Connective Tissue Growth Factor Transcription Research Articles

Epigenetic activation of CTGF transcription by high glucose in renal tubular epithelial cells is mediated by myocardin-related transcription factor A.

Diabetic nephropathy (DN) is one of the most devastating complications of diabetes. Connective tissue growth factor (CTGF) levels are up-regulated in patients with DN and in renal tubular epithelial cells (RTECs) exposed to high glucose (HG). The underlying epigenetic mechanism remains to be elucidated. In the present study, we investigate the role of myocardin-related transcription factor A (MRTF-A) in HG-induced CTGF transcription in RTECs. We report that in two different animal models of DN, one induced by streptozotocin (STZ) injection and the other induced by high-fat diet (HFD) feeding, MRTF-A deficiency attenuated CTGF induction in the kidneys. In cultured RTECs, MRTF-A knockdown similarly ameliorated CTGF induction by HG treatment. Upon CTGF induction, there was an increase in acetylated histone H3 (AcH3) and trimethylated H3K4 (H3K4Me3) on the CTGF promoter region accompanying a decrease in dimethylated H3K9 (H3K9Me2). MRTF-A ablation in vivo or depletion in vitro comparably dampened the accumulation of AcH3 and H3K4Me3 but restored H3K9Me2 on the CTGF promoter. Further analyses revealed that MRTF-A interacted with and recruited histone demethylase KDM3A to the CTGF promoter to activate transcription. KDM3A silencing equivalently weakened HG-induced CTGF induction in RTECs. In conclusion, MRTF-A contributes to HG-induced CTGF transcription via an epigenetic mechanism.

MKL1 mediates TGF-β-induced CTGF transcription to promote renal fibrosis.

Aberrant fibrogenesis impairs the architectural and functional homeostasis of the kidneys. It also predicts poor diagnosis in patients with end-stage renal disease (ESRD). Renal tubular epithelial cells (RTEC) can trans-differentiate into myofibroblasts to produce extracellular matrix proteins and contribute to renal fibrosis. Connective tissue growth factor (CTGF) is a cytokine upregulated in RTECs during renal fibrosis. In the present study, we investigated the regulation of CTGF transcription by megakaryocytic leukemia 1 (MKL1). Genetic deletion or pharmaceutical inhibition of MKL1 in mice mitigated renal fibrosis following the unilateral ureteral obstruction procedure. Notably, MKL1 deficiency in mice downregulated CTGF expression in the kidneys. Likewise, MKL1 knockdown or inhibition in RTEs blunted TGF-β induced CTGF expression. Further, it was discovered that MKL1 bound directly to the CTGF promoter by interacting with SMAD3 to activate CTGF transcription. In addition, MKL1 mediated the interplay between p300 and WDR5 to regulate CTGF transcription. CTGF knockdown dampened TGF-β induced pro-fibrogenic response in RTEs. MKL1 activity was reciprocally regulated by CTGF. In conclusion, we propose that targeting the MKL1-CTGF axis may generate novel therapeutic solutions against aberrant renal fibrogenesis.

A non-autonomous role of MKL1 in the activation of hepatic stellate cells

Although hepatic stellate cells (HSC) represent the major source of fibrogenesis in the liver under various pathological conditions, other cell types including hepatic parenchymal cells (hepatocytes) also contribute to HSC activation and hence liver fibrosis. The underlying mechanism, however, is poorly defined. Here we report that hepatocytes exposed to high concentrations of glucose (HG) emit a pro-fibrogenic cue as evidenced by the observation that primary HSCs cultured in conditioned media (CM) collected from hepatocytes exposed to HG up-regulated the production of extracellular matrix (ECM) proteins compared to CM collected from hepatocytes exposed to low glucose. We further identified the pro-fibrogenic cue from hepatocytes to be connective tissue growth factor (CTGF) because either depletion of endogenous CTGF in hepatocytes with siRNA or the addition of a CTGF-specific neutralizing antibody to the CM blunted the pro-fibrogenic effect elicit by HG treatment. Of interest, we discovered that genetic ablation or pharmaceutical inhibition of the transcriptional modulator MKL1 in hepatocytes also abrogated the HG-induced pro-fibrogenic effects. Mechanistically, MKL1 interacted with AP-1 and SMAD3 to trans-activate CTGF in hepatocytes in response to HG treatment. In conclusion, our data suggest that MKL1 contribute to HSC activation in a non-autonomous fashion by promoting CTGF transcription in hepatocytes.

