Connective Tissue Growth Factor Promoter Research Articles

Correlation of CTGF gene promoter methylation with CTGF expression in type 2 diabetes mellitus with or without nephropathy

Increasing evidence shows that DNA methylation is involved in the development and progression of diabetes mellitus (DM) and its complications. Previous studies conducted by our group have indicated that high glucose levels may induce the demethylation process of the connective tissue growth factor (CTGF) gene promoter and increase the expression of CTGF in human glomerular mesangial cells. Based on these findings, the aim of the present study was to investigate the methylation level of genomic DNA and the CTGF promoter in patients with type 2 DM and to analyze its possible correlation with CTGF expression. Methylation levels of the whole genomic DNA were detected by high-performance liquid chromatography in a non-diabetes control (NDM) group (n=29), a diabetes without nephropathy (NDN) group (n=37) and a diabetes with nephropathy (DN) group (n=38). CTGF promoter methylation levels were detected by methylation-specific polymerase chain reaction and bisulfite sequencing. The levels of serum CTGF were assessed using the enzyme-linked immunosorbent assay. The methylation levels of the whole genomic DNA were not significantly different among the three groups. However, the CTGF methylation levels in the two diabetes groups were significantly lower than those in the NDM group (P<0.05), with the lowest methylation level in the DN group (P<0.05). The CTGF protein levels in the DN group were significantly higher than those in the NDM and NDN groups (P<0.05). Levels of CTGF were negatively correlated with the estimated glomerular filtration rate (eGFR) and the methylation level of the promoter, while they were positively correlated with age, urinary albumin-to-creatinine ratio (UACR), blood urea nitrogen, creatinine, fasting blood sugar and postprandial blood glucose. Multiple stepwise regression analysis showed that CTGF expression was associated with the UACR, CTGF methylation level and eGFR. DNA methylation is a regulatory mechanism of CTGF expression, which is decreased in patients with DM, particularly in those with DN, and may contribute to the pathogenesis of nephropathy.

Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor

Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-β, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-β-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.

Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by ETAR-dependent JNK/AP-1 pathway

Endothelin-1 (ET-1) acts as a key mediator of vasoconstriction and tissue repair. Overproduction of connective tissue growth factor (CTGF) underlies the development of lung fibrosis. ET-1 induces expression of matrix-associated genes in lung fibroblasts, however, little is known about the signaling pathway of CTGF expression caused by ET-1. In this study, we found that ET-1 caused concentration- and time-dependently increases in CTGF expression in human embryonic lung fibroblast cell line (WI-38). ET-1-induced CTGF expression was inhibited by BQ123 (ETAR antagonist), but not BQ788 (ETBR antagonist). Moreover, ET-1-induced CTGF expression was significantly reduced by JNK inhibitor (SP600125), the dominant-negative mutants of JNK1/2 (JNK1/2 DN), and AP-1 inhibitor (curcumin). ET-1 induced phosphorylations of JNK and c-Jun in time-dependent manners. AP-1 luciferase activity was concentration-dependently increased by ET-1, and this effect was attenuated by SP600125. We also found that ET-1-induced CTGF expression was most controlled by the AP-1 binding region of CTGF promoter. ET-1-indiced CTGF luciferase activity was predominately controlled by the sequence −747 to −408bp upstream of the transcription start site on the human CTGF promoter. Furthermore, ET-1 caused the formation of AP-1-specific DNA-protein complex and the recruitment of c-Jun to the CTGF promoter. Moreover, we found that ET-1 induced α-smooth muscle actin (α-SMA) expression, which was inhibited by BQ123, SP600125, curcumin, and anti-CTGF antibody. These results suggest that ET-1 stimulates expressions of CTGF and α-SMA through ETAR/JNK/AP-1 signaling pathway, and CTGF is required for ET-1-induced α-SMA expression in human lung fibroblasts.

