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The matricellular protein CCN1 promotes mucosal healing in murine colitis through IL-6.

ABSTRACT: The matricellular protein CCN1 (CYR61) is known to function in wound healing and is upregulated in colons of patients with Crohn's disease and ulcerative colitis, yet its specific role in colitis is unknown. Here we have used Ccn1(dm/dm) knockin mice expressing a CCN1 mutant unable to bind integrins ?6?1 and ?M?2 as a model to probe CCN1 function in dextran sodium sulfate (DSS)-induced colitis. Ccn1(dm/dm) mice exhibited high mortality, impaired mucosal healing, and diminished interleukin-6 (IL-6) expression during the repair phase of DSS-induced colitis compared with wild-type mice, despite having comparable severity of initial inflammation and tissue injury. CCN1-induced IL-6 expression in macrophages through integrin ?M?2 and in fibroblasts through ?6?1, and IL-6 promoted intestinal epithelial cell (IEC) proliferation. Administration of purified CCN1 protein fully rescued Ccn1(dm/dm) mice from DSS-induced mortality, restored IEC proliferation and enhanced mucosal healing, whereas delivery of IL-6 partially rectified these defects. CCN1 therapy accelerated mucosal healing and recovery from DSS-induced colitis even in wild-type mice. These findings reveal a critical role for CCN1 in restoring mucosal homeostasis after intestinal injury in part through integrin-mediated induction of IL-6 expression, and suggest a therapeutic potential for activating the CCN1/IL-6 axis for treating inflammatory bowel disease.

SUBMITTER: Choi JS

PROVIDER: S-EPMC4583322 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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The matricellular protein CCN1 promotes mucosal healing in murine colitis through IL-6.

Choi J S JS Kim K-H KH Lau L F LF

Mucosal immunology 20150325 6

The matricellular protein CCN1 (CYR61) is known to function in wound healing and is upregulated in colons of patients with Crohn's disease and ulcerative colitis, yet its specific role in colitis is unknown. Here we have used Ccn1(dm/dm) knockin mice expressing a CCN1 mutant unable to bind integrins α6β1 and αMβ2 as a model to probe CCN1 function in dextran sodium sulfate (DSS)-induced colitis. Ccn1(dm/dm) mice exhibited high mortality, impaired mucosal healing, and diminished interleukin-6 (IL- ...[more]

PMID: 25807183

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Project description:Effects of the matricellular protein CCN1 on macrophage inflammatory gene expression was tested in a macrophage cell line I-13.35. CCN1 (CYR61) is a matricellular protein that is highly expressed at sites of inflammation and wound repair. In these contexts, CCN1 can modify the activities of specific cytokines, enabling TNF-alpha to be cytotoxic without blocking NFkB activity and enhancing the apoptotic activity of FasL and TRAIL. Here we show that CCN1 supports the adhesion of macrophages through integrin alphaMbeta2 and syndecan-4, activates NFkB-mediated transcription, and induces a pro-inflammatory genetic program characteristic of classically activated M1 macrophages that participates in Th1 responses. The effects of CCN1 include upregulation of cytokines (TNFa, IL-1a, IL-1b, IL-6, IL-12b), chemokines (MIP-1a, MCP-3, Gro1, Gro2, IP-10), regulators of oxidative stress and complement (iNOS, C3), and downregulation of specific receptors (TLR4, IL-10rb) and anti-inflammatory factors (TGF-b1). CCN1 regulates this genetic program through at least two distinct mechanisms: an immediate-early response resulting from direct activation of NFkB by CCN1, leading to the synthesis of cytokines including TNFa and IP-10; and a delayed response resulting from CCN1-induced TNFa, which acts as an autocrine/paracrine mediator to activate the expression of other cytokines including IL-1b and IL-6. These results identify CCN1 as a novel component of the extracellular matrix that activates pro-inflammatory genes in macrophages, implicating its role in regulating macrophage function during inflammation. Macrophage cell line I-13.35 (from Tlr4-defective C3H/HeJ) was treated with purified recombinant CCN1 protein for 6 hr. Total RNA was isolated, converted to cRNA probes, and hybridized to a oligonucleotide array representing 113 murine inflammation-related genes (from SA Biosciences at www.sabiosciences.com, Cat. Number OMM-011.) The array is a nylon membrane based DNA microarray on which 60-mer oligonucleotides probes were printed. Gene expression profile of resting cells incubated in serum-free medium (containing albumin, BSA) was used as a control for basal levels of gene expression.