Effects of connective tissue growth factor on prostate cancer bone metastasis and osteoblast differentiation.

Previous studies have demonstrated that connective tissue growth factor (CTGF) is expressed at increased levels in prostate cancer bone metastasis mouse models and patients with prostate cancer which metastasizes to the bone; however, the underlying molecular mechanism(s) remain unknown. The present study investigated the function of CTGF in osteoblast differentiation and its effect on prostate cancer bone metastasis by analyzing CTGF gene expression and transcription at different levels of invasion, metastasis of prostate cancer cells, and the influence of CTGF on proliferation and xenotransplantation. A mouse model demonstrating bone metastasis was used to investigate the function(s) of CTGF in bone metastasis and osteoblast differentiation. Results demonstrated that CTGF expression was increased in association with high bone metastasis in prostate cancer cells, and its expression was significantly decreased in whole cell lysates. CTGF expression in prostate cancer cells with high levels of bone metastasis was increased 1.9-fold compared with prostate cancer cells with low levels of bone metastasis. The expression of CTGF in mesenchymal cells was markedly increased compared with epithelial cells. Results indicated that the increased expression of CTGF does not affect the proliferation of tumor cells and possesses no influence on tumor volume. Control and CTGF plasmids were transfected into RM1 cells and led to 4 and 17% bone lesions, respectively. Increased expression of CTGF significantly enlarged the tumor area in the bone metastatic position compared with the control. Positive areas of alkaline phosphatase were significantly decreased as the concentration of CTGF increased. The results of the present study demonstrated that CTGF promotes prostate carcinoma to metastasize in the bone by dysregulating osteoblast differentiation.

Decreased expression of connective tissue growth factor in non-small cell lung cancer is associated with clinicopathological variables and can be restored by epigenetic modifiers.

PurposeRecent studies indicated undisputed contribution of connective tissue growth factor (CTGF) in the development of many cancers, including non-small cell lung cancer (NSCLC). However, the functional role and regulation of CTGF expression during tumorigenesis remain elusive. Our goal was to determine CTGF transcript and protein levels in tumoral and matched control tissues from 98 NSCLC patients, to correlate the results with clinicopathological features and to investigate whether the CTGF expression can be epigenetically regulated in NSCLC.MethodsWe used quantitative PCR, Western blotting and immunohistochemistry to evaluate CTGF expression in lung cancerous and histopathologically unchanged tissues. We tested the impact of 5-Aza-2′-deoxycytidine (5-dAzaC) and trichostatin A (TSA) on CTGF transcript and protein levels in NSCLC cells (A549, Calu-1). DNA methylation status of the CTGF regulatory region was evaluated by bisulfite sequencing. The influence of 5-dAzaC and TSA on NSCLC cells viability and proliferation was monitored by the trypan blue assay.ResultsWe found significantly decreased levels of CTGF mRNA and protein (both p < 0.0000001) in cancerous tissues of NSCLC patients. Down-regulation of CTGF occurred regardless of gender in all histological subtypes of NSCLC. Moreover, we showed that 5-dAzaC and TSA were able to restore CTGF mRNA and protein contents in NSCLC cells. However, no methylation within CTGF regulatory region was detected. Both compounds significantly reduced NSCLC cells proliferation.ConclusionsDecreased expression of CTGF is a common feature in NSCLC; however, it can be restored by the chromatin-modifying agents such as 5-dAzaC or TSA and consequently restrain cancer development.Electronic supplementary materialThe online version of this article (doi:10.1007/s00432-016-2195-3) contains supplementary material, which is available to authorized users.

