Connective Tissue Growth Factor Promoter Research Articles

CTGF regulated by ATF6 inhibits vascular endothelial inflammation and reduces hepatic ischemia–reperfusion injury

Vascular endothelial inflammation is crucial in hepatic ischemia–reperfusion injury (IRI). Our previous research has shown that connective tissue growth factor (CTGF), secreted by endothelial cells, protects against acute liver injury, but its upstream mechanism is unclear. We aimed to clarify the protective role of CTGF in endothelial cell inflammation during IRI and reveal the regulation between endoplasmic reticulum stress-induced activating transcription factor 6 (ATF6) and CTGF. Hypoxia/reoxygenation in endothelial cells, hepatic IRI in mice and clinical specimens were used to examine the relationships between CTGF and inflammatory factors and determine how ATF6 regulates CTGF and reduces damage. We found that activating ATF6 promoted CTGF expression and reduced liver damage in hepatic IRI. In vitro, activated ATF6 upregulated CTGF and downregulated inflammation, while ATF6 inhibition had the opposite effect. Dual-luciferase assays and chromatin immunoprecipitation confirmed that activated ATF6 binds to the CTGF promoter, enhancing its expression. Activated ATF6 increases CTGF and reduces extracellular regulated protein kinase 1/2 (ERK1/2) phosphorylation, decreasing inflammatory factors. Conversely, inhibiting ATF6 decreases CTGF and increases the phosphorylation of ERK1/2, increasing inflammatory factor levels. ERK1/2 inhibition reverses this effect. Clinical samples have shown that CTGF increases after IRI, inversely correlating with inflammatory cytokines. Therefore, ATF6 activation during liver IRI enhances CTGF expression and reduces endothelial inflammation via ERK1/2 inhibition, providing a novel target for diagnosing and treating liver IRI.

Long noncoding RNA AI662270 promotes kidney fibrosis through enhancing METTL3-mediated m6 A modification of CTGF mRNA.

The sustained release of profibrotic cytokines, mainly transforming growth factor-β (TGF-β), leads to the occurrence of kidney fibrosis and chronic kidney disease (CKD). Connective tissue growth factor (CTGF) appears to be an alternative target to TGF-β for antifibrotic therapy in CKD. In this study, we found that long noncoding RNA AI662270 was significantly increased in various renal fibrosis models. In vivo, ectopic expression of AI662270 alone was sufficient to activate interstitial fibroblasts and drive kidney fibrosis, whereas inhibition of AI662270 blocked the activation of interstitial fibroblasts and ameliorated kidney fibrosis in various murine models. Mechanistic studies revealed that overexpression of AI662270 significantly increased CTGF product, which was required for the role of AI662270 in driving kidney fibrosis. Furthermore, AI662270 binds to the CTGF promoter and directly interacts with METTL3, the methyltransferase of RNA N6 -methyladenosine (m6 A) modification. Functionally, AI662270-mediated recruitment of METTL3 increased the m6 A methylation of CTGF mRNA and consequently enhanced CTGF mRNA stability. In conclusion, our results support that AI662270 promotes CTGF expression at the posttranscriptional stage by recruiting METTL3 to the CTGF promoter and depositing m6 A modifications on the nascent mRNA, thereby, uncovering a novel regulatory mechanism of CTGF in the pathogenesis of kidney fibrosis.

Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

BackgroundReduction of histone deacetylase (HDAC) 2 expression and activity may contribute to amplified inflammation in patients with severe asthma. Connective tissue growth factor (CTGF) is a key mediator of airway fibrosis in severe asthma. However, the role of the HDAC2/Sin3A/methyl-CpG-binding protein (MeCP) 2 corepressor complex in the regulation of CTGF expression in lung fibroblasts remains unclear.MethodsThe role of the HDAC2/Sin3A/MeCP2 corepressor complex in endothelin (ET)-1-stimulated CTGF production in human lung fibroblasts (WI-38) was investigated. We also evaluated the expression of HDAC2, Sin3A and MeCP2 in the lung of ovalbumin-induced airway fibrosis model.ResultsHDAC2 suppressed ET-1-induced CTGF expression in WI-38 cells. ET-1 treatment reduced HDAC2 activity and increased H3 acetylation in a time-dependent manner. Furthermore, overexpression of HDAC2 inhibited ET-1-induced H3 acetylation. Inhibition of c-Jun N-terminal kinase, extracellular signal-regulated kinase, or p38 attenuated ET-1-induced H3 acetylation by suppressing HDAC2 phosphorylation and reducing HDAC2 activity. Overexpression of both Sin3A and MeCP2 attenuated ET-1-induced CTGF expression and H3 acetylation. ET-1 induced the disruption of the HDAC2/Sin3A/MeCP2 corepressor complex and then prompted the dissociation of HDAC2, Sin3A, and MeCP2 from the CTGF promoter region. Overexpression of HDAC2, Sin3A, or MeCP2 attenuated ET-1-stimulated AP-1-luciferase activity. Moreover, Sin3A- or MeCP2-suppressed ET-1-induced H3 acetylation and AP-1-luciferase activity were reversed by transfection of HDAC2 siRNA. In an ovalbumin-induced airway fibrosis model, the protein levels of HDAC2 and Sin3A were lower than in the control group; however, no significant difference in MeCP2 expression was observed. The ratio of phospho-HDAC2/HDAC2 and H3 acetylation in the lung tissue were higher in this model than in the control group. Overall, without stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex inhibits CTGF expression by regulating H3 deacetylation in the CTGF promoter region in human lung fibroblasts. With ET-1 stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex is disrupted and dissociated from the CTGF promoter region; this is followed by AP-1 activation and the eventual initiation of CTGF production.ConclusionsThe HDAC2/Sin3A/MeCP2 corepressor complex is an endogenous inhibitor of CTGF in lung fibroblasts. Additionally, HDAC2 and Sin3A may be of greater importance than MeCP2 in the pathogenesis of airway fibrosis.

Abstract 431: FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor

Abstract Metastatic melanoma is one of the most aggressive skin cancers and is associated with poor prognosis. BRAF and MEK inhibitors are used to treat patients with BRAFV600E-mutated advanced melanoma. However, the development of resistances to these treatments compromises therapeutic success. Our lab previously demonstrated that forkhead box D1 (FOXD1) plays a critical role in melanoma migration and invasion. Here, we found that FOXD1 was highly expressed in melanoma cells. Immunohistochemical assessment of 105 samples from patients with metastatic melanoma revealed that high FOXD1 expression in tumors was associated with poor survival and correlated with low MITF, SOX10 and high AXL expression. Upregulation of FOXD1 expression enhanced the resistance of melanoma to vemurafenib (BRAF inhibitor) or combinatorial treatment with vemurafenib and cobimetinib (MEK inhibitor). On the other hand, loss of FOXD1 increased the sensitivity of naïve melanoma cells towards vemurafenib or combinatorial treatment with vemurafenib and cobimetinib. Furthermore, high FOXD1 expression levels were found in BRAF inhibitor (BRAFi)-resistant cells. Downregulation of FOXD1 resulted in a resensitization of BRAFi-resistant cells to vemurafenib. By using microarray analysis, connective tissue growth factor (CTGF) was found to be one of the most downregulated genes in FOXD1 knockdown (KD) cells while its expression was highly increased upon FOXD1 overexpression. Thus, CTGF was identified as a downstream factor of FOXD1. In addition, in vitro expression analysis and evaluation of clinical samples demonstrated that CTGF and FOXD1 expression were positively correlated. By using a CHIP assay and a dual reporter luciferase assay, we discovered that FOXD1 could regulate the expression of CTGF by directly binding to the CTGF promoter. This result was confirmed with RT-PCR and western blot. In addition, we found that the protein level of CTGF was highly increased in BRAFi-resistant cells. Similar to FOXD1 knockdown, the knockdown of CTGF resensitized BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, we conclude that the transcription factor FOXD1 could promote dedifferentiation and targeted therapy-resistance in melanoma cells by regulating the expression of CTGF. FOXD1 might be a promising new diagnostical marker and a therapeutic target of targeted therapy resistant melanoma. Citation Format: Qian Sun, Daniel Novak, Laura Hüser, Juliane Poelchen, Huizi Wu, Karol Granados, Aniello Federico Federico, Ke Liu, Tamara Steinfass, Marlene Vierthaler, Viktor Umansky, Jochen Utikal. FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 431.

FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor.

Metastatic melanoma is an aggressive skin cancer and associated with a poor prognosis. In clinical terms, targeted therapy is one of the most important treatments for patients with BRAFV600E -mutated advanced melanoma. However, the development of resistance to this treatment compromises its therapeutic success. We previously demonstrated that forkhead box D1 (FOXD1) regulates melanoma migration and invasion. Here, we found that FOXD1 was highly expressed in melanoma cells and was associated with a poor survival of patients with metastatic melanoma. Upregulation of FOXD1 expression enhanced melanoma cells' resistance to vemurafenib (BRAF inhibitor [BRAFi]) or vemurafenib and cobimetinib (MEK inhibitor) combination treatment whereas loss of FOXD1 increased the sensitivity to treatment. By comparing gene expression levels between FOXD1 knockdown (KD) and overexpressing (OE) cells, we identified the connective tissue growth factor (CTGF) as a downstream factor of FOXD1. Chromatin immunoprecipitation and luciferase assay demonstrated the direct binding of FOXD1 to the CTGF promoter. Similar to FOXD1, knockdown of CTGF increased the sensitivity of BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, we conclude that FOXD1 might be a promising new diagnostic marker and a therapeutic target for the treatment of targeted therapy resistant melanoma.

TGF-β Induced CTGF Expression in Human Lung Epithelial Cells through ERK, ADAM17, RSK1, and C/EBPβ Pathways

Background: Lung epithelial cells play critical roles in idiopathic pulmonary fibrosis. Methods: In the present study, we investigated whether transforming growth factor-β (TGF-β)-induced expression of connective tissue growth factor (CTGF) was regulated by the extracellular signal-regulated kinase (ERK)/a disintegrin and metalloproteinase 17 (ADAM17)/ribosomal S6 kinases 1 (RSK1)/CCAAT/enhancer-binding protein β (C/EBPβ) signaling pathway in human lung epithelial cells (A549). Results: Our results revealed that TGF-β-induced CTGF expression was weakened by ADAM17 small interfering RNA (ADAM17 siRNA), TNF-α processing inhibitor-0 (TAPI-0, an ADAM17 inhibitor), U0126 (an ERK inhibitor), RSK1 siRNA, and C/EBPβ siRNA. TGF-β-induced ERK phosphorylation as well as ADAM17 phosphorylation was attenuated by U0126. The TGF-β-induced increase in RSK1 phosphorylation was inhibited by TAPI-0 and U0126. TGF-β-induced C/EBPβ phosphorylation was weakened by U0126, ADAM17 siRNA, and RSK1 siRNA. In addition, TGF-β increased the recruitment of C/EBPβ to the CTGF promoter. Furthermore, TGF-β enhanced fibronectin (FN), an epithelial–mesenchymal transition (EMT) marker, and CTGF mRNA levels and reduced E-cadherin mRNA levels. Moreover, TGF-β-stimulated FN protein expression was reduced by ADAM17 siRNA and CTGF siRNA. Conclusion: The results suggested that TGF-β induces CTGF expression through the ERK/ADAM17/RSK1/C/EBPβ signaling pathway. Moreover, ADAM17 and CTGF participate in TGF-β-induced FN expression in human lung epithelial cells.

Hypomethylation of CTGF Promoter in Placenta and Peripheral Blood of Pre-eclampsia Women.

