CCN3 is dynamically regulated by treatment and disease state in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated disease that damages myelin in the central nervous system (CNS). We investigated the profile of CCN3, a known regulator of immune function and a potential mediator of myelin regeneration, in multiple sclerosis in the context of disease state and disease-modifying treatment. METHODS: CCN3 expression was analysed in plasma, immune cells, CSF and brain tissue of MS patient groups and control subjects by ELISA, western blot, qPCR, histology and in situ hybridization. RESULTS: Plasma CCN3 levels were comparable between collective MS cohorts and controls but were significantly higher in progressive versus relapsing-remitting MS and between patients on interferon-ß versus natalizumab. Higher body mass index was associated with higher CCN3 levels in controls as reported previously, but this correlation was absent in MS patients. A significant positive correlation was found between CCN3 levels in matched plasma and CSF of MS patients which was absent in a comparator group of idiopathic intracranial hypertension patients. PBMCs and CD4+ T cells significantly upregulated CCN3 mRNA in MS patients versus controls. In the CNS, CCN3 was detected in neurons, astrocytes and blood vessels. Although overall levels of area immunoreactivity were comparable between non-affected, demyelinated and remyelinated tissue, the profile of expression varied dramatically. CONCLUSIONS: This investigation provides the first comprehensive profile of CCN3 expression in MS and provides rationale to determine if CCN3 contributes to neuroimmunological functions in the CNS.

Yin/Yang expression of CCN family members: Transforming growth factor beta 1, via ALK5/FAK/MEK, induces CCN1 and CCN2, yet suppresses CCN3, expression in human dermal fibroblasts.

The role of the microenvironment in driving connective tissue disease is being increasingly appreciated. Matricellular proteins of the CCN family are signaling modifiers that are secreted by cells into the extracellular matrix microenvironment where they have profound, context-dependent effects on organ development, homeostasis and disease. Indeed, CCN proteins are emergent targets for therapeutic intervention. Recent evidence suggests that, in vivo, CCN3 has effects opposing CCN2. Moreover, when CCN3 expression is high, CCN2 expression is low. That is, they appear to be regulated in a yin/yang fashion, leading to the hypothesis that the CCN2:CCN3 ratio is important to control tissue homeostasis. To begin to test the hypothesis that alterations in CCN2:CCN3 expression might be important in skin biology in vivo, we evaluated the relative ex vivo effects of the profibrotic protein TGFbeta1 on dermal fibroblasts on protein and RNA expression of CCN3 and CCN2, as well as the related protein CCN1. We also used signal transduction inhibitors to begin to identify the signal transduction pathways controlling the ability of fibroblasts to respond to TGFbeta1. As anticipated, CCN1 and CCN2 protein and mRNA were induced by TGFbeta1 in human dermal fibroblasts. This induction was blocked by TAK1, FAK, YAP1 and MEK inhibition. Conversely, TGFbeta1 suppressed CCN3 mRNA expression in a fashion insensitive to FAK, MEK, TAK1 or YAP1 inhibition. Unexpectedly, CCN3 protein was not detected in human dermal fibroblasts basally. These data suggest that, in dermal fibroblasts, the profibrotic protein TGFbeta1 has a divergent effect on CCN3 relative to CCN2 and CCN1, both at the mRNA and protein level. Given that the major source in skin in vivo of CCN proteins are fibroblasts, our data are consistent that alterations in CCN2/CCN1: CCN3 ratios in response to profibrotic agents such as TGFbeta1 may play a role in connective tissue pathologies including fibrosis.

Reduced immunohistochemical expression of CCN3 in vitiligo.

BACKGROUND: Defective adhesion seems to be involved in the chronic loss of melanocytes observed in vitiligo. Recent findings showed an association of genetic variants of an adhesion gene with vitiligo and reduced immunohistochemical expression of some adhesion molecules in vitiligo skin. AIMS: To compare CCN3 immunohistochemical expression in lesional and non-lesional epidermis of individuals with vitiligo. METHODS: A total of 66 skin specimens from 33 volunteers with vitiligo were analyzed by immunohistochemistry using anti-CCN3 antibodies. Absence of topical or systemic treatment for vitiligo over the previous 30 days and availability of an area of non-lesional skin for biopsy at least 15 cm away from any vitiliginous macules were the main inclusion criteria. RESULTS: A significant reduction of CCN3 expression was observed in lesional skin as compared to non-lesional skin (P = 0.001). LIMITATIONS: Paraffin embedded skin samples do not allow investigation by molecular biology methods. Not all samples allowed analysis due to the lamina preparation technique. Complete clinical data was not available for all patients. CONCLUSION: Our results support the hypothesis of impaired cell adhesion in vitiligo suggested by genetic studies. The pattern of immunohistochemical expression suggests that vitiligo might be an epithelial disease and not just a melanocyte disorder.

Differential expression of CCN family members CYR611, CTGF and NOV in gastric cancer and their association with disease progression.

