Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta

Connective-tissue growth factor (CTGF) is a secreted protein implicated in multiple cellular events including angiogenesis, skeletogenesis and wound healing. It is a member of the CCN family of secreted proteins, named after CTGF, cysteine-rich 61 (CYR61), and nephroblastoma overexpressed (NOV) proteins. The molecular mechanism by which CTGF or other CCN proteins regulate cell signalling is not known. CTGF contains a cysteine-rich domain (CR) similar to those found in chordin and other secreted proteins, which in some cases have been reported to function as bone morphogenetic protein (BMP) and TGF-beta binding domains. Here we show that CTGF directly binds BMP4 and TGF-beta 1 through its CR domain. CTGF can antagonize BMP4 activity by preventing its binding to BMP receptors and has the opposite effect, enhancement of receptor binding, on TGF-beta 1. These results show that CTGF inhibits BMP and activates TGF-beta signals by direct binding in the extracellular space.     

Regulation of bone morphogenetic protein-4 by matrix GLA protein in vascular endothelial cells involves activin-like kinase receptor 1

Matrix GLA protein (MGP) has previously been shown to enhance expression of vascular endothelial growth factor (VEGF) through the activin-like kinase receptor 1 (ALK1) in bovine aortic endothelial cells. MGP has also been identified as an inhibitor of bone morphogenetic protein-2 (BMP-2). This study showed that the effect of MGP on ALK1 signaling and VEGF expression in bovine aortic endothelial cells was dose-dependent, that a progressive increase of MGP levels ceased to be stimulatory and instead turned inhibitory. We identified a new regulatory pathway involving BMP that may explain this response. BMP-2 and BMP-4 induced expression of ALK1 in a dose-dependent fashion as determined by real-time PCR and immunoblotting. Activation of ALK1 signaling induced expression of MGP in addition to that of VEGF, allowing for negative feedback regulation of BMP by MGP. MGP inhibited BMP-4 activity similarly to that of BMP-2 and interacted with BMP-4 on a protein level as determined by co-immunoprecipitation. The dose-dependent effect on ALK1 expression and the stimulation of MGP and VEGF expression were dependent on signaling by transforming growth factor-beta (TGF-beta) and ALK1. Inhibition of TGF-beta by neutralizing antibodies abolished the inhibitory effect of high BMP-4 levels on ALK1 expression and the induction of MGP and VEGF. Depletion of ALK1 by small interfering RNA abolished the induction of MGP and VEGF. MGP promoter activity was also stimulated by BMP-4 in a TGF-beta-dependent fashion. The results suggest that the effects of BMP on endothelial cells occur in part through induction of ALK1, an effect that may be limited by ALK1-induced MGP.     

The tryptophan-rich motifs of the thrombospondin type 1 repeats bind VLAL motifs in the latent transforming growth factor-beta complex

Transforming growth factor-beta (TGF-beta) is secreted as a latent complex of the latency-associated peptide (LAP) and the mature domain, which must be activated for TGF-beta to signal. We previously identified thrombospondin 1 (TSP1) as a physiologic activator of TGF-beta in vitro and in vivo. The WSXW sequences in the type 1 repeats of TSP1 interact with the mature domain of TGF-beta, and WSXW peptides inhibit TSP1-mediated activation by blocking TSP1 binding to the TGF-beta latent complex. However, the binding site for the WSXW sequence was not identified. In this report, we show that the WSXW sequences bind the (61)VLAL sequence in mature TGF-beta and also bind (77)VLAL in LAP. A glutathione S-transferase (GST) fusion protein of the second TSP1 type 1 repeat (GST-TSR2) binds immobilized VLAL peptide. VLAL peptides inhibit binding of LAP and mature TGF-beta to soluble GST-TSR2 and immobilized WSXW peptide. VLAL peptide inhibits TSP1-mediated activation of recombinant and endothelial cell-derived latent TGF-beta. Furthermore, TGF-beta or LAP deleted in the VLAL sequence fails to bind immobilized WSXW or soluble GST-TSR2, indicating that binding to both VLAL sequences is important for association of TSP1 and the latent complex. Additionally, TSP1 is unable to activate latent TGF-beta when VLAL is deleted from the mature domain. These data show that the WSXW motif binds VLAL on both LAP and mature TGF-beta, and these interactions are critical for TSP1-mediated activation of the TGF-beta latent complex.     

