Induced Connective Tissue Growth Factor Expression Research Articles

CD248-expressing cancer-associated fibroblasts induce non-small cell lung cancer metastasis via Hippo pathway-mediated extracellular matrix stiffness.

Metastasis is a crucial stage in tumour progression, and cancer-associated fibroblasts (CAFs) support metastasis through their participation in extracellular matrix (ECM) stiffness. CD248 is a possible biomarker for non-small cell lung cancer (NSCLC)-derived CAFs, but its role in mediating ECM stiffness to promote NSCLC metastasis is unknown. We investigated the significance of CD248+ CAFs in activating the Hippo axis and promoting connective tissue growth factor (CTGF) expression, which affects the stromal collagen I environment and improves ECM stiffness, thereby facilitating NSCLC metastasis. In this study, we found that higher levels of CD248 in CAFs induced the formation of collagen I, which in turn increased extracellular matrix stiffness, thereby enabling NSCLC cell infiltration and migration. Hippo axis activation by CD248+ CAFs induces CTGF expression, which facilitates the formation of the collagen I milieu in the stromal matrix. In a tumour lung metastasis model utilizing fibroblast-specific CD248 gene knockout mice, CD248 gene knockout mice showed a significantly reduced ability to develop tumour lung metastasis compared to that of WT mice. Our findings demonstrate that CD248+ CAFs activate the Hippo pathway, thereby inducing CTGF expression, which in turn facilitates the collagen I milieu of the stromal matrix, which promotes NSCLC metastasis.

Mechanical stress-induced connective tissue growth factor plays a critical role in intestinal fibrosis in Crohn's-like colitis.

Crohn's disease (CD) is an inflammatory bowel disease characterized by transmural inflammation and intestinal fibrosis. Mechanisms of fibrosis in CD are not well understood. Transmural inflammation is associated with inflammatory cell infiltration, stenosis, and distention, which present mechanical stress (MS) to the bowel wall. We hypothesize that MS induces gene expression of profibrotic mediators such as connective tissue growth factor (CTGF), which may contribute to fibrosis in CD. A rodent model of CD was induced by intracolonic instillation of TNBS to the distal colon. TNBS instillation induced a localized transmural inflammation (site I), with a distended colon segment (site P) proximal to site I. We detected significant fibrosis and collagen content not only in site I but also in site P in CD rats by day 7. CTGF expression increased significantly in sites P and I, but not in the segment distal to the inflammation site. Increased CTGF expression was detected mainly in the smooth muscle cells (SMCs). When rats were fed exclusively with clear liquid diet to prevent mechanical distention in colitis, expression of CTGF in sites P and I was blocked. Direct stretch led to robust expression of CTGF in colonic SMC. Treatment of CD rats with anti-CTGF antibody FG-3149 reduced fibrosis and collagen content in both sites P and I and exhibited consistent trends toward normalizing expression of collagen mRNAs. In conclusion, our studies suggest that mechanical stress, by upregulating profibrotic mediators, i.e., CTGF, may play a critical role in fibrosis in CD.NEW & NOTEWORTHY We found that CTGF expression increased significantly not only in the inflammation site but in the distended segment proximal to inflammation in a rodent model of CD-like colitis. Release of mechanical distention prevented CTGF expression in CD rats, whereas direct stretch induced CTGF expression. Treatment with anti-CTGF antibody reduced fibrosis and collagen contents in CD rats. Thus, mechanical stress, via upregulating profibrotic mediators, i.e., CTGF, may play a critical role in fibrosis in CD.

Increased CTGF expression in alveolar epithelial cells by cyclic mechanical stretch: Its mechanism and the therapeutic effect of pirfenidone

The mechanisms of fibrosis onset and development remain to be elucidated. However, it has been reported that mechanical stretch promotes fibrosis in various organs and cells, and may be involved in the pathogenesis of pulmonary fibrosis. We demonstrated that ventilator-induced lung hyperextension stimulation in mice increased the expression of connective tissue growth factor (CTGF), a profibrotic cytokine, in lung tissue. Increased CTGF expression induced by cyclic mechanical stretch (CMS) was also observed in vitro using A549 human alveolar epithelial cells. Pathway analysis revealed that the induction of CTGF expression by CMS involved MEK phosphorylation. Furthermore, early growth response 1 (Egr-1) was identified as a transcription factor associated with CTGF expression. Finally, the antifibrotic drug pirfenidone significantly reduced CTGF expression, MEK phosphorylation, and Egr-1 levels induced by CMS. Thus, our results demonstrated that profibrotic cytokine CTGF induced by CMS may be a therapeutic target of pirfenidone.

