CCN2 Promoter Research Articles

Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at -286bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Analyses of the Posttranscriptional Regulation of CCN Genes: Approach to Multiple Steps of CCN2 Gene Expression.

Cells generally control the concentration of mRNA via transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation (decay) cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, degradation of the target mRNA, and inhibition of mRNA translation are performed either individually or in combination. From our experience, measurement of the steady-state levels of mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying the ccn2 gene expression. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. The stability of ccn2 mRNAs is then evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Finally, repression of ccn2 mRNA translation can be estimated by comparing the expression of mRNA and protein changes. We herein report the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can, in theory, be used to elucidate the posttranscriptional regulation of other genes belonging to the CCN family.

Zika virus promotes CCN1 expression via the CaMKIIα-CREB pathway in astrocytes

ABSTRACT Zika virus (ZIKV) infection in the human central nervous system (CNS) causes Guillain–Barre syndrome, cerebellum deformity, and other diseases. Astrocytes are immune response cells in the CNS and an important component of the blood–brain barrier. Consequently, any damage to astrocytes facilitates the spread of ZIKV in the CNS. Connective tissue growth factor/Nephroblastoma overexpressed gene family 1 (CCN1), an important inflammatory factor secreted by astrocytes, is reported to regulate innate immunity and viral infection. However, the mechanism by which astrocyte viral infection affects CCN1 expression remains undefined. In this study, we demonstrate that ZIKV infection up-regulates CCN1 expression in astrocytes, thus promoting intracellular viral replication. Other studies revealed that the cAMP response element (CRE) in the CCN1 promoter is activated by the ZIKV NS3 protein. The cAMP-responsive element-binding protein (CREB), a transacting factor of the CRE, is also activated by NS3 or ZIKV. Furthermore,a specific inhibitor of CREB, i.e. SGC-CBP30, reduced ZIKV-induced CCN1 up-regulation and ZIKV replication. Moreover, co-immunoprecipitation, overexpression, and knockdown studies confirmed that the interaction between NS3 and the regulatory domain of CaMKIIα could activate the CREB pathway, thus resulting in the up-regulation of CCN1 expression and enhancement of virus replication. In conclusion, the findings of our investigations on the NS3-CaMKIIα-CREB-CCN1 pathway provide a foundation for understanding the infection mechanism of ZIKV in the CNS.

Imatinib independent aberrant methylation of NOV/CCN3 in chronic myelogenous leukemia patients: a mechanism upstream of BCR-ABL1 function?

BackgroundThe NOV gene product, CCN3, has been reported in a diverse range of tumors to serve as a negative growth regulator, while acting as a tumor suppressor in Chronic Myelogenous Leukemia (CML). However, the precise mechanism of its silencing in CML is poorly understood. In the current study, we aimed to query if the gene regulation of CCN3 is mediated by the promoter methylation in the patients with CML. In addition, to clarify whether the epigenetic silencing is affected by BCR-ABL1 inhibition, we assessed the methylation status in the patients at different time intervals following the tyrosine kinase inhibition using imatinib therapy, as the first-line treatment for this type of leukemia.MethodsTo address this issue, we applied bisulfite-sequencing technique as a high-resolution method to study the regulatory segment of the CCN3 gene. The results were analyzed in newly diagnosed CML patients as well as following imatinib therapy. We also evaluated the correlation of CCN3 promoter methylation with BCR-ABL1 levels.ResultsOur findings revealed that the methylation occurs frequently in the promoter region of CML patients showing a significant increase of the methylated percentage at the CpG sites compared to normal individuals. Interestingly, this hypermethylation was indicated to be independent of BCR-ABL1 titers in both groups, which might suggest a mechanism beyond the BCR-ABL1 function.ConclusionDespite suggesting that the CCN3 hypermethylation acts as a molecular mechanism independent of BCR-ABL1 function in CML patients, this scenario requires further validation by complementary experiments. In the case of acting upstream of BCR-ABL1 signaling, the methylation marker can provide early detection and a novel platform for targeted epigenetic modifiers for efficient treatment in imatinib resistant patients.

Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells

ObjectiveThe objective of our study was to verify the hypothesis that the expression of connective tissue growth factor (CTGF/CCN2), a key molecule essential for the maintenance of nucleus pulposus (NP) matrix homeostasis, is regulated by osmolarity and intracellular calcium in NP cells. MethodsGene and protein expression levels of CCN2 were assessed using quantitative real-time PCR and western blot. Transfections and dual luciferase assays were performed to measure the effect of hyperosmolarity, tonicity enhancer binding protein (TonEBP) and Ca2+-calcineurin (Cn)-NFAT signaling on CCN2 promoter activity. ResultsCultured in hyperosmotic media, there was a significant decrease in the levels of CCN2 promoter activity, gene and protein expression in NP cells. The JASPAR database was used to analyze the construction of human CCN2 promoter, we found conserved TonE and NFAT binding sites. We then investigated whether TonEBP controlled CCN2 expression. Forced expression of TonEBP in NP cells showed that TonEBP negatively regulated CCN2 promoter activity, while suppression of TonEBP induced CCN2 promoter activity and expression. We then examined if Ca2+-Cn-NFAT signaling participated in the regulation of CCN2 expression. Co-expression of CCN2 reporter with individual NFAT1–4 expression plasmids and/or calcineurin A/B constructs suggested this signaling pathway played a role in the regulation of CCN2expression in NP cells. ConclusionsResults of these studies illustrated that the expression of CCN2 in NP cells was regulated by the NFAT family through a signaling pathway network involving both activator (Ca2+-Cn-NFAT signaling) and suppressor (Hyperosmolarity-TonEBP) molecules.

Interplay between the Yes‐Associated protein and the matricellular protein CCN1 Regulates the phenotypical plasticity of endothelial cells in developing blood vessels

CCN1 is a heparin and integrin‐binding matricellular protein which localizes pericellularly and acts primarily on cells that produce it. CCN1 is highly expressed at sites of active angiogenesis and tissue repair. Our studies in conditional knockout mouse models, demonstrate that endothelium‐specific deletion of the CCN1 gene results in severe vascular defects, most notably uncontrolled angiogenic sprouting and increased endothelial cell (EC) proliferation, resulting in the formation of a denser, widely lumenized vascular network. In this study, we investigated the regulatory mechanisms and signaling pathways involved in CCN1 gene expression and activity during angiogenesis. We found that the CCN1 gene is a target of the transcriptional coactivator Yes‐Associated protein (YAP), which acts in concert with other co‐activators such as myocardin‐related transcription factor (MRTF)‐A to activate the CCN1 promoter in angiogenic ECs. Loss of YAP function in mice is associated with reduced CCN1 promoter enrichment with YAP and decreased CCN1 gene expression. YAP is a key checkpoint of the Hippo pathway controlling cell growth, differentiation and cell‐cell junctions. It comprises a kinase cascade that inactivates by phosphorylation YAP leading to its cytoplasmic sequestration and growth inhibition. Loss of function of CCN1 in mice was associated with accumulation of active nonphosphorylated YAP in developing blood vessels. Consequently, growing ECs incorporate into preformed vessels and are no longer directed toward angiogenic sprouts, resulting in the formation of a thicker denser vasculature. In cultured ECs, a sustained expression of CCN1 through adenoviral gene transfer increases YAP phosphorylation and reduced its localization into the nucleus. CCN1‐integrin signaling through focal adhesion and the actin cytoskeleton provides the cells with a soft compliant matrix putting them in in low contractility, YAP repressive cytoskeletal states. This crosstalk between CCN1 and YAP regulates phenotypical plasticity of ECs and promotes correct sprouting and branching patterns of developing blood vessels.Support or Funding InformationR01EY022091‐05A1R01EY024998‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CCN family member 2/connective tissue growth factor (CCN2/CTGF) is regulated by Wnt\u2013\u03b2-catenin signaling in nucleus pulposus cells