Matrix GLA protein stimulates VEGF expression through increased transforming growth factor-beta1 activity in endothelial cells

Matrix GLA protein (MGP) is expressed in endothelial cells (EC), and MGP deficiency results in developmental defects suggesting involvement in EC function. To determine the role of MGP in EC, we cultured bovine aortic EC with increasing concentrations of human MGP (hMGP) for 24 h. The results showed increased proliferation, migration, tube formation, and increased release of vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF). HMGP, added endogenously or transiently expressed, increased VEGF gene expression dose-dependently as determined by real-time PCR. To determine the mechanism by which hMGP increased VEGF expression, we studied the effect of MGP on the activity of transforming growth factor (TGF)-beta1 compared with that of bone morphogenetic protein (BMP)-2 using transfection assays with TGF-beta- and BMP-response element reporter genes. Our results showed a strong enhancement of TGF-beta1 activity by hMGP, which was paralleled by increased VEGF expression. BMP-2 activity, on the other hand, was inhibited by hMGP. Neutralizing antibodies to TGF-beta blocked the effect of MGP on VEGF expression. The enhanced TGF-beta1 activity specifically activated the Smad1/5 pathway indicating that the TGF-beta receptor activin-like kinase 1 (ALK1) had been stimulated. It occurred without changes in expression of TGF-beta1 or ALK1 and was mimicked by transfection of constitutively active ALK1, which increased VEGF expression. Expression of VEGF and MGP was induced by TGF-beta1, but the induction of MGP preceded that of VEGF, consistent with a promoting effect on VEGF expression. Together, the results suggest that MGP plays a role in EC function, altering the response to TGF-beta superfamily growth factors.     

Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2.

Matrix GLA protein (MGP) has been identified as a calcification inhibitor in cartilage and vasculature. Part of this effect may be attributed to its influence on osteoinductive activity of bone morphogenetic protein-2 (BMP-2). To detect binding between MGP and BMP-2, we performed immunoprecipitation using MGP and BMP-2 tagged with FLAG and c-Myc. The results showed co-precipitation of BMP-2 with MGP. To quantify the effect of MGP on BMP-2 activity, we assayed for alkaline phosphatase activity and showed a dose-dependent effect. Low levels of MGP relative to BMP-2 (<1-fold excess) resulted in mild enhancement of osteoinduction, whereas intermediate levels (1-15-fold excess) resulted in strong inhibition. High levels of MGP (>15-fold excess), however, resulted in pronounced enhancement of the osteoinductive effect of BMP-2. Cross-linking studies showed that inhibitory levels of MGP abolished BMP-2 receptor binding. Immunoblotting showed a corresponding decrease in activation of Smad1, part of the BMP signaling system. Enhancing levels of MGP resulted in increased Smad1 activation. To determine the cellular localization of BMP-2 in the presence of MGP, binding assays were performed on whole cells and cell-synthesized matrix. Inhibitory levels of MGP yielded increased matrix binding of BMP-2, suggesting that MGP inhibits BMP-2 in part via matrix association. These results suggest that MGP is a BMP-2 regulatory protein.     

Regulation of cell adhesion receptors by transforming growth factor-beta. Regulation of vitronectin receptor and LFA-1

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) to regulate the expression of members of the alpha beta 2 and alpha beta 3 families of integrins. TGF-beta 1 elevates the expression of vitronectin receptors (alpha v beta 3 integrin) in all cells examined including WI-38 human lung fibroblasts, 3T3-L1 mouse fibroblasts, and MG-63 human osteogenic sarcoma cells. TGF-beta 1 action increases the level of mRNA and the synthesis of vitronectin receptor subunits with t1/2 o 3-4 h and 6 h, respectively. TGF-beta 1 up-regulates expression of the intercellular adhesion receptor, LFA-1 (alpha L beta 2), in THP-1 human monocytic leukemia cells by increasing the synthesis of alpha L subunit but not beta 2 subunit. The increase in alpha L synthesis and assembly into LFA-1 complexes induced by TGF-beta 1 occurs in parallel with elevated fibronectin receptor synthesis in THP-1 cells. These responses to TGF-beta 1 are lost upon phorbol ester-induced differentiation of THP-1 cells into the macrophage phenotype. The results suggest a role of TGF-beta in the regulation of cell-matrix interactions mediated by vitronectin receptors and cell-cell interactions mediated by LFA-1 in the immune system.     

Transforming growth factor beta 1 stimulates the production of basic fibroblast growth factor binding proteoglycans in Balb/c3T3 cells.