Effects of simvastatin on pulmonary fibrosis, pulmonary hypertension and exercise capacity in bleomycin‐treated rats

Pulmonary fibrosis is often complicated by pulmonary hypertension. Statins reduce fibroblast activity in vitro and pulmonary hypertension in vivo. We investigated whether Simvastatin exerts beneficial effects on pulmonary fibrosis and pulmonary hypertension in Bleomycin-treated rats in vivo. Rats were randomly assigned to controls, Bleomycin, Bleomycin plus Simvastatin from day 1 to 28 and Bleomycin plus Simvastatin from day 13 to 28. 28 days after Bleomycin instillation, right ventricular systolic pressure (RVSP), right ventricular mass (RV/(LV+S)), right ventricular and circulating brain natriuretic peptide (BNP) levels were determined to assess pulmonary hypertension. Pulmonary hydroxyproline content (HPC), pulmonary connective tissue growth factor (CTGF) transcription and lung compliance (LC) were analysed to characterize pulmonary fibrosis. Exercise capacity was determined by treadmill tests. Compared with controls, Bleomycin increased RVSP, RV/(LV+S), BNP levels, HPC and CTGF transcription and decreased LC significantly. Simvastatin administered from day 1 to 28 normalized all these parameters. Simvastatin administered from day 13 to 28 had no effect on HPC and LC, but reduced RV/(LV+S) significantly and induced a strong trend to lower RVSP and BNP levels. Exercise capacity was reduced by Bleomycin. Simvastatin significantly improved exercise intolerance in both treatment groups. Simvastatin prevents the development of pulmonary fibrosis, but fails to attenuate already established pulmonary fibrosis. In contrast, it ameliorates pulmonary hypertension and thereby exercise capacity in the prevention and the treatment group regardless of its effects on pulmonary fibrosis. Whether statins are a treatment option in humans with pulmonary fibrosis needs to be investigated by further study.

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.

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.

Increased connective tissue growth factor associated with cardiac fibrosis in the mdx mouse model of dystrophic cardiomyopathy

Cardiomyopathy contributes to morbidity and mortality in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disorder. A major feature of the hearts of DMD patients and the mdx mouse model of the disease is cardiac fibrosis. Connective tissue growth factor (CTGF) is involved in the fibrotic process in many organs. This study utilized the mdx mouse model to assess the role of CTGF and other extracellular matrix components during the development of fibrosis in the dystrophic heart. Left ventricular function of mdx and control mice at 6, 29 and 43 weeks was measured by echocardiography. Young (6 weeks old) mdx hearts had normal function and histology. At 29 weeks of age, mdx mice developed cardiac fibrosis and increased collagen expression. The onset of fibrosis was associated with increased CTGF transcript and protein expression. Increased intensity of CTGF immunostaining was localized to fibrotic areas in mdx hearts. The upregulation of CTGF was also concurrent with increased expression of tissue inhibitor of matrix metalloproteinases (TIMP-1). These changes persisted in 43 week old mdx hearts and were combined with impaired cardiac function and increased gene expression of transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMP-2, MMP-9). In summary, an association was observed between cardiac fibrosis and increased CTGF expression in the mdx mouse heart. CTGF may be a key mediator of early and persistent fibrosis in dystrophic cardiomyopathy.

Connective tissue growth factor expression is increased in collagenous colitis and coeliac disease

Subepithelial collagen deposition is a classical feature of collagenous colitis (CC), but is also seen in untreated coeliac disease. The end-stage mediator of excess cellular collagen production is connective tissue growth factor (CTGF). The aim of this study was to investigate CTGF expression by in situ hybridization (ISH) and polymerase chain reaction (PCR) in CC and coeliac disease as well as lymphocytic colitis (LC), Crohn's colitis and ulcerative colitis (UC). For coeliac disease we analysed fresh frozen material by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and archival material for ISH. PCR transcripts in coeliac disease were moderately elevated and labelled cells were significantly increased in the subepithelial zone. For CC, LC and UC we investigated archival material because of the rarity of the first two conditions. There was a marked increase in CTGF expression in the subepithelial zone in CC, localizing to cells with the morphology of smooth muscle cells, which was not seen in LC. The colocalization of CTGF transcripts with areas of excessive collagen deposition in coeliac disease and CC suggest that it might be the end-stage mediator of local fibrosis in these conditions.

