Platelet-derived growth factor or basic fibroblast growth factor induce anchorage-independent growth of human fibroblasts

Anchorage-independent growth, i.e., growth in semi-solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage-independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage-independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage-independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet-derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage-independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF-beta) decreased the growth induced by PDGF. EGF combined with TGF-beta induced colony formation in semi-solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage-independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF-beta can stimulate anchorage-independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.     

Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain

Integrin-mediated cell attachment and growth factor stimulation often act synergistically on cell proliferation, differentiation, migration, and survival. Some of these synergistic effects depend on the physical interaction of integrins with growth factor receptors. Here we examine the nature of the physical interaction between the alpha(v)beta(3) integrin and two receptor tyrosine kinases (RTKs), the platelet-derived growth factor receptor beta (PDGF-Rbeta) and the vascular endothelial growth factor receptor 2 (VEGF-R2, also known as KDR and flk-1). Both of these RTKs associate with the alpha(v)beta(3) integrin but do not associate with beta(1) integrins. Furthermore, growth factor stimulation of these RTKs promotes increased cell proliferation and migration when cells are attached to the alpha(v)beta(3) ligand, vitronectin. We show that alpha(v)beta(3) in which the beta(3) cytoplasmic domain is deleted or replaced with the beta(1) cytoplasmic domain coimmunoprecipitates with PDGF-Rbeta and VEGF-R2. The beta(3) extracellular domain alone was sufficient for the PDGF-Rbeta association whereas the VEGF-R2 association required the presence of the alpha(v) subunit. Activation of the RTKs by their ligands was not required for them to associate with the integrin. Cell migration to PDGF was enhanced in the cells transfected with the chimeric subunit containing the beta(3) extracellular domain but not when that domain came from the beta(1) subunit. These results show that the interactions that lead to the association of the alpha(v)beta(3) integrin with PDGF-Rbeta and VEGF-R2 and enhancement of RTK activity take place outside the cell.     

The pattern of enhancement of Src kinase activity on platelet-derived growth factor stimulation of glioblastoma cells is affected by the integrin engaged

Enhanced expression of both integrin alpha v beta 3 and platelet-derived growth factor receptor (PDGFr) has been described in glioblastoma tumors. We therefore explored the possibility that integrin alpha v beta 3 cooperates with PDGFr to promote cell migration in glioblastoma cells, and extended the study to identify the Src family members that are activated on PDGF stimulation. Glioblastoma cells utilize integrins alpha v beta 3 and alpha v beta 5 to mediate vitronectin attachment. We found that physiologic PDGF stimulation (83 pm, 10 min) of vitronectin-adherent cells promoted the specific recruitment of integrin alpha v beta 3-containing focal adhesions to the cell cortex and alpha v beta 3-mediated cell motility. Analysis of PDGFr immunoprecipitates indicated an association of the PDGFr beta with integrin alpha v beta 3, but not integrin alpha v beta 5. Cells plated onto collagen or laminin, which engage different integrins, exhibited significantly less migration on PDGF stimulation, indicating a cooperation of alpha v beta 3 and the PDGFr beta in glioblastoma cells that promotes migration. Further analysis of the cells plated onto vitronectin indicated that PDGF stimulation caused an increase in Src kinase activity, which was associated with integrin alpha v beta 3. In the vitronectin-adherent cells, Lyn was associated preferentially with alpha v beta 3 both in the presence and absence of PDGF stimulation. In contrast, Fyn was associated with both alpha v beta 3 and alpha v beta 5. Moreover, PDGF stimulation increased the activity of Lyn, but not Fyn, in vitronectin-adherent cells, and the activity of Fyn, but not Lyn, in laminin-adherent cells. Using cells attached to mAb anti-alpha v beta 3 or mAb anti-integrin alpha 6, we confirmed the activation of specific members of the Src kinase family with PDGF stimulation. Down-regulation of Lyn expression by siRNA significantly inhibited the cell migration mediated by integrin alpha v beta 3 in PDGF-stimulated cells, demonstrating the PDGFr beta cooperates with integrin alpha v beta 3 in promoting the motility of vitronectin-adherent glioblastoma cells through a Lyn kinase-mediated pathway. Notably, the data indicate that engagement of different integrins alters the identity of the Src family members that are activated on stimulation with PDGF.     

