Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins alpha 11beta 1 and alpha 2beta 1.
(1/261)
Polymerization of the ECM proteins fibronectin and laminin has been shown to take place in close vicinity to the cell surface and be facilitated by beta(1) integrins (Lohikangas, L., Gullberg, D., and Johansson, S. (2001) Exp. Cell Res. 265, 135-144 and Wennerberg, K., Lohikangas, L., Gullberg, D., Pfaff, M., Johansson, S., and Fassler, R. (1996) J. Cell Biol. 132, 227-238). We have studied the role of collagen receptors, integrins alpha(11)beta(1) and alpha(2)beta(1), and fibronectin in collagen polymerization using fibronectin-deficient mouse embryonic fibroblast cell lines. In contrast to the earlier belief that collagen polymerization occurs via self-assembly of collagen molecules we show that a preformed fibronectin matrix is essential for collagen network formation and that collagen-binding integrins strongly enhance this process. Thus, collagen deposition is regulated by the cells, both indirectly through integrin alpha(5)beta(1)-dependent polymerization of fibronectin and directly through collagen-binding integrins. (+info)
c-erbB2-induced disruption of matrix adhesion and morphogenesis reveals a novel role for protein kinase B as a negative regulator of alpha(2)beta(1) integrin function.
(2/261)
Overexpression of the growth factor receptor subunit c-erbB2, leading to its ligand-independent homodimerization and activation, has been implicated in the pathogenesis of mammary carcinoma. Here, we have examined the effects of c-erbB2 on the adhesive properties of a mammary epithelial cell line, HB2/tnz34, in which c-erbB2 homodimerization can be induced by means of a transfected hybrid "trk-neu" construct. trk-neu consists of the extracellular domain of the trkA nerve growth factor (NGF) receptor fused to the transmembrane and cytoplasmic domains of c-erbB2, allowing NGF-induced c-erbB2 homodimer signaling. Both spreading and adhesion on collagen surfaces were impaired on c-erbB2 activation in HB2/tnz34 cells. Antibody-mediated stimulation of alpha(2)beta(1) integrin function restored adhesion, suggesting a direct role for c-erbB2 in integrin inactivation. Using pharmacological inhibitors and transient transfections, we identified signaling pathways required for suppression of integrin function by c-erbB2. Among these was the MEK-ERK pathway, previously implicated in integrin inactivation. However, we could also show that downstream of phosphoinositide-3-kinase (PI3K), protein kinase B (PKB) acted as a previously unknown, potent inhibitor of integrin function and mediator of the disruptive effects of c-erbB2 on adhesion and morphogenesis. The integrin-linked kinase, previously identified as a PKB coactivator, was also found to be required for integrin inactivation by c-erbB2. In addition, the PI3K-dependent mTOR/S6 kinase pathway was shown to mediate c-erbB2-induced inhibition of adhesion (but not spreading) independently of PKB. Overexpression of MEK1 or PKB suppressed adhesion without requirement for c-erbB2 activation, suggesting that these two pathways partake in integrin inhibition by targeting common downstream effectors. These results demonstrate a major novel role for PI3K and PKB in regulation of integrin function. (+info)
Distinct roles of GPVI and integrin alpha(2)beta(1) in platelet shape change and aggregation induced by different collagens.
(3/261)
1. Various platelet membrane glycoproteins have been proposed as receptors for collagen, in some cases as receptors for specific collagen types. In this study we have compared the ability of a range of collagen types to activate platelets. 2. Bovine collagen types I-V, native equine tendon collagen fibrils and collagen-related peptide (CRP) all induced platelet aggregation and shape change. 3. Responses were abolished in FcRgamma chain-deficient platelets, which also lack GPVI, indicating a critical dependence on the GPVI/FcRgamma chain complex. 4. Responses to all collagens were unaffected in CD36-deficient platelets. 5. A monoclonal antibody (6F1) which binds to the alpha(2) integrin subunit of human platelets had a minimal effect on the rate and extent of aggregation induced by the collagens; however, it delayed the onset of aggregation following addition of all collagens. For shape change, 6F1 abolished the response induced by collagen types I and IV, substantially attenuated that to collagen types II, III and V, but only partially inhibited Horm collagen. 6. Simultaneous blockade of the P2Y(1) and P2Y(12) receptors, and inhibition of cyclo-oxygenase demonstrated that CRP can activate platelets independently of ADP and TxA(2); however, responses to the collagens were dependent on these mediators. 7. This study confirms the importance of the GPVI/FcRgamma chain complex in platelet responses induced by a range of collagen agonists, while providing no evidence for collagen type-specific receptors. It also provides evidence for a modulatory role of alpha(2)beta(1), the significance of which depends on the collagen preparation. (+info)
The reprolysin jararhagin, a snake venom metalloproteinase, functions as a fibrillar collagen agonist involved in fibroblast cell adhesion and signaling.
(4/261)
The integrins alpha(2)beta(1) and alpha(1)beta(1) have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to alpha(2)beta(1) regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the alpha(2)beta(1) integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor alpha(2)beta(1) in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (a disintegrin-like and metalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins. (+info)
Distribution of laminin 5, integrin receptors, and branching morphogenesis during human fetal lung development.