Aldosterone-Specific Activation of Cardiomyocyte Mineralocorticoid Receptor In Vivo

Inappropriate mineralocorticoid receptor (MR) activation is involved in cardiac diseases. Whether and how aldosterone is involved in the deleterious effects of cardiac mineralocorticoid activation is still unclear. Mice overexpressing MR in cardiomyocytes and their controls were treated for 7 days with aldosterone, and cardiac transcriptome was analyzed. Aldosterone regulated 265 genes in cardiomyocyte-targeted MR overexpression mice. Forty three of these genes were also differentially expressed between untreated cardiomyocyte-targeted MR overexpression and controls mice, thus representing putative aldosterone-regulated genes in cardiomyocytes. Among these genes, we focused on connective tissue growth factor (CTGF). In vivo, in cardiomyocyte-targeted MR overexpression mice, aldosterone (but not corticosterone) induced CTGF expression (mRNA and protein) in cardiomyocytes. Ex vivo, aldosterone induced the binding of mineralocorticoid receptor to CTGF promoter and increased the expression of its transcript. Aldosterone induction of CTGF synthesis in cardiomyocytes seems pathologically relevant as the increase in CTGF observed in a model of heart failure (transverse aortic constriction) in rats was prevented by eplerenone, a mineralocorticoid receptor blocker. This study demonstrates that aldosterone specifically regulates gene expression in cardiomyocytes despite large prevalence of glucocorticoids in plasma.

Abstract 3082: TGF-β synergistically stimulates malignant mesothelioma growth with defects in the Hippo pathway by inducing CTGF expression

Abstract Malignant mesothelioma (MM) arises from the serosal cells of the pleural, peritoneal and pericardial cavities with poor prognosis because of its frequent diagnosis in advanced stages. The primary cause of this disease has often been linked to asbestos exposure, and the number of patients worldwide is expected to peak in the next two decades. There is no known curative therapy for MM and further understanding in molecular mechanism is needed to identify new candidates for targeted treatment. We found that the TGF-β pathway is associated with oncogenic mutations and loss of the tumor suppressor NF2, and induces pro-oncogenic biological activities. Loss of NF2 is one of the genetic lesions which have been related to the growth of human MM. The NF2 gene is responsible for neurofibromatosis type II syndrome and encodes Merlin which negatively regulates YAP function. Nearly 75% MM cases have inactivating mutations in the NF2 gene or in downstream signaling molecules of Hippo signaling cascade. We examined a functional interaction between the Hippo and TGF-β pathways and found that endothelin-1 and connective tissue growth factor (CTGF) are the only proteins affected by these two pathways in MM cells. The CTGF promoter contains both a YAP/TEAD-binding site and a consensus Smad-binding site adjacent to each other. The expression of CTGF induced by TGF-β in MM cell lines was greatly affected by the existence of YAP, by its binding with Smad3, forming a complex on CTGF promoter lesion. Knocking down CTGF expression in MM cells prolonged the survival of mice, and a significant association was seen between CTGF expression and extracellular matrix deposition in MM xenografts and in patient tissue specimens. This study elucidates a novel molecular mechanism induced by the TGF-β pathway, which may be applicable to malignancies with defective NF2 and of mesenchymal origin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3082. doi:1538-7445.AM2012-3082

MZF‐1 regulates the CTGF gene in the hematopoietic compartment

Connective Tissue Growth Factor (CTGF), an extracellular protein with key roles in wound healing and tissue homeostasis, has recently been shown to be in abundance in thrombocytes. Paradoxically, megakaryocytes, the platelet precursors in the bone marrow, contain no detectable levels of CTGF, and the source of CTGF in platelets is yet to be determined. It has been proposed that cells in the vicinity of the megakaryocyte secrete CTGF upon cellular signaling, at which point it is then internalized by fragmenting platelets entering the vasculature.In this study, we report the discovery of a possible functional binding site for Myeloid Zinc Finger‐1 (MZF‐1), a transcription factor localized in the bone marrow, in the promoter region of the human CTGF gene, using bioinformatics. We further show that, in HeLa, HEK‐293, and select hematopoietic cells, binding of MZF‐1 to the CTGF promoter can be detected with Chromatin Immunoprecipitation (ChIP). Furthermore, we investigated the induction of CTGF by calcitriol (1,25‐ dihydroxyvitamine D3) via MZF‐1, through the PI‐3 kinase pathway in HS‐5 stromal fibroblasts, a possible candidate for the supply of CTGF to platelets entering the bloodstream. Finally, expression of CTGF is attenuated following ablation of MZF1 by miRNA. Taken together, our results argue that MZF‐1 is a key regulator of CTGF in the hematopoietic compartment, and may be a factor in the process of CTGF acquisition by thrombocytes.

