In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFbeta1-Smad (transforming growth factor-beta1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFbeta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFbeta1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, alpha-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFbeta1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGFbeta1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFbeta1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

The transforming growth factor (TGF)-beta/Smad3 signaling pathway is considered a central mediator of pathological organ fibrosis; however, contribution of Smad2/3-independent TGF-beta signaling has not been fully explored. The present study utilized previously a described model of scleroderma (SSc) fibrosis based on forced expression of the TGF-betaRI (ALK5) (Pannu, J., Gardner, H., Shearstone, J. R., Smith, E., and Trojanowska, M. (2006) Arthritis Rheum. 54, 3011-3021). This study was aimed at determining the molecular mechanisms underlying the profibrotic program in this model. We demonstrate that the TGF-betaRI-dependent up-regulation of collagen and CCN2 (CTGF) does not involve Smad2/3 activation but is mediated by ALK1/Smad1 and ERK1/2 pathways. The following findings support this conclusion: (i) Smad2 and -3 were not phosphorylated in response to TGF-betaRI, (ii) a TGF-betaRI mutant defective in Smad2/3 activation, ALK5(3A), potently stimulated collagen production, (iii) elevation of TGF-betaRI triggered sustained association of ALK5 with ALK1 and high levels of Smad1 phosphorylation, (iv) blockade of Smad1 via small interfering RNA abrogated collagen and CCN2 up-regulation in this model, (v) elevated TGF-betaRI led to a prolonged activation of ERK1/2, (vi) the pharmacologic inhibitor of ERK1/2 inhibited Smad1 phosphorylation and abrogated profibrotic effects of elevated TGFbeta-RI. Additional experiments demonstrated that a GC-rich response element located -6 to -16 (upstream of the transcription start site) in the CCN2 promoter mediated Smad1-dependent increased promoter activity in this model. This element was shown previously to mediate up-regulation of the CCN2 promoter in SSc fibroblasts. In conclusion, this study defines a novel ALK1/Smad1- and ERK1/2-dependent, Smad3-independent mode of TGF-beta signaling that may operate during chronic stages of fibrosis in SSc.

In vivo, CCN2 (connective tissue growth factor) promotes angiogenesis, osteogenesis, tissue repair, and fibrosis, through largely unknown mechanisms. In vitro, CCN2 promotes cell adhesion in a variety of systems via integrins and heparin sulfate proteoglycans (HSPGs). However, the physiological relevance of CCN2-mediated cell adhesion is unknown. Here, we find that HSPGs and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade are required for adult human dermal fibroblasts to adhere to CCN2. Endogenous CCN2 directly binds fibronectin and the fibronectin receptors integrins alpha4 beta1 and alpha5 and syndecan 4. Using Ccn2-/- mouse embryonic fibroblasts, we show that loss of endogenous CCN2 results in impaired spreading on fibronectin, delayed alpha-smooth muscle actin stress fiber formation, and reduced ERK and focal adhesion kinase phosphorylation. These results suggest that a physiological role of CCN2 is to potentiate the ability of fibroblasts to spread on fibronectin, which may be important in modulating fibroblast adhesion to the provisional matrix during tissue development and wound healing. These results are consistent with the notion that a principal function of CCN2 is to modulate receptor/ligand interactions in vivo.

