Integrin alpha1beta1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes

During endochondral ossification, type I collagen is synthesized by osteoblasts together with some hypertrophic chondrocytes. Type I collagen has also been reported to be progressively synthesized in degenerative joints. Because Matrix Metalloproteinase-13 (MMP-13) plays an active role in remodeling cartilage in fetal development and osteoarthritic cartilage, we investigated whether type I collagen could activate MMP-13 expression in chondrocytes. We used a well-established chondrocytic cell line (MC615) and we found that MMP-13 expression was induced in MC615 cells cultured in type I collagen gel. We also found that alpha1beta1 integrin, a major collagen receptor, was expressed by MC615 cells and we further assessed the role of alpha1beta1 integrin in conducting MMP-13 expression. Induction of MMP-13 expression by collagen was potently and synergistically inhibited by blocking antibodies against alpha1 and beta1 integrin subunits, indicating that alpha1beta1 integrin mediates the MMP-13-inducing cellular signal generated by three-dimensional type I collagen. We also determined that activities of tyrosine kinase and ERK and JNK MAP kinases were required for this collagen-induced MMP-13 expression. Interestingly, bone morphogenetic protein (BMP)-2 opposed this induction, an effect that may be related to a role of BMP-2 in the maintenance of cartilage matrix.     

Effect of glycosaminoglycans on matrix metalloproteinases in type II collagen-induced experimental arthritis

Matrix metalloproteinases (MMPs) are a family of neutral proteinases that are involved in tissue remodeling by mediating degradation of extracellular matrix components in both physiology and pathology. As MMPs appear to play a key role in the degradation of cartilage matrix in the progression of arthritic disease, MMPs are considered as potential therapeutic targets. The effect of chondroitin sulfate A (CSA) on MMPs in type II collagen-induced experimental arthritis was studied. The anti-arthritic effect of CSA was evidenced by a decrease in marker activities like lysosomal beta-hexosaminidase and beta-glucuronidase. Arthritic animals showed significantly higher activity of MMP2 and MMP9 and increased levels of other MMPs, including MMP3 and MT-1 MMP in cartilage and serum. Treatment with CSA significantly decreased the activity of MMPs, particularly MMP9 in serum and synovial effusate and cartilage. The effect of CSA was further studied by fragmenting CSA into low-molecular-weight oligosaccharides. The oligosaccharide-treated animals showed considerably lower MMP activity (particularly MMP9) compared with arthritic controls. The CSA (and the oligosaccharides derived from it) not only reduced the activity of MMPs but also decreased the protein level expression of MMPs, indicating that the production of MMPs is affected. These results indicate that the antiarthritic effect of CSA involves down-regulation of MMPs, which are critically involved in the progression of arthritic disease.     

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.     

Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.     

Enhanced expression of the human chitinase 3-like 2 gene (YKL-39) but not chitinase 3-like 1 gene (YKL-40) in osteoarthritic cartilage

The knowledge of molecular alterations in osteoarthritic cartilage is important to identify novel therapeutic targets or to develop new diagnostic tools. We aimed to characterize the molecular response to cartilage degeneration by identification of differentially expressed genes in human osteoarthritic versus normal cartilage. Gene fragments selectively amplified in osteoarthritic cartilage by cDNA representational difference analysis included YKL-39 and the oesophageal-cancer-related-gene-4 (ECRG4). YKL-39 expression was significantly upregulated in cartilage from patients with osteoarthritis (n=14) versus normal subjects (n=8) according to real-time PCR (19-fold, p=0.009) and cDNA array analysis (mean 15-fold, p<0.001) and correlated with collagen 2 up-regulation. In contrast, the homologous cousin molecule YKL-40 (chitinase 3-like 1), which is elevated in serum and synovial fluid of patients with arthritis, showed no significant regulation in OA cartilage. Enhanced levels of YKL-40 may, therefore, be derived from synovial cells while modulation of YKL-39 and collagen 2 expression reflected the cartilage metabolism in response to degradation.     