BackgroundThe aims of this study were to investigate the gene expression of CCN family members in rat intervertebral disc (IVD) cells and to examine whether Wnt–β-catenin signaling regulates the expression of CCN family 2 (CCN2)/connective tissue growth factor (CTGF) in rat nucleus pulposus (NP) cells.MethodsThe gene expression of CCN family members were assessed in rat IVD cells using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression pattern of CCN2 was also assessed in rat IVD cells using western blot and immunohistochemical analyses. Gain-of-function and loss-of-function experiments were performed to identify the mechanisms by which Wnt–β-catenin signaling influences the activity of the CCN2 promoter. To further determine if the mitogen-activated protein kinase (MAPK) pathway is required for the Wnt–β-catenin signaling-induced regulation of CCN2 expression in the NP cells, CCN2 expression was analyzed by reporter assay, RT-PCR and western blot analysis.ResultsCCN2 messenger RNA (mRNA) and protein were expressed in rat IVDs. Expression of CCN2 was significantly higher than for mRNA of other CCN family members in both rat NP and annulus fibrosus (AF) cells. The relative activity of the CCN2 promoter decreased 24 h after treatment with 6-bromoindirubin-3′-oxime (1.0 μM) (0.773 (95% 0.735, 0.812) P = 0.0077) in NP cells. In addition, treatment with the WT–β-catenin vector (500 ng) significantly decreased CCN2 promoter activity (0.688 (95% 0.535, 0.842) P = 0.0063), whereas β-catenin small interfering RNA (500 ng) significantly increased CCN2 promoter activity (1.775 (95% 1.435, 2.115) P < 0.001). Activation of Wnt–β-catenin signaling decreased the expression of CCN2 mRNA and protein by NP cells. Regulation of CCN2 by Wnt–β-catenin signaling involved the MAPK pathway in rat NP cells.ConclusionsThis study shows that Wnt–β-catenin signaling regulates the expression of CCN2 through the MAPK pathway in NP cells. Understanding the balance between Wnt–β-catenin signaling and CCN2 is necessary for developing therapeutic alternatives for the treatment of IVD degeneration.

Metabolic regulation of the CCN family genes by glycolysis in chondrocytes.

The CCN family consists of 6 genes in the mammalian genome and produces multifunctional proteins involved in a variety of biological processes. Recent reports indicate the profound roles of CCN2 in energy metabolism in chondrocytes, and Ccn2 deficiency is known to alter the expression of 2 other family members including Ccn3. However, almost nothing is known concerning the regulation of the CCN family genes by energy metabolism. In order to gain insight into this critical issue, we initially and comprehensively evaluated the effect of inhibition of glycolysis on the expression of all of the CCN family genes in chondrocytic cells. Upon the inhibition of a glycolytic enzyme, repression of CCN2 expression was observed, whereas CCN3 expression was conversely induced. Similar repression of CCN2 was conferred by the inhibition of aerobic ATP production, which, however, did not induce CCN3 expression. In contrast, glucose starvation significantly enhanced the expression of CCN3 in those cells. The results of a reporter gene assay using a molecular construct containing a CCN3 proximal promoter revealed a dose-dependent induction of the CCN3 promoter activity by the glycolytic inhibitor in chondrocytic cells. These results unveiled a critical role of glycolytic activity in the regulation of CCN2 and CCN3, which activity mediated the mutual regulation of these 2 major CCN family members in chondrocytes.

