alpha(1A) adrenergic receptor induces eukaryotic initiation factor 4E-binding protein 1 phosphorylation via a Ca(2+)-dependent pathway independent of phosphatidylinositol 3-kinase/Akt

Phosphorylation of the translation repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is thought to be partly responsible for increased protein synthesis induced by growth factors. This study investigated the effect of a G(q)-coupled receptor on protein synthesis and the phosphorylation state and function of 4E-BP1 in Rat-1 fibroblasts expressing the human alpha(1A) adrenergic receptor. Treatment of cells with phenylephrine (PE), a specific alpha(1) adrenergic receptor agonist, increased protein synthesis and induced the phosphorylation of 4E-BP1 and its release from translation initiation factor 4E. Although the PE-induced phosphorylation of 4E-BP1 was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002, neither phosphatidylinositol 3-kinase nor Akt, its downstream effector, is activated in cells treated with PE (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z., J. Biol. Chem. 275, 4803-4809). The effect of PE on 4E-BP1 phosphorylation was also abolished in cells depleted of intracellular Ca(2+) and in cells pretreated with calmodulin antagonists. By contrast, phosphorylation of 4E-BP1 still occurred in cells in which the Ca(2+)- and diacylglycerol-dependent isoforms of protein kinase C were down-regulated by prolonged exposure to a phorbol ester. We conclude that activation of the alpha(1A) adrenergic receptor in Rat-1 fibroblasts leads to phosphorylation of 4E-BP1 via a pathway that is Ca(2+)- and calmodulin-dependent. Phosphatidylinositol 3-kinase, Akt, and phorbol ester-sensitive protein kinase C isoforms do not appear to be required in this signaling pathway.     

alpha5beta1 integrin protects intestinal epithelial cells from apoptosis through a phosphatidylinositol 3-kinase and protein kinase B-dependent pathway

Renewal of the gastrointestinal epithelium involves a coordinated process of terminal differentiation and programmed cell death. Integrins have been implicated in the control of apoptotic processes in various cell types. Here we examine the role of integrins in the regulation of apoptosis in gastrointestinal epithelial cells with the use of a rat small intestinal epithelial cell line (RIE1) as a model. Overexpression of the integrin alpha5 subunit in RIE1 cells conferred protection against several proapoptotic stimuli. In contrast, overexpression of the integrin alpha2 subunit had no effect on cell survival. The antiapoptotic effect of the alpha5 subunit was partially retained by a mutated version that had a truncation of the cytoplasmic domain. The antiapoptotic effects of the full-length or truncated alpha5 subunit were reversed upon treatment with inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase), suggesting that the alpha5beta1 integrin might interact with the PI-3-kinase/Akt survival pathway. When cells overexpressing alpha5 were allowed to adhere to fibronectin, there was a moderate activation of protein kinase B (PKB)/Akt, whereas no such effect was seen in alpha2-overexpressing cells adhering to collagen. Furthermore, in cells overexpressing alpha5 and adhering to fibronectin, there was a dramatic enhancement of the ability of growth factors to stimulate PKB/Akt; again, this was not seen in cells overexpressing alpha2 subunit and adhering to collagen or fibronectin. Expression of a dominant negative version of PKB/Akt in RIE cells blocked to ability of alpha5 to enhance cell survival. Thus, the alpha5beta1 integrin seems to protect intestinal epithelial cells against proapoptotic stimuli by selectively enhancing the activity of the PI-3-kinase/Akt survival pathway.     

Beta ig-h3 induces keratinocyte differentiation via modulation of involucrin and transglutaminase expression through the integrin alpha3beta1 and the phosphatidylinositol 3-kinase/Akt signaling pathway

Beta ig-h3 is an extracellular matrix protein whose expression is highly induced by transforming growth factor (TGF)-beta1. Whereas beta ig-h3 is known to mediate keratinocyte adhesion and migration, its effects on keratinocyte differentiation remain unclear. In the present study, it was demonstrated that expression of both beta ig-h3 and TGF-beta1 was enhanced during keratinocyte differentiation and that expression of the former was strongly induced by that of the latter. This study also asked whether changes in beta-h3 expression would affect keratinocyte differentiation. Indeed, down-regulation of beta ig-h3 by transfection with antisense beta ig-h3 cDNA constructs effectively inhibited keratinocyte differentiation by decreasing the promoter activities and thus expression of involucrin and transglutaminase. The result was an approximately 2-fold increase in mitotic capacity of the cells. Conversely, overexpression of beta ig-h3, either by transfection with beta ig-h3 expression plasmids or by exposure to recombinant beta ig-h3, enhanced keratinocyte differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant beta ig-h3 also promoted keratinocyte adhesion through interaction with integrin alpha3beta1. Changes in beta ig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant beta ig-h3 but also blockage of Akt phosphorylation by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced beta ig-h3, induced by enhanced TGF-beta during keratinocyte differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin alpha3beta1 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that beta ig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation.     