TGF-β synergizes with defects in the Hippo pathway to stimulate human malignant mesothelioma growth

Malignant mesothelioma (MM) is an incurable malignancy that is caused by exposure to asbestos and is accompanied by severe fibrosis. Because MM is usually diagnosed at an advanced stage and clinical identification of early lesions is difficult, its molecular pathogenesis has not been completely elucidated. Nearly 75% of MM cases have inactivating mutations in the NF2 (neurofibromatosis type 2; Merlin) gene or in downstream signaling molecules of the Hippo signaling cascade, which negatively regulates the transcription factor Yes-associated protein (YAP). In this study, we demonstrate a functional interaction between the Hippo and TGF-β pathways in regulating connective tissue growth factor (CTGF). Expression of CTGF in MM cells was induced by the formation of a YAP-TEAD4-Smad3-p300 complex on the CTGF promoter. Knocking down CTGF expression in MM cells prolonged the survival of xenografted mice, and a significant association was seen between CTGF expression and extracellular matrix deposition in MM xenografts and in patient tissue specimens. We further suggest that CTGF may influence the malignancy of mesothelioma because of the different histological expression patterns observed in human MM tissues. These data suggest that CTGF is an important modulator of MM growth and pathology and represents a novel therapeutic target for this disease.

Aldosterone stimulates nuclear factor-kappa B activity and transcription of intercellular adhesion molecule-1 and connective tissue growth factor in rat mesangial cells via serum- and glucocorticoid-inducible protein kinase-1

Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. To determine the signaling pathway of aldosterone in relation to fibrosis and inflammation in mesangial cells, we investigated the effects of aldosterone on expression and activation of serum- and glucocorticoid-inducible protein kinase-1 (SGK1), the activation of nuclear factor-kappa B (NF-κB activation, and the expressions of intercellular adhesion molecule-1 (ICAM-1) and connective tissue growth factor (CTGF). Aldosterone stimulated SGK1 expression, phosphorylation (Ser-256), and kinase activity. The increments of phosphorylation and expression of SGK1 induced by aldosterone were inhibited by mineralocorticoid receptor (MR) inhibitor (eplerenone). Aldosterone stimulated NF-κB activity measured by NF-κB responsive elements, luciferase assay, and the levels of inhibitor of kappa B (IκB) phosphorylation. This aldosterone-induced activation of NF-κB was inhibited by the transfection of dominant-negative SGK1. Furthermore, aldosterone augmented the promoter activities and protein expressions of ICAM-1 and CTGF. The effects of aldosterone on ICAM-1 and CTGF promoter activities and protein expressions were inhibited by the transfection of dominant-negative SGK1 and dominant-negative IκBα. We also found that the MR antagonist significantly ameliorated the glomerular injury and enhancements in SGK1, ICAM-1, and CTGF expressions induced by 1% sodium chloride and aldosterone in vivo. In conclusion, our findings suggest that aldosterone stimulates ICAM-1 and CTGF transcription via activation of SGK1 and NF-κB, which may be involved in the progression of aldosterone-induced mesangial fibrosis and inflammation. MR antagonists may serve as useful therapeutic targets for the treatment of glomerular inflammatory disease.