PTU-034 Expression of connective tissue growth factor by human intestinal myofibroblasts and effect of rho-kinase inhibition

<h3>Introduction</h3> Connective tissue growth factor (CTGF) has been implicated in the development of fibrosis in different organs, including stricture formation in Crohn9s disease. As a profibrotic mediator, CTGF acts downstream and together with transforming growth factor β (TGF-β). Expression of TGF-β has previously been shown to be altered in myofibroblasts isolated from mucosal samples affected by inflammatory bowel disease. Rho-kinase is a downstream mediator of RhoA, a GTP-binding protein that is believed to regulate a number of myofibroblast functions. The aims of this study were to investigate (i) the expression of CTGF by human intestinal myofibroblasts isolated from mucosal samples obtained from different individuals and (ii) the effect of the Rho-kinase inhibitor Y-27632 on CTGF expression by intestinal myofibroblasts. <h3>Methods</h3> Myofibroblasts were isolated from normal colonic mucosal samples (obtained from operation resection specimens) and established in pure culture. Myofibroblasts were cultured in medium containing 0.1% or 10% fetal calf serum (FCS). CTGF transcript expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of protein (in cell lysates) by Western blot analysis. Following incubation with Y-27632 (final concentration 100 μM), changes in myofibroblast cell morphology, expression of CTGF and programmed cell death (analysis of the sub-G1 region of the cell cycle by flow cytometry) were studied. <h3>Results</h3> Studies by RT-PCR showed that when grown in 10% FCS, intestinal myofibroblast cultures from 5 mucosal tissue donors expressed CTGF transcript. Expression of CTGF protein was confirmed by western blot analysis of intestinal myofibroblasts grown in 10% FCS (n=7 donors). By contrast, when cultured in 0.1% FCS, only 2 out 5 donors expressed CTGF protein (0.1% FCS vs 10% FCS: p&lt;0.03). Exposure to Y-27632 led to change in morphology to spindle-shaped cells, with all the myofibroblasts affected by 24 h. When cultured for 24 h with 10% FCS, Y-27632 led to a reduction in expression of CTGF protein by intestinal myofibroblasts isolated from 6 donors. However, there was no significant difference in the number of myofibroblasts undergoing programmed cell death between those cultured in medium only, compared to cells exposed to Y-27632 (events in sub-G1 region of cells cycle: 203.8 (SEM 51.62) vs 393.0 (64.91), p=0.10). <h3>Conclusion</h3> 1. Myofibroblasts isolated from normal human colonic mucosal samples are capable of expressing CTGF. 2. The Rho-kinase inhibitor Y-27632 reduces expression of CTGF protein by colonic myofibroblasts. 3. The Rho-kinase pathway may be a suitable therapeutic target in stricturing Crohn9s disease.

CREBtrans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

Direct Aldosterone Action as a Profibrotic Factor via ROS-Mediated SGK1 in Peritoneal Fibroblasts

Background/Aims: Peritoneal fibrosis leads to discontinuation of peritoneal dialysis. Although aldosterone promotes tissue fibrosis in many organs, its contribution to peritoneal fibrosis and the underlying mechanism are poorly understood. The present study investigated the direct effect of aldosterone on cultured rat peritoneal fibroblasts (RPFs). Methods: The expression of aldosterone synthase (CYP11B2), mineralocorticoid receptors (MRs), 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), serum- and glucocorticoid-inducible protein kinase 1 (SGK1), and connective tissue growth factor (CTGF) mRNA was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). To determine the role of reactive oxygen species (ROS) induced by aldosterone, an active oxygen assay with several inhibitors was used. The ability of RPFs to produce aldosterone was examined by enzyme immunoassay. Small interfering RNA (siRNA) of SGK1 was transfected into cultured cells using lipofectamine. Results: CYP11B2, MRs, and 11β-HSD2 were expressed in RPFs. The release of aldosterone from RPFs into the culture medium was confirmed. Aldosterone increased the expression of SGK1 mRNA via ROS generation. Spironolactone, apocynin, and tempol significantly reduced SGK1 expression. Aldosterone upregulated CTGF transcripts significantly. SGK1 gene silencing suppressed aldosterone-induced CTGF expression. Conclusion: The local aldosterone system acts directly as a profibrotic factor via ROS-mediated SGK1 in RPFs.