Basic fibroblast growth factor (bFGF) binds to cell surface receptors and to heparin sulfate proteoglycans. Heparan sulfate binding may limit bFGF degradation and be an obligatory step for bFGF cell interaction. Transforming growth factor-beta 1 (TGF-beta 1) is a potent regulator of proteoglycan production and composition. The possibility that TGF-beta 1 synergistically regulates bFGF activity by altering bFGF-proteoglycan interactions was investigated. TGF-beta 1 increased 125I-bFGF binding to the extracellular matrix (ECM) of Balb/c3T3 cells 2-4-fold by increasing the number of bFGF binding sites. Increased bFGF binding correlated with a 2-5-fold increase in the production of sulfated proteoglycans, including heparan sulfate proteoglycans. TGF-beta 1 selectively stimulated production of high molecular mass proteoglycans (190-300 kDa) in conditioned medium and stimulated all proteoglycans in ECM. 125I-bFGF bound to TGF-beta 1 induced proteoglycans immobilized onto cationic nylon filters. Furthermore, ECM isolated from TGF-beta 1-treated cells incorporated more mitogenically active bFGF than native ECM. The mitogenic potential of the ECM was significantly reduced by treatment with heparinase. These results suggest that the ability of TGF-beta 1 to stimulate binding of bFGF to ECM, increase ECM heparan sulfate proteoglycan, and potentiate the mitogenic activity of bFGF are linked. Thus one aspect of TGF-beta 1/bFGF synergy may involve modulation of the ECM.     

Bone morphogenetic proteins signal through the transforming growth factor-beta type III receptor

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-beta (TGF-beta) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-beta type III receptor (TbetaRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-beta and inhibin receptor. Here we demonstrate that TbetaRIII functions as a BMP cell surface receptor. TbetaRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-beta. TbetaRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TbetaRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TbetaRIII function, we demonstrate that BMP-2 specifically stimulates TbetaRIII-mediated epithelial to mesenchymal cell transformation. The ability of TbetaRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-beta signaling suggests a broader role for TbetaRIII in orchestrating TGF-beta superfamily signaling.     

The contributions of integrin affinity and integrin-cytoskeletal engagement in endothelial and smooth muscle cell adhesion to vitronectin

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50-75%. Because PAI-1 binds vitronectin with approximately 10-100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins alphavbeta3 and alphavbeta5 at the cell-matrix interface.     

Transient production and secretion of human transforming growth factor TGF-beta 2

Transient transfection of simian COS cells with a recombinant plasmid encoding the human transforming growth factor TGF-beta 2 precursor protein results in the production of a latent, biologically inactive protein. Upon acidification, recombinant TGF-beta 2 exhibits full biological activity, including inhibition of mink lung epithelial cell growth, stimulation of anchorage-independent growth of murine embryonic fibroblasts, and competition for TGF-beta receptor binding. Further analysis of conditioned media with antiserum to either a pro- [amino acid (aa) residues 1-220] or mature [aa 297-414] peptide of the TGF-beta 2 precursor suggests that TGF-beta 2, similar to TGF-beta 1 production in Chinese hamster ovary cells [Gentry et al., Mol. Cell. Biol. 7 (1987) 3418-3427], is initially synthesized as a larger precursor protein which is proteolytically cleaved to yield the mature 112-aa transforming growth factor.     

CD36 peptides enhance or inhibit CD36-thrombospondin binding. A two-step process of ligand-receptor interaction.

CD36 (glycoprotein IV or IIIB) is an integral plasma membrane protein of wide cellular distribution and functions as a receptor site for thrombospondin (TSP), an adhesive protein important in cell-cell and cell-matrix interactions. OKM5, a monoclonal anti-CD36 antibody, has been reported to block CD36 cell adhesive functions suggesting that the OKM5 epitope on CD36 is functionally important. A panel of 10 synthetic CD36 peptides was made. One peptide, P139-155, specifically inhibited the immunoadsorption of CD36 by OKM5, and P139-155 was directly immunoadsorbed by OKM5, indicating that CD36 sequence 139-155 represents part of the OKM5 epitope. TSP bound to immobilized P139-155 in a dose-dependent and saturable manner. Surprisingly, P139-155 significantly augmented, instead of inhibited, binding of CD36 to TSP. This peptide did not induce platelet aggregation but augmented ADP- and collagen-induced aggregation in platelet-rich plasma. Another CD36 peptide, P93-110, which had no effect on OKM5 immunoadsorption, blocked binding of CD36 to immobilized TSP and partially inhibited collagen-induced platelet aggregation. P93-110 by itself did not bind to TSP; however, in the presence of P139-155, there was a marked enhancement of P93-110 binding to TSP, with a stoichiometry consistent with the trimeric nature of TSP. The data suggest that CD36-TSP interaction is a two-step process; the sequence 139-155 region of CD36 binds first to TSP, triggering a change in TSP to reveal a second site, which binds the 93-110 region of CD36 with high affinity. CD36 peptides can be used as stimulators or inhibitors in cellular adhesive events involving TSP-CD36 interaction. Conformational changes leading to the exposure or activation of high affinity binding sites may occur in both the receptor and the ligand upon cell-cell and cell-matrix adhesion.     