Coordinate induction of collagenase-1, stromelysin-1 and urokinase plasminogen activator (uPA) by the 120-kDa cell-binding fibronectin fragment in fibrocartilaginous cells: uPA contributes to activation of procollagenase-1.

Specific fibronectin (Fn) fragments found in synovial fluid of arthritic joints potentially contribute to the loss of cartilage proteoglycans by inducing matrix metalloproteinase (MMP) expression. However, whether or not the Fn fragment-modulated changes in expression of MMPs result in a net increase in matrix-degradative activity through alterations in the balance between MMP activation and inhibition has not been established. To understand the mechanisms by which proteolytic Fn fragments may contribute to joint degeneration, conditioned medium from fibrocartilaginous cells exposed to Fn, its 30-kDa fragment containing the collagen/gelatin-binding domain, its 120-kDa fragment containing the central cell-binding domain, and the RGD peptide were assayed for MMPs, and MMP activators and inhibitors. We found that the 120-kDa fragment of Fn (but not intact Fn), the 30-kDa fragment, and the RGD peptide, dose-dependently induced procollagenase-1 and prostromelysin-1 and decreased levels of the tissue inhibitor of metalloproteinases (TIMPs) -1 and -2. The alpha5beta1 integrin was implicated in the induction of collagenase by the 120-kDa Fn fragment, since collagenase induction was abrogated in the presence of blocking antibody to this integrin. Conditioned medium from cells exposed to the 120-kDa Fn fragment also demonstrated increased levels of the activated collagenase-1, which resulted in significantly elevated collagen degradative activity. That the urokinase plasminogen activator (uPA) was involved in the activation of procollagenase-1 was suggested by findings that the 120-kDa Fn fragment induced uPA coordinately with procollagenase-1, and the activation of procollagenase-1 was dose-dependently inhibited in the presence of plasminogen activator inhibitor-1. These data demonstrate that the 120-kDa cell-binding fragment of Fn induces a net increase in matrix-degradative activity in fibrocartilaginous cells by concomitantly inducing MMPs and their activator, uPA, while decreasing TIMPs.     

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.     

Beta 1 and beta 3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix.

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of beta 1 and beta 3 integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning beta 1 integrins with little, if any, contribution by the alpha V beta 3 integrin; on the other hand, cell migration over these substrates depended to a large extent on the alpha V beta 3 receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the beta 1 integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the beta 3 receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of beta 1 integrins binding to each matrix component: FN (alpha 5 beta 1, alpha 3 beta 1, alpha V beta 1), LN (alpha 1 beta 1, alpha 7 beta 1), VN (alpha V beta 1), I (alpha 1 beta 1, alpha 2 beta 1), and IV (alpha 1 beta 1). In contrast, the beta 3 integrin, alpha V beta 3, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that beta 1 and beta 3 integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.     

Enhancement of cell adhesion and spreading by a cartilage-specific noncollagenous protein, cartilage matrix protein (CMP/Matrilin-1), via integrin alpha1beta1

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10-20 micrograms/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.     

Extracellular matrix regulates induction of alkaline phosphatase expression by ascorbic acid in human fibroblasts

During wound healing and inflammation, fibroblasts express elevated alkaline phosphatase (ALP), but are not in contact with collagen fibrils in the fibronectin (FN)-rich granulation tissue. We hypothesized that the extracellular matrix (ECM) environment might influence the induction of ALP in fibroblasts. Here we tested this hypothesis by studying the ALP-inductive response of normal human gingival fibroblasts to ascorbic acid (AsA). AsA induced ALP activity and protein in cells in conventional monolayer culture. This induction was inhibited by blocking-antibodies to the FN receptor alpha 5 beta 1 integrin and by the proline analog 3,4-dehydroproline (DHP). DHP prevented cells from arranging FN fibrils into a pericellular network and reduced the activity of cell spreading on FN. Plating of cells on FN facilitated the up-regulation by AsA of ALP expression, but did not substitute for AsA. In contrast, AsA did not cause ALP induction in cells cultured on and in polymerized type I collagen gels. Collagen fibrils inhibited the up-regulation by AsA of ALP expression in cells plated on FN. These results indicate that the ECM regulates the induction of ALP expression by AsA in fibroblasts: FN enables them to express ALP in response to AsA through interaction with integrin alpha 5 beta 1, whereas type I collagen fibrils cause the suppression of ALP expression and overcome FN.     

