Cyr61, CTGF, Nov Family Research Articles

Endogenous CCN family member WISP1 inhibits trauma-induced heterotopic ossification.

Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin, resulting in pathologic cartilage and bone matrix deposition. Cyr61, CTGF, Nov (CCN) family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation, including the regulation of chondrogenesis. However, little is known regarding CCN family member expression or function in HO. Here, a combination of bulk and single-cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins, Wisp1 (also known as Ccn4) was most upregulated during the evolution of HO, and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1 expression across traumatic and genetic HO mouse models as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally, the transcriptome of Wisp1-null tenocytes revealed enrichment in signaling pathways, such as the STAT3 and PCP signaling pathways, that may explain increased HO in the context of Wisp1 deficiency. In sum, CCN family members, and in particular Wisp1, are spatiotemporally associated with and negatively regulate trauma-induced HO formation.

Adenoviral CCN gene transfers induce in vitro and in vivo endoplasmic reticulum stress and unfolded protein response

The endoplasmic reticulum (ER) is primarily recognized as the site of synthesis and folding of secreted membrane-bound and certain organelle-targeted proteins. Optimum protein folding requires several factors, including ATP, Ca2+ and an oxidizing environment to allow disulphide-bond formation. ER is highly sensitive to stress that perturb cellular energy levels, the redox state or the Ca2+ concentration. Such stresses reduce the protein folding capacity of the ER, resulting in the accumulation and aggregation of unfolded proteins, a condition referred to as unfolded protein response (UPR). Matricellular proteins of the CCN (CYR61, CTGF, NOV) family play essential roles in extracellular matrix signaling and turnover. They exhibit a similar type of organization and share a closely related primary structure, including 38 conserved cysteine residues. Since CCN1/CYR61 overexpression in hepatic stellate cells (HSC) induces ER stress-related apoptosis, we endeavored to investigate whether the adenovirus mediated gene transfer of other members of CCN proteins incurs ER stress in primary HSC and hepatocytes. We found Ad5-CMV-CCN2, Ad5-CMV-CCN3 and Ad5-CMV-CCN4 to induce ER stress and UPR comparable to Ad5-CMV-CCN1. UPR is a pro-survival response to reduce accumulation of unfolded proteins and restore normal ER functioning. If, however protein aggregation is persistent and the stress cannot be resolved, signaling switches from pro-survival to pro-apoptosis. The observed CCN-induced UPR is relevant in wound healing responses and essential for hepatic tissue repair following liver injury. Adenoviral gene transfer induced massive amounts of matricellular proteins proving to effectively mitigate liver fibrosis if targeted cell specific in HSC and myofibroblasts.

Abstract 669: Matricellular Protein CCN3 Mitigates the Progression of Abdominal Aortic Aneurysm

Background: Structural components within the vasculature, including extracellular matrix proteins, play an integral role in modulating vascular homeostasis in health and disease. Previous studies have identified important roles of a family of secreted extracellular matrix associated proteins termed CCN (Cyr61, Ctgf, Nov) in mediating matrix homeostasis. However, a role for this family of proteins in the initiation / progression of vascular diseases remains undetermined. Methods and Results: Herein, biochemical and genetic gain- and loss-of function studies reveal a novel role for CCN3 (a member of the CCN family) in abdominal aorta aneurysm (AAA) biology. Current studies suggest CCN3 levels are reduced in the rodent aorta following angiotensin II (AngII) infusion, findings recapitulated in human AAA tissues. To address whether CCN3 deficiency affects the development of AAA, control C57BL/6 and CCN3-/- mice (n=30 per group) were subjected to AngII infusion. In response to four-week AngII infusion, the aortas of CCN3-/- mice demonstrated exquisite sensitivity with more than seventy percent of CCN3-/- mice developing visible AAA. Pathologically, the AAA phenotype of CCN3-/- mice was manifested as follows: (1) severe disruption of medial architecture characterized by prominent elastin degradation and vessel dilation; (2) a significant loss of smooth muscle cells; (3) a profound inflammatory response as evidenced by enhanced immune cell infiltration, increase expression of inflammatory markers; (4) heightened reactive oxygen species generation. Moreover, the AAA phenotype of CCN3-/- mice was confirmed in a second model of AAA formation (elastase perfusion). Next, bone marrow transplant experiments indicate the AAA phenotype of CCN3-/- mice is intrinsic to the vasculature. Furthermore, lentiviral mediated overexpression of CCN3 is sufficient to mitigate elastase-mediate AAA progression in mice. Mechanistically, genetic and pharmacological studies suggest the ERK1/2 pathway serves as an important role in the regulation of CCN3-dependent AAA development. Conclusions: These results demonstrate CCN3 as an essential regulator of AAA biology and suggest the potential for future therapeutic strategies by manipulating CCN3 levels.