Pre-eclampsia (PE) is a serious pregnancy complication defined as hypertension combined with proteinuria. Previous report has indicated that the expression of connective tissue growth factor (CTGF) is upregulated in PE placentas. In this study, we aim to investigate the potential association between the methylation of the CTGF promoter and PE. The methylation status of CTGF promoter was assessed by methylation-specific PCR and the expression of CTGF was analyzed by real-time quantitative PCR and immunohistochemistry staining, in placental samples from PE patients and normal pregnant women. The methylation level of the CTGF promoter was also analyzed in peripheral blood DNA samples from 90 PE patients and 94 normal pregnant women and further confirmed by methylation-sensitive restriction enzyme digestion combined with real-time PCR. We found a decreased methylation rate of CTGF promoter in PE placentas compared with normal control. In addition, the frequency of hemimethylated (UM) promoter of CTGF in peripheral blood was significantly lower in the PE group (P = 0.019), while the frequency of overall methylated (MU + MM) promoter of CTGF in peripheral blood was lower (borderline significance P = 0.043) in the PE group. The percentage of methylated CpG in CTGF promoter was 30% lower in maternal blood of PE subjects than in controls. Moreover, the lower methylation frequency of CTGF promoter was in accordance with the higher expression of CTGF in trophoblast cells. Together, our data suggested an association between PE and the hypomethylation of CTGF promoter both in placentas and peripheral blood from a Chinese Han population.

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.

Mycobacterium tuberculosis induces connective tissue growth factor expression through the TLR2‐JNK‐AP‐1 pathway in human lung fibroblasts

Mycobacterium tuberculosis (M.tb) infection in lung causes pulmonary fibrosis, which leads to the irreversible reduction of pulmonary function. Fibrotic protein connective tissue growth factor (CTGF) expression has been confirmed to play a crucial role in lung fibrosis. However, the underlying signal pathway and effect of M.tb on CTGF expression in human lung fibroblasts are unclear. Our results revaled that M.tb caused time- and concentration-dependent increases in CTGF expression in human lung fibroblasts. A mechanistic investigation revealed that M.tb induced CTGF expression through TLR2 but not TLR4. The promoter activity assay indicated that M.tb-induced CTGF activity was mainly controlled by the promoter region at -747 to -184 bp, which contained signal transducer and activator of transcription 3 and activator protein 1 (AP-1) binding sites. Moreover, curcumin (AP-1 inhibitor) restrained M.tb-induced CTGF expression. M.tb also induced increases in AP-1 luciferase activity and DNA binding activity of c-Jun and c-Fos on the CTGF promoter. Furthermore, the knockdown of c-Jun by small interfering RNA attenuated M.tb-induced CTGF expression and AP-1 luciferase activity. A JNK inhibitor (SP600125) and a JNK dominant-negative mutant suppressed M.tb-induced CTGF expression. We also discovered that M.tb could induce the phosphorylation of JNK and c-Jun. Furthermore, SP600125 inhibited M.tb-induced c-Jun phosphorylation and AP-1- luciferase activity. M.tb-induced fibronectin expression was inhibited by anti-CTGF antibody. These results demonstrate that M.tb is activated through TLR2 to induce JNK activation, further increasing the DNA binding activity of c-Jun and c-Fos and finally inducing CTGF expression and extracellular matrix production.-Lee, H.-S., Hua, H.-S., Wang, C.-H., Yu, M.-C., Chen, B.-C., Lin, C.-H. Mycobacterium tuberculosis induces connective tissue growth factor expression through the TLR2-JNK-AP-1 pathway in human lung fibroblasts.

Activin a promotes myofibroblast differentiation of endometrial mesenchymal stem cells via STAT3-dependent Smad/CTGF pathway