A growth factor for cardiac myocytes is produced by cardiac nonmyocytes.

Cardiac nonmyocytes, primarily fibroblasts, surround cardiac myocytes in vivo. We examined whether nonmyocytes could modulate myocyte growth by production of one or more growth factors. Cardiac myocyte hypertrophic growth was stimulated in cultures with increasing numbers of cardiac nonmyocytes. This effect of nonmyocytes on myocyte size was reproduced by serum-free medium conditioned by the cardiac nonmyocytes. The majority of the nonmyocyte-derived myocyte growth-promoting activity bound to heparin-Sepharose and was eluted with 0.75 M NaCl. Several known polypeptide growth factors found recently in cardiac tissue, namely acidic fibroblast growth factor (aFGF), basic FGF (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF beta 1), also caused hypertrophy of cardiac myocytes in a dose-dependent manner. However, the nonmyocyte-derived growth factor (tentatively named NMDGF) could be distinguished from these other growth factors by different heparin-Sepharose binding profiles (TNF alpha, aFGF, bFGF, and TGF beta 1) by neutralizing growth factor-specific antisera (PDGF, TNF alpha, aFGF, bFGF, and TGF beta 1), by the failure of NMDGF to stimulate phosphatidylinositol hydrolysis (PDGF and TGF beta 1), and, finally, by the apparent molecular weight of NMDGF (45-50 kDa). This nonmyocyte-derived heparin-binding growth factor may represent a novel paracrine growth mechanism in myocardium.     

Migratory and proliferative effect of platelet-derived growth factor in rabbit retinal endothelial cells: Evidence of an autocrine pathway of platelet-derived growth factor

Angiogenesis is a crucial event in the progression of diabetic retinopathy. Migration and proliferation of endothelial cells (EC) are important steps in angiogenesis and are caused by angiogenic factors such as basic fibroblast growth factor (bFGF). In this work, capillary EC were isolated from rabbit retinal tissues and rabbit retinal EC (RREC) were found to secrete a migration factor for RREC in conditioned medium (CM). The activity was inhibited by an anti-platelet-derived growth factor (PDGF) antibody, but not by an anti-bFGF antibody. We also found that RREC showed a migratory response to PDGF. The response was induced by PDGF-BB and PDGF-AB dose dependently, but not by PDGF-AA, indicating that it was mediated by PDGF-β receptor-dependent pathways, and that the PDGF-like factor was PDGF-BB or -AB. In addition, PDGF-BB induced the proliferation of RREC as well as bFGF. These data indicate that RREC have an autocrine pathway of PDGF by the secretion of and the response to PDGF. PDGF may play significant parts in angiogenesis in the progression of diabetic retinopathy. © 1994 Wiley-Liss, Inc.     

Synergism among lysophosphatidic acid, beta1A integrins, and epidermal growth factor or platelet-derived growth factor in mediation of cell migration.

GD25 cells lacking the beta1 integrin subunit or expressing beta1A with certain cytoplasmic mutations have poor directed cell migration to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), ligands of receptor tyrosine kinases, or to lysophosphatidic acid (LPA), a ligand of G-protein-coupled receptors (Sakai, T., Zhang, Q., F?ssler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538 and Sakai, T., Peyruchaud, O., F?ssler, R., and Mosher, D. F. (1998) J. Biol. Chem. 273, 19378-19382). We demonstrate here that LPA synergizes with signals induced by beta1A integrins and ligated EGF or PDGF receptors to modulate migration. When LPA was mixed with EGF or PDGF, migration was greater than with EGF or PDGF alone. The enhancement was greater for beta1A-expressing cells than for beta1-null cells. Cells expressing beta1A with mutations of prolines or tyrosines in conserved cytoplasmic NPXY motifs had blunted migratory responses to mixtures of LPA and EGF or PDGF. The major effects on beta1A-expressing cells of LPA when combined with EGF or PDGF were to sensitize cells so that maximal responses were obtained with >10-fold lower concentrations of growth factor and increase the chemokinetic component of migration. Sensitization by LPA was lost when cells were preincubated with pertussis toxin or C3 exotransferase. There was no evidence for transactivation or sensitization of receptors for EGF or PDGF by LPA. EGF or PDGF and LPA caused activation of mitogen-activated protein kinase by pertussis toxin-insensitive and -sensitive pathways respectively, but activation was not additive. These findings indicate that signaling pathways initiated by the cytoplasmic domains of ligated beta1A integrins and tyrosine kinase receptors interact with signaling pathways initiated by LPA to facilitate directed cell migration.     