Significance: Fibroblasts play a critical role in normal wound healing. Various extracellular matrix (ECM) components, including collagens, fibrin, fibronectin, proteoglycans, glycosaminoglycans, and matricellular proteins, can be considered potent protagonists of fibroblast survival, migration, and metabolism. Recent Advances: Advances in tissue culture, tissue engineering, and ex vivo models have made the examination and precise measurements of ECM components in wound healing possible. Likewise, the development of specific transgenic animal models has created the opportunity to characterize the role of various ECM molecules in healing wounds. In addition, the recent characterization of new ECM molecules, including matricellular proteins, dermatopontin, and FACIT collagens (Fibril-Associated Collagens with Interrupted Triple helices), further demonstrates our cursory knowledge of the ECM in coordinated wound healing. Critical Issues: The manipulation and augmentation of ECM components in the healing wound is emerging in patient care, as demonstrated by the use of acellular dermal matrices, tissue scaffolds, and wound dressings or topical products bearing ECM proteins such as collagen, hyaluronan (HA), or elastin. Once thought of as neutral structural proteins, these molecules are now known to directly influence many aspects of cellular wound healing. Future Directions: The role that ECM molecules, such as CCN2, osteopontin, and secreted protein, acidic and rich in cysteine, play in signaling homing of fibroblast progenitor cells to sites of injury invites future research as we continue investigating the heterotopic origin of certain populations of fibroblasts in a healing wound. Likewise, research into differently sized fragments of the same polymeric ECM molecule is warranted as we learn that fragments of molecules such as HA and tenascin-C can have opposing effects on dermal fibroblasts.

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients.

OBJECTIVE

Leptin has profibrogenic effects in liver, although the mechanisms of this process are unclear. We sought to elucidate the direct and indirect effects of leptin on hepatic stellate cells (HSCs).

METHODS

HSCs from Sprague-Dawley rats were exposed to leptin and expression of collagen-I, tissue inhibitor of matrix metalloproteinases-1 (TIMP1), transforming growth factor beta1 (TGF-beta1), and connective tissue growth factor (CTGF/CCN2) was assessed. The effects of medium from Kupffer cells (KCs) and sinusoidal endothelial cells (SECs) following leptin were evaluated in HSCs; alpha-smooth muscle actin (alphaSMA) production and KC signaling were analyzed.

RESULTS

HSCs were not activated by incubation with leptin. However, HSCs cultured with medium taken from KCs that were incubated with leptin had increased expression of collagen I, TIMP1, TGF-beta1, and CTGF/CCN2, as well as alphaSMA protein levels and proliferation. These effects were leptin receptor dependent because conditioned medium from KCs isolated from leptin receptor-deficient Zucker (fa/fa) rats did not activate HSCs. In KCs incubated with leptin, messenger RNA and protein expression of TGF-beta1 and CTGF/CCN2 increased. Leptin potentiated signal transducer and activator of transcription 3, AKT, and extracellular signal-related kinase 1/2 phosphorylation in KCs and increased AP-1 and nuclear factor-kappaB DNA binding. Finally, addition of anti-TGF-beta to KC-conditioned medium inhibited HSC expression of collagen I, TIMP1, and CTGF/CCN2, whereas signal transducer and activator of transcription 3 inhibitor attenuated TGF-beta1 production by KC.

CONCLUSIONS

Leptin mediates HSC activation and liver fibrosis through indirect effects on KC; these effects are partly mediated by TGF-beta1.

The CCN family members cysteine-rich 61 (Cyr61/CCN1), connective tissue growth factor (CTGF/CCN2) and nephroblastoma over-expressed (Nov/CCN3) play diverse roles in cells, are known to regulate cell growth, adhesion, matrix production and migration and are involved in endocrine-regulated pathways in various cell types. The role of these molecules in cancer remains controversial. In a cohort of 122 human breast tumours (together with 32 normal breast tissues) we have analysed the expression of all three CCN members at the mRNA and protein levels. Significantly higher levels of Cyr61 (P = 0.02), but low levels of CTGF and Nov, were seen in tumour tissues compared with normal tissues. Significantly raised levels of Cyr61 were associated with poor prognosis (P = 0.02), nodal involvement (P = 0.03) and metastatic disease (P = 0.016). Patients who died of breast cancer also had high levels of Cyr61. In contrast, CTGF in patients with poor prognosis (P = 0.021), metastasis (P = 0.012), local recurrence (P = 0.0024) and mortality (P = 0.0072) had markedly reduced levels. Similar to CTGF, low levels of Nov were also seen in patients with poor prognosis and mortality and with significantly decreased survival (P = 0.033 and P = 0.0146, respectively). This result was fully supported by immunohistochemical analysis of frozen sectioned tissues. While fibroblasts and endothelial cells generally expressed good levels of all three CCN proteins, highly invasive MDA MB 231 cells expressed lower levels of CTGF and Nov, but higher levels of Cyr61, than the less invasive MCF-7. It is concluded that members of the CCN family are differentially expressed and may play important but contrasting roles in the progressive nature of human breast cancer. While Cyr61 appears to act as a factor stimulating aggressiveness, CTGF and Nov may act as tumour suppressors.

Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted s.c. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by positron emission tomography imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed colocalization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer.

Fibrotic diseases are a significant global burden for which there are limited treatment options. The effector cells of fibrosis are activated fibroblasts called myofibroblasts, a highly contractile cell type characterized by the appearance of α-smooth muscle actin stress fibers. The underlying mechanism behind myofibroblast differentiation and persistence has been under much investigation and is known to involve a complex signaling network involving transforming growth factor-β, endothelin-1, angiotensin II, CCN2 (connective tissue growth factor), and platelet-derived growth factor. This review addresses the contribution of these signaling molecules to cardiac fibrosis.

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up-regulation in fibrotic or steatotic livers, or in culture-activated or ethanol-treated primary mouse HSC, is associated with a reciprocal down-regulation of microRNA-214 (miR-214). By using protector or reporter assays to investigate the 3'-untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis-inducing stimuli was due to reduced expression of miR-214, which otherwise inhibited CCN2 expression by directly binding to the CCN2 3'-UTR. Additionally, miR-214 was present in HSC exosomes, which were bi-membrane vesicles, 50-150 nm in diameter, negatively charged (-26 mV), and positive for CD9. MiR-214 levels in exosomes but not in cell lysates were reduced by pretreatment of the cells with the exosome inhibitor, GW4869. Coculture of either quiescent HSC or miR-214-transfected activated HSC with CCN2 3'-UTR luciferase reporter-transfected recipient HSC resulted in miR-214- and exosome-dependent regulation of a wild-type CCN2 3'-UTR reporter but not of a mutant CCN2 3'-UTR reporter lacking the miR-214 binding site. Exosomes from HSC were a conduit for uptake of miR-214 by primary mouse hepatocytes. Down-regulation of CCN2 expression by miR-214 also occurred in human LX-2 HSC, consistent with a conserved miR-214 binding site in the human CCN2 3'-UTR. MiR-214 in LX-2 cells was shuttled by way of exosomes to recipient LX-2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3'-UTR activity or expression of CCN2 downstream targets, including alpha smooth muscle actin or collagen. Experimental fibrosis in mice was associated with reduced circulating miR-214 levels.

CONCLUSIONS

Exosomal transfer of miR-214 is a paradigm for the regulation of CCN2-dependent fibrogenesis and identifies fibrotic pathways as targets of intercellular regulation by exosomal miRs.

Connective tissue growth factor (CTGF, CCN2) is a key mediator of tissue fibrosis. CCN2 plays an important role in the development of glomerular and tubulointerstitial fibrosis in progressive kidney diseases. In this review, we discuss the biology of CCN2 with a focus on the regulation of CCN2 gene, cellular mechanisms of profibrotic CCN2 effects and the current in vivo and in vitro evidence for the role of CCN2 in the development of renal fibrosis. We also discuss the therapeutic potential of targeting CCN2 for the treatment of renal fibrosis.

Connective tissue growth factor (CTGF, CCN2) is a secreted matricellular protein, the functions of which depend on the interactions with other molecules in the microcellular environment. As an example of context-dependent activity of CTGF, this review will outline different aspects of CTGF function in relation to angiogenesis. CTGF is barely expressed in normal adult tissue, but is strongly upregulated in fibrotic tissue and is also increased during development, in wound healing, or in certain types of cancer. Accordingly, gene expression of CTGF is tightly regulated. To highlight the complexity of the regulation of CTGF gene expression, we discuss here the mechanisms involved in CTGF regulation by TGFbeta in different cell types, and the mechanisms related to CTGF gene expression in cells exposed to mechanical forces. Finally, we will touch upon novel aspects of epigenetic regulation of CTGF gene expression. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

The role of CTGF/CCN2 in osteolytic metastasis by breast cancer cells and its mechanism of action were studied. Osteolytic metastasis accompanied by CCN2 and PTHrP overproduction was efficiently inhibited by an anti-CCN2 antibody. Furthermore, we found that CCN2 was induced by PTHrP through PKA-, PKC-, and ERK-mediated pathways therein.