Calcitonin induces connective tissue growth factor through ERK1/2 signaling in renal tubular cells

Calcitonin (CT), a polypeptide hormone, plays important roles in a variety of physiological processes. CT has been used clinically to treat osteoporosis and humoral hypercalcemia of malignancy. In order to clarify the pharmacological effects of CT in the kidney, we identified potential downstream genes induced by CT in the renal cells. Using a cDNA subtraction hybridization method, we identified connective tissue growth factor (CTGF) as a CT-induced gene in the porcine renal cell line, LLC-PK1. Furthermore, we found that CT-mediated induction of the gene was not inhibited by cycloheximide, which suggests that CTGF gene was not induced by an increased synthesis of regulating proteins. Therefore, CTGF is an immediate early gene. We further demonstrated that the regulation of CTGF gene expression by CT involved the ERK1/2 pathway, because PD98059, a MEK1 inhibitor, partially inhibited the mRNA expression of CTGF induced by CT. CT-induced CTGF protein expression was also observed in vivo. Our present findings suggest that CT induces the transcription of CTGF through ERK1/2 phosphorylation. We also identified twelve other genes induced by CT that, like CTGF, were related to wound healing. These results suggest that CT may have an effect on renal differentiation and wound healing in the kidney.

Hypoxia/reoxygenation induces CTGF and PAI-1 in cultured human retinal pigment epithelium cells

Early age-related macular degeneration (AMD) is characterized by thickening of Bruch's membrane due to the accumulation of extracellular matrix (ECM). This finding could be related to hypoxia of the retinal pigment epithelium (RPE). In the present study, we investigated the effects of hypoxia and reoxygenation on the expression of connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), collagen type IV (Col IV) and fibronectin (Fn) in cultured human RPE cells. Cultured human RPE cells were kept for 12–36 h under hypoxic conditions (1% O 2). Reoxygenation was conducted for 24 h. Hypoxia-mediated CTGF and PAI-1 expression were analyzed by using immunohistochemistry, Northern and Western blot analysis. Actinomycin D was added to examine whether hypoxia induces the transcription of CTGF and PAI-1 mRNA. Furthermore, cells were transfected with siRNA against hypoxia-inducible factor-1alpha (HIF-1α) and kept under hypoxic conditions. The effects of antioxidants on hypoxia/reoxygenation-mediated CTGF and PAI-1 expression were tested by real-time PCR analysis. Production of Col IV and Fn were investigated by real-time PCR and Western blot analysis. Both hypoxia and hypoxia/reoxygenation increased the expression of CTGF, PAI-1, Col IV and Fn. Actinomycin D prevented the new transcription of CTGF and PAI-1 mRNA by hypoxia. Using siRNA against HIF-1α, the hypoxia-mediated increase of CTGF and PAI-1 was inhibited. Antioxidants attenuated the reoxygenation-mediated increase of CTGF and PAI-1. The process of hypoxia/reoxygenation in the RPE may lead to an increase of ECM in the RPE and thus may contribute to the accumulation of ECM in Bruch's membrane.