CCN is an acronym for cysteine-rich protein 61 (CYR61), connective tissue growth factor (CTGF) and nephroblastoma overexpressed (NOV). Aberrations of certain CCN members including CYR61, CTGF, Wnt1-inducible signalling pathway protein (WISP)-1 and -3 have been reported in gastric cancer. The present study aimed to examine the clinical relevance of NOV along with CYR61 and CTGF in gastric cancer by analysing their transcript levels. CYR61, CTGF and NOV transcript expression in 324 gastric cancer samples with paired adjacent normal gastric tissues were determined using real-time quantitative PCR and the results were statistically analysed against patient clinicopathological data using SPSS software. NOV mRNA levels in gastric cancer tissues were significantly elevated when compared with levels in their paired adjacent non-cancerous tissues. Local advanced tumours with invasive expansion (T3 and T4) expressed higher levels of NOV (p=0.013) compared with the less invasive tumours (T1 and T2). CYR61 transcript levels were also significantly increased in gastric cancers compared with levels in the adjacent non-cancerous tissues. Kaplan-Meier survival curves revealed that patients with CYR61-low transcript levels had longer overall survival (OS) (p=0.018) and disease-free survival (DFS) (p=0.015). NOV overexpression promoted the in vitro proliferation of AGS cells while the knockdown resulted in a reduced proliferation of HGC27 cells. A similar effect was observed for the invasion of these two gastric cancer cell lines. NOV expression was increased in gastric cancer which was associated with local invasion and distant metastases. Taken together, the expression of NOV and CYR61 was increased in gastric cancer. The elevated expression of CYR61 was associated with poorer survival. NOV promoted proliferation and invasion of gastric cancer cells. Further investigations may highlight their predictive and therapeutic potential in gastric cancer.

Myeloid Zinc Finger 1 (MZF-1) Regulates Expression of the CCN2/CTGF and CCN3/NOV Genes in the Hematopoietic Compartment.

Connective Tissue Growth Factor (CCN2/CTGF) and Nephroblastoma Overexpressed (CCN3/NOV) execute key functions within the hematopoietic compartment. Both are abundant in the bone marrow stroma, which is a niche for hematopoiesis and supports marrow function. Roles for 1,25-dihydroxyvitamin D3 (calcitriol) and all-trans retinoic acid in the bone marrow have also been elucidated. Interestingly, some of the annotated roles of these vitamins overlap with established functions of CCN2 and CCN3. Yet, no factor has been identified that unifies these observations. In this study, we report the regulation of the CTGF and NOV genes by Myeloid Zinc Finger-1 (MZF-1), a hematopoietic transcription factor. We show the interaction of MZF-1 with the CTGF and NOV promoters in several cell types. Up-regulation of MZF-1 via calcitriol and vitamin A induces expression of CTGF and NOV, implicating a role for these vitamins in the functions of these two genes. Lastly, knockdown of MZF1 reduces levels of CTGF and NOV. Collectively, our results argue that MZF-1 regulates the CTGF and NOV genes in the hematopoietic compartment, and may be involved in their respective functions in the stroma.

Expression of CCN family members correlates with the clinical features of hepatocellular carcinoma.

Studies have reported that the CCN family of proteins plays an important role in stimulating tumorigenesis. However, the relationship between the CCN protein family members and the features of hepatocellular carcinoma (HCC) remains unclear. The objective of this study was to determine the relationship between the expression levels of CCN protein family members and the features of HCC. Expression levels of the CCN family of proteins in 80-paired primary HCC samples and 11 normal liver samples were determined by a quantitative real-time PCR assay. Enhanced expression of nephroblastoma overexpressed protein (NOV) and decreased expression of Wnt-induced secreted protein 1 (WISP1), cysteine-rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were found in HCC samples when compared to levels in matched non-cancerous tissues. No significant difference in WISP2 was found between matched-pair samples; only a few samples showed WISP3 expression. Furthermore, the expression levels of NOV, WISP1 and CYR61 were closely correlated with certain clinical features, including venous invasion, cellular differentiation, pTNM stage, disease-free survival and overall survival. Our results suggest that HCC progression may be enhanced by NOV and suppressed by WISP1 and CYR61. Our statistical analysis suggests that these proteins may be valuable in determining the prognosis of this deadly disease and directs attention to modulating the levels of these proteins as a potential mode of therapy.

NOV is upregulated and promotes migration and invasion in bladder cancer.

