Increased Expression Of CTGF Research Articles

Intracellular signaling pathways involved in the regulation of gene expression by pilocarpine

ObjectivesPilocarpine is commonly used clinically to treat dry mouth. The long-term administration of pilocarpine reportedly improves salivary secretion more effectively than short-term administration. Therefore, we hypothesized that pilocarpine alters gene expression in salivary glands via muscarinic receptor stimulation. This study aimed to investigate the effects of pilocarpine use on gene expression mediated by mitogen-activated protein kinase (MAPK) activity. MethodsThe effects of pilocarpine on gene expression were investigated in rats and human salivary gland (HSY) cells using several inhibitors of intracellular signaling pathways. Gene expression in the rat submandibular gland and HSY cells was determined using reverse transcription–quantitative polymerase chain reaction analysis of total RNA. ResultsIn animal experiments, at 7 days after pilocarpine stimulation, Ctgf and Sgk1 expressions were increased in the submandibular gland. In cell culture experiments, pilocarpine increased Ctgf expression in HSY cells. The mitogen-activated protein kinase kinase inhibitor trametinib, the Src inhibitor PP2, and the muscarinic acetylcholine receptor antagonist atropine suppressed the effect of pilocarpine on gene expression. ConclusionsPilocarpine enhances Ctgf and Sgk1 expressions by activating Src-mediated MAPK activity. Although further studies are required to fully understand the roles of Ctgf and Sgk1, changes in gene expression may play an important role in improving salivary secretions.

PIN1 facilitates ubiquitin-mediated degradation of serine/threonine kinase 3 and promotes melanoma development via TAZ activation.

The Hippo signaling pathway controls cellular processes including growth, homeostasis, and apoptosis. The kinase STK3 acts upstream in this pathway to activate LATS1/2 kinase, which phosphorylates and inactivates the transcriptional coactivators YAP/TAZ. The dysregulation of Hippo signaling leads to human diseases including cancer; however, the molecular mechanisms underlying its dysregulation in melanoma are unknown. We aimed to determine the role of the PIN1 in Hippo signaling dysregulation and melanoma tumorigenesis. We report that PIN1 interacts with STK3 and induces ubiquitination-dependent proteasomal degradation of STK3. Furthermore, PIN1 plays a critical role in the nuclear translocation of TAZ, which forms a complex with TEAD to increase CTGF expression. PIN1 ablation blocks TAZ/TEAD complex formation and decreases CTGF expression. PIN1-mediated STK3 degradation is associated with enhanced cell growth, induction of cell transformation, and increased tumorigenicity. In clinical context, PIN1 and STK3 levels are inversely correlated in patient melanoma tissues. These findings indicate that PIN1-mediated STK3 destabilization contributes to the dysregulation of Hippo signaling, leading to oncogenic signaling and melanoma tumorigenesis. Our data suggest that inhibition of the PIN1-STK3 axis could be a novel treatment strategy for malignant melanoma.

Deletion of connective tissue growth factor ameliorates peritoneal fibrosis by inhibiting angiogenesis and inflammation.

Connective tissue growth factor (CTGF/CCN2) regulates the signalling of other growth factors and promotes fibrosis. CTGF is increased in mice and humans with peritoneal fibrosis. Inhibition of CTGF has not been examined as a potential therapeutic target for peritoneal fibrosis because systemic CTGF knockout mice die at the perinatal stage. To study the role of CTGF in peritoneal fibrosis of adult mice, we generated CTGF conditional knockout (cKO) mice by crossing CTGF floxed mice with RosaCreERT2 mice. We administered tamoxifen to Rosa-CTGF cKO mice to delete the CTGF gene throughout the body. We induced peritoneal fibrosis by intraperitoneal injection of chlorhexidine gluconate (CG) in wild-type and Rosa-CTGF cKO mice. Induction of peritoneal fibrosis in wild-type mice increased CTGF expression and produced severe thickening of the peritoneum. In contrast, CG-treated Rosa-CTGF cKO mice exhibited reduced thickening of the peritoneum. Peritoneal equilibration test revealed that the excessive peritoneal small-solute transport in CG-treated wild-type mice was normalized by CTGF deletion. CG-treated Rosa-CTGF cKO mice exhibited a reduced number of αSMA-, Ki67-, CD31- and MAC-2-positive cells in the peritoneum. Analyses of peritoneal mRNA showed that CG-treated Rosa-CTGF cKO mice exhibited reduced expression of Cd68, Acta2 (αSMA), Pecam1 (CD31) and Vegfa. These results indicate that a deficiency of CTGF can reduce peritoneal thickening and help to maintain peritoneal function by reducing angiogenesis and inflammation in peritoneal fibrosis. These results suggest that CTGF plays an important role in the progression of peritoneal fibrosis.

