14-3-3beta-Rac1-p21 activated kinase signaling regulates Akt1-mediated cytoskeletal organization, lamellipodia formation and fibronectin matrix assembly

Akt1 belongs to the three-gene Akt family and functions as a serine-threonine kinase regulating phosphorylation of an array of substrates and mediating cellular processes such as cell migration, proliferation, survival, and cell cycle. Our previous studies have established the importance of Akt1 in angiogenesis and absence of Akt1 resulted in impaired integrin activation, adhesion, migration, and extracellular matrix assembly by endothelial cells and fibroblasts. In this study, we identify the downstream signaling pathways activated by Akt1 in the regulation of these cellular events. We demonstrate here that Akt1 is necessary for the growth factor stimulated activation of 14-3-3beta-Rac1-p21 activated kinase (Pak) pathway in endothelial cells and fibroblasts. While activation of Akt1 resulted in translocation of Rac1 to membrane ruffles, enhanced Rac1 activity, Pak1 phosphorylation, and lamellipodia formation, resulting in enhanced adhesion and assembly of fibronectin, inhibition of Akt1 resulted in inhibition of these processes due to impaired Rac1-Pak signaling. Formation of lamellipodia, adhesion, and fibronectin assembly by myristoylated Akt1 expression in NIH 3T3 fibroblasts was inhibited by co-expression with either dominant negative Rac1 or dominant negative Pak1. In contrast, impaired lamellipodia formation, adhesion, and fibronectin assembly by dominant negative-Akt1 expression was rescued by co-expression with either constitutively active-Rac1 or -Pak1. Moreover, previously reported defects in adhesion and extracellular matrix assembly by Akt1(-/-) fibroblasts could be rescued by expression with either active-Rac1 or -Pak1, implying the importance of Rac1-Pak signaling in growth factor stimulated cytoskeletal assembly, lamellipodia formation and cell migration in endothelial cells and fibroblasts downstream of Akt1 activation.     

Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor alpha(v)beta(3) remains within focal contacts, the fibronectin receptor alpha(5)beta(1) translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 +/- 0.7 microm/h and is independent of cell migration. It is induced by ligation of alpha(5)beta(1) integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied alpha(5)beta(1) integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating alpha(5)beta(1) integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.     

Urokinase-type plasminogen activator receptor regulates a novel pathway of fibronectin matrix assembly requiring Src-dependent transactivation of epidermal growth factor receptor

Previous studies have indicated that the urokinase-type plasminogen activator receptor (uPAR) can functionally interact with integrins thereby modulating integrin activity. We have previously demonstrated that treatment of fibroblasts with the uPAR ligand, P25, results in an increase in the activation of the beta1 integrin and a 35-fold increase in fibronectin matrix assembly (Monaghan, E., Gueorguiev, V., Wilkins-Port, C., and McKeown-Longo, P. J. (2004) J. Biol. Chem. 279, 1400-1407). Experiments were conducted to address the mechanism of uPAR regulation of matrix assembly. Treatment of fibroblasts with P25 led to an increase in the activation of the epidermal growth factor receptor (EGFR) and a colocalization of activated EGFR with beta1 integrins in cell matrix contacts. The effects of P25 on matrix assembly and beta1 integrin activation were inhibited by pretreatment with EGFR or Src kinase inhibitors, suggesting a role for both Src and EGFR in integrin activation by uPAR. Phosphorylation of EGFR in response to P25 occurred on Tyr-845, an Src-dependent phosphorylation site and was inhibited by PP2, the Src kinase inhibitor, consistent with Src kinase lying upstream of EGFR and integrin activation. Cells null for Src kinases also showed a loss of P25-induced matrix assembly, integrin activation, and EGFR phosphorylation. These P25-induced effects were restored following Src re-expression. The effects of P25 were specific for uPAR as enhanced matrix assembly by P25 was not seen in uPAR-/- cells, but was restored upon uPAR re-expression. These data provide evidence for a novel pathway of fibronectin matrix assembly through the uPAR-dependent sequential activation of Src kinase, EGFR, and beta1 integrin.     

The receptor for urokinase-type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts

Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of alpha5beta1-dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the beta1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in alpha5beta1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6-10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the beta1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the alpha5beta1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.     