Role of phosphatidylinositol 3-kinase in platelet aggregation in type 1 diabetes mellitus

It was found that incubation of platelet rich plasma with wortmannin, an irreversible selective inhibitor of phosphoinositide 3-kinase (PI3K), leads to sharp drop in platelet aggregation ability in healthy donors, whereas in type 1 diabetes mellitus patients this effect was less manifested or not quite determined. Translocation dynamics of PI3K regulatory subunit into cytoskeleton fraction under induction of platelet aggregation by various ADP concentrations and after wortmannin treatment was studied. Reciprocal interaction of endothelial constitutive NO synthase with PI3K in mechanisms of platelet functional state regulation under studied pathological and normal conditions have been analyzed.     

Distinct contributions of glycoprotein VI and alpha(2)beta(1) integrin to the induction of platelet protein tyrosine phosphorylation and aggregation

Platelet activation by collagen depends principally on two receptors, alpha(2)beta(1) integrin (GPIa-IIa) and GPVI. During this activation, the nonreceptor protein tyrosine kinase pp72(syk) is rapidly phosphorylated, but the precise contribution of alpha(2)beta(1) integrin and GPVI to signaling for this phosphorylation is not clear. We have recently found that proteolysis of platelet alpha(2)beta(1) integrin by the snake venom metalloproteinase, jararhagin, results in inhibition of collagen-induced platelet aggregation and pp72(syk) phosphorylation. In order to verify whether the treatment of platelets with jararhagin had any effect on GPVI signaling, in this study we stimulated platelets treated with either jararhagin or anti-alpha(2)beta(1) antibody with two GPVI agonists, an antibody to GPVI and convulxin. Platelet shape change and phosphorylation of pp72(syk) by both GPVI agonists was preserved, as was the structure and function of GPVI shown by (125)I-labeled convulxin binding to immunoprecipitated GPVI from jararhagin-treated platelets. In contrast, defective platelet aggregation in response to GPVI agonists occurred in both jararhagin-treated and alpha(2)beta(1)-blocked platelets. This apparent cosignaling role of alpha(2)beta(1) integrin for platelet aggregation suggests the possibility of a topographical association of this integrin with GPVI. We found that both platelet alpha(2)beta(1) integrin and GPVI coimmunoprecipitated with alpha(IIb)beta(3) integrin. Since platelet aggregation requires activation of alpha(IIb)beta(3) integrin, defective aggregation in the absence of alpha(2)beta(1) suggests that this receptor may provide a signaling link between GPVI and alpha(IIb)beta(3). Our study therefore demonstrates that platelet signaling leading to pp72(syk) phosphorylation initiated with GPVI engagement by either convulxin or GPVI antibody does not depend on alpha(2)beta(1) integrin. However, alpha(IIb)beta(3) integrin may, in this model, require functional alpha(2)beta(1) integrin for its activation.     

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.     

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.     

Integrin alpha(IIb)beta(3) signaling in platelet adhesion and aggregation.

Intracellular signals are received and generated by the alpha(IIb)beta(3) integrin on platelets. Recent advances have been made in the areas of agonist receptors that initiate platelet activation, downstream signaling molecules (e.g. small G-proteins and kinases) and changes in ligand-occupied alpha(IIb)beta(3) that cause further signaling and clot retraction.     

The CK2 phosphorylation of vitronectin. Promotion of cell adhesion via the alpha(v)beta 3-phosphatidylinositol 3-kinase pathway

Phosphorylation of vitronectin (Vn) by casein kinase II was previously shown to occur at Thr50 and Thr57 and to augment a major physiological function of vitronectin-cell adhesion and spreading. Here we show that this phosphorylation increases cell adhesion via the alpha(v)beta3 (not via the alpha(v)beta5 integrin), suggesting that alpha(v)beta3 differs from alpha(v)beta5 in its biorecognition profile. Although both the phospho (CK2-PVn) and non-phospho (Vn) analogs of vitronectin (simulated by mutants Vn(T50E,T57E), and Vn(T50A,T57A), respectively) trigger the alpha(v)beta3 as well as the alpha(v)beta5 integrins, and equally activate the ERK pathway, these two forms are different in their activation of the focal adhesion kinase/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway. Specifically, we show (i) that, upon exposure of cells to Vn/CK2-PVn, their PKB activation depends on the availability of the alpha(v)beta3 integrin on their surface; (ii) that upon adhesion of the beta3-transfected cells onto the CK2-PVn, the extent of PKB activation coincides with the enhanced adhesion of these cells, and (iii) that both the PKB activation and the elevation in the adhesion of these cells is PI3K-dependent. The occurrence of a cell surface receptor that specifically distinguishes between a phosphorylated and a non-phosphorylated analog of Vn, together with the fact that it preferentially activates a distinct intra-cellular signaling pathway, suggest that extra-cellular CK2 phosphorylation may play an important role in the regulation of cell adhesion and migration.     

Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa)

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) binds fibrinogen via recognition sequences such as Arg-Gly-Asp (RGD). Fibrinogen binding requires agonist activation of platelets, whereas the binding of short synthetic RGD peptides does not. We now find that RGD peptide binding leads to changes in alpha IIb beta 3 that are associated with acquisition of high affinity fibrinogen-binding function (activation) and subsequent platelet aggregation. The structural specificities for peptide activation and for inhibition of ligand binding are similar, indicating that both are consequences of occupancy of the same site(s) on alpha IIb beta 3. Thus, the RGD sequence is a trigger of high affinity ligand binding to alpha IIb beta 3, and certain RGD-mimetics are partial agonists as well as competitive antagonists of integrin function.     

Selection and structure of ion-selective ligands for platelet integrin alpha IIb(beta) 3.

Integrins contain a number of divalent cation binding sites that control ligand binding affinity. Ions such as Ca(2+) and Mg(2+) bind to distinct sites on integrin and can have opposing effects on ligand binding. These effects are presumably brought about by alterations of the shape of the ligand binding pocket. To gain insight into the nature of these structural differences, we probed the integrin ligand binding site with an RGD-based library of unparalleled complexity. A cysteine-constrained phage library containing six random amino acids and the RGD motif present in seven different registers was used to select for ligands that exhibit ion-selective binding to integrin alpha(IIb)beta(3). The library was used to select for peptides that bind to the integrin alpha(IIb)beta(3) preferentially in Ca(2+) versus Mg(2+). Peptides were identified which bound selectively in each ion. The Ca(2+)-selective peptides had a range of sequences, with the only obvious consensus involving a motif that had four cysteine residues bonded in a 1,4:2,3 arrangement. Interestingly though, the Mg(2+)-selective peptides exhibited a well defined consensus motif containing Cys-X-aromatic-L/G-R-G-D-hydrophobic-R-R/K-Cys. As a first step toward understanding the structural basis for this selectivity, solution NMR structures were obtained for representatives of both sets of peptides. All peptides formed turns, with the RGD motif at the apex. The Mg(2+)-selected peptides contained a unique basic patch that protrudes from the base of the turn.     

Phosphoinositide 3-kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing alpha(IIb)beta(3) integrin activation

Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.     

Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1)

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.     

A role for phosphatidylinositol 3-kinase in the regulation of beta 1 integrin activity by the CD2 antigen

The rapid and reversible upregulation of the functional activity of integrin receptors on T lymphocytes is a vital step in the adhesive interactions that occur during successful T cell recognition of foreign antigen and transendothelial migration. Although the ligation of several different cell surface receptors, including the antigen- specific CD3/T cell receptor complex, the CD2, CD7, and CD28 antigens, as well as several chemokine receptors, has been shown to rapidly upregulate integrin function, the intracellular signaling events that initiate this increase in adhesion remain poorly defined. In this study, we have used DNA-mediated gene transfer to explore the role of phosphatidylinositol 3-kinase (PI 3-K) in the upregulation of beta 1 integrin functional activity mediated by the CD2 antigen. CD2 was expressed in the myelomonocytic cell line HL60, which expresses beta 1 integrins that mediate adhesion to fibronectin and VCAM-1 in an activation-dependent manner. Antibody stimulation of CD2 expressed on HL60 transfectants resulted within minutes in increased beta 1-mediated adhesion to fibronectin and VCAM-1 at levels comparable to that obtained upon stimulation with the phorbol ester PMA. A role for PI 3-K in CD2-mediated increases in beta 1 integrin function is suggested by: (a) the ability of the PI 3-K inhibitor wortmannin to completely inhibit CD2-induced increases in beta 1 integrin activity; (b) the association of PI 3-K with CD2; and (c) induced PI 3-K activity upon CD2 stimulation. The mode of association of PI 3-K with CD2 is not mediated by tyrosine phosphorylation-dependent binding of PI 3-K via SH2 domains, since: (a) PI 3-K is associated with CD2 in unstimulated cells; (b) CD2 stimulation fails to increase the amount of associated PI 3-K; and (c) the CD2 cytoplasmic domain lacks tyrosine residues. A role for both protein kinase C and cytoskeletal rearrangements in CD2 regulation of integrin activity is also suggested, since a PKC inhibitor partially inhibits CD2-induced increases in beta 1 integrin function, and CD2 stimulation increases F-actin content in a wortmannin- sensitive manner. Analysis of human peripheral T cells indicated that CD2 stimulation also results in PI 3-K-dependent upregulation of beta 1 integrin activity. Thus, these results demonstrate that CD2 can function as an adhesion regulator in the absence of expression of the CD3/T cell receptor complex; and directly implicate PI 3-K as a critical intracellular mediator involved in the regulation of beta 1 integrin functional activity by the CD2 antigen.     