Connective Tissue Growth Factor is a Target of Notch Signaling in Cells of the Osteoblastic Lineage

Connective tissue growth factor (Ctgf) or CCN2 is a protein synthesized by osteoblasts necessary for skeletal homeostasis, although its overexpression inhibits osteogenic signals and bone formation. Ctgf is induced by bone morphogenetic proteins, transforming growth factor β and Wnt; and in the present studies, we explored whether Notch regulated Ctgf expression in osteoblasts. We employed RosaNotch mice, where the Notch intracellular domain (NICD) is expressed following the excision of a STOP cassette, placed between the Rosa26 promoter and NICD. Notch was activated by transduction of adenoviral vectors expressing Cre recombinase (Ad-CMV-Cre). Notch induced Ctgf mRNA levels in a time dependent manner and increased Ctgf heterogeneous nuclear RNA. Notch also destabilized Ctgf mRNA shortening its half-life from 13h to 3h. The effect of Notch on Ctgf expression was lost following Rbpjκ downregulation, demonstrating that it was mediated by Notch canonical signaling. However, downregulation of the classic Notch target genes Hes1, Hey1 and Hey2 did not modify the effect of Notch on Ctgf expression. Wild type osteoblasts exposed to immobilized Delta-like 1 displayed enhanced Notch signaling and increased Ctgf expression. In addition to the effects of Notch in vitro, Notch induced Ctgf in vivo, and calvariae and femurs from RosaNotch mice mated with transgenics expressing the Cre recombinase in cells of the osteoblastic lineage exhibited increased expression of Ctgf. In conclusion, Ctgf is a target of Notch canonical signaling in osteoblasts, and may act in concert with Notch to regulate skeletal homeostasis.

Highlights of This Issue

Lung cancer is one of the most prevalent and deadliest cancers worldwide. A major risk factor in the disease comes from the carcinogens produced from cigarette smoking. As such, understanding the cellular mechanisms of detoxification is of general interest. In the current study, mRNA expression analysis indicated that the detoxification enzyme glutathione S-transferase GST-M2 and connective tissue growth factor CCN2 (CTGF) were reduced in lung tumor tissue. Overexpression of GST-M2 increased CTGF expression, reduced cellular migration, and inhibited tumor growth and metastasis in animal models. Importantly, higher expression of GST-M2 and CCN2 using real-time PCR is associated with a favorable prognosis in non–small cell lung cancer (NSCLC). Overall, this study reveals the potential of GST-M2 as a novel tumor suppressor via inhibiting tumor growth and metastasis, which has been demonstrated in vitro and in animal models. The antitumor effects of GST-M2 and CCN2 activity in animal models have provoked an intense interest in the development of small-molecule activators of GST-M2 and CCN2 (and their gene therapy) to prevent NSCLCs in the future.As tumors grow, there is an increased demand for nutrients and oxygen. Therefore, intratumoral hypoxia is considered a major driving force in breast cancer progression and has been linked to metastasis. Gilkes and colleagues demonstrated that hypoxia-inducible factor 1 (HIF-1) mediated increased expression of collagen hydroxylases (PLOD1 and PLOD2), which modify type I collagen prior to its secretion into the extracellular matrix (ECM). In addition, human breast cancer specimens stratified by elevated PLOD2 expression were associated significantly with increased patient mortality. In animal models, PLOD2 knockdown resulted in a marked decrease in tumor stiffness, local tissue invasion, and metastasis to lymph nodes and lungs without affecting primary tumor growth. Taken together, HIF-1 stimulates PLOD2 expression and collagen biogenesis to promote metastasis of hypoxic breast cancer cells. Thus, specific inhibitors of HIF-1 and/or collagen hydroxylases may be of clinical benefit for preventing breast cancer metastasis.The primary treatment for advanced/metastatic prostate cancer involves androgen deprivation therapy. Unfortunately, recurrence develops due to reactivation of androgen receptor (AR) activity resulting in castration-resistance and frequent osteoblastic bone metastasis. Based on genomic analyses, Zheng and colleagues mechanistically identified WNT7B as a novel target of AR in castration-resistant prostate cancer (CRPC). Manipulation of WNT7B levels promoted CRPC cell growth under androgen-deprived conditions and activated a noncanonical WNT pathway involving protein kinase C (PKC) isozymes. Critically, WNT7B was selectively upregulated in human xenografts that induce an osteoblastic reaction when grown in bone. These results establish a connection between AR and WNT signaling and suggest a unique opportunity to develop effective therapies for advanced prostate cancer by targeting the AR-WNT7B-PKCs axis, which would inhibit CRPC growth and prevent the formation of a metastatic reservoir in bone.Considered a major driver of invasive solid tumor progression, epithelial-mesenchymal transformation (EMT) is a cellular reprogramming by which malignant epithelial cells generally lose their cell–cell adhesion properties to become highly invasive. Austin and colleagues used mesenchymally transformed breast cancer cells to demonstrate that the small molecule sarasinoside A1, at noncytotoxic doses, reversed a number of aspects of EMT. Sarasinoside A1 inhibited cell invasion and promoted cell adhesion in three-dimensional cultures. Interestingly, sarasinoside A1 also initiated a morphologic re-epithelialization and stabilized the cytoskeleton at sites of nascent cell–cell contact. Combined, these findings indicate the potential to impede tumor invasion and metastasis without the need to reinduce the expression of cell adhesion molecules, such as E-cadherin, that are often lost or silenced during mesenchymal transformation.