BACKGROUND

Connective tissue growth factor (CTGF/CCN2) is a mediator of local angiogenesis induced by breast cancer, but its role in osteolytic metastasis has not been evaluated. PTH-related peptide (PTHrP) is another critical factor in the development of the osteolytic metastasis. Using both in vivo and in vitro approaches, we studied whether/how neutralization of CCN2 prevented bone metastasis and how PTHrP signaling is related.

METHODS

A mouse model of bone metastasis by human breast cancer cell line MDA231 was treated with a CCN2-neutralizing antibody, and osteolytic bone metastases were assessed on radiographs and immunohistochemistry. Ccn2 gene expression and transcription were examined by Northern blot and luciferase analysis. Immunoblot analysis and kinase inhibitors were used to identify the signaling pathways implicated. Anti-angiogenic/osteoclastogenic effects of ccn2 downregulation were also evaluated.

RESULTS

Treatment of mice with a CCN2-neutralizing antibody greatly decreased osteolytic bone metastasis, microvasculature, and osteoclasts involved. The antibody also suppressed the growth of subcutaneous tumor in vivo and proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. Downregulation of ccn2 also repressed osteoclastogenesis. CCN2 expression was specifically observed in cancer cells producing PTHrP and type I PTH/PTHrP receptor (PTH1R) invaded the bone marrow, and PTHrP strongly upregulated ccn2 in MDA231 cells in vitro. Activation of protein kinase C (PKC) and protein kinase A (PKA) was necessary and sufficient for the stimulation of ccn2 by PTHrP. Indeed, inhibition of the extracellular signal-regulated kinase (ERK1/2), PKC, or PKA by specific inhibitors counteracted the stimulation of ccn2 expression. Incubation of MDA231 cells with PTHrP induced the activation of ERK1/2. Consistent with these findings, inhibition of PKC prevented PTHrP-induced ERK1/2 activation, whereas 12-O-tetradecanoylphorbol13-acetate (TPA), a stimulator of PKC, upregulated it.

CONCLUSIONS

CCN2 was critically involved in osteolytic metastasis and was induced by PKA- and PKC-dependent activation of ERK1/2 signaling by PTHrP. Thus, CCN2 may be a new molecular target for anti-osteolytic therapy to shut off the PTHrP-CCN2 signaling pathway.

Connective tissue growth factor (CTGF or CCN2), a member of the CCN family, is involved in diverse biological processes such as cell adhesion, proliferation, and angiogenesis. In this study, we show that overexpression of CTGF occurred in a significant proportion of esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and metastatic. Forced expression of CTGF in Eca109 ESCC cells accelerated their growth in culture and significantly increased tumor formation in nude mice, whereas RNA interference-mediated knockdown of CTGF in ESCC cells significantly inhibited cell growth and colony formation, as well as tumorigenicity in vivo. Moreover, overexpression of CTGF in ESCC cells resulted in the accumulation and nuclear translocation of beta-catenin, leading to activation of beta-catenin-T-cell factor (TCF)/Lef signaling. Up-regulation of c-Myc and cyclin D1, two target genes of beta-catenin-TCF/Lef signaling, was also observed in the CTGF-overexpressing cells. These effects of CTGF in ESCC cells were abolished by transfection with either dominant negative beta-catenin or dominant negative TCF4. Furthermore, we identified a beta-catenin-TCF/Lef-binding site (TBE) in the promoter region of CTGF and found that CTGF is a transcriptional target of beta-catenin-TCF/Lef signaling. Taken together, these results revealed that the interaction of CTGF and beta-catenin-TCF/Lef forms a positive feedback loop, which could contribute to the tumorigenicity of ESCC.