MiR-133 and miR-30 Regulate Connective Tissue Growth Factor

The myocardium of the failing heart undergoes a number of structural alterations, most notably hypertrophy of cardiac myocytes and an increase in extracellular matrix proteins, often seen as primary fibrosis. Connective tissue growth factor (CTGF) is a key molecule in the process of fibrosis and therefore seems an attractive therapeutic target. Regulation of CTGF expression at the promoter level has been studied extensively, but it is unknown how CTGF transcripts are regulated at the posttranscriptional level. Here we provide several lines of evidence to show that CTGF is importantly regulated by 2 major cardiac microRNAs (miRNAs), miR-133 and miR-30. First, the expression of both miRNAs was inversely related to the amount of CTGF in 2 rodent models of heart disease and in human pathological left ventricular hypertrophy. Second, in cultured cardiomyocytes and fibroblasts, knockdown of these miRNAs increased CTGF levels. Third, overexpression of miR-133 or miR-30c decreased CTGF levels, which was accompanied by decreased production of collagens. Fourth, we show that CTGF is a direct target of these miRNAs, because they directly interact with the 3' untranslated region of CTGF. Taken together, our results indicate that miR-133 and miR-30 importantly limit the production of CTGF. We also provide evidence that the decrease of these 2 miRNAs in pathological left ventricular hypertrophy allows CTGF levels to increase, which contributes to collagen synthesis. In conclusion, our results show that both miR-133 and miR-30 directly downregulate CTGF, a key profibrotic protein, and thereby establish an important role for these miRNAs in the control of structural changes in the extracellular matrix of the myocardium.

Endothelin-1 Induces Expression of Connective Tissue Growth Factor in Bronchial Epithelial Cells

Endothelin-1 (ET-1) is a potent vaso and bronchoconstrictor. We have previously shown that in the presence of ET-1 bronchial epithelial cells migrate more slowly. Connective tissue growth factor (CTGF) is a cysteine rich secreted protein and regulates fibrosis. In addition, we have previously shown that CTGF is expressed in airway epithelial cells under the conditions of stress starvation. The aim of this study was to describe the effect of ET-1 on CTGF expression, us- ing a lung epithelial-fibroblast co-culture system. Bronchial epithelial cells (BEC) were grown and stimulated with ET-1, 100ng/ml of media, for 24 hours. At 0, 4 and 24 hours, total RNA was isolated. Reverse transcription PCR was performed and CTGF transcripts were semi-quantified. In addition, BEC were grown in co-culture, in the presence of human lung fi- broblasts (FB). The same experiment was conducted for FB grown in the presence or absence of ET-1. Control cells were grown only in the basal media. At both time points, following exposure to ET-1, BEC expressed more CTGF as compared to control. Cells in co-culture with FB, expressed no CTGF at 4 hours, and detectable CTGF at 24 hours. CTGF was de- tected at all time points for FB in both control and stimulated cells. ET-1 induces expression of CTGF in BEC as com- pared to control. ET-1 may be an important regulator of CTGF expression in the airway. Lung fibroblasts may secrete epithelial cell regulatory factors, controlling the expression of CTGF in the presence of ET-1.

Serial Analysis of Gene Expression Identifies Connective Tissue Growth Factor Expression as a Prognostic Biomarker in Gallbladder Cancer

Gallbladder cancer (GBC) is an uncommon neoplasm in the United States, but one with high mortality rates. This malignancy remains largely understudied at the molecular level such that few targeted therapies or predictive biomarkers exist. We built the first series of serial analysis of gene expression (SAGE) libraries from GBC and nonneoplastic gallbladder mucosa, composed of 21-bp long-SAGE tags. SAGE libraries were generated from three stage-matched GBC patients (representing Hispanic/Latino, Native American, and Caucasian ethnicities, respectively) and one histologically alithiasic gallbladder. Real-time quantitative PCR was done on microdissected epithelium from five matched GBC and corresponding nonneoplastic gallbladder mucosa. Immunohistochemical analysis was done on a panel of 182 archival GBC in high-throughput tissue microarray format. SAGE tags corresponding to connective tissue growth factor (CTGF) transcripts were identified as differentially overexpressed in all pairwise comparisons of GBC (P < 0.001). Real-time quantitative PCR confirmed significant overexpression of CTGF transcripts in microdissected primary GBC (P < 0.05), but not in metastatic GBC, compared with nonneoplastic gallbladder epithelium. By immunohistochemistry, 66 of 182 (36%) GBC had high CTGF antigen labeling, which was significantly associated with better survival on univariate analysis (P = 0.0069, log-rank test). An unbiased analysis of the GBC transcriptome by SAGE has identified CTGF expression as a predictive biomarker of favorable prognosis in this malignancy. The SAGE libraries from GBC and nonneoplastic gallbladder mucosa are publicly available at the Cancer Genome Anatomy Project web site and should facilitate much needed research into this lethal neoplasm.