Autonomous migration of human fetal skin fibroblasts into a denuded area in a cell monolayer is mediated by basic fibroblast growth factor and collagen

Summary Human fetal skin fibroblasts (TIG-3S) were found to migrate into a denuded area in a cell monolayer when cultured in both serum-depleted and serum-supplemented media, unlike adult-donor skin fibroblasts which migrated well only when cultured in serum-supplemented medium. Therefore, a series of experiments was carried out to determine whether autocrine factors are involved in their migration. The migration of TIG-3S cells in serum-depleted medium was suppressed by the addition of suramin, a factor with growth factor antagonist properties, which suggests that growth factors are important for cell migration. The suramin-induced inhibition was reversed completely by adding excess basic fibroblast growth factor (bFGF) to the culture medium and partially by platelet-derived growth factor (PDGF). Treatment with neutralizing anti-PDGF antibody did not suppress TIG-3S cell migration, whereas neutralizing anti-bFGF antibody did, which indicates that bFGF is an autocrine and PDGF a paracrine factor involved in cell migration. Next, an experiment was performed to ascertain whether the extracellular matrix is involved in TIG-3S cell migration. Monensin, an inhibitor of extracellular matrix secretion, inhibited cell migration, which was reversed by adding excess type I collagen, but not excess plasma fibronectin. In addition, further evidence for the involvement of collagen was provided by the observation that ethyl-3,4-dihydroxybenzoate, a specific inhibitor of collagen synthesis, suppressed cell migration. These results suggest that the autonomous migration of TIG-3S human fetal skin fibroblasts is mediated by bFGF and type I collagen, which they produce and secrete.     

Altered response to growth factors or retinoic acid in phenotypic transformation of normal rat kidney cells expressing human c-fos gene.

Anchorage-independent growth of normal rat kidney (NRK) fibroblast in soft agar depends on both transforming growth factor beta (TGF beta) and epidermal growth factor (EGF). To examine whether c-fos protein is involved in phenotypic transformation of NRK cells, we have transfected and isolated several NRK cell lines that carry the human c-fos gene fused to the metallothionein IIA promoter. A transfectant, Nf-1, had constitutive levels of the human c-fos expression. Anchorage-independent growth of Nf-1 was already stimulated by EGF alone, and the colony sizes of Nf-1 were comparable to those of the parental NRK in the presence of both EGF and TGF beta. Anchorage-independent growth of NRK could be observed in the presence of TGF beta or retinoic acid or platelet derived growth factor (PDGF) and EGF. No growth of NRK in soft agar appeared when basic fibroblast growth factor (bFGF) and EGF were present. By contrast, anchorage-independent growth of Nf-1 was surprisingly enhanced by EGF and TGF beta or retinoic acid or PDGF or bFGF. Expression of the human c-fos gene may compensate the signal to phenotypic transformation induced by TGF beta as well as retinoic acid or PDGF or bFGF.     

Modulation of growth factor responsiveness of murine mammary carcinoma cells by cell matrix interactions: correlation of cell proliferation and spreading.