Effect of dexamethasone on the assembly of the matrix of fibronectin and on its receptors organization in Ehlers-Danlos syndrome skin fibroblasts.

The effect of dexamethasone (DEX) on the expression of fibronectin (FN), proalpha(1)(I) collagen (Col1), integrin alpha(2), alpha(5)and beta(1)subunits mRNAs, were studied by quantitative in situ hybridization (ISH) with radiolabelled probes in relationship with the organization of the extracellular matrix (ECM) of FN in human skin fibroblasts. In particular, two fibroblast strains were analysed, one derived from a control donor, typically organizing a rich ECM of FN, and the other from a patient affected by Ehlers-Danlos syndrome (EDS), which did not assemble the FN-ECM. Treatment of both fibroblast strains with 10(-7) m DEX slightly enhanced the level of FN mRNA (by about 1.5-fold), did not influence the level of alpha(5)subunit mRNA and reduced Col1, alpha(2)and beta(1)integrin subunits mRNAs by 2-3-fold. These results show that, in these cells, DEX coordinately downregulates the expression of Col1 and its specific integrin alpha(2)beta(1). Moreover, DEX regulates in a different manner the alpha(5)and beta(1)subunits forming the main FN receptor (FNR) in skin fibroblasts. Immunofluorescence microscopy evidencing the FN-ECM and integrins containing alpha(5)and beta(1)subunits showed that in control cells DEX induced a slight enhancement of the FN-ECM and of the alpha(5)beta(1)receptors patches. Therefore, in these cells the decrease of beta(1)FN receptor subunit mRNA, as well as the decrease of Col1 and its receptor mRNAs, did not influence the FN-ECM assembly. In EDS fibroblasts, DEX decreased the cytoplasmic accumulation of FN and induced the assembly of a rich FN-ECM through the formation of large FNR integrin patches, codistributing with the FN-ECM. We suggest that in EDS skin fibroblasts DEX corrects the defective FN-ECM favouring the sorting and the organization of FN and its alpha(5)beta(1)integrin receptor.     

Fibronectin regulates latent transforming growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding protein-1

Latent transforming growth factor-beta-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGF beta in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-beta (TGF beta) into the ECM of osteoblasts and fibroblasts. Immunolocalization studies suggested that fibronectin provides an initial scaffold that precedes and patterns LTBP1 deposition but that LTBP1 and fibronectin are later localized in separate fibrillar networks, suggesting that the initial template is lost. Treatment of fetal rat calvarial osteoblasts with a 70-kDa N-terminal fibronectin fragment that inhibits fibronectin assembly impaired incorporation of LTBP1 and TGFbeta into the ECM. Consistent with this, LTBP1 failed to assemble in embryonic fibroblasts that lack the gene for fibronectin. LTBP1 assembly was rescued by full-length fibronectin and superfibronectin, which are capable of assembly into fibronectin fibrils, but not by other fibronectin fragments, including a 160-kDa RGD-containing fragment that activates alpha5beta1 integrins. This suggests that the critical event for LTBP1 assembly is the formation of a fibronectin fibrillar network and that integrin ligation by fibronectin molecules alone is not sufficient. Not only was fibronectin essential for the initial incorporation of LTBP1 into the ECM, but the continued presence of fibronectin was required for the continued assembly of LTBP1. These studies highlight a nonredundant role for fibronectin in LTBP1 assembly into the ECM and suggest a novel role for fibronectin in regulation of TGF beta via LTBP1 interactions.     

Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly

The ability of single subunit chimeric receptors containing various integrin beta intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the beta 1, beta 3, beta 3B, or beta 5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the beta 3 and beta 5 chimeras mimicked endogenous ligand-occupied integrins and, like the beta 1 chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the beta 3B intracellular domain (a beta 3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intracellular mechanism. Furthermore, when expressed at higher levels, the beta 1 and beta 3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The beta 5 chimera was a less effective inhibitor, and the beta 3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected beta 1 chimera and the endogenous beta 1 subunit indicated that in 10 to 15 h assays, the beta 1 chimera can inhibit cell spreading when expressed at levels approximately equal to the endogenous beta 1 subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit alpha 5 beta 1 localization to fibrils and matrix assembly. Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that beta intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.     