Loss of connective tissue growth factor permits mesenchymal transition in ovarian cancer (260)

<b>Objectives:</b> Epithelial-mesenchymal transition (EMT) is a characteristic feature of ovarian cancer (OC) metastasis. Our study aimed to determine the role of Connective Tissue Growth Factor (CTGF) in EMT. <b>Methods:</b> R182 and R2615 are epithelial OC cell lines that are CK18+, b-catenin+, TWIST -, and vimentin+. Time-course experiments +/- Transforming Growth Factor-b (TGF-b) and a TGF-b inhibitor were performed to evaluate the timing of CTGF expression in EMT. R182 and R2615 CTGF knockouts (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector. Phenotypic characterization of wild types (WT) and CTGF KO cell lines were performed with the assays, including scratch wound, proliferation, and anoikis resistance assays. Scratch wound assays were treated with 50 ng/mL and 100 ng/mL concentrations of recombinant CTGF for 48 hours (hr). Cells were plated in triplicate, and confluence was measured. Proliferation was determined by cell count using Biotek Cytation over 72 hr. For anoikis resistance, cells were plated in triplicate in an ultra-low adhesive cell plate with cell growth media. Using Promega CellTiter assay, cell viability was quantified by absorbance at 490 nm at the following time points: 1hr, 24hr, 48hr, and 120hr. Western blots were performed to evaluate the expression of epithelial and mesenchymal markers. RNA sequence analysis was performed, and pathway analysis was evaluated using the iPathway guide. <b>Results:</b> TGF-b induced CTGF expression in both OC cell lines tested. CTGF was induced as early as 30 minutes post-treatment, continued to increase until 6hr, and expression was maintained until 24 hr. No CTGF expression was seen in the mesenchymal mR182 and mR2615 OC cell lines. CTGF KO was confirmed by western blot analysis and indel sequencing. CTGF KO had no effect on the expression of epithelial markers, b-catenin, CK18, and vimentin, but SNAIL, a mesenchymal marker, was induced. Loss of CTGF also had no effect on cell proliferation but promoted anoikis resistance. R182 CTGF KO cells displayed 80% viability when grown in detached conditions for 48hr compared to only 20% viability in WT cells. At 120hr, 30% of KO cells remain viable compared to <10% for WT cells. R2615 WT and KO cells demonstrated similar results. Finally, the scratch assay showed that CTGF KO cells decrease the time to confluence compared to WT. Administration of recombinant CTGF does not rescue the phenotype to WT levels.Fig. 1 <b>Conclusions:</b> CTGF is expressed early in epithelial OC cells during EMT and is regulated by TGF-b. Our data suggest that CTGF permits maintenance of an epithelial phenotype through inhibition of SNAIL expression, and loss of CTGF should be evaluated as a poor prognosis indicator in OC.

Kahweol, a Diterpenoid Molecule, Inhibits CTGF-Dependent Synthetic Phenotype Switching and Migration in Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) phenotype switching from contractile to synthetic is essential for proliferation and migration in vascular pathophysiology. Connective tissue growth factor (CTGF) is a matricellular protein involved in cell adhesion, migration, and proliferation. Kahweol, a diterpene molecule in arabica coffee beans, has been reported to have anti-inflammatory, antiproliferative, and apoptotic effects in many cells. However, in VSMCs, the effects of kahweol on CTGF activities have not been investigated. Thus, in this study, the effects and associated mechanisms of kahweol in CTGF-dependent phenotype switching and migration in VSMCs were examined. Experiments were performed on primary rat aortic smooth muscle cells and a rat VSMC line, A7r5. Western blot analysis was used to determine the protein levels. The mRNA levels of synthetic markers were measured by qRT-PCR. Migration of VSMCs was evaluated by wound healing and transwell assays. Kahweol reduced the angiotensin II (Ang II)-induced CTGF expression. Further, kahweol inhibited expressions of synthetic phenotype markers of VSMC. The kahweol-reduced synthetic marker protein levels were reversed by the administration of rCTGF. However, expressions of contractile phenotype markers of VSMC were not affected. Kahweol suppressed Ang II-stimulated VSMC migration. Moreover, kahweol downregulated Ang II-induced p-FAK, p-Erk, and Yes-associated protein (YAP) protein expressions. Taken together, in Ang II-stimulated VSMCs, kahweol inhibited CTGF-dependent synthetic phenotype switching and migration, with focal adhesion kinase (FAK), Erk, and YAP involved in the underlying mechanisms of the kahweol effects. These results suggest that kahweol has a potential as a therapeutic agent to inhibit CTGF, which is a molecular target in sclerogenic vascular disease.