Adhesion properties of human bladder cell lines with extracellular matrix components: the role of integrins and glycosylation

Integrin subunits present on human bladder cells displayed heterogeneous functional specificity in adhesion to extracellular matrix proteins (ECM). The non-malignant cell line (HCV29) showed significantly higher adhesion efficiency to collagen IV, laminin (LN) and fibronectin (FN) than cancer (T24, Hu456) and v-raf transfected (BC3726) cell lines. Specific antibodies to the alpha(2), alpha(5) and beta(1) integrin subunits inhibited adhesion of the non-malignant cells, indicating these integrin participation in the adhesion to ECM proteins. In contrast, adhesion of cancer cells was not inhibited by specific antibodies to the beta(1) integrin subunit. Antibodies to alpha(3) integrin increased adhesion of cancer cells to collagen, LN and FN, but also of the HCV29 line with collagen. It seems that alpha(3) subunit plays a major role in modulation of other integrin receptors especially in cancer cells. Differences in adhesion to ECM proteins between the non-malignant and cancer cell lines in response to Gal and Fuc were not evident, except for the v-raf transfected cell line which showed a distinct about 6-fold increased adhesion to LN on addition of both saccharides. N-Acetylneuraminic acid inhibited adhesion of all cell lines to LN and FN irrespective of their malignancy.     

Functional genomic analysis in arthritis-affected cartilage: yin-yang regulation of inflammatory mediators by alpha 5 beta 1 and alpha V beta 3 integrins

Osteoarthritis-affected cartilage exhibits enhanced expression of fibronectin (FN) and osteopontin (OPN) mRNA in differential display and bioinformatics screen. Functional genomic analysis shows that the engagement of the integrin receptors alpha 5 beta 1 and alpha v beta 3 of FN and OPN, respectively, have profound effects on chondrocyte functions. Ligation of alpha 5 beta 1 using activating mAb JBS5 (which acts as agonist similar to FN N-terminal fragment) up-regulates the inflammatory mediators such as NO and PGE2 as well as the cytokines, IL-6 and IL-8. Furthermore, up-regulation of these proinflammatory mediators by alpha 5 beta1 integrin ligation is mediated via induction and autocrine production of IL-1 beta, because type II soluble IL-1 decoy receptor inhibits their production. In contrast, alpha v beta 3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of IL-1 beta, NO, and PGE2 (triggered by alpha 5 beta 1, IL-1 beta, IL-18, or IL-1 beta, TNF-alpha, plus LPS) in a dominant negative fashion by osteoarthritis-affected cartilage and activated bovine chondrocytes. These data demonstrate a cross-talk in signaling mechanisms among integrins and show that integrin-mediated "outside in" and "inside out" signaling very likely influences cartilage homeostasis, and its deregulation may play a role in the pathogenesis of osteoarthritis.     

Cholesterol alters the interaction of glycosphingolipid GM3 with alpha5beta1 integrin and increases integrin-mediated cell adhesion to fibronectin

Integrins bind to their ligand in the extracellular matrix (ECM), such as fibronectin (FN), through a specific interaction between the amino acid motifs in the ligand, and binding sites in the extracellular domains of the integrin molecule generated jointly by its alpha and beta subunits. It has been proposed that membrane cholesterol and glycosphingolipids (GSLs) can regulate integrin-ECM interactions and it has been demonstrated that increased membrane cholesterol leads to increased cell adhesion to FN. Here, we have shown that a specific glycosphingolipid GM3 binds directly to alpha5beta1 integrin and an increase in membrane cholesterol results in the redistribution of GM3-associated alpha5beta1 integrin molecules specifically on the surface that is in contact with the substratum. Our results suggest that GM3-associated alpha5beta1 integrins bind less avidly to FN than GM3-free integrins and that cholesterol and GM3 play an interdependent role in the distribution of alpha5beta1integrin molecules in the membrane and regulation of cell adhesion.     

