NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and -9.

BACKGROUND: Sustained neuroinflammation strongly contributes to the pathogenesis of pain. The clinical challenge of chronic pain relief led to the identification of molecules such as cytokines, chemokines and more recently matrix metalloproteinases (MMPs) as putative therapeutic targets. Evidence points to a founder member of the matricial CCN family, NOV/CCN3, as a modulator of these inflammatory mediators. We thus investigated the possible involvement of NOV in a preclinical model of persistent inflammatory pain. METHODS: We used the complete Freund's adjuvant (CFA)-induced model of persistent inflammatory pain and cultured primary sensory neurons for in vitro experiments. The mRNA expression of NOV and pro-inflammatory factors were measured with real-time quantitative PCR, CCL2 protein expression was assessed using ELISA, MMP-2 and -9 activities using zymography. The effect of drugs on tactile allodynia was evaluated by the von Frey test. RESULTS: NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. In vitro, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1ß- and TNF-α-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-α-induced MMP-9 expression through ß1 integrin engagement. In vivo, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia. CONCLUSIONS: This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.

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.

FGF1 nuclear translocation is required for both its neurotrophic activity and its p53-dependent apoptosis protection.

Fibroblast growth factor 1 (FGF1) is a differentiation and survival factor for neuronal cells both in vitro and in vivo. FGF1 activities can be mediated not only by paracrine and autocrine pathways involving FGF receptors but also by an intracrine pathway, which is an underestimated mode of action. Indeed, FGF1 lacks a secretion signal peptide and contains a nuclear localization sequence (NLS), which is consistent with its usual intracellular and nuclear localization. To progress in the comprehension of the FGF1 intracrine pathway in neuronal cells, we examined the role of the nuclear translocation of FGF1 for its neurotrophic activity as well as for its protective activity against p53-dependent apoptosis. Thus, we have transfected PC12 cells with different FGF1 expression vectors encoding wild type or mutant (Delta NLS) FGF1. This deletion inhibited both FGF1 nuclear translocation and FGF1 neurotrophic activity (including differentiation and serum-free cell survival). We also show that endogenous FGF1 protection of PC12 cells against p53-dependent cell death requires FGF1 nuclear translocation. Strikingly, wild type FGF1 is found interacting with p53, in contrast to the mutant FGF1 deleted of its NLS, suggesting the presence of direct and/or indirect interactions between FGF1 and p53 pathways. Thus, we present evidences that FGF1 may act by a nuclear pathway to induce neuronal differentiation and to protect the cells from apoptosis whether cell death is induced by serum depletion or p53 activation.

Nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3 (NOV/CCN3), a selective adrenocortical cell proapoptotic factor, is down-regulated in childhood adrenocortical tumors.

CONTEXT: Childhood adrenocortical tumors (ACTs) have a fetal adrenal phenotype and overexpress steroidogenic factor-1 (SF-1). Nephroblastoma overexpressed (NOV)/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3 mRNA is significantly down-regulated in childhood ACTs. OBJECTIVE: The objective of the study was to measure NOV protein levels in childhood ACTs and characterize NOV expression regulation and biological function in human adrenocortical cells. DESIGN AND SETTING: Protein extracts from ACT and normal adrenal cortex samples, human adrenocortical carcinoma H295R, primary adrenocortical tumors and fetal adrenal cultures, tissue culture supernatants, and cell lysates from H295R cells overexpressing SF-1 in an inducible fashion were used. MAIN OUTCOME MEASURES: NOV protein levels were measured by enzyme-linked immunoassay and immunoblot. Transient transfection assays were used to study the activity of NOV promoter. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, caspase assays, and flow cytometry were used to assess the proapoptotic activity of NOV on cells in culture. RESULTS: NOV mRNA and protein expression is lower in childhood ACTs than in normal adrenal cortex. No significant difference was observed between adenomas and carcinomas. SF-1 overexpression down-regulates NOV at the transcriptional level. NOV has a selective proapoptotic activity toward human adrenocortical cells. The C-terminal domain of NOV is responsible for its proapoptotic effect. NOV protein is expressed in DAX-1-positive human fetal adrenal cells. CONCLUSIONS: NOV is a selective proapoptotic factor for human adrenocortical cells. Reduced expression of NOV in ACTs may play an important role in the process of childhood ACT tumorigenesis, accounting at least in part for the defect of apoptotic regression of the fetal adrenal that has been proposed to be responsible for tumor formation.