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.

Transforming growth factor-β1 induces connective tissue growth factor expression and promotes peritoneal metastasis of gastric cancer.

Transforming growth factor-β1 (TGF-β1) is involved in human cancer development and progression. Nonetheless, the role of TGF-β1 as regards peritoneal metastasis of gastric cancer has not been completely characterized. In the present study, we investigated the exact role of TGF-β1 on peritoneal metastasis of gastric cancer. The results indicated that human peritoneal mesothelial cells (HPMCs) exposed to TGF-β1 or serum-free conditional medium (SF-CM) of SGC7901 that produced a large amount of TGF-β1 became exfoliated, apoptosis and exhibited signs of injury, and the tumor-mesothelial cell adhesion significantly increased. Connective tissue growth factor (CTGF) expression was also increased when HPMCs were exposed to TGF-β1 or SF-CM of SGC7901. However, these effects were significantly decreased when HPMCs were exposed to SF-CM of SGC7901-TGFβS, a TGF-β1 knockdown stable cell line. Animal studies revealed that nude mice injected with SGC7901-TGFβS cells featured a smaller number of peritoneal seeding nodules and lower expression of CTGF in ascites than the control cell lines. These findings suggest that TGF-β1 promotes peritoneal metastasis of gastric cancer and induces CTGF expression. Therefore, blockage of TGF-β1 or TGF-β1 signaling pathway might prevent and treat peritoneal metastasis of gastric cancer.

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.

Involvement of lncR-30245 in Myocardial Infarction–Induced Cardiac Fibrosis Through Peroxisome Proliferator-Activated Receptor-γ–Mediated Connective Tissue Growth Factor Signalling Pathway

BackgroundLong noncoding RNAs (lncRNAs) are emerging as important mediators of cardiac pathophysiology. The aim of the present study is to investigate the effects of lncR-30245, an lncRNA, on cardiac fibrogenesis and the underlying mechanism. MethodsMyocardial infarction (MI) and transforming growth factor (TGF)-β1 were used to induce fibrotic phenotypes. Cardiac fibrosis was detected by Masson’s trichrome staining. Cardiac function was evaluated by echocardiography. Western blot, quantitative reverse transcription-polymerase chain reaction, and pharmacological approaches were used to investigate the role of lncR-30245 in cardiac fibrogenesis. ResultsExpression of lncR-30245 was significantly increased in MI hearts and TGF-β1–treated cardiac fibroblasts (CFs). LncR-30245 was mainly located in the cytoplasm. Overexpression of lncR-30245 promoted collagen production and CF proliferation. Knockdown of lncR-30245 significantly inhibited TGF-β1–induced collagen production and CF proliferation. LncR-30245 overexpression inhibited the antifibrotic role of peroxisome proliferator-activated receptor (PPAR)-γ and increased connective tissue growth factor (CTGF) expression, whereas lncR-30245 knockdown exerted the opposite effects. Rosiglitazone, a PPAR-γ agonist, significantly inhibited lncR-30245–induced CTGF upregulation and collagen production in CFs. In contrast, T0070907, a PPAR-γ antagonist, attenuated the inhibitory effects of lncR-30245 small interfering RNA (siRNA) on TGF-β1–induced CTGF expression and collagen production. LncR-30245 knockdown significantly enhanced ejection fraction and fractional shortening and attenuated cardiac fibrosis in MI mice. ConclusionOur study indicates that the lncR-30245/PPAR-γ/CTGF pathway mediates MI-induced cardiac fibrosis and might be a therapeutic target for various cardiac diseases associated with fibrosis.