Recombinant expression and characterization of a novel fibronectin isoform expressed in cartilaginous tissues

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.     

Membrane-associated IL-1 contributes to chronic synovitis and cartilage destruction in human IL-1 alpha transgenic mice

IL-1 molecules are encoded by two distinct genes, IL-1alpha and IL-1beta. Both isoforms possess essentially identical activities and potencies, whereas IL-1alpha, in contrast to IL-1beta, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1alpha (hIL-1alpha) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys(64) to Ala(271) of the hIL-1alpha amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1alpha. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1alpha activity or concentration of serum hIL-1alpha. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1alpha, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.     

Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin

Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors.     

Coordination of chondrocyte differentiation and joint formation by alpha5beta1 integrin in the developing appendicular skeleton

The control point by which chondrocytes take the decision between the cartilage differentiation program or the joint formation program is unknown. Here, we have investigated the effect of alpha5beta1 integrin inhibitors and bone morphogenetic protein (BMP) on joint formation. Blocking of alpha5beta1 integrin by specific antibodies or RGD peptide (arginine-glycine-aspartic acid) induced inhibition of pre-hypertrophic chondrocyte differentiation and ectopic joint formation between proliferating chondrocytes and hypertrophic chondrocytes. Ectopic joint expressed Wnt14, Gdf5, chordin, autotaxin, type I collagen and CD44, while expression of Indian hedgehog and type II collagen was downregulated in cartilage. Expression of these interzone markers confirmed that the new structure is a new joint being formed. In the presence of BMP7, inhibition of alpha5beta1 integrin function still induced the formation of the ectopic joint between proliferating chondrocytes and hypertrophic chondrocytes. By contrast, misexpression of alpha5beta1 integrin resulted in fusion of joints and formation of pre-hypertrophic chondrocytes. These facts indicate that the decision of which cell fate to make pre-joint or pre-hypertrophic is made on the basis of the presence or absence of alpha5beta1 integrin on chondrocytes.     

Accessibility to the fibronectin synergy site in a 3D matrix regulates engagement of alpha5beta1 versus alphavbeta3 integrin receptors

Cell adhesion and migration on fibronectin (FN) extracellular matrix are mediated by integrin receptors. Integrins alpha5beta1 and alphavbeta3 require the RGD cell-binding sequence in FN, but alpha5beta1 also requires the nearby synergy site for maximal binding. In this study, we investigated how differences in the numbers of RGD or synergy sites within a three-dimensional (3D) FN-rich matrix influence cell adhesion and migration. CHO cell adhesion, spreading, and migration were reduced on 3D chimeric matrix containing FN lacking RGD (FN(RGD-)). Incorporation of FN with mutation of the synergy site (FN(syn-)), however, resulted in selective usage of integrins. CHO cells expressing alpha5beta1 showed decreased interactions with FN(syn-) chimeric matrix. In contrast, the presence of FN(syn-) had no effect on CHOalphavbeta3 cell migration. Interestingly, CHOalpha5/alphavbeta3 cells expressing both integrins selectively used alpha5beta1 for migration on wild type FN matrix but preferred alphavbeta3 for migration on FN(syn-) chimeric matrix. Thus sequestration or exposure of the FN synergy site within a 3D matrix may represent a novel mechanism for regulating cell functions through differential usage of integrin receptors. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resource: a video recording shows migration of HT1080 cells on 3D matrix. HT1080 cells were allowed to attach to the matrix in serum-free DMEM for 2 h. FBS was then added to the medium to a final concentration of 10% and video recording was started. Images were taken every 5 min for 2 h. The video plays at 6 frames/s.].