WISP-1 a novel angiogenic regulator of the CCN family promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression.

Oral squamous cell carcinoma (OSCC), which accounts for nearly 90% of head and neck cancers, is characterized by poor prognosis and a low survival rate. VEGF-A is the most established angiogenic factor involved in the angiogenic-regulated tumor progression. WISP-1/CCN4 is an extracellular matrix-related protein that belongs to the Cyr61, CTGF, Nov (CCN) family and regulates many biological functions, such as angiogenesis. Previous studies indicated the role of WISP-1 in tumor progression. However, the angiogenic property of WISP-1 in the cancer microenvironment has never been discussed. Here, we provide novel insights regarding the role of WISP-1 in the angiogenesis through promoting VEGF-A expression. In this study, the correlation of WISP-1 and VEGF-A was confirmed by IHC staining of specimens from patients with OSCC. In vitro results indicated that WISP-1 induced VEGF-A expression via the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signaling pathway in OSCC. This pathway in turn induces the recruitment of endothelial progenitor cells and triggers the neovascularization in the tumor microenvironment. Our in vivo data revealed that tumor-secreted WISP-1 promoted the angiogenesis through VRGF expression and increased angiogenesis-related tumor growth. Our study offers new information that highlights WISP-1 as a potential novel therapeutic target for OSCC.

Ras activation mediates WISP-1-induced increases in cell motility and matrix metalloproteinase expression in human osteosarcoma

WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matrix cellular proteins. Osteosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effect of WISP-1 on migration activity in human osteosarcoma cells is mostly unknown. In this study, we first found that the expression of WISP-1 in osteosarcoma patients was significantly higher than that in normal bone and corrected with tumor stage. Exogenous treatment of osteosarcoma cells with WISP-1 promoted cell motility and matrix metalloproteinase (MMP)-2 and MMP-9 expression. In addition, the Ras and Raf-1 inhibitor or siRNA abolished WISP-1-induced cell migration and MMP expression. On the other hand, activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after WISP-1 treatment was demonstrated, and WISP-1-induced expression of MMPs and migration activity were inhibited by the specific inhibitor, and mutant of MEK, ERK, and NF-κB cascades. Taken together, our results indicated that WISP-1 enhances the migration of osteosarcoma cells by increasing MMP-2 and MMP-9 expression through the integrin receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway.

Temporal and spatial expression of CCN3 during retina development

NOV/CCN3 is one of the founding members of the CCN (Cyr61 CTGF NOV) family. In the avian retina, CCN3 expression is mostly located within the central region of the inner nuclear layer. As retinal development progresses and this retinal layer differentiates and matures, CCN3 expression forms a dorsal-ventral and a central-peripheral gradient. CCN3 is produced by two glial cell types, peripapillary cells and Müller cells, as well as by horizontal, amacrine, and bipolar interneurons. In retinal neurons and Müller cell cultures, CCN3 expression is induced by activated BMP signaling, whereas Notch signaling decreases CCN3 mRNA and protein levels in Müller cells and has no effect in retinal neurons. In Müller cells, the CCN3 expression detected may thus result from a balance between the Notch and BMP signaling pathways.