CRE and SRE mediate LPA-induced CCN1 transcription in mouse aortic smooth muscle cells

Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein (ox-LDL), is a potent bioactive phospholipid. Our recent data reveal that LPA induces matricellular protein CCN1 (also known as Cyr61) expression in aortic smooth muscle cells (SMCs) and that CCN1 bridges LPA and integrin signaling pathways leading to SMC migration. Whether and how LPA regulates the transcriptional machinery of the CCN1 gene are unknown. In this study, we found that LPA markedly induces CCN1 mRNA expression in SMCs. Using deleting mutation and reporter gene strategies, we demonstrated regions from -2038 to -1787 and from -101 to +63 of the CCN1 promoter contain the essential regulatory elements. The serum response element (SRE) and cyclic AMP-response element (CRE) are located in these regions. LPA induced time-dependent phosphorylation of serum response factor (SRF) and CRE-binding protein (CREB) in mouse SMCs. Luciferase assays of a series of deleted, mutated CCN1 promoter-reporter gene constructs and dominant negative construct revealed the distal SRE and the proximal CRE in the CCN1 promoter are required for LPA-induced CCN1 gene expression. Our results imply that elevated LPA levels may trigger SMC migration and exacerbate restenosis and atherosclerotic lesions through the induced CCN1, which communicates with a set of plasma membrane proteins and intracellular kinases.

Analysis of Posttranscriptional Regulation of CCN Genes.

Cells generally control the concentration of mRNA by transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation ("decay") cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, and degradation of the target mRNA are performed either individually, or in combination. From our experience, measurement of the steady-state levels of the mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying ccn2 gene expression level. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. Finally, the stability of ccn2 mRNAs is evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Here, we describe the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can in theory be applied to elucidating the posttranscriptional regulation of other genes belonging to the CCN family.

Promoter Analyses of CCN Genes.

Promoter analysis is the most basics in the analysis of gene regulation. Luciferase gene is the most commonly used reporter gene in promoter analysis. Luciferase is an enzyme that is used when firefly and Renilla reniformis (sea pansy) emit light. The first experimental step in this reporter gene assay is to connect a particular DNA segment to a luciferase gene. The second step is to transfect the reporter construct into the cells. Thereafter, stable luciferase will be produced with the help of transcriptional machinery, mRNA transporters, and translational machinery in the cells. Luciferase assay measures the quantity of light that is emitted by luciferin-luciferase reaction. Consistent with the fact that CCN2 expression has been shown to be altered by a variety of stimuli, the CCN2 promoter region also haa been shown to be bound and regulated by multiple transcription factors such as Smad, MMP3, NF-κB, AP1, TCF/LEF, and Sox9.

AFAP1 Is a Novel Downstream Mediator of TGF-β1 for CCN2 Induction in Osteoblasts.

BackgroundCCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-β1; however, the molecular mechanisms that control CCN2 induction in osteoblasts are poorly understood. AFAP1 binds activated forms of Src and can direct the activation of Src in certain cell types, however a role for AFAP1 downstream of TGF-β1 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-β1 in primary osteoblasts.ResultsWe demonstrated that AFAP1 expression in osteoblasts occurs in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production/maturation (~day 14–21), an a further increase in expression during mineralization (~day 21). AFAP1 expression is induced by TGF-β1 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-β1 and CCN2 promoter activity and protein induction by TGF-β1 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-β1. We also demonstrated that TGF-β1 induction of extracellular matrix protein collagen XIIa occurs in an AFAP1 dependent fashion.ConclusionsThis study demonstrates that AFAP1 is an essential downstream signaling component of TGF-β1 for Src activation, CCN2 induction and collagen XIIa in osteoblasts.

Cell-based assay system for high-throughput screening of anti-photo-aging agents in fibroblast transfectants.