Oral application of 1,7‐dimethylxanthine (paraxanthine) attenuates the formation of experimental cholestatic liver fibrosis

Several epidemiological studies suggest a beneficial effect of coffee consumption on the formation and progression of fibrogenic diseases, particularly of the liver. Recent data now point to a modulation of transforming growth factor-β (TGF-β) signaling by paraxanthine (1,7-dimethylxanthine [1,7-DMX]), the demethylated primary metabolite of caffeine Twenty adult Sprague-Dawley rats were bile duct ligated (BDL) or sham operated with or without concomitant oral 1,7-DMX (1 mM) application. Serum was investigated by standard biochemical analysis, in-house connective tissue growth factor (CTGF), enzyme linked immunosorbent assay (ELISA) or liquid chromatography-mass spectrometry analysis. Liver tissue was stained using hematoxylin-eosin (HE) and Sirius-red staining. Whole liver lysates, primary rat hepatocytes (PC) and hepatic stellate cells (HSC) were investigated by CTGF, and total Smad2/3 Western blot analysis, CTGF reporter gene assay or an in-house malondialdehyde ELISA. The in vitro 50% inhibitory dose (ID50) of 1,7-DMX was 0.95 mM by for CTGF promoter activity and protein expression in PC and 1.25 mM for protein expression in HSC. Oral 1,7-DMX application (1 mM) attenuated cholestatic hepatocellular injury in vivo as determined by biochemical serum analysis and reduced intercellular collagen deposition in the cholestatic rat liver (HE- and Sirius-red staining). Western Blot analysis of whole liver lysates revealed a reduction of intrahepatic concentrations of Smad2/3 and CTGF following oral 1,7-DMX intake. However, serum CTGF concentrations were not reduced in 1,7-DMX treated BDL rats. Oral 1,7-DMX furthermore led to a reduction of intrahepatic lipid peroxidation (malondialdehyde concentrations) as markers of oxidative stress in BDL rats. Our pilot study warrants further studies of 1,7-DMX as a potential new drug to fight fibrotic processes, not just of the liver.

FosB Regulates Stretch-Induced Expression of Extracellular Matrix Proteins in Smooth Muscle

Fibroproliferative remodeling in smooth muscle-rich hollow organs is associated with aberrant extracellular matrix (ECM) production. Although mechanical stimuli regulate ECM protein expression, the transcriptional mediators of this process remain poorly defined. Previously, we implicated AP-1 as a mediator of smooth muscle cell (SMC) mechanotransduction; however, its role in stretch-induced ECM regulation has not been explored. Herein, we identify a novel role for the AP-1 subunit FosB in stretch-induced ECM expression in SMCs. The DNA-binding activity of AP-1 increased after stretch stimulation of SMCs in vitro. In contrast to c-Jun and c-fos, which are also activated by the SMC mitogen platelet-derived growth factor, FosB was only activated by stretch. FosB silencing attenuated the expression of the profibrotic factors tenascin C (TNC) and connective tissue growth factor (CTGF), whereas forced expression of Jun~FosB stimulated TNC and CTGF promoter activity. Chromatin immunoprecipitation revealed enrichment of AP-1 at the TNC and CTGF promoters. Bladder distension in vivo enhanced nuclear localization of c-jun and FosB. Finally, the distension-induced expression of TNC and CTGF in the detrusor smooth muscle of bladders from wild-type mice was significantly attenuated in FosB-null mice. Together, these findings identify FosB as a mechanosensitive regulator of ECM production in smooth muscle.

Runx2/Smad3 Complex Negatively Regulates TGF-β-Induced Connective Tissue Growth Factor gene Expression in Vascular Smooth Muscle Cells

Connective tissue growth factor (CTGF), a direct target gene of transforming growth factor-β (TGF-β) signaling, plays an important role in the development of atherosclerosis. We previously showed that Runx2, a key transcription factor in osteoblast differentiation, regulates osteogenic conversion and dedifferentiation of vascular smooth muscle cells (VSMCs). In this study, we investigated the hypothesis that Runx2 modulates CTGF gene expression via the regulation of TGF-β signaling. Expression of the Runx2 gene was decreased, and CTGF mRNA levels were reciprocally increased by TGF-β in a time-dependent manner in cultured human aortic smooth muscle cells (HASMCs) and C3H10T1/2 cells. Forced expression of Runx2 decreased and the reduction of Runx2 expression by small interfering RNA enhanced both basal and TGF-β-stimulated CTGF gene expression in HASMCs. Site-directed mutation analysis of the CTGF promoter indicated that transcriptional repression by Runx2 was mediated by the Smad-binding element (SBE) under basal and TGF-β-stimulated conditions. Data obtained from immunoblots of Runx2-, Smad3- or Smad4-transfected cells and chromatin immunoprecipitation analysis indicated that Runx2 interacts with Smad3 at the SBE. Immunohistochemistry revealed that the expression of Runx2 and CTGF was distinct and almost mutually exclusive in human atherosclerotic plaque. These results for the first time demonstrate that Runx2/Smad3 complex negatively regulates endogenous and TGF-β-induced CTGF gene expression in VSMCs. Thus, the induction of Runx2 expression contributes to the phenotypic modulation of VSMCs, in which the TGF-β/Smad pathway plays a major role.