BackgroundEndometriosis, characterized by the presence of functional endometrial tissues outside the uterus, is one of the most common gynecological disorders. Endometrial mesenchymal stem cells (MSCs) are crucial for the occurrence and development of endometriosis. Ectopic endometrial MSCs exist in the peritoneal cavity. Thus, the bioactive factors in endometriotic peritoneal fluid may regulate the biological behaviors of endometrial MSCs.MethodsIn this study, after assessing the concentration of Activin A in peritoneal fluid using ELISA, we isolated and cultured endometrial MSCs and investigated whether Activin A stimulated endometrial MSCs to differentiate into myofibroblasts and clarified the underlying mechanisms by quantitative real-time PCR, Western blot analysis, immunofluorescent staining, RNA interference and Chromatin immunoprecipitation. We also employed the inhibitors of Activin A to explore the possibility of suppressing the development of fibrosis in endometriosis using primary endometrial MSCs cultures and a mouse model of endometriosis.ResultsHere, we revealed that Activin A significantly elevated in endometriotic peritoneal fluid and activin receptor-like kinase (ALK4), the specific receptor for Activin A, obviously enhanced in ectopic endometrial MSCs compared with eutopic endometrial MSCs from women with or without endometriosis. Next, we found that Activin A drived myofibroblast differentiation of endometrial MSCs, with extremely enhanced expression of connective tissue growth factor (CTGF). CTGF was shown to be required for Activin A-induced expression of ACTA2, COL1A1 and FN1 in endometrial MSCs. CTGF induction by Activin A in endometrial MSCs involved the activation of Smad2/3, as evidenced by the phosphorylation and nuclear translocation of Smad2/3 as well as the binding of Smad2/3 to CTGF promoter. Furthermore, Smad/CTGF pathway in endometrial MSCs required activation of STAT3 while independent of PI3K, JNK and p-38 pathways. In addition, we also demonstrated that inhibition of Activin A pathway impeded myofibroblast differentiation of endometrial MSCs and ameliorated fibrosis in endometriosis mice.ConclusionsActivin A promotes myofibroblast differentiation of endometrial mesenchymal stem cells via STAT3-dependent Smad/CTGF pathway. The results provided the first evidence that STAT3 acted as a crucial Activin A downstream mediator to regulate CTGF production. Our data may supplement the stem cell theory of endometriosis and provide the experimental basis to treat endometriosis-associated fibrosis by manipulating Activin A signaling.

MiR-221 stimulates breast cancer cells and cancer-associated fibroblasts (CAFs) through selective interference with the A20/c-Rel/CTGF signaling

BackgroundMicroRNA (miRNAs) are non-coding small RNA molecules that regulate gene expression by inhibiting the translation of target mRNAs. Among several dysregulated miRNAs in human cancer, the up-regulation of miR-221 has been associated with development of a variety of hematologic and solid malignancies. In this study, we investigated the involvement of miR-221 in breast cancer.MethodsTaqMan microRNA assay was used to detect the miR-221 levels in normal cells and in MDA-MB 231 and SkBr3 breast cancer cells as well as in main players of the tumor microenvironment, namely cancer-associated fibroblasts (CAFs). miR-221 mimic sequence and locked nucleic acid (LNA)-i-miR-221 construct were used to induce or inhibit, respectively, the miR-221 expression in cells used. Quantitative PCR and western blotting analysis were performed to evaluate the levels of the miR-221 target gene A20 (TNFAIP3), as well as the member of the NF-kB complex namely c-Rel and the connective tissue growth factor (CTGF). Chromatin immunoprecipitation (ChIP) assay was performed to ascertain the recruitment of c-Rel to the CTFG promoter. Finally, the cell growth and migration in the presence of LNA-i-miR-221 or silencing c-Rel and CTGF by specific short hairpin were assessed by cell count, colony formation and boyden chambers assays. Statistical analysis was performed by ANOVA.ResultsWe first demonstrated that LNA-i-miR-221 inhibits both endogenous and ectopic expression of miR-221 in our experimental models. Next, we found that the A20 down-regulation, as well as the up-regulation of c-Rel induced by miR-221 were no longer evident using LNA-i-miR-221. Moreover, we established that the miR-221 dependent recruitment of c-Rel to the NF-kB binding site located within the CTGF promoter region is prevented by using LNA-i-miR-221. Furthermore, we determined that the up-regulation of CTGF mRNA and protein levels by miR-221 is no longer evident using LNA-i-miR221 and silencing c-Rel. Finally, we assessed that cell growth and migration induced by miR-221 in MDA-MB 231 and SkBr3 breast cancer cells as well as in CAFs are abolished by LNAi-miR-221 and silencing c-Rel or CTGF.ConclusionsOverall, these data provide novel insights into the stimulatory action of miR-221 in breast cancer cells and CAFs, suggesting that its inhibition may be considered toward targeted therapeutic approaches in breast cancer patients.

Unique mechanisms of connective tissue growth factor regulation in airway smooth muscle in asthma: Relationship with airway remodelling.

