Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes

Epiligrin is a new glycoprotein in most epithelial basement membranes (BMs) and is a ligand for cell adhesion via integrin alpha 3 beta 1. In the extracellular matrix of human foreskin keratinocytes (HFKs), epiligrin contains three disulfide-bonded, glycoprotein subunits, E170, E145, and E135, based on molecular size in kilodaltons. Epiligrin, immunopurified with MAb P1E1, induced cell adhesion and localization of integrin alpha 3 beta 1 in focal adhesions (FAs). Cell adhesion to epiligrin was inhibited with an anti-alpha 3 beta 1 MAb. Epiligrin also colocalized with integrin alpha 6 beta 4 in hemidesmosome-like stable anchoring contacts (SACs). alpha 3 beta 1-FAs encircled alpha 6 beta 4-SACs in a complex adhesion structure. alpha 3 beta 1 and epiligrin localized in BM junctions of epithelial cells primarily in organs of endodermal/ectodermal origin. In epidermis, epiligrin was detected in the lamina lucida of BMs. alpha 3 beta 1 localized in plasma membranes of basal cells in contact with epiligrin and also in lateral/apical membranes. Epiligrin is the ligand of an adhesion super complex composed of alpha 3 beta 1-FAs and alpha 6 beta 4-SACs (hemidesmosomes).     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.     

Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.     

The alpha 6 beta 4 integrin and epithelial cell migration.

Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.     

Functions of alpha3beta1 integrin

alpha3beta1 integrin is a laminin receptor with apparently diverse functions. In epithelial cells it acts as a receptor for the basement membrane, whereas in neuronal and possibly tumor cells it mediates migration. Interactions of alpha3beta1 integrin with tetraspanin proteins may provide clues to how it transduces signals that affect cell behavior.     

Inhibitory effects of MLDG-containing heterodimeric disintegrins reveal distinct structural requirements for interaction of the integrin alpha 9beta 1 with VCAM-1, tenascin-C, and osteopontin

The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.     

Cross-talk of integrin alpha3beta1 and tissue factor in cell migration

In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is beta1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing alphavbeta3 or certain beta1 integrin heterodimers (alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha9beta1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates alpha3beta1-dependent migration on laminin 5. Expression of TF suppresses alpha3beta1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of alpha3beta1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility.     

Axolotl pronephric duct migration requires an epidermally derived, laminin 1-containing extracellular matrix and the integrin receptor alpha6beta1

The epidermis overlying the migrating axolotl pronephric duct is known to participate in duct guidance. This epidermis deposits an extracellular matrix onto the migrating duct and its pathway that is a potential source of directional guidance cues. The role of this matrix in pronephric duct guidance was assayed by presenting matrix deposited on microcarriers directly to migrating pronephric ducts in situ. We found that reorientation of extracellular-matrix-bearing carriers prior to their presentation to migrating ducts caused a corresponding reorientation of pronephric duct migration. Subepidermal microinjection of function-blocking antibodies against alpha6 integrin, beta1 integrin or the laminin-1/E8 domain recognized by alpha6beta1 integrin, all of which were detected and localized here, inhibited pronephric duct migration. Moreover, pre-exposure to anti-laminin-1/E8 function-blocking antibody prevented reoriented carriers of epidermally deposited matrix from reorienting pronephric duct migration. These results are incorporated into an integrated model of pronephric duct guidance consistent with all present evidence, proposing roles for the previously implicated glial cell-line derived neurotrophic factor and its receptor as well as for laminin 1 and alpha6beta1 integrin.     

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.     

Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.     

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin.     

Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

Inhibitory role of alpha 6 beta 4-associated erbB-2 and phosphoinositide 3-kinase in keratinocyte haptotactic migration dependent on alpha 3 beta 1 integrin

Keratinocytes and other epithelial cells express two receptors for the basement membrane (BM) extracellular matrix component laminin-5 (Ln-5), integrins alpha 3 beta 1 and alpha 6 beta 4. While alpha 3 beta 1 mediates adhesion, spreading, and migration (Kreidberg, J.A. 2000. Curr. Opin. Cell Biol. 12:548--553), alpha 6 beta 4 is involved in BM anchorage via hemidesmosomes (Borradori, L., and A. Sonnenberg. 1999. J. Invest. Dermatol. 112:411--418). We investigated a possible regulatory interplay between alpha 3 beta 1 and alpha 6 beta 4 in cell motility using HaCaT keratinocytes as a model. We found that alpha 6 beta 4 antibodies inhibit alpha 3 beta 1-mediated migration on Ln-5, but only when migration is haptotactic (i.e., spontaneous or stimulated by alpha 3 beta 1 activation), and not when chemotactic (i.e., triggered by epidermal growth factor receptor). Inhibition of migration by alpha 6 beta 4 depends upon phosphoinositide 3-kinase (PI3-K) since it is abolished by PI3-K blockers and by dominant-negative PI3-K, and constitutively active PI3-K prevents haptotaxis. In HaCaT cells incubated with anti-alpha 6 beta 4 antibodies, activation of PI3-K is mediated by alpha 6 beta 4-associated erbB-2, as indicated by erbB-2 autophosphorylation and erbB-2/p85 PI3-K coprecipitation. Furthermore, dominant-negative erbB-2 abolishes inhibition of haptotaxis by anti-alpha 6 beta 4 antibodies. These results support a model whereby (a) haptotactic cell migration on Ln-5 is regulated by concerted action of alpha 3beta 1 and alpha 6 beta 4 integrins, (b) alpha 6 beta 4-associated erbB-2 and PI3-K negatively affect haptotaxis, and (c) chemotaxis on Ln-5 is not affected by alpha 6 beta 4 antibodies and may require PI3-K activity. This model could be of general relevance to motility of epithelial cells in contact with BM.     

Identification of a novel integrin alpha 6 beta 1 binding site in the angiogenic inducer CCN1 (CYR61)

The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including alpha 6 beta 1. Previous studies have shown that CCN1 interaction with integrin alpha 6 beta 1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin alpha 6 beta 1. In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-alpha 6 and anti-beta 1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating alpha 6 beta 1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other alpha 6 beta 1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits alpha 6 beta 1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin alpha 6 beta 1 directly, we perform affinity chromatography and show that integrin alpha 6 beta 1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin alpha 6 beta 1, providing the basis for the development of peptide mimetics to examine the functional role of alpha 6 beta 1 in angiogenesis.