Lovastatin Inhibits TGF-β–Induced Myofibroblast Transdifferentiation in Human Tenon Fibroblasts

The transdifferentiation of Tenon fibroblasts to myofibroblasts is a pivotal step in filtering bleb scarring. It is mediated by the cytokine TGF-beta, Rho-dependent contractility, and cell-matrix interactions in an interdependent fashion. HMG-CoA-reductase inhibitors (statins) have been shown to inhibit Rho-GTPase signaling; therefore, the authors studied the influence of lovastatin on TGF-beta-mediated myofibroblast transdifferentiation to assess the potential use of statins in wound healing modulation. Human Tenon fibroblasts were grown in culture, pretreated with lovastatin, lovastatin and mevalonate, or specific inhibitors of farnesyl transferase or geranylgeranyl transferase and were stimulated with TGF-beta1. alpha-Smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) transcription were assessed by real-time PCR. alpha-SMA protein expression and localization were studied by Western blot and confocal immunofluorescence microscopy. Cell contractility was determined in collagen gel contraction assays. Phosphorylation of the signaling proteins Smad-2/3 and p38 were detected by Western blot, and Smad-2/3 localization was determined by confocal immunofluorescence microscopy. Lovastatin inhibited TGF-beta-induced CTGF transcription, alpha-SMA expression and incorporation into actin stress fibers, and subsequent collagen gel contraction. These effects were reversed by mevalonate. The inhibition of geranylgeranyl transferase but not farnesyl transferase blocked TGF-beta-induced alpha-SMA expression. Lovastatin decreased TGF-beta-induced p38 activation, whereas Smad-2/3 phosphorylation and nuclear translocation were preserved. Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human Tenon fibroblasts, most likely by interfering with Rho-signaling. Statins may, therefore, serve to inhibit scarring after filtering glaucoma surgery.

Hypoxia in Human Trophoblasts Stimulates the Expression and Secretion of Connective Tissue Growth Factor

The mechanisms underlying cellular injury when human placental trophoblasts are exposed to hypoxia are unclear. Connective tissue growth factor (CTGF) mediates cell injury and fibrosis in diverse tissues. We hypothesized that hypoxia enhances the production of CTGF in primary term human trophoblasts. Using cultured term primary human trophoblasts as well as villous biopsies from term human placentas, we showed that CTGF protein is expressed in trophoblasts. When compared with cells cultured in standard conditions (FiO2 = 20%), exposure of primary human trophoblasts to low oxygen concentration (FiO2 = 8% or <or= 1%) enhanced the expression of CTGF mRNA in a time-dependent manner, with a significant increase in CTGF levels after 16 h (2.7 +/- 0.7-fold; P < 0.01), reaching a maximum of 10.9 +/- 3.2-fold at 72 h. Whereas exposure to hypoxia had no effect on cellular CTGF protein levels, secretion of CTGF to the medium was increased after 16 h in hypoxia and remained elevated through 72 h. The increase in cellular CTGF transcript levels and CTGF protein secretion was recapitulated by exposure of trophoblasts to agents that enhance the activity of hypoxia-inducible factor (HIF)1alpha, including cobalt chloride or the proline hydroxylase inhibitor dimethyloxaloylglycine, and attenuated using the HIF1alpha inhibitor 2-methoxyestradiol. Although all TGFbeta isoforms stimulated the expression of CTGF in trophoblasts, only the expression of TGFbeta1 mRNA was enhanced by hypoxia. We conclude that hypoxia increases cellular CTGF mRNA levels and CTGF protein secretion from cultured trophoblasts, likely in a HIF1alpha-dependent manner.