(5/261)
The role of the epithelial adhesion ligand laminin 5 (LN5) in lung development has been poorly investigated. To determine its potential involvement in lung organogenesis, we used immunofluorescence microscopy to investigate the distribution of LN5 and its integrin (Int) receptors alpha2beta1, alpha3beta1, alpha6beta1, and alpha6beta4 during human fetal airway branching morphogenesis and respiratory epithelium differentiation. At the pseudoglandular and canalicular stages of airway development, LN5 and its constituent chains were localized in the basement membrane (BM) of the proximal respiratory tubules and in the cytoplasm of the epithelial cells forming the growing epithelial buds, which expressed Int alpha2beta1, alpha3beta1, and, transiently, alpha6beta1. At the alveolar and adult stages, LN5 and its constituent chains were localized both in the BM of evolving and differentiated bronchioles and in the alveolar parenchyma. The bronchiolar epithelium markedly expressed Int alpha2beta1 and alpha3beta1, whereas the alveolar parenchyma strongly expressed Int alpha2beta1, alpha3beta1, and alpha6beta1. Throughout fetal development and in the adult, LN5 and its constituent chains were detected both in the tracheal BM, regardless of the degree of epithelial differentiation, and in the cytoplasm of the cells at the invading front of the growing glandular ducts. Ultrastructural studies showed that nucleation of the hemidesmosomes (HDs) correlated with the differentiation of the tracheal epithelium. These results suggest that LN5 may play multiple roles during branching morphogenesis, by modulating proliferation and/or migration of the epithelial cells in the respiratory buds and by establishing branch points, through interaction initially with Int alpha6beta1 and later with Int alpha2beta1 and alpha3beta1. We also propose that LN5 may regulate the differentiation of the tracheal epithelium by means of Int-beta4, which governs HD nucleation. (+info)
Fibronectin fragments and blocking antibodies to alpha2beta1 and alpha5beta1 integrins stimulate mitogen-activated protein kinase signaling and increase collagenase 3 (matrix metalloproteinase 13) production by human articular chondrocytes.
(6/261)
OBJECTIVE: To determine if integrin-mediated signaling results in activation of chondrocyte mitogen-activated protein (MAP) kinases that lead to increased expression of matrix metalloproteinase 13 (MMP-13; collagenase 3), a potent mediator of cartilage matrix degradation. METHODS: Human articular chondrocytes isolated from normal ankle and knee cartilage obtained from tissue donors were cultured in monolayers. The cells were treated with a 120-kd fibronectin fragment (FN-f) that binds the alpha5beta1 integrin or with antibodies to specific integrin receptors. Activation of MAP kinases was determined by immunoblotting with phosphospecific antibodies. MMP production was measured by gelatin zymography, and MMP-13 production and activation were determined by immunoblotting and by a fluorogenic peptide assay. RESULTS: Human articular chondrocytes were found to respond to the 120-kd FN-f and to adhesion-blocking antibodies to the alpha2beta1 and alpha5beta1 integrins with increased phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2, c-Jun N-terminal kinase (JNK), and p38 MAP kinases. Intact FN and integrin-blocking antibodies to alpha1, alpha3, and alphaVbeta3 and a nonblocking alpha5 antibody had no effect. After MAP kinase activation, increased phosphorylation of c-Jun and the nuclear factor kappaB inhibitor was noted, followed by increased pro- and activated MMP-13 in the conditioned media. Inhibitors of mitogen-activated protein kinase kinase, p38, and JNK were each able to inhibit increased MMP-13 production, while the interleukin-1 receptor antagonist (IL-1Ra) protein did not. However, the IL-1Ra partially inhibited FN-f-induced activation of MMP-13. CONCLUSION: Integrin-mediated MAP kinase signaling stimulated by FN-f is associated with increased production and release of pro- and active MMP-13. Autocrine production of IL-1 appears to result in additional MMP-13 activation. These processes may play a key role in feedback loops responsible for progressive cartilage degradation in arthritis. (+info)
Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI-mediated Rap1 activation in platelets.
(7/261)
Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin alpha(IIb)beta(3) activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcRgamma but independent of another platelet collagen receptor, alpha(2)beta(1). Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y(12) and not the P2Y(1) receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y(1) or the P2Y(12)-associated G-protein, Galphai(2), indicate that human and murine platelets also have a significant P2Y(12)-independent component of GPVI-mediated Rap1 activation. The P2Y(12)-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y(12)-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling. (+info)
Collagen I initiates endothelial cell morphogenesis by inducing actin polymerization through suppression of cyclic AMP and protein kinase A.
(8/261)
Collagen I provokes endothelial cells to assume a spindle-shaped morphology and to align into solid cord-like assemblies. These cords closely imitate the solid pre-capillary cords of embryonic angiogenesis, raising interesting questions about underlying mechanisms. Studies described here identify a critical mechanism beginning with collagen I ligation of integrins alpha(1)beta(1) and alpha(2)beta(1), followed by suppression of cyclic AMP and cyclic AMP (cAMP)-dependent protein kinase A, and marked induction of actin polymerization to form prominent stress fibers. In contrast to collagen I, laminin-1 neither suppressed cAMP nor protein kinase A activity nor induced actin polymerization or changes in cell shape. Moreover, fibroblasts did not respond to collagen I with changes in cAMP, actin polymerization, or cell shape, thus indicating that collagen signaling, as observed in endothelial cells, does not extend to all cell types. Pharmacological elevation of cAMP blocked collagen-induced actin polymerization and formation of cords by endothelial cells; conversely, pharmacological suppression of either cAMP or protein kinase A induced actin polymerization. Collectively, these studies identify a previously unrecognized and critical mechanism, involving suppression of cAMP-dependent protein kinase A and induction of actin polymerization, through which collagen I drives endothelial cell organization into multicellular pre-capillary cords. (+info)