Rho-Kinase Induces CTGF Expression through Actin Dynamics in Mesangial Cells

Introduction: Transforming growth factor (TGF)-β promotes the development of diabetic nephropathy by enhancing excessive production of extracellular matrix such as connective tissue growth factor (CTGF) in mesangial cells. However, the mechanism by which TGF-β induces CTGF expression has not been elucidated. In this study, we aimed to investigate contribution of small GTPase Rho and Rho-kinase to TGF-β-mediated CTGF expression in mesangial cells. Methods and Results: Exposure of mouse mesangial cells (MES-13) to TGF-β induced activation of Rho and Rho-kinase. Y-27632, a specific Rho-kinase inhibitor, reduced TGF-β-mediated CTGF protein expression, indicating that TGF-β induces CTGF expression via Rho-kinase activation. Western blot analysis demonstrated that Y-27632 attenuates TGF-β-mediated phosphorylation of Erk1/2 and JNK/SAPK and nuclear translocation of NF-κB. Rho-kinase has been shown to regulate cytoskeleton reorganization. We therefore investigated whether Rho-kinase is involved in actin dynamics-induced NF-κB activation. Immunostaining studies demonstrated that Y-27632 and Cytochalasin D, an inhibitor of actin polymerization, attenuated TGF-β-mediated nuclear translocation of NF-κB, indicating that Rho-kinase-mediated actin reorganization regulates NF-κB activation. Furthermore, siRNA-mediated knockdown of Rho-kinase isoforms demonstrated ROCK2 but not ROCK1 is involved in these observations. In the present study, we demonstrate for the first time that Rho-kinase, especially ROCK2, mediates TGF-β-induced expression of CTGF via phosphorylation of Erk1/2 and JNK/SAPK and subsequent actin reorganization-mediated nuclear translocation of NF-κB in mesangial cells. Conclusion: Rho-kinase could be an important therapeutic target against diabetic nephropathy. Disclosure Y. Nagai: None. D. Kawanami: None. K. Matoba: None. Y. Takeda: None. T. Akamine: None. S. Ishizawa: None. Y. Kanazawa: None. T. Yokota: None. K. Utsunomiya: None.

Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells.

Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate (S1P), which acts as a key regulator of inflammatory and fibrotic reactions, mainly via S1P receptor activation. Here, we show that in the human renal proximal tubular epithelial cell line HK2, the profibrotic mediator transforming growth factor β (TGFβ) induces SK-1 mRNA and protein expression, and in parallel, it also upregulates the expression of the fibrotic markers connective tissue growth factor (CTGF) and fibronectin. Stable downregulation of SK-1 by RNAi resulted in the increased expression of CTGF, suggesting a suppressive effect of SK-1-derived intracellular S1P in the fibrotic process, which is lost when SK-1 is downregulated. In a further approach, the S1P transporter Spns2, which is known to export S1P and thereby reduces intracellular S1P levels, was stably downregulated in HK2 cells by RNAi. This treatment decreased TGFβ-induced CTGF and fibronectin expression, and it abolished the strong induction of the monocyte chemotactic protein 1 (MCP-1) by the pro-inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1β. Moreover, it enhanced the expression of aquaporin 1, which is an important water channel that is expressed in the proximal tubules, and reverted aquaporin 1 downregulation induced by IL-1β/TNFα. On the other hand, overexpression of a Spns2-GFP construct increased S1P secretion and it resulted in enhanced TGFβ-induced CTGF expression. In summary, our data demonstrate that in human renal proximal tubular epithelial cells, SK-1 downregulation accelerates an inflammatory and fibrotic reaction, whereas Spns2 downregulation has an opposite effect. We conclude that Spns2 represents a promising new target for the treatment of tubulointerstitial inflammation and fibrosis.

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.

Differential expression of transforming growth factor-beta1, connective tissue growth factor, phosphorylated-SMAD2/3 and phosphorylated-ERK1/2 during mouse tooth development.