NOV/CCN3 impairs muscle cell commitment and differentiation.

NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10(-6) M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts.

NOV/CCN3 induces adhesion of muscle skeletal cells and cooperates with FGF2 and IGF-1 to promote proliferation and survival.

During mammalian development, expression of the Nephroblastoma overexpressed gene (NOV/CCN3) is tightly regulated in skeletal muscles. Ex vivo, ectopic expression of NOV blocks myogenic differentiation. NOV also supports endothelial cell adhesion and angiogenesis through interactions with integrins. Integrins play fundamental roles during myogenesis. In this study, we show that NOV mediates adhesion and spreading of myoblasts. Myoblasts adhesion to NOV does not require proteoglycans and is dependent on integrin beta1, whereas spreading involves another RGD-sensitive integrin. The C-Terminal part of NOV as well as full-length is able to support adhesion of myoblasts; in addition, both increase focal-adhesion kinase (FAK) phosphorylation. Furthermore, NOV is an adhesive substrate that, combined with FGF2 or IGF-1, promotes cell specific proliferation and survival, respectively, in a better way than fibronectin. Taken together, these results identify NOV as an adhesion substrate for myoblasts which, in concert with growth factors, could play a role in the physiology of muscle cells.

New target genes for NOV/CCN3 in chondrocytes: TGF-beta2 and type X collagen.

UNLABELLED: We studied the involvement of NOV/CCN3, whose function is poorly understood, in chondrocyte differentiation. NOV was found to upregulate TGF-beta2 and type X collagen and to act as a downstream effector of TGF-beta1 in ATDC5 and primary chondrocytes. Thus, NOV is a positive modulator of chondrogenesis. INTRODUCTION: NOV/CCN3 is a matricellular protein that belongs to the CCN family. A growing body of evidence indicates that NOV could play a role in cell differentiation, particularly in chondrogenesis. During chick embryo development, NOV expression is tightly regulated in cartilage, and a high expression of NOV has been associated with cartilage differentiation in Wilms' tumors. However, a precise role for NOV and potential target genes of NOV in chondrogenesis are unknown. MATERIALS AND METHODS: ATDC5 cells and primary chondrocytes were either treated with NOV recombinant protein or transfected with a NOV-specific siRNA to determine, using quantitative RT-PCR, the effect of NOV on the expression of several molecules involved in chondrocyte differentiation. Stable ATDC5 clones expressing NOV were also established to show that NOV was a downstream effector of TGF-beta1. RESULTS: We established that NOV/CCN3 expression increases in ATDC5 cells at early stages of chondrogenic differentiation and precedes the appearance of TGF-beta2 and of several chondrocytic markers such as SOX9 or type X collagen. When exogenously administered, NOV recombinant protein up-regulates TGF-beta2 and type X collagen mRNA levels both in ATDC5 cells and in primary mouse chondrocytes but does not influence SOX9 expression. This regulation also occurs at the endogenous level because downregulation of NOV expression is correlated with an inhibition of TGF-beta2 and type X collagen in primary chondrocytes. Furthermore, we found that NOV expression is downregulated when chondrocytes are exposed to TGF-beta1-dedifferentiating treatment in chondrocytes, further providing evidence that NOV may counteract TGF-beta1 effects on chondrocytes. CONCLUSIONS: This study provides the first characterization of two new targets of NOV involved in chondrocyte differentiation, shows that NOV acts with TGF-beta1 in a cascade of gene regulation, and indicates that NOV is a positive modulator of chondrogenesis.

The expression of novH in adrenocortical cells is down-regulated by TGFbeta 1 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.