Neovascularization, increased basal membrane thickness and increased airway smooth muscle (ASM) bulk are hallmarks of airway remodelling in asthma. In this study, we examined connective tissue growth factor (CTGF) dysregulation in human lung tissue and animal models of allergic airway disease. Immunohistochemistry revealed that ASM cells from patients with severe asthma (A) exhibited high expression of CTGF, compared to mild and non‐asthmatic (NA) tissues. This finding was replicated in a sheep model of allergic airways disease. In vitro, transforming growth factor (TGF)‐β increased CTGF expression both in NA‐ and A‐ASM cells but the expression was higher in A‐ASM at both the mRNA and protein level as assessed by PCR and Western blot. Transfection of CTGF promoter‐luciferase reporter constructs into NA‐ and A‐ASM cells indicated that no region of the CTGF promoter (−1500 to +200 bp) displayed enhanced activity in the presence of TGF‐β. However, in silico analysis of the CTGF promoter suggested that distant transcription factor binding sites may influence CTGF promoter activation by TGF‐β in ASM cells. The discord between promoter activity and mRNA expression was also explained, in part, by differential post‐transcriptional regulation in A‐ASM cells due to enhanced mRNA stability for CTGF. In patients, higher CTGF gene expression in bronchial biopsies was correlated with increased basement membrane thickness indicating that the enhanced CTGF expression in A‐ASM may contribute to airway remodelling in asthma.

MEKK1, JNK, and SMAD3 mediate CXCL12-stimulated connective tissue growth factor expression in human lung fibroblasts

BackgroundIn idiopathic pulmonary fibrosis, the interaction of CXCL12 and CXC receptor 4 (CXCR4) plays a critical role in lung fibrosis. Connective tissue growth factor (CTGF) overexpression underlies the development of pulmonary fibrosis. Our previous report showed that the Rac1-dependent extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein (AP)-1 pathways are involved in CXCL12-generated CTGF expression in human lung fibroblasts (WI-38). In present study, we additionally inspected the involvement of mitogen-activated protein kinase kinase kinase 1 (MEKK1)/JNK-dependent SMAD3 in CXCL12-triggered CTGF expression in WI-38 cells.MethodsWI-38 cells were stimulated with CXCL12 in the absence or presence of specific inhibitors or small interfering RNAs (siRNAs). CTGF expression and signaling transduction molecules were assessed by Western blot, luciferase activity assay, or ChIP assay.ResultsCXCL-12-induced CTGF expression was attenuated by SIS3 (a SMAD3 inhibitor) and SMAD3 siRNA, but not by SB431542 (an activin receptor-like kinase 5, ALK5, inhibitor). CXCL12-stimulated CTGF expression was also attenuated by MEKK1 siRNA. Treatment of cells with CXCL12 caused an increase in SMAD3 phosphorylation at Ser208, translocation to nuclei, SMAD3-luciferase activity, and recruitment of SMAD3 to the CTGF promoter. Stimulation of cells with CXCL12 resulted in increase in JNK phosphorylation at Thr183/Tyr185 and MEKK1 phosphorylation at Thr261. Moreover, CXCL12-mediated SMAD3 phosphorylation or SMAD3-luciferase activity was inhibited by MEKK1 siRNA or SP600125. Finally, CXCL12-mediated JNK phosphorylation was attenuated by MEKK1 siRNA.ConclusionIn conclusion, results of this study suggest that CXCL12 activates the MEKK1/JNK signaling pathway, which in turn initiates SMAD3 phosphorylation, its translocation to nuclei, and recruitment of SMAD3 to the CTGF promoter, which ultimately induces CTGF expression in human lung fibroblasts.