Kallikrein-related Peptidase 12 Hydrolyzes Matricellular Proteins of the CCN Family and Modifies Interactions of CCN1 and CCN5 with Growth Factors

Kallikrein-related peptidases (KLKs) are an emerging group of secreted serine proteases involved in several physiological and pathological processes. We used a degradomic approach to identify potential substrates of KLK12. MDA-MB-231 cells were treated either with KLK12 or vehicle control, and the proteome of the overlying medium was analyzed by mass spectrometry. CCN1 (cyr61, ctgf, nov) was among the proteins released by the KLK12-treated cells, suggesting that KLK12 might be responsible for the shedding of this protein from the cell surface. Fragmentation of CCN1 by KLK12 was further confirmed in vitro, and the main cleavage site was localized in the hinge region between the first and second half of the recombinant protein. KLK12 can target all six members of the CCN family at different proteolytic sites. Limited proteolysis of CCNs (cyr61, ctgf, nov) was also observed in the presence of other members of the KLK family, such as KLK1, KLK5, and KLK14, whereas KLK6, KLK11, and KLK13 were unable to fragment CCNs. Because KLK12 seems to have a role in angiogenesis, we investigated the relations between KLK12, CCNs, and several factors known to be involved in angiogenesis. Solid phase binding assays showed that fragmentation of CCN1 or CCN5 by KLK12 prevents VEGF(165) binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-β1 and FGF-2, either as intact or truncated forms, was found to be concentration-dependent. These findings suggest that KLK12 may indirectly regulate the bioavailability and activity of several growth factors through processing of their CCN binding partners.

First Structural Glimpse of CCN3 and CCN5 Multifunctional Signaling Regulators Elucidated by Small Angle X-ray Scattering

The CCN (cyr61, ctgf, nov) proteins (CCN1-6) are an important family of matricellular regulatory factors involved in internal and external cell signaling. They are central to essential biological processes such as adhesion, proliferation, angiogenesis, tumorigenesis, wound healing, and modulation of the extracellular matrix. They possess a highly conserved modular structure with four distinct modules that interact with a wide range of regulatory proteins and ligands. However, at the structural level, little is known although their biological function(s) seems to require cooperation between individual modules. Here we present for the first time structural determinants of two of the CCN family members, CCN3 and CCN5 (expressed in Escherichia coli), using small angle x-ray scattering. The results provide a description of the overall molecular shape and possible general three-dimensional modular arrangement for CCN proteins. These data unequivocally provide insight of the nature of CCN protein(s) in solution and thus important insight into their structure-function relationships.

Abstract 1387: WISP-1 increases MMP-2 expression and cell motility in human chondrosarcoma cells

Abstract Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matricellular proteins. However, the effect of WISP-1 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that WISP-1 increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells (JJ012 cells). We also found that human chondrosarcoma tissues had significant expression of the WISP-1 which was higher than that in normal cartilage. α5β1 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) inhibited the WISP-1-induced increase of the migration and MMP-2 up-regulation of chondrosarcoma cells. WISP-1 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors also suppressed the cell migration and MMP-2 expression enhanced by WISP-1. Moreover, WISP-1 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-2 promoter. Taken together, our results indicated that WISP-1 enhances the migration of chondrosarcoma cells by increasing MMP-2 expression through the α5β1 integrin receptor, FAK, MEK, ERK, p65 and NF-κB signal transduction pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1387. doi:10.1158/1538-7445.AM2011-1387

WISP-1 increases MMP-2 expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matricellular proteins. However, the effect of WISP-1 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that WISP-1 increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells (JJ012 cells). We also found that human chondrosarcoma tissues had significant expression of the WISP-1 which was higher than that in normal cartilage. α5β1 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) inhibited the WISP-1-induced increase of the migration and MMP-2 up-regulation of chondrosarcoma cells. WISP-1 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors also suppressed the cell migration and MMP-2 expression enhanced by WISP-1. Moreover, WISP-1 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-2 promoter. Taken together, our results indicated that WISP-1 enhances the migration of chondrosarcoma cells by increasing MMP-2 expression through the α5β1 integrin receptor, FAK, MEK, ERK, p65 and NF-κB signal transduction pathway.