Abstract 3474: Connective tissue growth factor promotes tumor angiogenesis by modulating multiple angiogenic factors via binding to cystine knot motifs

Abstract Introduction Connective tissue growth factor (CTGF), a member of the CCN (Cyr61/CTGF/Nov) protein family, may function as both a positive and negative regulator of angiogenesis. However, the mechanism(s) underlying the regulatory behavior of CTGF is poorly understood. Here, we have undertaken a multipronged approach to provide insight into how CTGF enhances tumor angiogenesis. Methods Lewis lung carcinomas (LLC) cells were subcutaneously injected in syngeneic transgenic mice expressing green fluorescent protein (GFP) under the control of CTGF promoter (CTGFp-GFP). Recombinant lentivirus was generated to overexpress FLAG tagged CTGF while recombinant adenovirus expressing CTGF shRNA was used to knock down CTGF expression in LLCs. The levels of CTGF mRNA were examined by RT-PCR analysis. Tumor sizes were measured within two weeks and intratumoral microvessel density was quantified by immunochemical detection of Meca-32+ blood vessels. In addition, CTGF binding proteins were identified using the yeast two-hybrid approach. Immunoprecipitation assay was performed to verify these interactions. In vitro angiogenesis activity was indicated by tubule length formation by human umbilical vein endothelial cells (HUVECs). The migratory activity was measured in Boyden chamber assay. Activation of downstream signal effectors including cell division cycle 42 GTP binding protein (Cdc42) and extracellular signal-regulated kinase (ERK)1/2 was detected in Western blotting analysis. Results CTGF was expressed in Meca-32+ endothelial cells of intratumoral vessels growing in CTGFp-GFP transgenic mice. CTGF expression was also highly induced in hypoxic cultured LLC cells. Ectopic expression of CTGF promoted LLC tumor growth (n=3) and the microvessel density. Knocking down CTGF expression in LLC tumors (n=3) inhibited the growth of LLC tumors and decreased the microvessel density compared to those with scramble shRNA. Several proangiogenic factors were identified and shown to bind CTGF via cystine knot motifs. The factors include vascular endothelial growth factor (VEGF)-A, von Willebrand factor (vWF), platelet derived growth factor (PDGF)-B and Slit3. The combination of CTGF and Slit3 together resulted in an increased tubule length and an enhanced activation of Cdc42. Elevated cell migration and enhanced activation of ERK1/2 were observed when NIH3T3 fibroblasts were cultured with both CTGF and PDGF-B proteins. Although CTGF has been shown to bind to VEGF-A and inhibits VEGF-A mediated angiogenesis, our findings demonstrate that CTGF functions in concert with angiogenic factors Slit3 and PDGF-B to promote angiogenesis. Conclusion This study shows a critical role for CTGF in LLC tumor angiogenesis. CTGF binding to cystine knot motifs of key angiogenic factors represent a novel mechanism to regulate angiogenesis through PDGF-B and Slit3 signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3474. doi:10.1158/1538-7445.AM2011-3474

The Notch ligand Delta-like 1 integrates inputs from TGFbeta/Activin and Wnt pathways

Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor ( CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.

