Chronic kidney disease is reaching epidemic proportions worldwide and there is no effective treatment. Connective tissue growth factor (CCN2) has been suggested as a risk biomarker and a potential therapeutic target for renal diseases, but its specific receptor has not been identified. Epidermal growth factor receptor (EGFR) participates in kidney damage, but whether CCN2 activates the EGFR pathway is unknown. Here, we show that CCN2 is a novel EGFR ligand. CCN2 binding to EGFR extracellular domain was demonstrated by surface plasmon resonance. CCN2 contains four distinct structural modules. The carboxyl-terminal module (CCN2(IV)) showed a clear interaction with soluble EGFR, suggesting that EGFR-binding site is located in this module. Injection of CCN2(IV) in mice increased EGFR phosphorylation in the kidney, mainly in tubular epithelial cells. EGFR kinase inhibition decreased CCN2(IV)-induced renal changes (ERK activation and inflammation). Studies in cultured tubular epithelial cells showed that CCN2(IV) binds to EGFR leading to ERK activation and proinflammatory factors overexpression. CCN2 interacts with the neurotrophin receptor TrkA, and EGFR/TrkA receptor crosstalk was found in response to CCN2(IV) stimulation. Moreover, endogenous CCN2 blockade inhibited TGF-β-induced EGFR activation. These findings indicate that CCN2 is a novel EGFR ligand that contributes to renal damage through EGFR signalling.

Connective tissue growth factor (CCN2) is a 349-residue mosaic protein that contains four structural modules (modules 1-4), which are presumptive domains for interactions with regulatory binding proteins and receptors. Module 3, corresponding to residues 199-243, is a thrombospondin structural homology repeat (TSR) and is flanked by regions that are highly susceptible to proteolytic cleavage. To test whether CCN2 module 3 (CCN2(3)) has intrinsic biological properties, it was produced recombinantly in Escherichia coli (E. coli) and examined for its effects on the function of hepatic stellate cells (HSC), the principal fibrogenic cell type in the liver. CCN2(3) stimulated dose-dependent HSC adhesion and activity of p42/p44 mitogen activated protein kinase, the latter of which was antagonized by blocking the activity of focal adhesion kinase. HSC adhesion to immobilized CCN2(3) was attributed to binding interactions with cell surface integrin alpha6beta1. As assessed by RT-PCR or Western blotting, CCN2(3) stimulated production of fibronectin and pro-collagen type IV(alpha5), both of which are downstream components of HSC-mediated fibrogenesis and which are constituents of high density matrix in fibrotic lesions. These data show that while the full length CCN2 protein is strongly associated with fibrosis and stellate cell function, key integrinbinding properties, signaling, and fibrogenic pathways are exhibited by module 3 alone. These data indicate that module 3 of CCN2 is intrinsically active and suggest that liberation of module 3 following CCN2 proteolysis may contribute to HSC-mediated fibrogenesis, as well as other CCN2-dependent processes.

Fibrosis affects organs such as the skin, liver, kidney and lung and is a cause of significant morbidity. There is no therapy for fibrosis. Recent significant molecular insights into the signaling underlying fibrosis have been made. Transforming growth factor beta (TGF beta) signaling is a major contributor to fibrogenesis. The signaling mechanisms through which TGF beta induces fibrogenic responses have been under intense scrutiny. Moreover, the potent pro-fibrotic proteins endothelin-1 (ET-1) and CCN2 (connective tissue growth factor, CTGF) are believed to play an essential role in this process as downstream regulators or co-factors of TGF beta signaling. This review summarizes these recent crucial observations with emphasis on the disease scleroderma.

Adhesive signaling plays a key role in cellular differentiation, including in chondrogenesis. Herein, we probe the contribution to early chondrogenesis of two key modulators of adhesion, namely focal adhesion kinase (FAK)/Src and CCN2 (connective tissue growth factor, CTGF). We use the micromass model of chondrogenesis to show that FAK/Src signaling, which mediates cell/matrix attachment, suppresses early chondrogenesis, including the induction of Ccn2, Agc, and Sox6. The FAK/Src inhibitor PP2 elevates Ccn2, Agc, and Sox6 expression in wild-type mesenchymal cells in micromass culture, but not in cells lacking CCN2. Our results suggest a reduction in FAK/Src signaling is a critical feature permitting chondrogenic differentiation and that CCN2 operates downstream of this loss to promote chondrogenesis.