The matricellular protein CCN1 is significantly elevated in acutely ultraviolet-irradiated human skin and negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation. In this study, we established a stable cell line, termed CCN1-GFs, by transfection of the pAcGFP1-1-CCN1 promoter plasmid and examined its usefulness as a cell-based assay system for screening anti-aging ingredients. The promoter of the reporter plasmid pAcGFP1-1-CCN1 promoter was transfected into NIH3T3 cells using the Lipofectamine reagent. G418-resistant cells were selected and further cloned. To confirm whether AcGFP1-1-CCN1 promoter plasmid recombined in the NIH3T3 cells, the level of AcGFP1-1-CCN1 was measured by PCR analysis. To determine if NIH3T3 cells expressed the gene encoding green fluorescence protein in a CCN1 promoter-dependent manner, the reporter enzyme activities were assayed using a fluorimeter and flow cytometer. To determine if CCN1 inhibitor, which was selected through this system, exerted a direct effect on the downstream signal, mRNA expression of collagen1 and MMP1A was checked by using real-time PCR. UVB irradiation of CCN1-GFs resulted in increased CCN1 promoter activity. Treatment with retinoic acid, a CCN1 inhibitor, inhibited UV-induced CCN1 promoter activity. Subsequent use of this assay system to screen anti-aging ingredients revealed that CCN1-GFs treated with sclareol showed decreased levels of UVB-induced CCN1 expression. Sclareol attenuated UVB-induced photo-aging by an increase in collagen synthesis and decrease in MMP-1 activity.

A possible contribution of endothelial CCN1 downregulation due to Fli1 deficiency to the development of digital ulcers in systemic sclerosis.

CCN1 is a pleiotropic molecule involved in angiogenesis and postnatal vasculogenesis, both of which are impaired in systemic sclerosis (SSc). To elucidate the potential role of CCN1 in the development of SSc, we investigated CCN1 expression in the lesional skin of SSc patients and SSc animal models and the clinical correlation of serum CCN1 levels. CCN1 expression was markedly decreased in dermal small blood vessels of SSc patients compared with those of healthy controls, while comparable between normal and SSc dermal fibroblasts. Transcription factor Fli1, whose deficiency due to epigenetic suppression is implicated in the pathogenesis of SSc, occupied the CCN1 promoter and gene silencing of Fli1 resulted in the reduction of CCN1 expression in human dermal microvascular endothelial cells. Consistently, CCN1 expression was suppressed uniformly and remarkably in dermal blood vessels of Fli1(+/-) mice and partially in those of endothelial cell-specific Fli1 knockout mice. Furthermore, serum CCN1 levels were significantly decreased in SSc patients with previous and current history of digital ulcers as compared to those without. Collectively, these results suggest that endothelial CCN1 downregulation at least partially due to Fli1 deficiency may contribute to the development of digital ulcers in SSc patients. This study further supports the idea that epigenetic downregulation of Fli1 is a potential predisposing factor in the pathogenesis of SSc.

CAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1

Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel cAMP-dependent mechanism controlling VSMC behaviour.

CCN2 Functions as Anti-Inflammatory Molecule in the Intervertebral Disc

The aim of the study was to delineate regulation of CTGF/CCN2 expression by cytokines, IL-1β and TNF-α. In addition, we addressed if CCN2 modulates cytokine action on NP cells. Our studies showed that cytokines suppress CCN2 expression and that this effect was mediated in an NF-κB dependent but MAPK independent fashion. Site-directed mutagenesis and genomic chromatin immunoprecipitation (ChIP) analyses showed that the conserved κB sites located at −93/−86 and −546/−537 bp in the CCN2 promoter interacted with p65/RelA and mediated this suppressive effect of cytokines on CCN2 expression. Concerning role of CCN2 under inflammatory state, it was seen that the treatment of NP cells with IL-1β along with CCN2 blocked the stimulatory effect of IL-1β on MMP-3, ADAMTS5, prolyl hydroxylase 3 and syndecan 4 expression. Furthermore, silencing of CCN2 in human NP cells resulted in increased expression of catabolic genes and inflammatory cytokines such as IL-6, IL-4, and IL-12 as measured by gene expression analysis and cytokine protein array, respectively, under basal state. However, reporter analysis clearly showed that the inhibitory effect of CCN2 on IL-1β was independent of regulation of NF-κB signaling pathway. Using echistatin and VLO4, peptide inhibitors to αvβ3 and α5β1 integrins, we also showed that CCN2 interaction with these integrins was necessary for the inhibition of IL-1β-induced catabolic gene expression. Finally, analysis of human tissues showed a trend of altered expression of these integrins during degeneration. Collective, our results suggest that CCN2 and cytokines may form a functional negative feedback loop in NP cells important in the pathogenesis of disc degeneration. Disclosure of Interest None declared