Rad GTPase inhibits cardiac fibrosis through connective tissue growth factor

Our previous studies documented that Rad (Ras associated with diabetes), a member of the RGK (Rad, Gem, and Kir) family of Ras-related small G protein, is significantly decreased in human failing hearts and plays an important role in attenuating cardiac hypertrophy. The goal of this study is to identify the effect of Rad on cardiac fibrosis and the underlying mechanisms. Rad knockout (KO) mice showed more severe cardiac fibrosis compared with wild-type littermate controls as detected by Sirius Red staining. Western blot analyses demonstrated that the expression of connective tissue growth factor (CTGF), a key mediator of fibrosis, increased dramatically in Rad KO mice. Overexpression of Rad in cultured neonatal cardiomyocytes suppressed both basal and transforming growth factor-β1-induced CTGF expression. Elevated CTGF expression was observed in cardiomyocytes when Rad was reduced by RNA interference. Moreover, cardiac fibroblasts produced greater extracellular matrix (ECM) when stimulated with conditioned medium from Rad-knockdown cardiomyocytes. ECM production was completely abolished by adding a CTGF-neutralizing antibody into the medium. CCAAT/enhancer-binding protein δ (C/EBP-δ) was demonstrated to activate CTGF in cardiomyocytes. Chromatin immunoprecipitation assay and co-immunoprecipitation further demonstrated that Rad inhibited the binding of C/EBP-δ to the CTGF promoter via direct interaction with C/EBP-δ. Our data reveal that Rad deficiency can lead to cardiac fibrosis. Rad inhibits CTGF expression through binding with C/EBP-δ, thus regulating ECM production in the heart. This study suggests a potential link between decreased Rad levels and increased cardiac fibrosis in human failing hearts.

Ethanol-stimulated differentiated functions of human or mouse hepatic stellate cells are mediated by connective tissue growth factor

Connective tissue growth factor (CTGF) expression is intimately associated with hepatic fibrotic pathophysiology. In this study, CTGF production and action was investigated in ethanol-treated mouse primary hepatic stellate cells (HSC) or human LX-2 cells. CTGF, transforming growth factor-beta1 (TGF-β1), alpha-smooth muscle actin (α-SMA) or collagen α1(I) mRNA were quantified by real-time PCR after treatment of HSC with ethanol or acetaldehyde. CTGF protein production was assessed by immunoprecipitation or ELISA. Ethanol-stimulated CTGF transcription was investigated using CTGF promoter reporter constructs. The TGF-β1- or CTGF-dependency of ethanol-induced CTGF, α-SMA, or collagen α1(I) was determined using small interfering RNA (siRNA) to TGF-β1 or CTGF. In human steatohepatitis, CTGF was produced by presumptive activated HSC. In cultured human or mouse HSC, production of CTGF, α-SMA and/or collagen was increased by ethanol treatment, an effect mimicked by acetaldehyde and blocked by 4-methylpyrazole (4-MP) or N-acetylcysteine (NAC). CTGF promoter activity was stimulated in a sustained fashion by ethanol or TGF-β1. Mutation of the Smad site or basal control element (BCE-1) in the CTGF promoter caused a 5-fold reduction in ethanol-stimulated CTGF promoter activity. Administration of TGF-β1 siRNA or CTGF siRNA significantly decreased ethanol- or acetaldehyde-stimulated mRNA or protein levels of CTGF, α-SMA or collagen I in LX-2 cells. In mouse HSC, TGF-β1- or ethanol-stimulated CTGF, α-SMA or collagen I were significantly attenuated by CTGF siRNA. Ethanol-induced α-SMA or collagen α1(I) in HSC are mediated via TGF-β-dependent CTGF production, highlighting potential therapeutic benefits of targeting CTGF in alcoholic liver disease.

Tachycardia of atrial myocytes induces collagen expression in atrial fibroblasts through transforming growth factor 1