BACKGROUND

Connective tissue growth factor (CTGF) has been identified as a key factor in the pathogenesis of diseases with significant fibrosis-related complications such as hepatitis, diabetes and renal transplantation. Increasing evidence shows that CTGF levels in plasma, serum and urine have promising biomarker applicability in these disorders.

OBJECTIVE

To present an overview of current knowledge on CTGF in various patient populations and the technical aspects of CTGF measurement by enzyme-linked immunosorbent assay (ELISA).

METHODS

We performed a comprehensive literature search by using electronic bibliographic databases.

CONCLUSIONS

CTGF is associated with disease severity parameters and outcome in fibrotic disease and may have diagnostic and prognostic values. However, CTGF ELISA needs standardization.

OBJECTIVE

Transforming Growth Factor beta1 (TGF-beta1) and its downstream effector Connective Tissue Growth Factor (CTGF/CCN2), are well known fibrogenic activators and we previously showed that the Rho/ROCK pathway controls CTGF expression in intestinal smooth muscle cells isolated from patients with delayed radiation enteritis. The aim of the present work was to investigate the balance between Smad and Rho signalling pathways in the TGF-beta1 CTGF induction and modulation of radiation-induced fibrogenic differentiation after addition of pravastatin, an inhibitor of Rho isoprenylation.

METHODS

Primary human smooth muscle cells isolated from normal (N-SMC) or radiation enteritis (RE-SMC) biopsies were incubated with TGF-beta1 (10 ng/ml). Induction of CTGF, as well as nucleo-cytoplasmic distribution of phospho-Smad2/3, Smad2/3 and Smad4 were analysed by Western blot and immunocytochemistry. Smad DNA binding was assessed by EMSA and Rho activation was measured by pull-down assay.

RESULTS

After TGF-beta1 addition, Smads were translocated to the nucleus in both cell types. Nuclear accumulation of Smad as well as their DNA-binding activity were higher in N-SMC than in RE-SMC, whereas the opposite was observed for Rho activation, suggesting a main involvement of Rho pathway in sustained fibrogenic differentiation. This hypothesis was further supported by the antifibrotic effect observed in vitro after cell treatment with pravastatin (i.e. decreased expression of CTGF, TGF-beta1 and Collagen Ialpha2).

CONCLUSIONS

Our results suggest that TGF-beta1-induced CTGF transactivation mainly depends on the Smad pathway in N-SMC, whereas in RE-SMC, Smad and Rho pathways are involved. Inhibition of Rho activity by pravastatin alters fibrogenic differentiation in vitro which opens up new therapeutic perspectives.

Variant CCN proteins have been identified over the past decade in several normal and pathological situations. The production of CCN truncated proteins have been reported in the case of CCN2(ctgf), CCN3(nov), CCN4(wisp-1) and CCN6(wisp-3). Furthermore, the natural CCN5 is known to miss the C-terminal domain that is present in all other members of the CCN family of proteins. In spite of compelling evidence that assign important biological activities to these truncated CCN variants, their potential regulatory functions have only recently begun to be widely accepted. The report of CCN1(cyr61) intron 3 retention in breast cancer cells now confirms that, in addition to well documented post-translational processing of full length CCN proteins, alternative splicing is to be regarded as another effective way to generate CCN variants. These observations add to a previous bulk of evidence that support the existence of alternative splicing for other CCN genes. It has become clearly evident that we need to recognize these mechanisms as a means to increase the biological diversity of CCN proteins.

OBJECTIVE

Fibrotic diseases such as SSc (systemic sclerosis, scleroderma) are characterized by the abnormal presence of the myofibroblast, a specialized type of fibroblast that overexpresses the highly contractile protein α-smooth muscle actin. Myofibroblasts display excessive adhesive properties and hence exert a potent mechanical force. We aim to identify the precise contribution of adhesive signalling, which requires integrin-mediated activation of focal adhesion kinase (FAK)/src, to fibrogenic gene expression in normal and fibrotic SSc fibroblasts.

