrins, active Rap1 is required for cell adhesion induced by direct activation of integrins LFA-1 and VLA-4. We suggest that Rap1 determines the functional availability of integrins for productive binding to integrin ligands.     

Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1

Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the alpha3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the alpha2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-alpha2beta1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-alpha3beta1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.     

The chemokine stromal cell-derived factor-1 alpha modulates alpha 4 beta 7 integrin-mediated lymphocyte adhesion to mucosal addressin cell adhesion molecule-1 and fibronectin

The interaction between the integrin alpha(4)beta(7) and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that alpha(4)beta(7)-mediated adhesion of CD4(+) T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1alpha in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4(+) T cells were targets of SDF-1alpha-triggered increased adhesion. In addition, SDF-1alpha augmented alpha(4)beta(7)-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4(+) and RPMI 8866 cells to SDF-1alpha, enhanced alpha(4)beta(7)-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Galpha(i)-dependent as well as Galpha(i)-independent signaling. Accordingly, we show that SDF-1alpha induced a rapid and transient association between its receptor CXCR4 and Galpha(i), whereas association of pertussis toxin-insensitive Galpha(13) with CXCR4 was slower and of a lesser extent. SDF-1alpha also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of alpha(4)beta(7)-mediated lymphocyte adhesion in response to SDF-1alpha, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1alpha of lymphocyte adhesion mediated by alpha(4)beta(7) could contribute to lymphocyte homing to secondary lymphoid tissues.     

Dual functionality of the anti-beta1 integrin antibody, 12G10, exemplifies agonistic signalling from the ligand binding pocket of integrin adhesion receptors

Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.     

Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha 5 beta 1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.     

Integrin alpha4beta1 involvement in stromal cell-derived factor-1alpha-promoted myeloma cell transendothelial migration and adhesion: role of cAMP and the actin cytoskeleton in adhesion

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is expressed by bone marrow (BM) stromal cells and plays key roles in cell homing to and retention into the bone marrow. In multiple myeloma, blood-borne malignant plasma cells home to the BM and accumulate in contact with stromal cells, implicating myeloma cell migration across endothelium. Myeloma cells express the SDF-1alpha receptor CXCR4, as well as the integrin alpha4beta1, which mediates their attachment to BM stroma. We show here that SDF-1alpha promotes transendothelial migration of purified BM myeloma cells and myeloma-derived NCI-H929 cells, involving a transient upregulation of alpha4beta1-dependent cell adhesion to the endothelium. Characterization of intracellular signaling pathways involved in the modulation by SDF-1alpha of alpha4beta1-mediated myeloma cell adhesion revealed that intracellular cAMP amounts associated with the activation of protein kinase A play key roles in this modulation. Furthermore, a functional link between cAMP actions on the dynamics of actin cytoskeleton, RhoA activation, and alpha4beta1-dependent cell adhesion in response to SDF-1alpha has been found. The regulation of alpha4beta1-mediated myeloma cell adhesion by SDF-1alpha could play key roles during myeloma cell homing into and trafficking inside the BM, and characterization of the molecular events involved in SDF-1alpha-activated modulation of this adhesion will contribute to a better understanding of mechanisms participating in cell migration.     

The slingshot family of phosphatases mediates Rac1 regulation of cofilin phosphorylation, laminin-332 organization, and motility behavior of keratinocytes

The motility of keratinocytes is an essential component of wound closure and the development of epidermal tumors. In vitro, the specific motile behavior of keratinocytes is dictated by the assembly of laminin-332 tracks, a process that is dependent upon alpha6beta4 integrin signaling to Rac1 and the actin-severing protein cofilin. Here we have analyzed how cofilin phosphorylation is regulated by phosphatases (slingshot (SSH) or chronophin (CIN)) downstream of signaling by alpha6beta4 integrin/Rac1 in human keratinocytes. Keratinocytes express all members of the SSH family (SSH1, SSH2, and SSH3) and CIN. However, expression of phosphatase-dead versions of all three SSH proteins, but not dominant inactive CIN, results in phosphorylation/inactivation of cofilin, changes in actin cytoskeleton organization, loss of cell polarity, and assembly of aberrant arrays of laminin-332 in human keratinocytes. SSH activity is regulated by 14-3-3 protein binding, and intriguingly, 14-3-3/alpha6beta4 integrin protein interaction is required for keratinocyte migration. We wondered whether 14-3-3 proteins function as regulators of Rac1-mediated keratinocyte migration patterns. In support of this hypothesis, inhibition of Rac1 results in an increase in 14-3-3 protein association with SSH. Thus, we propose a novel mechanism in which alpha6beta4 integrin signaling via Rac1, 14-3-3 proteins, and SSH family members regulates cofilin activation, cell polarity, and matrix assembly, leading to specific epidermal cell migration behavior.     