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta 1 (TGF-β1) and TGF-β1-induced CTGF expression is regulated through SMAD and mitogen-activated protein kinase (MAPK) signaling pathways. The fine modulation of TGF-β1 signaling is very important to the process of tooth development. However, little is known about the localization of CTGF, MAPK and SMAD in the context of TGF-β1 signaling during odontogenesis. Hence, we aimed to investigate the expression of TGF-β1, CTGF, phosphorylated-SMAD2/3 (p-SMAD2/3) and phosphorylated-ERK1/2 (p-ERK1/2). ICR mice heads of embryonic (E) day 13.5, E14.5, E16.5, postnatal (PN) day 0.5 and PN3.5 were processed for immunohistochemistry. Results revealed that at E13.5, TGF-β1 and CTGF were strongly expressed in dental epithelium (DE) and dental mesenchyme (DM), while p-SMAD2/3 was intensely expressed in the internal side of DE. p-ERK1/2 was not present in DE or DM. At E14.5 and E16.5, strong staining for TGF-β1 and CTGF was detected in enamel knot (EK) and dental papilla (DPL). DPL was intensely stained for p-ERK1/2 but negatively stained for p-SMAD2/3. There was no staining for p-SMAD2/3 and p-ERK1/2 in EK. At PN0.5 and PN3.5, moderate to intense staining for TGF-β1 and CTGF was evident in preameloblasts (PA), secretary ameloblasts (SA) and dental pulp (DP). p-SMAD2/3 was strongly expressed in SA and DP but sparsely localized in PA. p-ERK1/2 was intensely expressed in DP, although negative staining was observed in PA and SA. These data demonstrate that TGF-β1 and CTGF show an identical expression pattern, while p-SMAD2/3 and p-ERK1/2 exhibit differential expression, and indicate that p-SMAD2/3 and p-ERK1/2 might play a regulatory role in TGF-β1 induced CTGF expression during tooth development.

Angiotensin II induces connective tissue growth factor expression in human hepatic stellate cells by a transforming growth factor \u03b2-independent mechanism

Angiotensin II (Ang II) promotes hepatic fibrosis by increasing extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF) plays a crucial role in the pathogenesis of hepatic fibrosis and emerges as downstream of the profibrogenic cytokine transforming growth factor-β (TGF-β). We aimed to investigate the molecular events that lead from the Ang II receptor to CTGF upregulation in human hepatic stellate cells, a principal fibrogenic cell type. Ang II produced an early, AT1 receptor-dependent stimulation of CTGF expression and induced a rapid activation of PKC and its downstream p38 MAPK, thereby activating a nuclear factor-κB (NF-κB) and Smad2/3 cross-talk pathway. Chemical blockade of NF-κB and Smad2/3 signaling synergistically diminished Ang II-mediated CTGF induction and exhibited an additive effect in abrogating the ECM accumulation caused by Ang II. Furthermore, we demonstrated that CTGF expression was essential for Ang II-mediated ECM synthesis. Interestingly, the ability of dephosphorylated, but not phosphorylated JNK to activate Smad2/3 signaling revealed a novel role of JNK in Ang II-mediated CTGF overexpression. These results suggest that Ang II induces CTGF expression and ECM accumulation through a special TGF-β-independent interaction between the NF-κB and Smad2/3 signals elicited by the AT1/PKCα/p38 MAPK pathway.

Kahweol decreases hepatic fibrosis by inhibiting the expression of connective tissue growth factor via the transforming growth factor-beta signaling pathway.

Kahweol is a diterpene molecule found in Coffea Arabica beans. Previous studies have shown that coffee reduces liver fibrosis, but it is not clear which component of coffee has the protective effect. In this study, we examined whether kahweol has a protective effect on hepatic fibrosis in vivo and in vitro. Kahweol decreased hepatic fibrosis by inhibiting connective tissue growth factor (CTGF) expression in thioacetamide (TAA)-treated mice. The expression of phospho-Smad3, signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal protein kinase (JNK) increased in the livers of TAA-treated mice and decreased in the kahweol-treated group. Kahweol significantly decreased the expression of transforming growth factor beta (TGF-β)-stimulated type I collagen and CTGF expression in vitro. In addition, kahweol significantly decreased the expression of Smad3, STAT3, ERK and JNK, which are involved in the induction of CTGF expression by TGF-β in hepatocytes, but not in HSCs. These results suggest that kahweol may be a new candidate for treatment of liver fibrosis.