METHODS

We subject either FAK wild-type and knockout fibroblasts or normal and SSc fibroblasts treated with FAK/src inhibitors to real-time polymerase chain, western blot, cell migration and collagen gel contraction analyses.

RESULTS

FAK operates downstream of both integrin β1 and reactive oxygen species (ROS) to promote the expression of genes involved in matrix production and remodelling, including CCN2, α-smooth muscle actin and type I collagen. Blocking either FAK/src with PP2 or ROS with N-acetyl cysteine alleviates the elevated contractile and migratory capability of lesional SSc dermal fibroblasts.

CONCLUSIONS

Excessive adhesive signalling is intimately involved with the fibrotic phenotype of lesional SSc fibroblasts; blocking adhesive signalling or ROS generation may be beneficial in controlling the fibrosis observed in SSc.

BACKGROUND

Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.

RESULTS

Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.

CONCLUSIONS

Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.

Endothelin (ET)-1 is a vasoconstrictor involved in cardiovascular diseases. Connective tissue growth factor/CCN2 (CTGF) is a fibrotic mediator overexpressed in human atherosclerotic lesions, myocardial infarction, and hypertension. In different cell types CTGF regulates cell proliferation/apoptosis, migration, and extracellular matrix (ECM) accumulation and plays important roles in angiogenesis, chondrogenesis, osteogenesis, tissue repair, cancer and fibrosis. In the present study, we investigated the ET-1 signaling which triggers CTGF expression in cultured adult mouse atrial-muscle HL-1 cells used as a model system. ET-1 activated the CTGF promoter and induced CTGF expression at both mRNA and protein levels. Real-time PCR analysis revealed CTGF induction also in isolated rat heart preparations perfused with ET-1. Several intracellular signals elicited by ET-1 via ET receptors and even Epidermal Growth Factor Receptor (EGFR) contributed to the up-regulation of CTGF, including ERK activation and induction of the AP-1 components c-fos and c-jun, as also evaluated by ChIP analysis. Moreover, in cells treated with ET-1 the expression of ECM component decorin was abolished by CTGF silencing, indicating that CTGF is involved in ET-1 induced ECM accumulation not only in a direct manner but also through downstream effectors. Collectively, our data indicate that CTGF could be a mediator of the profibrotic effects of ET-1 in cardiomyocytes. CTGF inhibitors should be considered in setting a comprehensive pharmacological approach towards ET-1 induced cardiovascular diseases.

Key roles for connective tissue growth factor (CTGF/CCN2) are demonstrated in the wound repair process where it promotes myofibroblast differentiation and angiogenesis. Similar mechanisms are active in tumor-reactive stroma where CTGF is expressed. Other potential roles include prevention of hypoxia-induced apoptosis and promoting epithelial-mesenchymal transistion (EMT). CTGF expression in tumors has been associated to both tumor suppression and progression. For example, CTGF expression in acute lymphoblastic leukemia, breast, pancreas and gastric cancer correlates to worse prognosis whereas the opposite is true for colorectal, lung and ovarian cancer. This discrepancy is not yet understood. High expression of CTGF is a hallmark of ileal carcinoids, which are well-differentiated endocrine carcinomas with serotonin production originating from the small intestine and proximal colon. These tumors maintain a high grade of differentiation and low proliferation. Despite this, they are malignant and most patients have metastatic disease at diagnosis. These tumors demonstrate several phenotypes potentially related to CTGF function namely: cell migration, absent tumor cell apoptosis, as well as, reactive and well vascularised myofibroblast rich stroma and fibrosis development locally and in distal organs. The presence of CTGF in other endocrine tumors indicates a role in the progression of well-differentiated tumors.

Yes-associated protein (YAP) is a key transcriptional coactivator of Hippo pathway and has been shown to be an oncoprotein in ovarian cancer (OC). Verteporfin (VP), clinically used in photodynamic therapy for neovascular macular degeneration, has been recently proven to be a suppressor of YAP-TEAD complex and has shown potential in anticancer treatment. In this study, we aimed to explore the potential effect of VP in the treatment of OC. Our results showed that VP led to inhibition of proliferation in a time- and dose-dependent manner and to the suppression of migratory and invasive capacities of OC cells. Western blot and real-time polymerase chain reaction demonstrated that VP induced YAP cytoplasmic retention and deregulated inducible YAP and CCNs in OC cells. In vivo, VP exerted a significant effect on tumor growth in OVCAR8 xenograft mice, resulting in tumor nodules with lower average weight and reduced volume of gross ascites. In addition, VP treatment remarkably upregulated cytoplasmic YAP and phosphorylation YAP and downregulated CCN1 and CCN2, but exerted little effect on YAP-upstream components in Hippo pathway. In conclusion, our results suggested that VP may be a promising agent for OC, acting by suppressing YAP-TEAD complex.