Angiotensin II plays a critical role in diabetic pulmonary fibrosis most likely via activation of NADPH oxidase-mediated nitrosative damage

Diabetic patients have a high risk of pulmonary disorders that are usually associated with restrictive impairment of lung function, suggesting a fibrotic process (van den Borst B, Gosker HR, Zeegers MP, Schols AM. Chest 138: 393-406, 2010; Ehrlich SF, Quesenberry CP Jr, Van Den Eeden SK, Shan J, Ferrara A. Diabetes Care 33: 55-60, 2010). The present study was undertaken to define whether and how diabetes causes lung fibrosis. Lung samples from streptozotocin-induced type 1 diabetic mice, spontaneously developed type 1 diabetic OVE26 mice, and their age-matched controls were investigated with histopathological and biochemical analysis. Signaling mechanism was investigated with cultured normal human lung fibroblasts in vitro. In both diabetes models, histological examination with Sirius red and hemotoxylin and eosin stains showed fibrosis along with massive inflammatory cell infiltration. The fibrotic and inflammatory processes were confirmed by real-time PCR and Western blotting assays for the increased fibronectin, CTGF, PAI-1, and TNFα mRNA and protein expressions. Diabetes also significantly increased NADPH oxidase (NOX) expression and protein nitration along with upregulation of angiotensin II (Ang II) and its receptor expression. In cell culture, exposure of lung fibroblasts to Ang II increased CTGF expression in a dose- and time-dependent manner, which could be abolished by inhibition of superoxide, NO, and peroxynitrite accumulation. Furthermore, chronic infusion of Ang II to normal mice at a subpressor dose induced diabetes-like lung fibrosis, and Ang II receptor AT1 blocker (losartan) abolished the lung fibrotic and inflammatory responses in diabetic mice. These results suggest that Ang II plays a critical role in diabetic lung fibrosis, which is most likely caused by NOX activation-mediated nitrosative damage.

A mouse strain where basal connective tissue growth factor gene expression can be switched from low to high.

Connective tissue growth factor (CTGF) is a signaling molecule that primarily functions in extracellular matrix maintenance and repair. Increased Ctgf expression is associated with fibrosis in chronic organ injury. Studying the role of CTGF in fibrotic disease in vivo, however, has been hampered by perinatal lethality of the Ctgf null mice as well as the limited scope of previous mouse models of Ctgf overproduction. Here, we devised a new approach and engineered a single mutant mouse strain where the endogenous Ctgf-3′ untranslated region (3′UTR) was replaced with a cassette containing two 3′UTR sequences arranged in tandem. The modified Ctgf allele uses a 3′UTR from the mouse FBJ osteosarcoma oncogene (c-Fos) and produces an unstable mRNA, resulting in 60% of normal Ctgf expression (Lo allele). Upon Cre-expression, excision of the c-Fos-3′UTR creates a transcript utilizing the more stable bovine growth hormone (bGH) 3′UTR, resulting in increased Ctgf expression (Hi allele). Using the Ctgf Lo and Hi mutants, and crosses to a Ctgf knockout or Cre-expressing mice, we have generated a series of strains with a 30-fold range of Ctgf expression. Mice with the lowest Ctgf expression, 30% of normal, appear healthy, while a global nine-fold overexpression of Ctgf causes abnormalities, including developmental delay and craniofacial defects, and embryonic death at E10-12. Overexpression of Ctgf by tamoxifen-inducible Cre in the postnatal life, on the other hand, is compatible with life. The Ctgf Lo-Hi mutant mice should prove useful in further understanding the function of CTGF in fibrotic diseases. Additionally, this method can be used for the production of mouse lines with quantitative variations in other genes, particularly with genes that are broadly expressed, have distinct functions in different tissues, or where altered gene expression is not compatible with normal development.