KLF 15 Works as an Early Anti-Fibrotic Transcriptional Regulator in Ang II-Induced Renal Fibrosis via Down-Regulation of CTGF Expression

Background/Aims: Angiotensin II (Ang II) has been regarded as an important profibrogenic cytokine in renal fibrosis. Krüppel-like factor 15 (KLF15) has been identified as an important negative transcription factor in renal fibrosis. However, little is known about the role of KLF15 in Ang II-induced renal fibrosis. Methods: In this study, we randomized mice into a control group, Ang II group or Ang II plus losartan group. KLF15 expression was examined with real-time PCR and immunofluorescence in these groups. In vitro, KLF15 expression was examined by Western blot in rat renal fibroblasts (NRK-49F) stimulated with Ang II, and the effect of altered KLF15 expression on the regulation of the profibrotic factor connective tissue growth factor (CTGF) was further explored with co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) analyses. Results: Compared with the control group, the murine model of Ang II-induced renal fibrosis demonstrated a significant decrease in renal KLF15 expression at 4 weeks and presented with progressive renal fibrosis at 6 weeks. Meanwhile, losartan, an angiotensin type 1 (AT1) receptor antagonist, effectively prevented the down-regulation of KLF15 expression induced by Ang II infusion. In vitro, NRK-49F cells stimulated with Ang II exhibited a significant decrease in KLF15 expression, accompanied by a marked increase in the expression of profibrotic factors and in the production of extracellular matrix. The up-regulation of CTGF expression induced by Ang II stimulation was inhibited by KLF15 overexpression in NRK-49F cells, and losartan treatment prevented the down-regulation of KLF15 expression and the up-regulation of CTGF expression induced by Ang II stimulation. Furthermore, CoIP and ChIP assays revealed that the transcription regulator KLF15 directly bound to the co-activator P/CAF and repressed its recruitment to the CTGF promoter. Conclusions: Ang II down-regulates KLF15 expression via the AT1 receptor, and KLF15 is likely to inhibit Ang II-induced CTGF expression by repressing the recruitment of the co-activator P/CAF to the CTGF promoter.

High glucose-induced cytoplasmic translocation of Dnmt3a contributes to CTGF hypo-methylation in mesangial cells.

Connective tissue growth factor (CTGF) plays an essential role in the pathogenesis of diabetic nephropathy and we have previously identified that high glucose induced the expression of CTGF by decreasing DNA methylation. The aim of the present study was to investigate the underlying mechanisms of the high glucose-induced CTGF hypo-methylation. Human glomerular mesangial cells (hMSCs) were treated with low glucose (5 mM), mannitol (30 mM) or high glucose (30 mM) respectively. Immunofluorescence staining, real-time quantitative PCR and western blotting were performed to determine the subcellular distribution and expression of CTGF and Dnmt3a. ChIP-PCR assay was applied to investigate the capability of Dnmt3a to bind the CpG island of CTGF. Our results showed that high glucose induced both mRNA and protein expressions of CTGF, and led to increased cytoplasmic translocation of Dnmt3a in cultured hMSCs. The nuclear Dnmt3a protein was significantly reduced after high glucose treatment, although the expression of total Dnmt3a protein was not altered. We further discovered that ERK/MAPK signalling contributed to the high glucose-induced cytoplasmic translocation of Dnmt3a. Consequently, less Dnmt3a protein was bound to the CpG island of CTGF promoter, which induced an increase in CTGF expression by epigenetic regulation in the presence of high glucose. In conclusion, high glucose induces cytoplasmic translocation of Dnmt3a, possibly through activating ERK/MAPK signalling pathway, which contributes to the decreased binding of Dnmt3a on CTGF promoter and the subsequent CTGF hypo-methylation in diabetic nephropathy.

Chemical chaperon 4-phenylbutyrate protects against the endoplasmic reticulum stress-mediated renal fibrosis in vivo and in vitro.

Renal tubulointerstitial fibrosis is the common and final pathologic change of kidney in end-stage renal disease. Interesting, endoplasmic reticulum (ER) stress is known to contribute to the pathophysiological mechanisms during the development of renal fibrosis. Here, we investigated the effects of chemical chaperon sodium 4-phenylbutyrate (4-PBA) on renal fibrosis in vivo and in vitro. In a rat unilateral ureteral obstruction (UUO) model, 4-PBA mimicked endogenous ER chaperon in the kidneys and significantly reduced glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), activating transcription factor 4 (ATF4), and phosphorylated JNK protein expressions as well as restored spliced X-box-binding protein 1 (XBP1) expressions in the kidneys of UUO rats. 4-PBA also attenuated the increases of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) protein expressions, tubulointerstitial fibrosis, and apoptosis in the kidneys of UUO rats. Moreover, transforming growth factor (TGF)-β markedly increased ER stress-associated molecules, profibrotic factors, and apoptotic markers in the renal tubular cells (NRK-52E), all of which could be significantly counteracted by 4-PBA treatment. 4-PBA also diminished TGF-β-increased CTGF promoter activity and CTGF mRNA expression in NRK-52E cells. Taken together, our results indicated that 4-PBA acts as an ER chaperone to ameliorate ER stress-induced renal tubular cell apoptosis and renal fibrosis.