Clinical and experimental studies have demonstrated that connective-tissue growth factor (CTGF) expression is increased in fibrotic human liver and experimental animal models of liver fibrogenesis. CTGF has been linked to transforming growth factor-beta (TGF-beta) pathways in fibroproliferative diseases and specific polymorphisms within the CTGF gene may predispose for fibrosis in systemic sclerosis. As CTGF is detectable in various human fluids (serum, plasma and urine), it may provide information about fibrotic remodelling processes and reflect hepatic TGF-beta bioactivity. We established a novel ELISA for the measurement of serum CTGF and tested its clinical value in patients with chronic hepatitis C virus (HCV) infection and chronic liver disease (CLD). HCV infected patients (n = 138) had significantly higher serum CTGF levels than healthy controls. CTGF was linked to the histological degree of liver fibrosis. To expand the results to other aetiologies, a separate cohort of CLD patients (n = 129) was evaluated, showing higher serum CTGF than healthy controls and again an association with advanced stages of liver cirrhosis (Child B and C). Although independent of the underlying aetiology, serum CTGF was most powerful in indicating fibrosis/advanced disease states in HCV-related disorders. The genotyping of six polymorphisms (rs6917644, rs9399005, rs6918698, rs9493150, rs2151532 and rs11966728) covering the CTGF locus in 365 patients suffering from chronic hepatitis C revealed that none of these polymorphisms showed a genotypic or allelic association with the severity of hepatic fibrosis. Taken together, serum CTGF is suitable for determination of hepatic fibrosis and most powerful in patients with chronic HCV infection.

Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor beta1 (TGF-beta1) where it acts as a downstream mediator of TGF-beta1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk, and Smad signaling for CTGF induction by TGF-beta1 in osteoblasts; however, the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-beta1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-beta1. Additionally, inhibiting Src activation prevented Erk activation, Smads 2 and 3 activation and nuclear translocation by TGF-beta1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway directly by mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF-beta1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. These data demonstrate that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-beta1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts.

The pressure-loaded right ventricle (RV) adversely affects left ventricular (LV) function. We recently found that these ventricular-ventricular interactions lead to LV myocardial fibrosis through transforming growth factor-β1 (TGF-β1) signaling. We investigated the mechanisms mediating biventricular fibrosis in RV afterload and their potential modification by angiotensin receptor blockade. An adjustable pulmonary artery band (PAB) was placed in rabbits. In sham-operated control rabbits, the band was left uninflated (n = 6). In the RV afterload group, the PAB was sequentially inflated to generate systemic RV pressure at 28 days (n = 8). In a third group, the PAB was inflated to systemic levels, and the angiotensin receptor blocker losartan was added (n = 6). Five weeks after surgery, the animals were killed for assessments of biventricular hypertrophy, fibrosis, apoptosis, and the components of their signaling pathways. PAB animals developed biventricular hypertrophy, fibrosis, and apoptosis, versus sham rabbits, in which these conditions were decreased with losartan. RV and LV TGF-β1, connective tissue growth factor (CTGF) (CCN2), endothelin-1 (ET-1), endothelin receptor B, and matrix metalloproteinase 2/9 mRNA levels were increased in PAB animals versus sham animals, and decreased with losartan. Given the marked biventricular CTGF up-regulation in PAB and down-regulation with losartan, we investigated CTGF signaling. RV and LV Smad 2/3/4 protein levels and LV RhoA mRNA levels were increased with PAB and reduced with losartan. In conclusion, isolated RV afterload induces biventricular fibrosis and apoptosis, which are reduced by angiotensin receptor blockade. Adverse ventricular-ventricular interactions induced by isolated RV afterload appear to be mediated through TGF-β1-CTGF and ET-1 pathways.