Abstract 5077: Signaling through the cytoplasmic tail of MUC1 directly activates transcription of the metastasis promoting growth factor CTGF in pancreatic adenocarcinoma

Abstract Transmembrane mucin MUC1 is highly expressed in pancreatic adenocarcinoma and contributes to tumor growth, invasion, and metastasis. Differential phosphorylation of the cytoplasmic tail of MUC1 (MUC1.CT) by growth factor receptor tyrosine kinases is associated with morphogenetic signal transduction, as it enables interactions with signal transducers and leads to nuclear accumulation of MUC1.CT. MUC1.CT contains no known DNA binding motif but localizes to promoters of genes implicated in tumorigenesis in complex with transcription factors, modulating their regulatory activity. Using microarray analysis, we identified genes differentially transcribed in S2013 pancreatic adenocarcinoma cells stably overexpressing MUC1. These data were correlated with transcriptional targets of MUC1.CT identified on a genome-wide scale in pancreatic adenocarcinoma cells using a combination of chromatin immunoprecipitation and promoter array methodologies (ChIP-on-chip). We further investigated the transcriptional regulation of one novel target, connective tissue growth factor (CTGF/CCN2), by MUC1.CT. CTGF is highly expressed in pancreatic cancer and is a known mediator of tumor growth, angiogenesis, and metastasis. Overexpression of MUC1 significantly increased CTGF expression in pancreatic tumor cells in response to EGF, PDGF-BB, or HGF stimulation. The MUC1.CT becomes phosphorylated by EGFR, PDGFR-beta, and c-Met and overexpression of MUC1 constructs harboring mutations within the target phosphorylation motifs for these receptor tyrosine kinases reduced CTGF expression. We determined that MUC1.CT directly regulated the CTGF gene transcription through co-occupancy with transcription factors at several regions within and surrounding the CTGF promoter. MUC1 overexpression promoted localization of mutant p53 (R273H) and beta-catenin to the CTGF promoter, while decreasing occupancy by a repressor of CTGF transcription, c-Jun. We propose that MUC1, when overexpressed in pancreatic adenocarcinoma, enables expression of CTGF in response to growth factor stimulations by promoting mutant p53 and beta-catenin transcriptional activity and alleviating CTGF transcriptional repression by c-Jun. Taken together, these results reveal a novel signaling pathway that selectively induces CTGF expression in MUC1-overexpressing pancreatic tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5077.

Increased CTGF expression occurs in human and mouse hepatocytes of damaged livers and is mediated by TGF-β signaling

Increased CTGF expression occurs in human and mouse hepatocytes of damaged livers and is mediated by TGF-β signaling

Fetal fibroblasts have greater connective tissue growth factor expression after TGF-beta stimulation than adult fibroblasts

Abstract Introduction: Scarless wound healing in fetal skin is mediated by the dermal fibroblast, but the mechanism remains largely unknown. We examined the differences of fetal and adult fibroblasts to respond to TGF-beta isoform stimulation. CTGF expression, a TGF-beta target, was quantitated after TGF-beta isoform treatment. Methods: Primary mouse fibroblast cultures at gestational days 16.5 (E17), 18.5 (E19), and 6 weeks (adult) were stimulated with 10ng/mL of TGF-beta 1, TGF-beta 2, or TGF-beta 3 for 24 hours. Quantitative real-time PCR was performed for CTGF mRNA expression. Results: At baseline, CTGF was reduced 0.5 and 0.7 fold in E17 and E19 fibroblasts compared to adult fibroblasts (p 70 fold in E17 fibroblasts. TGF-beta 1 also increased CTGF expression in E19 and adult fibroblasts, but expression was less than in the E17 fibroblasts (p 80 fold, >60 fold, and 10 fold in E17, E19, and adult fibroblasts respectively (p Conclusions: E17 and E19 fibroblasts have lower CTGF expression at baseline compared to adult fibroblasts. Early gestation fibroblasts increase CTGF expression to a greater degree than late gestation and adult fibroblasts after TGF-beta isoform stimulation, suggesting the signaling pathway for TGF-beta is functional early in development.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization.

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.