Project description:Activation of RhoA, a low molecular-weight G-protein, plays an important role in protecting the heart against ischemic stress. Studies using non-cardiac cells demonstrate that the expression and subsequent secretion of the matricellular protein CCN1 is induced by GPCR agonists that activate RhoA. In this study we determined whether and how CCN1 is induced by GPCR agonists in cardiomyocytes and examined the role of CCN1 in ischemic cardioprotection in cardiomyocytes and the isolated perfused heart. In neonatal rat ventricular myocytes (NRVMs), sphingosine 1-phosphate (S1P), lysophosphatidic acid (LPA) and endothelin-1 induced robust increases in CCN1 expression while phenylephrine, isoproterenol and carbachol had little or no effect. The ability of agonists to activate the small G-protein RhoA correlated with their ability to induce CCN1. CCN1 induction by S1P was blocked when RhoA function was inhibited with C3 exoenzyme or a pharmacological RhoA inhibitor. Conversely overexpression of RhoA was sufficient to induce CCN1 expression. To delineate the signals downstream of RhoA we tested the role of MRTF-A (MKL1), a co-activator of SRF, in S1P-mediated CCN1 expression. S1P increased the nuclear accumulation of MRTF-A and this was inhibited by the functional inactivation of RhoA. In addition, pharmacological inhibitors of MRTF-A or knockdown of MRTF-A significantly diminished S1P-mediated CCN1 expression, indicating a requirement for RhoA/MRTF-A signaling. We also present data indicating that CCN1 is secreted following agonist treatment and RhoA activation, and binds to cells where it can serve an autocrine function. To determine the functional significance of CCN1 expression and signaling, simulated ischemia/reperfusion (sI/R)-induced apoptosis was assessed in NRVMs. The ability of S1P to protect against sI/R was significantly reduced by the inhibition of RhoA, ROCK or MRTF-A or by CCN1 knockdown. We also demonstrate that ischemia/reperfusion induces CCN1 expression in the isolated perfused heart and that this functions as a cardioprotective mechanism, evidenced by the significant increase in infarct development in response to I/R in the cardiac specific CCN1 KO relative to control mice. Our findings implicate CCN1 as a mediator of cardioprotection induced by GPCR agonists that activate RhoA/MRTF-A signaling.

Project description:Tetraspanins are a family of proteins with four transmembrane domains that associate between themselves and cluster with other partner proteins, conforming a distinct class of membrane domains, the tetraspanin-enriched microdomains (TEMs). These TEMs constitute macromolecular signaling platforms that regulate key processes in several cellular settings controlling signaling thresholds and avidity of receptors. In this study, we investigated the role of CD9, a tetraspanin that regulates major biological processes such as cell migration and immunological responses, in two mouse models of colitis that have been used to study the pathogenesis of inflammatory bowel disease (IBD). Previous in vitro studies revealed an important role in the interaction of leukocytes with inflamed endothelium, but in vivo evidence of the involvement of CD9 in inflammatory diseases is scarce. Here, we studied the role of CD9 in the pathogenesis of colitis in vivo. Colitis was induced by administration of dextran sodium sulfate (DSS), a chemical colitogen that causes epithelial disruption and intestinal inflammation. CD9-/- mice showed less severe colitis than wild-type counterparts upon exposure to DSS (2% solution) and enhanced survival in response to a lethal DSS dose (4%). Decreased neutrophil and macrophage cell infiltration was observed in colonic tissue from CD9-/- animals, in accordance with their lower serum levels of TNF-?, IL-6, and other proinflammatory cytokines in the colon. The specific role of CD9 in IBD was further dissected by transfer of CD4+ CD45RBhi naive T cells into the Rag1-/- mouse colitis model. However, no significant differences were observed in these settings between both groups, ruling out a role for CD9 in IBD in the lymphoid compartment. Experiments with bone marrow chimeras revealed that CD9 in the non-hematopoietic compartment is involved in colon injury and limits the proliferation of epithelial cells. Our data indicate that CD9 in non-hematopoietic cells plays an important role in colitis by limiting epithelial cell proliferation. Future strategies to repress CD9 expression may be of therapeutic benefit in the treatment of IBD.

Dataset Information

The matricellular protein CCN1 promotes mucosal healing in murine colitis through IL-6.

Publications

The matricellular protein CCN1 promotes mucosal healing in murine colitis through IL-6.

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