Connective tissue growth factor (CTGF/CCN2) is a highly profibrogenic molecule which is overexpressed in many fibrotic lesions, including those of the liver. CTGF/CCN2 is transcriptionally activated by transforming growth factor-beta (TGF-beta) and appears to mediate some of the extracellular matrix (ECM)-inducing properties that have been previously attributed to TGF-beta. CTGF/CCN2 and TGF-beta stimulate connective tissue cell proliferation and ECM synthesis in vitro and exhibit shared fibrogenic and angiogenic properties in vivo. In fibrotic liver, CTGF/CCN2 mRNA and protein are produced by fibroblasts, myofibroblasts, hepatic stellate cells (HSCs), endothelial cells, and bile duct epithelial cells. CTGF/CCN2 is also produced at high levels in hepatocytes during cytochrome P-4502E1-mediated ethanol oxidation. CTGF/CCN2 expression in cultured HSCs is enhanced following their activation or stimulation by TGF-beta while exogenous CTGF/CCN2 is able to promote HSC adhesion, proliferation, locomotion, and collagen production. Collectively, these data suggest that during initiating or downstream fibrogenic events in the liver, production of CTGF/CCN2 is regulated primarily by TGF-beta in one or more cell types and that CTGF/CCN2 plays important roles in HSC activation and progression of fibrosis. This article reviews the data that support the importance of CTGF/CCN2 in hepatic fibrosis and highlights the concept that CTGF/CCN2 may represent a new therapeutic target in this disease.

The endothelins are a family of endothelium-derived peptides that possess a variety of biological activities, including potent vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and pulmonary fibrosis. Here, we use genome-wide expression array analysis to show that the addition of ET-1 (100 nm, 4 h) to normal lung fibroblasts directly induces expression of matrix and matrix-associated genes, including the profibrotic protein CCN2 (connective tissue growth factor, or CTGF). ET-1 induces the MEK/ERK MAP kinase pathway in fibroblasts. Blockade of the MEK/ERK kinase pathway with U0126 abrogates the ability of ET-1 to induce expression of matrix and matrix-associated mRNAs and the CCN2 protein. The CCN2 promoter possesses an ET-1 response element, which maps to the previously identified basal control element-1 (BCE-1) site. Our results suggest that ET-1 induces a program of matrix synthesis in lung fibroblasts and that ET-1 may play a key role in connective tissue deposition during wound repair and in pulmonary fibrosis.

OBJECTIVE

Fibrosis is excessive scarring caused by the accumulation and contraction of extracellular matrix proteins and is a common end pathway in many chronic diseases, including scleroderma (systemic sclerosis [SSc]). Indeed, pulmonary fibrosis is a major cause of death in SSc. Transforming growth factor beta (TGFbeta) induces endothelin 1 (ET-1) in human lung fibroblasts by a Smad-independent, JNK-dependent mechanism. The goal of this study was to assess whether ET-1 is a downstream mediator of the profibrotic effects of TGFbeta in lung fibroblasts.

METHODS

We used a specific endothelin receptor antagonist to determine whether ET-1 is a downstream mediator of TGFbeta responses in lung fibroblasts, using microarray technology, real-time polymerase chain reaction, and Western blot analyses.

RESULTS

The ability of TGFbeta to induce the expression of a cohort of profibrotic genes, including type I collagen, fibronectin, and CCN2, and to contract a collagen gel matrix, depends on ET-1.

CONCLUSIONS

ET-1 contributes to the ability of TGFbeta to promote a profibrotic phenotype in human lung fibroblasts, consistent with the notion that endothelin receptor antagonism may be beneficial in controlling fibrogenic responses in lung fibroblasts.