BACKGROUND

The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-beta (TGF-beta) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-beta, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-beta. To explore this notion, we characterized TGF-beta-induced activation of fibroblasts from CCN2-null (CCN2(-/-)) mouse embryos.

METHODS

The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-beta signal transduction and regulation of collagen gene expression were examined in CCN2(-/-) MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays.

RESULTS

Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2(-/-) MEFs was markedly reduced compared to wild type MEFs, TGF-beta-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2(-/-) MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2.

CONCLUSIONS

Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-beta-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.

The objective of the study was to examine the regulation of CCN2 by inflammatory cytokines, IL-1β, and TNF-α and to determine whether CCN2 modulates IL-1β-dependent catabolic gene expression in nucleus pulposus (NP) cells. IL-1β and TNF-α suppress CCN2 mRNA and protein expression in an NF-κB-dependent but MAPK-independent manner. The conserved κB sites located at -93/-86 and -546/-537 bp in the CCN2 promoter mediated this suppression. On the other hand, treatment of NP cells with IL-1β in combination with CCN2 suppressed the inductive effect of IL-1β on catabolic genes, including MMP-3, ADAMTS-5, syndecan 4, and prolyl hydroxylase 3. Likewise, silencing of CCN2 in human NP cells resulted in elevated basal expression of several catabolic genes and inflammatory cytokines like IL-6, IL-4, and IL-12 as measured by gene expression and cytokine protein array, respectively. Interestingly, the suppressive effect of CCN2 on IL-1β was independent of modulation of NF-κB signaling. Using disintegrins, echistatin, and VLO4, peptide inhibitors to αvβ3 and α5β1 integrins, we showed that CCN2 binding to both integrins was required for the inhibition of IL-1β-induced catabolic gene expression. It is noteworthy that analysis of human tissues showed a trend of altered expression of these integrins during degeneration. Taken together, these results suggest that CCN2 and inflammatory cytokines form a functional negative feedback loop in NP cells that may be important in the pathogenesis of disc disease.

Osteoclasts are unique cells responsible for the resorption of bone matrix. MicroRNAs (miRNAs) are involved in the regulation of a wide range of physiological processes. Here, we examined the role of miR-26a in RANKL-induced osteoclastogenesis. The expression of miR-26a was up-regulated by RANKL at the late stage of osteoclastogenesis. Ectopic expression of an miR-26a mimic in osteoclast precursor cells attenuated osteoclast formation, actin-ring formation, and bone resorption by suppressing the expression of connective tissue growth factor/CCN family 2 (CTGF/CCN2), which can promote osteoclast formation via up-regulation of dendritic cell-specific transmembrane protein (DC-STAMP). On the other hand, overexpression of miR-26a inhibitor enhanced RANKL-induced osteoclast formation and function as well as CTGF expression. In addition, the inhibitory effect of miR-26a on osteoclast formation and function was prevented by treatment with recombinant CTGF. Collectively, our results suggest that miR-26a modulates osteoclast formation and function through the regulation of CTGF.

BACKGROUND

Connective tissue growth factor (CCN2; CTGF) is a newly identified growth factor, which is involved in the regulation of wound repair and fibrosis. Because there is variation among individuals with respect to tissue response to injury, genetic factors might be involved in the final outcome of tissue repair or scarring. For example, polymorphisms in the promoter region of genes, such as those encoding transforming growth factor beta1 (TGF-beta1), interleukin 10 (IL-10), and tumour necrosis factor alpha (TNF-alpha), influence transcriptional responses and are thought to contribute to the dysregulation of these genes in pathological conditions.

OBJECTIVE

To investigate whether the promoter region of the ccn2 (ctgf) gene contains polymorphic sequences that might account for differential expression.

METHODS

Seventy seven human DNA samples were sequenced-45 were from healthy controls and 32 were from patients with ischaemic heart disease (IHD)-using M13 tailed sequence specific ccn2 (ctgf) primers for amplification of a 600 bp fragment upstream of the transcription start site. Amplicons were bidirectionally sequenced with a dye primer M13 forward and reverse sequencing kit.