A novel role of CCN3 in regulating endothelial inflammation

The vascular endothelium plays a fundamental role in the health and disease of the cardiovascular system. The molecular mechanisms regulating endothelial homeostasis, however, remain incompletely understood. CCN3, a member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators, has been shown to play an important role in numerous cell types. The function of CCN3 in endothelial cells has yet to be elucidated. Immunohistochemical analysis of CCN3 expression in mouse tissues revealed robust immunoreactivity in the endothelium of large arteries, small resistance vessels, and veins. We found that CCN3 expression in human umbilical vein endothelial cells (HUVECs) is transcriptionally induced by laminar shear stress (LSS) and HMG CoA-reductase inhibitors (statins). Promoter analyses identified the transcription factor Kruppel-like factor 2 (KLF2) as a direct regulator of CCN3 expression. In contrast to LSS, proinflammatory cytokines reduced CCN3 expression. Adenoviral overexpression of CCN3 in HUVEC markedly inhibited the cytokine-mediated induction of vascular adhesion molecule-1 (VCAM-1). Consistent with this observation, CCN3 significantly reduced monocyte adhesion. Conversely, CCN3 knockdown in HUVECs resulted in enhancement of cytokine-induced VCAM-1 expression. Concordant effects were observed on monocyte adhesion. Gain and loss-of-function mechanistic studies demonstrated that CCN3 negatively regulates nuclear factor kappaB (NF-κB) activity by reducing its translocation into the nucleus and subsequent binding to the VCAM-1 promoter, suggesting that CCN3's anti-inflammatory effects occur secondary to inhibition of NF-κB nuclear accumulation. This study identifies CCN3 as a novel regulator of endothelial proinflammatory activation.

Abstract 4954: A Novel Role of CCN3 in Regulating Endothelial Inflammation

Background : The vascular endothelium plays a fundamental role in the health and disease of the cardiovascular system. However, the molecular mechanisms regulating endothelial homeostasis remain incompletely understood. CCN3, a member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators, has been shown to play an important role in numerous cell types. While CCN3 expression in endothelial cells has been described, its function in this cell type has yet to be elucidated. Methods and Results : A detailed immunohistochemical analysis of CCN3 expression in mouse tissues revealed robust immunoreactivity in the endothelium of large arteries, small resistance vessels, as well as veins. Likewise, CCN3 expression is observed in cultured human endothelial cells derived from a variety of vascular beds. CCN3 expression in human umbilical vein endothelial cells (HUVECs) is transcriptionally induced by laminar shear stress (LSS) by ~27-fold. Similarly, treatment of HUVEC with HMG CoA-reductase inhibitors (statins) induced CCN3 expression by ~32-fold. A combination of promoter deletion and mutational analyses coupled with chromatin immuno-precipitation studies identified the Kruppel-like factor 2 as a direct regulator of CCN3 expression. In contrast to LSS, proinflammatory cytokines such as TNF α reduced CCN3 expression. Functionally, adenoviral overexpression of CCN3 in HUVEC markedly inhibited the cytokine-mediated induction of VCAM-1. Consistent with this observation, CCN3 overexpression significantly reduced monocyte adhesion. Conversely, siRNA mediated “knockdown” of CCN3 protein in HUVECs resulted in enhancement of cytokine-induced VCAM-1 expression. Concordant effects were observed on monocyte adhesion. Mechanistically, gain and loss-of-function studies demonstrate that CCN3 negatively regulates NF- κ B activity by reducing its translocation into nucleus and DNA binding to VCAM-1 promoter, suggesting that CCN3’s anti-inflammatory effects occur secondary to inhibition of NF- κ B nuclear accumulation. Conclusions: This study identifies CCN3 as a novel regulator of endothelial proinflammatory activation.