CCN2 induction by TGF‐β1 requires osteoblast specific promoter motifs (541.6)

Connective tissue growth factor (CCN2; formerly CTGF) is a 38kDa, cysteine rich, extracellular matrix protein that belongs to the CCN family of proteins. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF‐B1 and acts as a mediator of TGF‐B1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of CCN2 promoter motifs involved in CCN2 induction by TGF‐B1 in primary osteoblasts. We used a combination of luciferase promoter reporter assays and gel shift studies to demonstrate the novel requirement of both the TGF‐B1 response element (TRE) and Smad binding element (SBE) for CCN2 promoter trans‐activation in osteoblasts. We propose that the TRE and SBE function as cognate motifs for CCN2 induction by TGF‐B1 in an osteoblast specific manner.Grant Funding Source: Supported by NIAMS

CCN2 Suppresses Catabolic Effects of Interleukin-1β through α5β1 and αVβ3 Integrins in Nucleus Pulposus Cells: IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION

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.

Tumour cells down-regulate CCN2 gene expression in co-cultured fibroblasts in a Smad7- and ERK-dependent manner

BackgroundRecent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix components found in the stroma. The aim of this study was to investigate mechanisms involved in tumour cell-mediated regulation of extracellular matrix and adhesion molecules in co-cultured fibroblasts. To this end, microarray analysis was performed on CCD-1068SK human fibroblast cells after direct co-culture with MDA-MB-231 human breast tumour cells.ResultsWe found that the expression of both connective tissue growth factor (CTGF/CCN2) and type I collagen was negatively regulated in CCD-1068SK fibroblast cells under direct co-culture conditions. Further analysis revealed that Smad7, a known negative regulator of the Smad signalling pathway involved in CCN2 promoter regulation, was increased in directly co-cultured fibroblasts. Inhibition of Smad7 expression in CCD-1068SK fibroblasts resulted in increased CCN2 expression, while Smad7 overexpression had the opposite effect. Silencing CCN2 gene expression in fibroblasts led, in turn, to a decrease in type I collagen mRNA and protein levels. ERK signalling was also shown to be impaired in CCD-1068SK fibroblasts after direct co-culture with MDA-MB-231 tumour cells, with Smad7 overexpression in fibroblasts leading to a similar decrease in ERK activity. These effects were not, however, seen in fibroblasts that were indirectly co-cultured with tumour cells.ConclusionWe therefore conclude that breast cancer cells require close contact with fibroblasts in order to upregulate Smad7 which, in turn, leads to decreased ERK signalling resulting in diminished expression of the stromal proteins CCN2 and type I collagen.

Nov/Ccn3, a Novel Transcriptional Target of FoxO1, Impairs Pancreatic β-Cell Function

Type 2 diabetes is characterized by both insulin resistance and progressive deterioration of β-cell function. The forkhead transcription factor FoxO1 is a prominent mediator of insulin signaling in β-cells. We reasoned that identification of FoxO1 target genes in β-cells could reveal mechanisms linking β-cell dysfunction to insulin resistance. In this study, we report the characterization of Nov/Ccn3 as a novel transcriptional target of FoxO1 in pancreatic β-cells. FoxO1 binds to an evolutionarily conserved response element in the Ccn3 promoter to regulate its expression. Accordingly, CCN3 levels are elevated in pancreatic islets of mice with overexpression of a constitutively active form of FoxO1 or insulin resistance. Our functional studies reveal that CCN3 impairs β-cell proliferation concomitantly with a reduction in cAMP levels. Moreover, CCN3 decreases glucose oxidation, which translates into inhibition of glucose-stimulated Ca2+ entry and insulin secretion. Our results identify CCN3, a novel transcriptional target of FoxO1 in pancreatic β-cells, as a potential target for therapeutic intervention in the treatment of diabetes.