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.     

Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation

The α(6)β(4) integrin promotes carcinoma invasion through its ability to promote directed migration and polarization of carcinoma cells. In this study, we explore how the α(6)β(4) integrin cooperates with lysophosphatidic acid (LPA) to activate Rho and Rac small GTPases. Through the use of dominant negative Rho constructs, C3 exotransferase, and Rho kinase inhibitor, we find that Rho is critical for LPA-dependent chemotaxis and lamellae formation. However, utilization of specific Rho isoforms depends on integrin α(6)β(4) expression status. Integrin α(6)β(4)-negative MDA-MB-435 cells utilize only RhoC for motility, whereas integrin α(6)β(4)-expressing cells utilize RhoC but additionally activate and utilize RhoA for LPA-dependent cell motility and lamellae formation. Notably, the activation of RhoA by cooperative LPA and integrin α(6)β(4) signaling requires the Rho guanine nucleotide exchange factor AKAP-Lbc. We also determine that integrin α(6)β(4) cannot activate Rac1 directly but promotes LPA-mediated Rac1 activation that is dependent on RhoA activity and de novo β(1) integrin ligation. Finally, we find that the regulation of Rac1 and RhoA in response to LPA is differentially regulated by phosphodiesterases, PKA, and phosphatidylinositol 3-kinase, thus supporting their spatially distinct compartmentalization. In summary, signaling from integrin α(6)β(4) facilitates LPA-stimulated chemotaxis through preferential activation of RhoA, which, in turn, facilitates activation of Rac1.     

Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells

Chemokines are secreted into the tumor microenvironment by tumor-infiltrating inflammatory cells as well as by tumor cells. Chemokine receptors mediate agonist-dependent cell responses, including migration and activation of several signaling pathways. In the present study we show that several human melanoma cell lines and melanoma cells on macroscopically infiltrated lymph nodes express the chemokine receptors CXCR3 and CXCR4. Using the highly invasive melanoma cell line BLM, we demonstrate that the chemokine Mig, a ligand for CXCR3, activates the small GTPases RhoA and Rac1, induces a reorganization of the actin cytoskeleton, and triggers cell chemotaxis and modulation of integrin VLA-5- and VLA-4-dependent cell adhesion to fibronectin. Furthermore, the chemokine SDF-1alpha, the ligand of CXCR4, triggered modulation of beta(1) integrin-dependent melanoma cell adhesion to fibronectin. Additionally, Mig and SDF-1alpha activated MAPKs p44/42 and p38 on melanoma cells. Expression of functional CXCR3 and CXCR4 receptors on melanoma cells indicates that they might contribute to cell motility during invasion as well as to regulation of cell proliferation and survival.     

Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by alpha 5 beta 1 and alpha v beta 3 integrins

Fibrillins are the major glycoprotein components of microfibrils that form a template for tropoelastin during elastic fibrillogenesis. We have examined cell adhesion to assembled purified microfibrils, and its molecular basis. Human dermal fibroblasts exhibited Arg-Gly-Asp and cation-dependent adhesion to microfibrils and recombinant fibrillin-1 protein fragments. Strong integrin alpha 5 beta 1 interactions with fibrillin ligands were identified, but integrin alpha v beta 3 also contributed to cell adhesion. Fluorescence-activated cell sorting analysis confirmed the presence of abundant alpha 5 beta 1 and some alpha v beta 3 receptors on these cells. Adhesion to microfibrils and to Arg-Gly-Asp containing fibrillin-1 protein fragments induced signaling events that led to cell spreading, altered cytoskeletal organization, and enhanced extracellular fibrillin-1 deposition. Differences in cell shape when plated on fibrillin or fibronectin implied substrate-specific alpha 5 beta 1-mediated cellular responses. An Arg-Gly-Asp-independent cell adhesion sequence was also identified within fibrillin-1. Adhesion and spreading of smooth muscle cells on fibrillin ligands was enhanced by antibody-induced beta1 integrin activation. A375-SM melanoma cells bound Arg-Gly-Asp-containing fibrillin-1 protein fragments mainly through alpha v beta 3, whereas HT1080 cells used mainly alpha 5 beta 1. This study has shown that fibrillin microfibrils mediate cell adhesion, that alpha 5 beta 1 and alpha v beta 3 are both important but cell-specific fibrillin-1 receptors, and that cellular interactions with fibrillin-1 influence cell behavior.