NADPH oxidase 4 contributes to connective tissue growth factor expression through Smad3-dependent signaling pathway

Transforming growth factor-β (TGF-β)/Smad signaling has been implicated in connective tissue growth factor (CTGF) expression in vascular smooth muscle cells (VSMC). Reactive oxygen species (ROS) are involved in activation of TGF-β/Smad signaling. However, detailed mechanisms underlying the process remain unclear. In present study, we demonstrated TGF-β1 strongly induced CTGF expression, Smad3 activation, NADPH oxidase 4 (Nox4) expression and increased ROS production in primary rat VSMC in vitro. NADPH oxidases inhibitor diphenylene iodonium (DPI) eliminated TGF-β1-induced CTGF expression and ROS generation. In addition, small-interfering RNA (siRNA) silencing of Smad3 or Nox4 significantly suppressed TGF-β1-mediated CTGF expression in VSMC. Furthermore, Nox4 silencing or inhibition eliminated TGF-β1-induced Smad3 activation and interaction between Nox4 and Smad3. In vivo studies further identified a positive correlation of Nox4 levels with Smad3 activation and CTGF expression in atherosclerotic arteries of patients and animal models. These data established that a novel mechanistic link of Nox4-dependent activation of Smad3 to increased TGF-β1-induced CTGF in the process of vascular remodeling, which suggested a new potential pathway for therapeutic interventions.

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.

Serum Biomarkers as Predictors of Stage of Work-related Musculoskeletal Disorders

Musculoskeletal disorders (MSDs) are a leading worldwide cause of long-term pain and physical disability,1 with diagnoses including tendinopathies, nerve compression syndromes, and muscular and joint disorders.2,3 Studies in people with upper extremity work-related MSDs (WMSDs) find evidence of inflammation, fibrosis, and degeneration in serum and musculotendinous tissues, although the timing of each is unknown.4-7 Serum biomarkers that might aid in pinpointing the stage of these disorders are being investigated. Several risk factors have been identified, including forceful exertions, repetitive motion, and nonneutral body postures. A recent systematic review showed a consistent pattern of force-repetition interaction for musculoskeletal disorder risk, with low-force repetitive tasks demonstrating a modest increase in MSD risk, whereas high-force repetitive tasks result in rapid escalation in MSD risk, which is indicative of tissue fatigue failure.8 We have developed a rat model of voluntary reaching and handlepulling for food reward,9 in which reach rates and force levels were determined from epidemiologic studies. One goal of our laboratory is to identify biomarkers for monitoring disease progression of WMSDs and appropriate targeting of treatments. We recently examined whether serum inflammatory cytokines exhibit force × repetition interaction responses using this rat model.10 Serum tumor necrosis factor-α (TNF-α) shows a significant force × repetition interaction (P = 0.0003) with task performance (Figure 1, A). No increases in TNF-α were seen in low-force groups, but high increases were observed in rats performing high-repetition, high-force tasks.11-13 These results indicate that serum TNF-α follows the fatigue-failure theory during acute phases of ≤3 months. TNF-α and related cytokines may provide the best gauge of overall acute tissue damage resulting from repetitive, forceful exertions and may be the best biomarkers of this phase of WMSDs.Figure 1: Graph of serum biomarkers in a rat model of upper extremity work-related musculoskeletal disorders. A, Serum levels of tumor necrosis factor (TNF)-α in rats performing one of four tasks for 12 weeks. B, Schematic illustration showing temporal changes in serum levels of TNF-α and interleukin-10 (IL-10) (left axis) and connective tissue growth factor (CTGF) (right axis) in rats performing high-repetition, low-force (HRLF) tasks for 24 weeks. HRHF = high-repetition, high-force; LRHF = low-repetition, high-force; LRLF = low-repetition, low-forceThe timing of the inflammatory versus fibrotic responses with WMSDs is also of clinical interest. Studies have detected serum biomarkers of inflammation in patients with upper extremity WMSDs of short duration (≤3 months), including TNF-α,4-6 again suggesting a role for inflammatory cytokines early in the course of upper extremity WMSDs. However, studies examining tissues from patients with upper extremity WMSDs during surgical intervention show increased tissue fibrogenic proteins and fibrotic histopathology, which is indicative of deranged extracellular matrix production and degeneration in tissues by this time point.7 Therefore, we extended our rat studies to examine the effects of performing a high-repetition, low-force task for 24 weeks. Serum TNF-α levels increased early after training but declined by week 18 (Figure 1, B). In contrast, serum interleukin-10 (IL-10), an anti-inflammatory cytokine, increased steadily until week 18. This may be one reason for the decreasing TNF-α because IL-10 plays a key role in limiting immune responses. We next examined serum for connective tissue growth factor (CTGF), an important mediator of fibrosis. Studies have shown that CTGF increases in tissues under conditions of overload or injury and that it is a key player in the pathogenesis of fibrosis.14 We observed increased serum and tissue CTGF by week 24, compared with controls (P = 0.03) (Figure 1, B). It is known that transforming growth factor-β1 induces CTGF expression, leading to fibroblast proliferation and collagen deposition.14 However, CTGF production is also regulated by TNF-α,15 which is an interesting fact considering their temporal relationship (Figure 1, B). Importantly, because the CTGF level is normally low in sera of healthy individuals, it may serve as a predictive biomarker for patients in the fibrotic stage of WMSDs. Our results indicate that most serum inflammatory cytokines, including TNF-α, demonstrate force-repetition interactions. These findings support the use of key pro-inflammatory cytokines as biomarkers of acute tissue damage and the fatigue failure hypothesis as a mechanism underlying WMSDs. We have also observed significantly increased serum and tissue CTGF levels that correlated with tissue fibrosis. Additional studies are underway to determine whether elevated CTGF is an essential mediator of fibrotic events. If so, one could envision CTGF as a potential therapeutic target to prevent fibrosis and reduced function and as a serum biomarker of tissue fibrogenic changes occurring with WMSDs.