NOV is pro-tumourigenic via epithelial-mesenchymal transition (EMT) in several malignancies but is not studied in bladder cancer (BCa). Whether NOV is responsible for bladder carcinogenesis and the underlying mechanism is unclear. Using immunohistochemical staining, we quantified expressions of NOV, pS6, Vimentin and E-cadherin in 66 bladder cancer and 10 normal bladder urothelium samples. EMT was profiled by EMT index (EMTi) calculated as the ratio of Vimentin to E-cadherin. In vitro and in vivo studies were carried out to profile the role of NOV in the tumourigenesis of BCa. NOV was upregulated in bladder cancer compared to normal tissue, and its expression was correlated to pS6 and EMTi. Expression of NOV was higher in recurrent and multiple tumours and was increased with progression of tumour grade. NOV expression was also higher in BCa cell lines. Silence of NOV attenuated EMT, decreased invasion and migration of BCa cells. Silence of NOV also inhibited xenograft tumour growth and decreased tumour EMT. NOV is pro-tumourigenic in bladder cancer especially in nonmuscle-invasive entities (NMIBC). NOV may promote carcinogenesis via promotion of EMT and association with increased mTOR activity.

CCN3/NOV gene expression in human prostate cancer is directly suppressed by the androgen receptor.

Androgen receptor (AR) has essential roles during prostate cancer progression. With genome-wide AR-binding sites mapped to high resolution, studies have recently reported AR as a transcriptional repressor. How AR inhibits gene expression and how this contributes to prostate cancer, however, are incompletely understood. Through meta-analysis of microarray data, here we nominate nephroblastoma overexpressed (NOV) as a top androgen-repressed gene. We show that NOV is directly suppressed by androgen through the AR. AR occupies the NOV enhancer and communicates with the NOV promoter through DNA looping. AR activation recruits the polycomb group protein EZH2, which subsequently catalyzes histone H3 lysine 27 tri-methylation around the NOV promoter, thus leading to repressive chromatin remodeling and epigenetic silencing. Concordantly, AR and EZH2 inhibition synergistically restored NOV expression. NOV is downregulated in human prostate cancer wherein AR and EZH2 are upregulated. Functionally, NOV inhibits prostate cancer cell growth in vitro and in vivo. NOV reconstitution reverses androgen-induced cell growth and NOV knockdown drives androgen-independent cell growth. In addition, NOV expression is restored by hormone-deprivation therapies in mice and prostate cancer patients. Therefore, using NOV as a model gene we gained further understanding of the mechanisms underlying AR-mediated transcriptional repression. Our findings establish a tumor-suppressive role of NOV in prostate cancer and suggest that one important, but previously underestimated, manner by which AR contributes to prostate cancer progression is through inhibition of key tumor-suppressor genes.

CCN3 protein participates in bone regeneration as an inhibitory factor.

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.

The gene encoding the hematopoietic stem cell regulator CCN3/NOV is under direct cytokine control through the transcription factors STAT5A/B.

Cytokines control the biology of hematopoietic stem cells (HSCs) and progenitor cells in part through the transcription factors STAT5A/B. To investigate the target genes of STAT5A/B activated by cytokines in HSCs and progenitors, we performed microarray analyses using Lineage(-) Sca-1(+) c-Kit(+) (KSL) cells in the presence and absence of STAT5A/B. Stimulation with a mixture containing IL-3, IL-6, stem cell factor, thrombopoietin, and Flt3 ligand induced Ccn3/Nov mRNA over 100-fold in WT (control) but not Stat5a/b-null KSL cells. CCN3/NOV is a positive regulator of human HSC self-renewal and development of committed blood cells. Without stimulation, the Ccn3/Nov signal level was low in control KSL cells similar to Stat5a/b-null KSL cells. To determine which cytokine activates the Ccn3/Nov gene, we analyzed Lineage(-) c-Kit(+) (KL) and 32D cells using quantitative PCR and ChIP assays. Although stimulation with a mixture lacking IL-3 prevented the induction of Ccn3/Nov in control KL cells, IL-3 alone could induce Ccn3/Nov mRNA in control KL and 32D cells. ChIP assays using 32D cells revealed IL-3-induced binding of STAT5A/B to a γ-interferon-activated sequences site in the Ccn3/Nov gene promoter. This is the first report that Ccn3/Nov is directly induced by cytokines through STAT5A/B.

Prognostic relevance of CCN3 in Ewing sarcoma.

Ewing sarcoma is a highly aggressive malignant bone tumor occurring preferentially in children and young adults. At present, only clinical features, such as patient age, presence of clinically evident metastases at diagnosis, and poor response to neoadjuvant chemotherapy, are widely accepted as prognostic indicators in Ewing sarcoma. In this study, we assessed the prognostic value of CCN3 (Nov), a matricellular protein that play crucial roles in bone formation. Polyclonal antibodies directed against each of the different CCN3 modules were used to identify variant CCN3 proteins in tumors and to draw potential relationships between the expression of these variants and the outcome of patients with Ewing sarcoma. Our results confirmed that expression of the full-length CCN3 in Ewing sarcoma is associated to a worse prognostic. Furthermore, we report a possible relationship between the expression of a CCN3 protein lacking an internal module (von Willebrand factor type C) and sensitivity to radiotherapy. We hypothesize that the increased level of variant CCN3 in the tumor cells reduces their tumorigenic potential and results in better outcome.