Connective tissue growth factor (CTGF, CCN2) gene regulation: a potent clinical bio‐marker of fibroproliferative disease?

The CCN (cyr61, ctgf, nov) family of modular proteins regulate diverse biological affects including cell adhesion, matrix production, tissue remodelling, proliferation and differentiation. Recent targeted gene disruption studies have demonstrated the CCN family to be developmentally essential for chondrogenesis, osteogenesis and angiogenesis. CCN2 is induced by agents such as angiotensin II, endothelin-1, glucocorticoids, HGF, TGFβ, and VEGF, and by hypoxia and biomechanical and shear stress. Dysregulated expression of CCN2 has also been widely documented in many fibroproliferative diseases. This mini-review will focus on CCN2, and the recent progress in understanding CCN2 gene regulation in health and disease. That CCN2 should be considered a novel and informative surrogate clinical bio-marker for fibroproliferative disease is discussed.

Cysteine-rich 61 (CYR61) inhibits cisplatin-induced apoptosis in ovarian carcinoma cells

Cysteine-rich 61 (CYR61), a member of the connective tissue factor CCN (Cyr61, CTGF, Nov) family, facilitates angiogenesis by interacting with integrins. Recent observations have indicated that CYR61 also rescues cells from anti-cancer drug-mediated apoptosis but the detailed mechanism underlying the role of CYR61 during apoptosis has not been identified. To better understand the role of CYR61 during cisplatin-induced apoptosis in tumor cells, we overexpressed or inhibited CYR61 expression in human cervical cancer cells (HeLa cells) and measured cisplatin-mediated apoptosis. The results from these experiments clearly demonstrate that CYR61 prevents cisplatin-induced apoptosis by inhibiting caspase-3 activity in HeLa cells. Therefore, CYR61 may be a useful therapeutic target for cisplatin-resistant tumors.

Dickkopf homolog 1 mediates endothelin-1-stimulated new bone formation.

Tumor-produced endothelin-1 (ET-1) stimulates osteoblasts to form new bone and is an important mediator of osteoblastic bone metastasis. The anabolic actions of ET-1 in osteoblasts were investigated by gene microarray analyses of murine neonatal calvarial organ cultures. Targets of ET-1 action were validated by real-time RT-PCR in murine primary osteoblast cultures. IL-6, IL-11, the CCN (CYR61, CTGF, NOV) family members cysteine-rich protein 61 and connective tissue growth factor, inhibin beta-A, serum/glucocorticoid regulated kinase, receptor activator of nuclear factor kappaB ligand, snail homolog 1, tissue inhibitor of metalloproteinase 3, and TG-interacting factor transcripts were increased by ET-1. ET-1 decreased the transcript for the Wnt signaling pathway inhibitor, dickkopf homolog 1 (Dkk1). Calvarial organ cultures treated with ET-1 had lower concentrations of DKK1 protein in conditioned media than control cultures. High DKK1 concentrations in bone marrow suppress bone formation in multiple myeloma. We hypothesized that the converse occurs in osteoblastic bone metastasis, where ET-1 stimulates osteoblast activity by reducing autocrine production of DKK1. Recombinant DKK1 blocked ET-1-mediated osteoblast proliferation and new bone formation in calvarial organ cultures, whereas a DKK1-neutralizing antibody increased osteoblast numbers and new bone formation. ET-1 directed nuclear translocation of beta-catenin in osteoblasts, indicating activation of the Wnt signaling pathway. The data suggest that ET-1 increases osteoblast proliferation and new bone formation by activating the Wnt signaling pathway through suppression of the Wnt pathway inhibitor DKK1.