Abstract 316: A Potential Mechanism Involving mTOR For The Expression Of CTGF In Agonist-stimulated Adult Cardiomyocytes

Cardiac hypertrophy ensues as a response to multiple stimuli, such as mechanical stress, neurohumoral activation, growth factors and cytokines. Connective Tissue Growth Factor (CTGF), a potent fibrogenic cytokine, regulates a wide range of biological functions including ECM deposition, wound repair, angiogenesis, migration, differentiation, survival and proliferation. While CTGF overexpression in fibroblasts has been shown to be responsible for fibrosis in various organs, controversy exists about the source of CTGF. Since interstitial fibrosis contributes to ventricular wall stiffness and impairs diastolic function, understating how key factors such as CTGF are expressed and released for the genesis of fibrosis in the hypertrophying heart is important to develop new treatment options. To this end, we explored the signaling pathway(s) involved in the phenylephrine (PE), a hypertrophic agonist, induced expression of CTGF by cardiomyocytes (CMs). Since mammalian target of rapamycin (mTOR) is reported to regulate PE-induced hypertrophic signaling, we hypothesize that mTOR plays a role in PE induced CTGF expression in CMs. To test if CMs produce CTGF, we treated adult feline CMs with phenylephrine. PE stimulated CTGF mRNA expression in a dose and time dependent manner. mTOR forms two distinct complexes, mTORC1 and mTORC2. Whereas both complexes are sensitive to a pharmacological inhibitor Torin1, only mTORC1 is sensitive to Rapamycin inhibition. Our results indicate that PE stimulated CTGF expression could be substantially enhanced by torin1 pretreatment of CMs. Moreover, shRNA mediated silencing of Rictor in CMs, one of the components of mTORC2, significantly augmented the PE induced CTGF mRNA expression. But mTORC1 inhibition using Rapamycin or activation of its downstream target S6K1 using Rapamycin resistant S6K1 adenovirus had no impact in PE -stimulated CTGF expression. The same trend was also observed in the level of secreted CTGF. In conclusion, these results strongly indicate that mTORC2 plays a repressive role in CTGF mRNA expression in adult CMs, and that the loss of such repression in PO myocardium might be a potential mechanism for the onset of cardiac fibrosis in hypertrophying myocardium.