We investigated the molecular mechanism of rapid-depolarization-induced atrial fibrosis. We used a direct atrial myocyte-fibroblast contact co-culture and a fibroblast-specific transforming growth factor β1 (TGF-β1), connective tissue growth factor (CTGF) and procollagen type I α-1 (COL1A1) luciferase reporter system to investigate the possible molecular mechanism of rapid-depolarization-induced atrial fibrosis. Mouse atrial fibroblasts were first transfected with promoter-luciferase reporters, and then co-cultured with HL-1 atrial myocytes. Rapid depolarization of atrial myocytes by rapid electrical field stimulation induced increased TGF-β1, CTGF and COL1A1 promoter activities in the co-cultured atrial fibroblasts (2.4 ± 0.3-fold increase, P= 0.008 for TGF-β1; 2.9 ± 0.4-fold increase, P< 0.001 for CTGF; and 2.1 ± 0.2-fold increase, P= 0.008 for COL1A1). Rapid depolarization of atrial myocytes increased paracrine secretion of angiotensin II (Ang II) and reactive oxygen species in the co-culture medium. Rapid electrical field stimulation-induced ROS generation in atrial myocytes was attenuated by the membrane NADPH oxidase inhibitor, apocynin. Atrial myocyte-induced expression of TGF-β1, CTGF and COL1A1 in atrial fibroblasts was attenuated by co-treatment with the Ang II receptor blocker, losartan, and apocynin. Atrial myocyte-induced COL1A1 expression in atrial fibroblasts was attenuated by anti-TGF-β1 antibody and RNA interference knockdown of the TGF-β1 receptor. We first demonstrated that tachycardia of atrial myocytes induced paracrine secretion of Ang II and reactive oxygen species, which in turn induced expression of CTGF and procollagen in co-cultured atrial fibroblasts through increasing TGF-β1 expression. The results may imply that use of an AngII receptor blocker, in combination with an anti-oxidant, blocks rapid-depolarization-induced atrial fibrosis.

Regulation of CCN2/Connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: Role of Smad and activator protein 1 signaling

To investigate transforming growth factor beta (TGFbeta) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGFbeta-mediated CTGF promoter activity. CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGFbeta treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGFbeta-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGFbeta was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGFbeta expression in the degenerated state. TGFbeta, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc.

Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosis

Connective tissue growth factor (CTGF) is a cysteine-rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor beta (TGFbeta) signaling or through distinct signal transduction pathways. We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast-specific collagen alpha2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis. Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK-1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild-type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGFbeta. These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis.

Lipopolysaccharide induced connective tissue growth factor gene expression in human bronchial epithelial cells

Connective tissue growth factor (CTGF) is up-regulated in the lungs of patients with chronic obstructive pulmonary disease (COPD). Cigarette smoke and repeated bacterial infections, both of which are rich sources of LPS, are major causes of COPD. The high levels of LPS in lung epithelial lining fluid also suggest that it may have a considerable impact on the airway epithelium, in terms of cytokine and growth factor production. The aim of this study was to clarify the mechanism of LPS-induced CTGF expression in bronchial epithelial cells. The expression and transcriptional regulation of the CTGF gene were assessed using the cultured human bronchial epithelial cell line, BEAS-2B. LPS significantly up-regulated CTGF mRNA expression in a dose-dependent fashion, with 100 microg/mL LPS causing a twofold increase after 2 h. CTGF protein expression was also up-regulated by LPS after 8 h. Transforming growth factor-beta1 mRNA expression was not changed by LPS treatment. A pharmacological inhibitor of nuclear factor (NF)-kappaB, MG132, inhibited LPS-induced CTGF mRNA expression. Furthermore, luciferase assays demonstrated that deletion of base pairs -253 to -53 from the CTGF promoter, where the Smad and proximal NF-kappaB binding sites are located, decreased the induction of CTGF by LPS. After stimulation with LPS, the p65 subunit of NF-kappaB was shown to be bound to the CTGF promoter in vitro and in situ. LPS directly induced CTGF expression in bronchial epithelial cells, independently of transforming growth factor-beta1, suggesting a possible mechanism for airway remodelling in COPD that is induced by smoking and repeated bacterial infections.

AS-201: Connective Tissue Growth Factor Promoter Polymorphism in Patients with Acute Coronary Syndromes

Connective tissue growth factor (CTGF) has been associated with several disease states, such as atherosclerosis, diabetic nephropathy, or systemic sclerosis. Single nucleotide polymorphism G-945C (rs6918698) localized in the promoter region of the CTGF gene has been shown to have an influence on gene expression. The aim of our study was to analyze this functional polymorphism in patients with acute coronary syndromes.