Involvement of platelet glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin) in thrombin-induced synthesis of phosphatidylinositol 3',4'-bisphosphate.

32P-Labeled human platelets were incubated with thrombin (1 unit/ml) for 5 min at 37 degrees C under conditions allowing maximal synthesis of [32P]phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2). Incorporation of 32P into the latter phosphoinositide was dose-dependently reduced (to a maximal level averaging 60%) by the tetrapeptide RGDS, an inhibitor of fibrinogen binding to activated glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin). Identical results were obtained with the fibrinogen gamma-chain dodecapeptide HHLGGAKQAGDV, whereas the tripeptide RGD and the tetrapeptide RGES displayed reduced or undetectable effects on 32P labeling of PtdIns(3,4)P2, respectively, in good correlation with their ability to inhibit platelet aggregation and fibrinogen binding to activated alpha IIb-beta 3 integrin. In addition, pathological platelets from three patients suffering thrombasthenia, which lack alpha IIb-beta 3 integrin and fail to aggregate in response to thrombin, displayed hardly detectable increases in the 32P labeling of PtdIns(3,4)P2. In contrast, thrombin-stimulated synthesis of PtdIns(3,4)P2 was unaltered in other deficient platelets lacking the glycoprotein Ib-IX complex (Bernard-Soulier syndrome). Although additional pathways seem to be involved in the regulation of phosphatidylinositol-3-kinase, these data indicate a strong relationship between platelet aggregation involving fibrinogen binding to activated alpha IIb-beta 3 integrin and the synthesis of the novel phosphoinositides phosphorylated at position D-3 of the inositol ring.     

Transforming growth factor beta1 induces alphavbeta3 integrin expression in human lung fibroblasts via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway

In response to transforming growth factor beta1 (TGFbeta) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFbeta has been shown to increase the expression of specific alphav integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFbeta1 increased both beta3 integrin subunit mRNA and protein levels as well as surface expression of alphavbeta3 in human lung fibroblasts. TGFbeta1-induced alphavbeta3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of beta3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or alphavbeta3 blocking antibody prevented the increase in beta3 but not beta5 integrin expression. In addition, echistatin inhibited TGFbeta1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFbeta1-induced expression of alphavbeta3 and p38 MAPK phosphorylation but not beta5 integrin expression and Smad3 activation. The TGFbeta1-induced alphavbeta3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-kappaB-dependent pathways. Our results demonstrate that TGFbeta1 induces alphavbeta3 integrin expression via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFbeta1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.     

A streptococcal collagen-like protein interacts with the alpha2beta1 integrin and induces intracellular signaling

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing alpha2beta1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-alpha2beta1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125(FAK) of C2C12 cells expressing alpha2beta1 integrin, whereas parental cells do not. Furthermore, C2C12 adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125FAK, p130CAS, and p68Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the alpha2 integrin interacts with p176 with a relatively high affinity (K(D) = 17 nm). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.     

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 an alpha(3)beta(1) integrin recognition sequence in thrombospondin-1.

A synthetic peptide containing amino acid residues 190-201 of thrombospondin-1 (TSP1) promoted adhesion of MDA-MB-435 breast carcinoma cells when immobilized and inhibited adhesion of the same cells to TSP1 when added in solution. Adhesion to this peptide was enhanced by a beta(1) integrin-activating antibody, Mn(2+), and insulin-like growth factor I and was inhibited by an alpha(3)beta(1) integrin function-blocking antibody. The soluble peptide inhibited adhesion of cells to the immobilized TSP1 peptide or spreading on intact TSP1 but at the same concentrations did not inhibit attachment or spreading on type IV collagen or fibronectin. Substitution of several residues in the TSP1 peptide with Ala residues abolished or diminished the inhibitory activity of the peptide in solution, but only substitution of Arg-198 completely inactivated the adhesive activity of the immobilized peptide. The essential residues for activity of the peptide as a soluble inhibitor are Asn-196, Val-197, and Arg-198, but flanking residues enhance the inhibitory activity of this core sequence, either by altering the conformation of the active sequence or by interacting with the integrin. This functional sequence is conserved in all known mammalian TSP1 sequences and in TSP1 from Xenopus laevis. The TSP1 peptide also inhibited adhesion of MDA-MB-435 cells to the laminin-1 peptide GD6, which contains a potential integrin-recognition sequence Asn-Leu-Arg and is derived from a similar position in a pentraxin module. Adhesion studies using recombinant TSP1 fragments also localized beta1 integrin-dependent adhesion to residues 175-242 of this region, which contain the active sequence.