Nuclear addressing provides a clue for the transforming activity of amino‐truncated CCN3 proteins

CCN3 is a founding member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators. These secreted proteins are key regulators in embryonic development, and are associated with severe pathologies including fibrotic diseases and cancers. CCN3 was discovered as a MAV integration site in an avian nephroblastoma. Previous work established that the amino-truncated protein expressed in this tumor was inducing morphological transformation of chicken embryo fibroblasts, whereas the full-length secreted CCN3 protein was inhibiting cell growth. Amino-truncated variants were identified in cancer cell lines. Since the lack of signal peptide was expected to alter the fate of the truncated proteins, we hypothesized that modifications of CCN3 subcellular addressing could be responsible for the oncogenic activities of CCN3. The CCN proteins are composed of four structural modules (IGFBP, TSP1, VWC, and CT). We report that amino-truncated variants of CCN3 are addressed to the nucleus and that the carboxyterminal (CT) module of CCN3 is responsible for the nuclear addressing. Furthermore, our data identify nuclear CCN3 variants as potential transcriptional regulators. In this context, the CT module confers on nuclear CCN3 proteins a negative regulatory effect on transcription. We propose that the nuclear localization of amino-truncated CCN3 proteins be correlated to oncogenicity.

Inhibition of CCN6 (WISP3) expression promotes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells

IntroductionCCN6/WISP3 belongs to the CCN (Cyr61, CTGF, Nov) family of genes that contains a conserved insulin-like growth factor (IGF) binding protein motif. CCN6 is a secreted protein lost in 80% of the aggressive inflammatory breast cancers, and can decrease mammary tumor growth in vitro and in vivo. We hypothesized that inhibition of CCN6 might result in the loss of a growth regulatory function that protects mammary epithelial cells from the tumorigenic effects of growth factors, particularly IGF-1.MethodWe treated human mammary epithelial (HME) cells with a CCN6 hairpin short interfering RNA.ResultsCCN6-deficient cells showed increased motility and invasiveness, and developed features of epithelial-mesenchymal transition (EMT). Inhibition of CCN6 expression promoted anchorage-independent growth of HME cells and rendered them more responsive to the growth effects of IGF-1, which was coupled with the increased phosphorylation of IGF-1 receptor and insulin receptor substrate-1 (IRS-1).ConclusionSpecific stable inhibition of CCN6 expression in HME cells induces EMT, promotes anchorage-independent growth, motility and invasiveness, and sensitizes mammary epithelial cells to the growth effects of IGF-1.

Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

The Expression of novH in Adrenocortical Cells Is Down-regulated by TGFβ1 through c-Jun in a Smad-independent Manner

The human NOV secreted glycoprotein (NOVH) is abundant in the fetal and adult adrenal cortex. The amount of NOVH increases in benign adrenocortical tumors and decreases in malignant adrenocortical tumors, suggesting that NOVH plays a role in tumorigenesis in the adrenal cortex. Transforming growth factor beta1 (TGFbeta1), fibroblast growth factor 2 (FGF2), and insulin growth factors (IGFs) play crucial roles in the physiology of the adrenal cortex. We investigated the effects of these factors on the expression of novH in the NCI H295R adrenocortical cell line. The amounts of NOVH protein and novH transcripts were down-regulated by TGFbeta1 and up-regulated by FGF2, whereas IGFs had no effect. Furthermore, the TGFbeta1-dependent inhibition of novH promoter activity was completely abrogated following site-directed mutation of two activating protein (AP-1) sequences (positions -473 and -447), whereas the stimulatory effect of FGF2 was not affected. Co-transfection with dominant negative forms of c-Jun and MEKK1 also abrogated novH-targeted regulation by TGFbeta1, whereas the overproduction of Smad proteins or dominant negative forms of Smad had no effect. Taken together, these results suggest that c-Jun and MEKK1 signaling but not Smad signaling are involved in the TGFbeta1-dependent decrease in NOVH in NCI H295R cells. In conclusion, our data provide evidence that novH is a new target of TGFbeta1; unlike other members of the CCN (cyr61, ctgf, nov) family, however, its expression is repressed rather than induced.