We have examined the role of growth factors and extracellular matrix in the proliferation and cell adhesion of a murine mammary carcinoma, SP1, and a stable highly metastatic variant, SP1-3M. On fibronectin, both cell types proliferated strongly in response to basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB (PDGF-BB) after culture for 24 h and 72 h. In contrast, on collagen type I, SP1 cells proliferated only weakly to PDGF-BB at either time, and SP1-3M cells showed a response to PDGF-BB only at 72 h. The proliferative response to bFGF was also consistently lower when the cells were cultured on collagen than on fibronectin. No significant proliferative responses were detected to epithelial growth factor (EGF), transforming growth factor-beta (TGF-beta), or estrogen on any substratum. The lack of responsiveness to PDGF-BB of cells cultured on collagen type I was not due to differences in numbers or affinity of PDGF receptors. We therefore examined the adhesion and spreading properties of SP1 and SP1-3M cells. Without exogenous growth factors, both cell lines adhered to fibronectin and laminin. SP1-3M cells did not bind to collagen type I, whereas SP1 cells did. Attachment to all three substrata was inhibited by anti-beta 1 integrin IgG, suggesting that the primary adhesion to these substrata is mediated by beta 1 integrins. SP1 and SP1-3M cells showed similar integrin patterns following immunoprecipitation by anti-beta 1 integrin IgG. bFGF stimulated increased adhesion and spreading of both SP1 and SP1-3M cells to collagen type I within 24 h, whereas PDGF-BB was less capable of this effect. Our results suggest that the proliferative response of SP1 and SP1-3M cells to PDGF-BB and bFGF is dependent on the extracellular matrix environment, and imply that modification of extracellular matrix and/or surface integrin receptors may regulate responsiveness to these growth factors in the SP1 tumor model.     

Basic fibroblast growth factor modulates the mitogenic potency of the platelet-derived growth factor (PDGF) isoforms by specific upregulation of the PDGF alpha receptor in vascular smooth muscle cells.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.     

Transforming growth factors beta(1) (TGF-beta(1)) and TGF-beta(2) promote glioma cell migration via Up-regulation of alpha(V)beta(3) integrin expression

The migratory behaviour of malignant gliomas relies on the interaction of integrins with extracellular matrix (ECM) components. Transforming growth factor-beta(1) (TGF-beta(1)) potently stimulates glioma cell motility whereas TGF-beta(2) is known for its immunosuppressive properties. Here, we show that both TGF-beta(1) and TGF-beta(2) promote migration of glioma cells. In parallel, TGF-beta(1) and TGF-beta(2) induce alpha(V) and beta(3) intergrin mRNA expression and enhance cell surface expression of alpha(V)beta(3) integrin. TGF-beta-mediated promotion of migration is abrogated by echistatin, a Arg-Gly-Asp (RGD) peptide antagonist of alpha(V)beta(3) integrin, and by a neutralizing anti-alpha(V)beta(3) integrin antibody. Taken together, we report a novel mechanism by which TGF-beta modulates cell ECM interactions and promotes glioma cell motility.     

Integrin alpha9beta1 directly binds to vascular endothelial growth factor (VEGF)-A and contributes to VEGF-A-induced angiogenesis

Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin alpha9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from alpha9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike alpha3beta1or alphavbeta3 integrins, alpha9beta1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because alpha9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that alpha9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin alpha9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.     

Receptor heterodimerization: essential mechanism for platelet-derived growth factor-induced epidermal growth factor receptor transactivation

Previous studies showed that the epidermal growth factor receptor (EGFR) can be transactivated by platelet-derived growth factor (PDGF) stimulation and that EGFR transactivation is required for PDGF-stimulated cell migration. To investigate the mechanism for cross talk between the PDGF beta receptor (PDGFbetaR) and the EGFR, we stimulated rat aortic vascular smooth muscle cells (VSMC) with 20 ng of PDGF/ml. Transactivation of the EGFR, defined by receptor tyrosine phosphorylation, occurred with the same time course as PDGFbetaR activation. Basal formation of PDGFbetaR-EGFR heterodimers was shown by coimmunoprecipitation studies, and interestingly, disruption of this receptor heterodimer abolished EGFR transactivation. Breakdown of the heterodimer was observed when VSMC were pretreated with antioxidants or with a Src family kinase inhibitor. Disruption of heterodimers decreased ERK1 and ERK2 activation by PDGF. Although PDGF-induced PDGFbetaR activation was abolished after pretreatment with 1 microM AG1295 (a specific PDGF receptor kinase inhibitor), EGFR transactivation was still observed, indicating that PDGFbetaR kinase activity is not required. In conclusion, our data demonstrate that the PDGFbetaR and the EGFR form PDGFbetaR-EGFR heterodimers basally, and we suggest that heterodimers represent a novel signaling complex which plays an important role in PDGF signal transduction.     

Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components.

We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor 1, bovine fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-beta blocked the cellular network formation. Conversely, addition of TGF-beta 1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF, EGF, and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-beta. Some TGF-beta remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior, suggesting caution in the interpretation of experiments on cellular activity related to Matrigel, collagen type IV, and possibly other extracellular matrix components.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis.