RESULTS

A C to G substitution was identified at position -132 in one of the patients with IHD. Moreover, in five of the 32 patients with IHD and in six of the 45 healthy controls, a G to C polymorphism was found at position -447. These substitutions at -132 and -447 are thought to lie within predicted binding domains for the transcription factors Pbx-1 and MZF1, respectively. In addition, insertions at position -43 (G), -47 (C), -71 (G) and a C to T substitution at position -198 were found in all DNA samples compared with the published ccn2 (ctgf) promoter sequence. These corrections do not involve sequences predicted to function as transcription factor binding sites.

CONCLUSIONS

Sequence analysis of the ccn2 (ctgf) promoter of 77 human DNA samples has revealed corrections and polymorphic sites. The latter lie within putative regulatory elements.

BACKGROUND

Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. CTGF is abundantly expressed in osteoarthritis (OA). However, the relationship between CTGF and IL-6 in OA synovial fibroblasts (OASFs) is mostly unknown.

RESULTS

OASFs showed significant expression of CTGF, and expression was higher than in normal SFs. OASFs stimulation with CTGF induced concentration-dependent increases in IL-6 expression. CTGF mediated IL-6 production was attenuated by αvβ5 integrin neutralized antibody and apoptosis signal-regulating kinase 1 (ASK1) shRNA. Pretreatment with p38 inhibitor (SB203580), JNK inhibitor (SP600125), AP-1 inhibitors (Curcumin and Tanshinone IIA), and NF-κB inhibitors (PDTC and TPCK) also inhibited the potentiating action of CTGF. CTGF-mediated increase of NF-κB and AP-1 luciferase activity was inhibited by SB203580 and SP600125 or ASK1 shRNA or p38 and JNK mutant.

CONCLUSIONS

Our results suggest that CTGF increased IL-6 production in OASFs via the αvβ5 integrin, ASK1, p38/JNK, and AP-1/NF-κB signaling pathways.

Regulation of connective tissue growth factor (CCN2/CTGF) in gingival fibroblasts is unique and may provide therapeutic opportunities to treat oral fibrotic diseases. RhoA was previously implicated in mediating the expression of CCN2/CTGF. We now present evidence that Rho family GTPases Rac1 and Cdc42 are the principal mediators of the transforming growth factor-beta1 (TGFbeta1)-stimulated expression of CCN2/CTGF in primary human gingival fibroblasts. TGFbeta1 does not stimulate RhoA activation in gingival fibroblasts, and the overexpression of dominant-negative RhoA does not reduce CCN2/CTGF expression in response to TGFbeta1. In contrast, the overexpression of dominant-negative forms of Cdc42 or Rac1 results in a dramatic reduction of CCN2/CTGF protein levels. Lovastatin and a geranylgeranyltransferase inhibitor reduce the TGFbeta1-stimulated levels of CCN2/CTGF protein by approximately 75 and 100%, respectively. We previously demonstrated that JNK1 phosphorylation by TGFbeta1 is also critical for TGFbeta1-induced CCN2/CTGF expression, and forskolin partially reduces levels of phosphorylated JNK1. Inhibition of geranylgeranyltransferase has no effect on levels of JNK phosphorylation in response to TGFbeta1 suggesting Rho-GTPases act independently of JNK1. The combination of lovastatin and forskolin results in a greater inhibitory effect than each agent alone and reduces CCN2/CTGF mRNA and protein expression by greater than 90%. This novel combination has additive inhibitory effects on the TGFbeta1-stimulated expression of CCN2/CTGF in human gingival fibroblasts through the simultaneous disruption of Rho- and JNK1-mediated pathways, respectively. This combination of available therapeutic compounds may therefore be useful in designing treatment strategies for oral fibrotic conditions in which gingival CCN2/CTGF is elevated.

OBJECTIVE

We have previously demonstrated elevated levels of connective tissue growth factor (CTGF/CCN2) in the aqueous humor (AqH) of pseudoexfoliation glaucoma (PXFG) patients when compared with cataract controls. Furthermore, there is a significant trabecular meshwork (TM) and lamina cribrosa (LC) fibrotic phenotype associated with glaucoma, possibly driven by CTGF. The purpose of this study was to investigate the potential of anti-CTGF immunotherapy in glaucoma.