Human fibroblasts with mutations in COL5A1 and COL3A1 genes do not organize collagens and fibronectin in the extracellular matrix, down-regulate alpha2beta1 integrin, and recruit alphavbeta3 Instead of alpha5beta1 integrin

Dermal fibroblasts derived from types I and IV Ehlers-Danlos syndrome (EDS) patients, carrying mutations in COL5A1 and COL3A1 genes, respectively, synthesize aberrant types V and III collagen (COLL) and show defective organization of these proteins into the extracellular matrix (ECM) and high reduction of their functional receptor, the alpha(2)beta(1) integrin, compared with control fibroblasts. EDS cells also show reduced levels of fibronectin (FN) in the culture medium and lack an FN fibrillar network. Finally, EDS cells prevalently organize alpha(v)beta(3) integrin instead of alpha(5)beta(1) integrin. The alpha(v)beta(3) integrin, distributed on the whole EDS cell surface, shows FN binding and assembly properties when the cells are treated with purified FN. Treatment of EDS cells with purified COLLV or COLLIII, but not with FN, restores the control phenotype (COLL(+), FN(+), alpha(v)beta(3)(-), alpha(5)beta(1)(+), alpha(2)beta(1)(+)). Function-blocking antibodies to COLLV, COLLIII, or alpha(2)beta(1) integrin induce in control fibroblasts an EDS-like phenotype (COLL(-), FN(-), alpha(v)beta(3)(+), alpha(5)beta(1)(-), alpha(2)beta(1)(-)). These results show that in human fibroblasts alpha(2)beta(1) integrin organization and function are controlled by its ligand, and that the alpha(2)beta(1)-COLL interaction, in turn, regulates FN integrin receptor recruitment: high alpha(2)beta(1) integrin levels induce alpha(5)beta(1) integrin organization, while low alpha(2)beta(1) integrin levels lead to alpha(v)beta(3) integrin organization.     

Regulation of cell adhesion receptors by transforming growth factor-beta. Concomitant regulation of integrins that share a common beta 1 subunit

Cell adhesion to extracellular matrices is mediated by a set of heterodimeric cell surface receptors called integrins that might be the subject of regulation by growth and differentiation factors. We have examined the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of the very late antigens or alpha beta 1 group of integrins in human cell lines. The six known members of this family share a common beta 1 subunit but have distinct alpha subunits that confer selective affinity toward type I collagen, fibronectin, laminin, and other as yet unknown cell adhesion proteins. Using a panel of specific antibodies and cDNA probes, we show that in WI-38 lung fibroblasts TGF-beta 1 elevates concomitantly the expression of alpha 1, alpha 2, alpha 3, alpha 5, and beta 1 integrin subunits at the protein and/or mRNA level, their assembly into the corresponding alpha beta 1 complexes, and their exposure on the cell surface. The rate of synthesis of total alpha subunits relative to beta 1 subunit is higher in TGF-beta 1-treated cells than in control cells. The characteristically slow (t1/2 approximately 10 h) rate of beta 1 conversion from precursor form to mature glycoprotein in untreated cells increases markedly (to t1/2 approximately 3 h) in response to TGF-beta 1. The results suggest that in WI-38 fibroblasts the beta 1 subunit is synthesized in excess over alpha subunits, and assembly of beta 1 subunits with rate-limiting alpha subunits is required for transit through the Golgi and exposure of alpha beta 1 complex on the cell surface. TGF-beta 1 does not induce the synthesis of integrin subunits that are not expressed in unstimulated cells, such as alpha 4 and alpha 6 subunits in WI-38 fibroblasts. However, alpha 4 and alpha 6 subunits can be regulated by TGF-beta in those cells that express them. The results suggest that TGF-beta regulates the expression of individual integrin subunits by parallel but independent mechanisms. By modifying the balance of individual alpha beta 1 integrins, TGF-beta 1 might modulate those aspects of cell migration, positioning, and development that are guided by adhesion to extracellular matrices.     

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.     

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.     

Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the alpha v beta 6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the beta 6 subunit into poorly invasive SCC9 cells to establish the SCC9 beta 6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of beta 6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to alpha v beta 6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-alpha 5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of beta 6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9 beta 6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-alpha v beta 6 antibodies. The expression of beta 6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9 beta 6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-alpha v beta 6 antibodies. These results demonstrate that expression of beta 6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin alpha v beta 6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.     

Efficient dendritic cell priming of T lymphocytes depends on the extracellular matrix protein mindin

Rho guanosine triphosphatases (GTPases) regulate multiple aspects of dendritic cell (DC) function, but what regulates the expression of Rho GTPases in DCs is unknown. Here, we show that the extracellular matrix protein mindin regulates the expression of Rho GTPases in DCs. Mindin(-/-) mice displayed defective CD4+ T-cell priming and impaired humoral immune responses to T-dependent antigens. Mindin(-/-) DCs had reduced expression of Rac1/2 and impaired priming capacity owing to inefficient engagement with T lymphocytes. Ectopic Rac1 expression restored the priming capability of Mindin(-/-) DCs. Furthermore, we show that DC adhesion to mindin matrix was blocked by antibodies to alpha4, alpha5 and beta1 integrins. DCs lacking beta1 integrin had reduced adhesion to mindin matrix, decreased expression of Rac1/2 and impaired priming capacity. These results suggest that mindin-integrin interactions play a key role in regulating Rho GTPase expression in DCs and DC priming of T lymphocytes.     

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.     

Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions

This study establishes that the physical state of the extracellular matrix can regulate integrin-mediated cytoskeletal assembly and tyrosine phosphorylation to generate two distinct types of cell-matrix adhesions. In primary fibroblasts, alpha(5)beta(1) integrin associates mainly with fibronectin fibrils and forms adhesions structurally distinct from focal contacts, independent of actomyosin-mediated cell contractility. These "fibrillar adhesions" are enriched in tensin, but contain low levels of the typical focal contact components paxillin, vinculin, and tyrosine-phosphorylated proteins. However, when the fibronectin is covalently linked to the substrate, alpha(5)beta(1) integrin forms highly tyrosine-phosphorylated, "classical" focal contacts containing high levels of paxillin and vinculin. These experiments indicate that the physical state of the matrix, not just its molecular composition, is a critical factor in defining cytoskeletal organization and phosphorylation at adhesion sites. We propose that molecular organization of adhesion sites is controlled by at least two mechanisms: 1) specific integrins associate with their ligands in transmembrane complexes with appropriate cytoplasmic anchor proteins (e.g., fibronectin-alpha(5)beta(1) integrin-tensin complexes), and 2) physical properties (e.g., rigidity) of the extracellular matrix regulate local tension at adhesion sites and activate local tyrosine phosphorylation, recruiting a variety of plaque molecules to these sites. These mechanisms generate structurally and functionally distinct types of matrix adhesions in fibroblasts.     

Integrins and cell signaling in chondrocytes

Integrins are adhesion receptor heterodimers that transmit information from the extracellular matrix (ECM) to the cell through activation of cell signaling pathways. Chondrocytes express several members of the integrin family including alpha5beta1 which is the primary chondrocyte receptor for fibronectin. Cell signaling mediated through integrins regulates several chondrocyte functions including differentiation, matrix remodeling, responses to mechanical stimulation and cell survival. Integrin-mediated activation of members of the mitogen-activated protein kinase family likely plays a key role in transmitting signals regulating chondrocyte gene expression. Upstream mediators of mitogen-activated protein kinase (MAP kinase) activation include focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (pyk2) which are both expressed by chondrocytes. A better understanding of chondrocyte integrin signaling is needed to define the mechanisms by which the ECM regulates chondrocyte function.     