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGF beta), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGF beta and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGF beta to bFGF determines the magnitude of the Schwann cell response.     

Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.     

Modulation, by transforming growth factor beta (TGF beta), of the mitogenic effect of fibroblast growth factor (FGF) on rat oligodendrocytes in culture

The transforming growth factor beta (TGF beta) is a weak mitogen for rat oligodendrocytes grown in serum-free chemically defined medium. When these cells were treated by basic fibroblast growth factor (bFGF), which is the most potent known mitogen for cultured oligodendrocytes, together with TGF beta we observed that at low doses TGF beta potentiates the mitogenic effect of bFGF while at higher concentrations it partly inhibits this effect.     

Regulation of vascular smooth muscle cell integrin expression by transforming growth factor beta1 and by platelet-derived growth factor-BB.

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the alpha v beta 3 integrin complex was induced by both growth factors, although TGF-beta 1 appeared to be the more potent inducer. mRNA level of the beta 3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the alpha v integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the alpha v beta 3 integrin protein complex after growth factor stimulation was due to increased expression of the beta 3 integrin subunit mRNA. The TGF-beta 1 induced increase in beta 3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in beta 3 mRNA level. By contrast, PDGF-BB induced a more rapid increase in beta 3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable beta 3 integrin mRNA remained after 24 h. Interestingly, the PDGF-BB induced elevation of beta 3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the alpha 5 integrin subunit in response to growth factors was very similar to beta 3. However, in contrast to beta 3 and alpha 5, neither TGF-beta 1 nor PDGF-BB were able to alter the expression of the beta 1 integrin subunit in vascular SMC. However, in TGF-beta 1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the beta 1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-beta 1 treated fibroblasts. The alpha 1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with beta 1. Analysis of the alpha 1 integrin subunit mRNA level indicated that it was indeed induced by TGF-beta 1, but not by PDGF-BB, suggesting that the 190 kDa polypeptide may be the alpha 1 integrin subunit. These results indicate that TGF-beta 1 and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC.     

Connective tissue growth factor (CCN2) induces adhesion of rat activated hepatic stellate cells by binding of its C-terminal domain to integrin alpha(v)beta(3) and heparan sulfate proteoglycan

Connective tissue growth factor (CCN2, also known as CTGF) is a matricellular protein that appears to play an important role in hepatic stellate cell (HSC)-mediated fibrogenesis. After signal peptide cleavage, the full-length CCN2 molecule comprises four structural modules (CCN2(1-4)) and is susceptible to proteolysis by HSC yielding isoforms comprising essentially modules 3 and 4 (CCN2(3-4)) or module 4 alone (CCN2(4)). In this study we show that rat activated HSC are capable of adhesion to all three CCN2 isoforms via the binding of module 4 to integrin alpha(v)beta(3), a process that is dependent on interactions between module 4 and cell surface heparan sulfate proteoglycans (HSPGs). These findings are based on several lines of evidence. First, integrin alpha(v)beta(3) was detected in HSC lysates by immunoprecipitation and Western blot, and CCN2(4)-mediated HSC adhesion was blocked by anti-integrin alpha(v)beta(3) antibody. Second, as assessed by immunoprecipitation and solid phase binding assay, CCN2(4) bound directly to integrin alpha(v)beta(3) in cell-free systems. Third, destruction or inhibition of synthesis of cell surface HSPGs with, respectively, heparinase or sodium chlorate abrogated HSC adhesion to CCN2(4). Fourth, prior occupancy of heparin-binding sites on CCN2(4) with soluble heparin completely blocked HSC adhesion. These findings indicate that integrin alpha(v)beta(3) functions as a co-receptor with HSPGs for CCN2(4)-mediated HSC adhesion. Furthermore, by peptide mapping and site-directed mutagenesis we demonstrated that the sequence IRTPKISKPIKFELSG within CCN2(4) is a unique binding domain for integrin alpha(v)beta(3) that is sufficient to mediate integrin alpha(v)beta(3)- and HSPG-dependent HSC adhesion. These findings offer the possibility of developing novel antifibrotic therapies that target the integrin-binding domain.