Connective tissue growth factor (CTGF) is implicated as a factor promoting tissue fibrosis in several disorders, including diabetic nephropathy. However, the molecular mechanism(s) by which it functions is not known. CTGF rapidly activates several intracellular signaling molecules in human mesangial cells (HMC), including extracellular signal-related kinase 1/2, Jun NH(2)-terminal kinase, protein kinase B, CaMK II, protein kinase Calpha, and protein kinase Cdelta, suggesting that it functions via a signaling receptor. Treating HMC with CTGF stimulated tyrosine phosphorylation of proteins 75 to 80 and 140 to 180 kD within 10 min, and Western blot analysis of anti-phosphotyrosine immunoprecipitates identified the neurotrophin receptor TrkA (molecular weight approximately 140 kD). Cross-linking rCTGF to cell surface proteins with 3,3'-dithiobis(sulfosuccinimidylpropionate) revealed that complexes formed with TrkA and with the general neurotrophin co-receptor p75(NTR). rCTGF stimulated phosphorylation of TrkA (tyr 490, 674/675). K252a, a known selective inhibitor of Trk, blocked this phosphorylation, CTGF-induced activation of signaling proteins, and CTGF-dependent induction of the transcription factor TGF-beta-inducible early gene in HMC. It is concluded that TrkA serves as a tyrosine kinase receptor for CTGF.

The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including alpha 6 beta 1. Previous studies have shown that CCN1 interaction with integrin alpha 6 beta 1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin alpha 6 beta 1. In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-alpha 6 and anti-beta 1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating alpha 6 beta 1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other alpha 6 beta 1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits alpha 6 beta 1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin alpha 6 beta 1 directly, we perform affinity chromatography and show that integrin alpha 6 beta 1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin alpha 6 beta 1, providing the basis for the development of peptide mimetics to examine the functional role of alpha 6 beta 1 in angiogenesis.

CCN2 (connective tissue growth factor), an important regulator of angiogenesis, chondrogenesis, and wound healing, is overexpressed in a majority of fibrotic diseases and in various tumors. This study investigated regulation of CCN2 gene expression by Ets family of transcription factors, focusing on two members, Fli1 and Ets1, with deregulated expression during fibrosis and tumorigenesis. We show that Ets1 and Fli1 have opposite effects on CCN2 gene expression. Ets1 functions as an activator of CCN2 transcription, whereas Fli1 acts as a repressor. A functional Ets binding site was mapped at -114 within the CCN2 promoter. This site not only mediates stimulation by Ets factors, including Ets1, Ets2, and GABPalpha/beta, but is also required for the transforming growth factor (TGF)-beta response. The contrasting functions of Ets1 and Fli1 in regulation of the CCN2 gene were confirmed by suppressing their endogenous levels using adenoviral vectors expressing specific small interfering RNAs. Additional experiments using chromatin immunoprecipitation assays have revealed that in fibroblasts both Ets1 and Fli1 occupy the CCN2 promoter. TGF-beta stimulation resulted in displacement of Fli1 from the CCN2 promoter and a transient inhibition of Fli1 synthesis. Moreover, reduction of Fli1 expression resulted in up-regulation of COL1A1 and COL1A2 genes and down-regulation of the MMP1 gene. Thus, inhibition of Fli1 recapitulated some of the key effects of TGF-beta, suggesting that Fli1 suppression is involved in activation of the profibrotic gene program in fibroblasts. On the other hand, activation of the CCN2 gene downstream of Ets1 is consistent with its role in angiogenesis and extracellular matrix remodeling. This study strongly supports a critical role of Fli1 and Ets1 in the pathological extracellular matrix regulation during fibrosis and cancer.

OBJECTIVE

To identify the components of conditioned medium obtained from intervertebral disc nucleus pulposus-derived canine notochord cells, and to evaluate the capacity of such factors to affect disc-derived chondrocyte gene expression of aggrecan, versican, and hyaluronic acid synthase 2 (HAS-2) as a function of culture conditions.

METHODS

Canine notochord cells obtained from nonchondrodystrophic dogs were cultured within alginate beads under conditions of serum deficiency (Dulbecco's modified Eagle's medium [DMEM]) to produce notochord cell-conditioned medium (NCCM). NCCM was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectroscopy. Bovine disc-derived chondrocytes were cultured with serum-deficient medium (DMEM) and NCCM and assayed for the effect of tissue culture conditions on aggrecan, versican, and HAS-2 gene expression. Next, chondrocyte gene expression for aggrecan was evaluated using DMEM containing recombinant connective tissue growth factor (rCTGF), and the results compared with those obtained using NCCM and DMEM.