SANE,a novel LEM domain protein,regulates bone morphogenetic protein signaling through interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.     

Sequence specific protein binding to and activation of the TGF-beta 3 promoter through a repeated TCCC motif.

We have previously characterized the TGF-beta 3 promoter and shown that the activity of this promoter is highly variable in different cell types. Although the promoter contains a proximal cAMP responsive element, which is critical to basal and forskolin-induced promoter activity, this element is not responsible for the variable, cell-specific regulation of the promoter. In this paper, we identify a 25 base pair sequence in the proximal region of the TGF-beta 3 promoter that binds a novel DNA-binding protein. This region includes the sequence T-CCCTCCCTCCC, (3 x TCCC), and mutation of these T-CCC repeats inhibits protein binding. Further, we show that in the cell line A375, which we have previously shown expresses high levels of TGF-beta 3 mRNA, this region is responsible for mediating high level TGF-beta 3 promoter activity. Immediately 3' to the 3 x TCCC sequence is a consensus AP-2 binding site, however, we show that this region does not bind AP-2, and AP-2 does not transactivate the TGF-beta 3 promoter. Therefore, we provide strong evidence that high level expression of TGF-beta 3 in A375 cells results from transactivation of the TGF-beta 3 promoter by a protein that binds to a repeated TCCC motif in the promoter and suggest that this DNA-binding protein likely also regulates aspects of developmental and tissue-specific expression of this cytokine.     

Transforming growth factor-beta complexes with thrombospondin.

Thrombospondin (TSP) was demonstrated to inhibit the growth of bovine aortic endothelial cells, an activity that was not neutralized by antibodies to TSP or by other agents that block TSP-cell interactions but that partially was reversed by a neutralizing antibody to transforming growth factor-beta (TGF-beta). Similar to TGF-beta, TSP supported the growth of NRK-49F colonies in soft agar in a dose-dependent manner, which required epidermal growth factor and was neutralized by anti-TGF-beta antibody. Chromatography of a TSP preparation did not separate the TGF-beta-like NRK colony-forming activity from high molecular weight protein. However, when chromatography was performed at pH 11, this activity was dissociated from TSP. These results suggest that at least some growth modulating activities of TSP are due to TGF-beta associated with TSP by strong non-covalent forces. Most of the active TGF-beta released from platelets after degranulation was associated with TSP, as demonstrated by anti-TSP immunoaffinity and gel permeation chromatography. 125I-TGF-beta binds to purified TSP in an interaction that is specific in the sense that bound TGF-beta could be displaced by TGF-depleted TSP but not significantly by native TSP, heparin, decorin, alpha 2-macroglobulin, fibronectin, or albumin. Hence, TGF-beta can bind to TSP, and the complex forms under physiological conditions. Furthermore, TSP-associated TGF-beta is biologically active, and the binding of TGF-beta to TSP may protect TGF-beta from extracellular inactivators.     

The peptide Glu-His-Ile-Pro-Ala binds fibrinogen and inhibits platelet aggregation and adhesion to fibrinogen and vitronectin.

Antiplatelet agents are clinically useful as antithrombotic entities. The importance of antiplatelet agents led us to design, synthesize, and characterize a new antiplatelet peptide. This peptide is a presumptive mimic of a ligand binding site on the platelet fibrinogen receptor. Unlike peptides related to Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val that bind to the fibrinogen receptor, this peptide binds to fibrinogen. The anticomplementarity hypothesis was used to design this presumptive peptide mimic of the vitronectin binding site on the fibrinogen receptor, glycoprotein IIb/IIIa complexes. The resulting peptide (Glu-His-Ile-Pro-Ala) has the characteristics of a fibrinogen binding site mimic: It binds fibrinogen and inhibits both the adhesion of platelets to fibrinogen and platelet aggregation. The peptide also inhibits the adhesion of platelets to vitronectin. The antiplatelet activity of this mimic peptide was dependent on its amino acid sequence, since closely related analogues were either inactive or less active inhibitors of platelet function than the original peptide. These results demonstrate that the peptide Glu-His-Ile-Pro-Ala has the characteristics expected of a mimic of a glycoprotein IIb/IIIa ligand binding site.     

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein–DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter–luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.