METHODS

Primary TM and LC cells were cultured from human donors with (GTM/GLC) and without (NTM/NLC) primary open angle glaucoma (POAG). Aqueous humor samples from PXFG, POAG, and control cataract patients were applied to N/GTM and N/GLC cells in the presence or absence of a therapeutic, humanized monoclonal anti-CTGF antibody FG-3019 (10 μg/mL). Hydrogen peroxide (H2O2) was also used as a stimulus. Expression of fibrotic genes (fibronectin-1, fibrillin-1, CTGF, collagen type I α1, and α-smooth muscle actin) was assessed by q-PCR. Protein expression of collagen 1A1 and α-smooth muscle actin was examined in N/G TM cells by SDS-PAGE. The modulatory effect of FG-3019 (10 μg/mL) and IgG (10 μg/mL) were also assessed.

RESULTS

Treatment of cells with AqH from PXFG and POAG patients and H2O2 induced a significant (P < 0.05) increase in expression of profibrotic genes, which was significantly reduced by pretreatment with FG-3019 (P < 0.05). FG-3019 also reduced expression of α-smooth muscle actin and collagen 1A1 protein expression in N/GTM cells.

CONCLUSIONS

FG-3019 is effective in blocking extracellular matrix production in TM and LC cells, thus supporting a role for the use of anti-CTGF immunotherapy in the treatment of glaucoma.

During mouse embryogenesis, CTGF/CCN2 is expressed in zones containing hypertrophic chondroctyes and calcifying cartilage such as long bones, ribs, vertebral column, and phalanges. But in fish, its expression is yet unclear. Development of the vertebrae is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process is highly conserved during evolution. Analysis of 3.2kb of the CTGF/CCN2 proximal promoter sequence revealed a consensus TATAA box, putative AP1, Brn-2, CdxA, C/EBP alpha, C/EBP beta, C-Ets-, delta E, HFH-2, and HSF2 binding sites. Transient expression experiments with a 5'-deletion revealed at least 4 regulatory regions in the zebrafish CTGF/CCN2 gene, 2 with a stimulatory effect on transcription and 2 with an apparent inhibitory effect after IGF-I treatment in the ZFL cell line. To study the promoter-specific expression, we constructed a series of CTGF/CCN2 (3.0-, 2.5-, 2.0-, 1.5-, 1.0-, and 0.4-kb) promoter-driven green fluorescent protein (GFP) fragments encoding the GFP cDNA transgene which was microinjected into zebrafish embryos. Morphological studies of transgenic zebrafish indicated that the CTGF/CCN2 promoter-driven GFP transcripts appeared in the notochord. Targeted knockdown of the CTGF/CCN2 gene by two antisense morpholino oligonucleotides resulted in disruptions to notochord development. From a comparative point of view, this study of the CTGF/CCN2 gene in zebrafish may correlate well with those previously published on the mouse. These molecular results suggest that CTGF/CCN2 plays an important role in notochord development and is required for general embryonic development.

CCN2/connective tissue growth factor (CCN2/CTGF) is known to promote both the proliferation and differentiation of chondrocytes, which actions are mediated by ERK and p38 MAPK, respectively. In this study, we first re-evaluated the involvement of multiple MAPKs therein and found that JNK also mediated such CCN2 signals. Thereafter, we further analyzed the roles of upstream kinases. The involvement of PKC, PI3K and PKA in the CCN2 signaling to promote the maturation, proliferation and terminal differentiation of a human chondrocytic cell line, HCS-2/8 and rabbit primary growth cartilage cells was investigated. As a result, the PKC inhibitor calphostin C repressed all of the effects of CCN2, which were represented by increased synthesis of DNA and proteoglycans and the display of alkaline phosphatase activity. In addition, evaluation of the effect of the PI3K inhibitor wortmannin disclosed the contribution of PI3K in transducing CCN2 signals to promote chondrocyte hypertrophy. This signal was known to be mediated by PKB, which was translocated into the nucleus upon CCN2 stimulation. Of note, calphostin C showed inhibitory effects on the activation of p38 MAPK, ERK and also PKB, whereas it exerted no effect on JNK activation. These results suggest that PKC is a driver of multiple signal transducing kinases that promote the proliferation and differentiation of chondrocytes. The requirement of PI3K in transmitting the signal for terminal differentiation and PKC-independent signaling pathways for the promotion of chondrocytic growth and differentiation, which was mediated by JNK, were also uncovered.