RESULTS

NCCM contained aggrecan, Cu/Zn superoxide dismutase, fibronectin, and CTGF precursor. Culture with NCCM caused an up-regulation of aggrecan, versican, and HAS-2 gene expression. NCCM induced aggrecan gene expression in chondrocytes at a level similar to that induced by 100-200 ng/ml rCTGF. Nonchondrodystrophic and chondrodystrophic canine notochord cells exhibited similar levels of CTGF gene expression.

CONCLUSIONS

Nucleus pulposus-derived notochord cells secrete CTGF (CCN2), a recently discovered multifunctional growth factor. There is no difference between CTGF gene expression in nonchondrodystrophic and chondrodystrophic canine notochord cells, suggesting a possible role of CTGF as an anabolic factor within the disc nucleus that is, to at least some degree, dependent on the population of notochord cells within the disc nucleus.

Matrix metalloproteinase 3 (MMP3) is well known as a secretory endopeptidase that degrades extracellular matrices. Recent reports indicated the presence of MMPs in the nucleus (A. J. Kwon et al., FASEB J. 18:690-692, 2004); however, its function has not been well investigated. Here, we report a novel function of human nuclear MMP3 as a trans regulator of connective tissue growth factor (CCN2/CTGF). Initially, we cloned MMP3 cDNA as a DNA-binding factor for the CCN2/CTGF gene. An interaction between MMP3 and transcription enhancer dominant in chondrocytes (TRENDIC) in the CCN2/CTGF promoter was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by overexpressed MMP3, whereas a TRENDIC mutant promoter lost the response. Also, the knocking down of MMP3 suppressed CCN2/CTGF expression. By cytochemical and histochemical analyses, MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 and six putative nuclear localization signals in MMP3 also were shown. Furthermore, we determined that heterochromatin protein gamma coordinately regulates CCN2/CTGF by interacting with MMP3. The involvement of this novel role of MMP3 in the development, tissue remodeling, and pathology of arthritic diseases through CCN2/CTGF regulation thus is suggested.

CCN1 (CYR61) is a matricellular inducer of angiogenesis essential for successful vascular development. Though devoid of the canonical RGD sequence motif recognized by some integrins, CCN1 binds to, and functions through integrin alphavbeta3 to promote pro-angiogenic activities in activated endothelial cells. In this study we identify a 20-residue sequence, V2 (NCKHQCTCIDGAVGCIPLCP), in domain II of CCN1 as a novel binding site for integrin alphavbeta3. Immobilized synthetic V2 peptide supports alphavbeta3-mediated cell adhesion; soluble V2 peptide inhibits endothelial cell adhesion to CCN1 and the homologous family members CCN2 (connective tissue growth factor, CTGF) or CCN3 (NOV) but not to collagen. These activities are obliterated by mutation of the aspartate residue in the V2 peptide to alanine. The corresponding D125A mutation in the context of the N-terminal half of CCN1 (domains I and II) greatly diminished direct solid phase binding to purified integrin alphavbeta3 and abolished alphavbeta3-mediated cell adhesion activity. Likewise, soluble full-length CCN1 with the D125A mutation is defective in binding purified alphavbeta3 and impaired in alphavbeta3-mediated pro-angiogenic activities in vascular endothelial cells, including stimulation of cell migration and enhancement of DNA synthesis. In contrast, immobilized full-length CCN1-D125A mutant binds alphavbeta3 and supports alphavbeta3-mediated cell adhesion similar to wild type CCN1. These results indicate that V2 is the primary alphavbeta3 binding site in soluble CCN1, whereas additional cryptic alphavbeta3 binding site(s) in the C-terminal half of CCN1 becomes exposed when the protein is immobilized. Together, these results identify a novel and functionally important binding site for integrin alphavbeta3 and provide a new approach for dissecting alphavbeta3-specific CCN1 functions both in cultured cells and in the organism.