Integrin alphavbeta3 regulates microfibril assembly in human periodontal ligament cells

Fibrillin-1 is the major structural component of extracellular microfibrils. However, the mechanism by which extracellular fibrillin-1 assembles into microfibrils is not fully understood. Fibrillin-1 contains the Arg-Gly-Asp (RGD) motif, which may allow binding to RGD-recognizing integrins. We hypothesized that integrin αvβ3 on the cell surface of human periodontal ligament (PDL) fibroblasts may influence fibrillin-1 assembly into cell/matrix layers. We treated PDL fibroblasts with an integrin αvβ3-specific antagonist to examine fibrillin-1 assembly. Western blotting and immunofluorescence analysis showed that treatment with the integrin αvβ3 antagonist at 5 μM clearly abolished fibrillin-1 deposition. These results provide for the first time evidence that integrin αvβ3 regulates extracellular assembly of fibrillin-1, thereby modulating cell-mediated homeostasis of microfibrils.     

Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway

A beta1 integrin phosphatidylinositol 3-kinase/Akt pathway regulates fibroblast survival in collagen matrices. When fibroblasts attach to collagen, Akt becomes phosphorylated, providing a survival signal. In contrast, in response to mechanical forces generated during collagen contraction, Akt is dephosphorylated and fibroblasts undergo apoptosis. The kinase(s) responsible for regulating Akt phosphorylation in response to matrix-derived mechanical signals are unclear. Integrin-linked kinase (ILK) is associated with the beta1 integrin in the focal adhesion complex and as such is a candidate kinase that may regulate Akt phosphorylation and fibroblast viability. Nevertheless, there is no direct evidence that matrix-derived mechanical forces regulate cell viability by modulating ILK activity. Here, we show that ILK activity decreased in response to collagen matrix contraction, which correlated with Akt dephosphorylation and induction of fibroblast apoptosis. In contrast, enforced activation of beta1 integrin by activating antibody preserved ILK and Akt activity during collagen matrix contraction, and this is associated with protection from collagen contraction-induced apoptosis. Knock-down of ILK by small, interfering RNA (siRNA) attenuated Akt phosphorylation in response to ligation of beta1 integrin by collagen or activating antibody and enhanced fibroblast apoptosis in response to collagen contraction. Kinase dead ILK attenuated Akt phosphorylation and enhanced fibroblast apoptosis, whereas hyperactive and wild type ILK augmented Akt phosphorylation and protected fibroblasts from apoptosis. Constitutively active Akt preserved Akt activity and rescued ILK siRNA-treated fibroblasts from collagen contraction-induced apoptosis. These data establish that matrix-derived mechanical forces sensed by beta1 integrin are capable of modulating ILK activity which regulates fibroblast viability via an Akt-dependent mechanism.     

The major outer sheath protein of Treponema denticola inhibits the binding step of collagen phagocytosis in fibroblasts

Bacterial infections contribfute to the chronicity of connective tissue lesions in part by perturbing extracellular matrix remodelling processes. We examined a novel mechanism by which the major outer sheath protein (Msp) of the spirochaete Treponema denticola disrupts matrix remodelling mediated by intracellular digestion of collagen. The initial collagen-binding step of phagocytosis was examined in human gingival fibroblasts and Rat-2 fibroblasts. Cells were pretreated with Msp or vehicle, and binding of collagen-coated beads was measured by flow cytometry. Exposure to Msp induced a dose- and time-dependent decrease in cells that bound collagen beads; the inhibition of binding was reversed by absorption with anti-Msp antibodies. Msp-treated fibroblasts remained viable but underwent actin reorganization, including the assembly of a dense meshwork of subcortical actin filaments. Shear force assays showed that Msp abrogated collagen-binding interactions in the minimal affinity range required for stable adhesion. Fluorescence microscopy and immunoblotting showed equivalent amounts of beta1 integrin associated with collagen beads bound to Msp- and vehicle-treated cells. Photobleaching experiments found a similar percentage mobile fraction of beta1 integrins recovered in bleached areas of the plasma membrane. In contrast, Msp-induced inhibition of collagen binding was reversed by beta1 integrin affinity-activating antibodies and by latrunculin B, which prevented subcortical actin assembly. We conclude that native Msp of T. denticola inhibits the binding step of collagen phagocytosis in fibroblasts by inducing subcortical actin filament assembly and restricting affinity modulation of beta1 integrins. We suggest that, like Msp, bacterial toxins that target the cytoskeleton may also perturb the signalling networks required for cellular engagement of matrix ligands.     

Cell adhesion to extracellular matrix regulates the life cycle of integrins.

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.