A promoter polymorphism -945C>G in the connective tissue growth factor in heart failure patients with mechanical circulatory support: a new marker for bridge to recovery?

Mechanical circulatory support (MCS) creates improvement of cardiac function in a small portion of patients with idiopathic dilated cardiomyopathy (iDCM). Among other factors, cardiomyocyte hypertrophy seems to represent an important prerequisite for MCS-related cardiac recovery. We have previously shown that connective tissue growth factor (CTGF) leads to adaptive cardiomyocyte hypertrophy associated with a protective cardiac function in transgenic mice. To test whether a functional genetic variant in the CTGF promoter impacts MCS-related cardiac recovery, three groups of iDCM patients with and without cardiac recovery on MCS were genotyped. The CTGF promoter variant (c.-945C>G) was analysed in 314 patients with iDCM receiving medical treatment only (Group I). Forty-nine iDCM patients who were either weaned from MCS for more than 6 months (Group II; n=20) or bridged to cardiac transplantation (Group III: n=29) were also genotyped. Patients on MCS were followed up for at least 12 months. Clinical characteristics and outcome on MCS were correlated with the respective genotypes. The c.-945C>G allele frequencies in 314 iDCM patients (Group I) were similar to controls deposited in the HapMap database or those published in a recent study. There were no differences in allele prevalence between patients with mild to moderate iDCM (Group I) compared with patients with severe iDCM requiring MCS (Groups II and III). Intriguingly, 50% of patients who were weaned from MCS (Group II) were homozygous for the G allele compared with only 17.2% of patients included in Group III, which is a significant difference (P=0.03). Homozygosity of the promoter-activating G allele in the CTGF_c.-945C>G variant is overrepresented in patients with cardiac recovery on MCS when compared with iDCM patients without cardiac recovery. Further studies are needed to evaluate c.-945C>G as a genetic predictor for clinical outcome on MCS.

CXCL12 induces connective tissue growth factor expression in human lung fibroblasts through the Rac1/ERK, JNK, and AP-1 pathways.

CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression.

Conditional Knockout of CTGF Affects Corneal Wound Healing

This study aimed to elucidate the role of connective tissue growth factor (CTGF) in healthy eyes and wounded corneas of mice and rabbits. Conditional knockout mice were used to determine the role of CTGF in corneal healing. CTGF expression was determined using transgenic mice carrying CTGF promoter driven-eGFP, quantitative RT-PCR, and immunofluorescent staining. Mice that carried two floxed CTGF alleles and a Cre/ERT2 transgene under the control of human ubiquitin C (ubc) promoter were used to conditionally delete CTGF gene in a tamoxifen-inducible manner. Phototherapeutic keratectomy (PTK) was used to generate an acute corneal wound and corneal re-epithelialization was assessed by fluorescein staining. Connective tissue growth factor expression was found in multiple ocular tissues with relatively high levels in the corneal endothelium, lens subcapsular epithelium, and in the vasculature of the iris and retina. Wounded corneas responded with an immediate upregulation of CTGF in the epithelium at the wound margin and a sustained CTGF induction during re-epithelialization. At the onset of haze formation, CTGF protein becomes more focused in the basal epithelium. Deletion of the CTGF gene caused a 40% reduction (P < 0.01) in the cornea re-epithelialization rate in knockout mice compared with wild-type mice. Connective tissue growth factor is expressed in the naïve cornea, lens, iris, and retina, and is expressed immediately after epithelial injury. Loss of CTGF impairs efficient re-epithelialization of corneal wounds.