Mechanical Stretch Modulates the Promoter Activity of the Profibrotic Factor CCN2 through Increased Actin Polymerization and NF-κB Activation

Abstract

The connective tissue growth factor known as CCN2 is an inducible, profibrotic molecule that becomes aberrantly expressed in mechanical overload-bearing tissues. In this study, we found that CCN2 gene expression is rapidly induced in cyclically stretched bladder smooth muscle cells (SMCs) in vitro and in the detrusor muscle of a mechanically overloaded bladder in a rat model of experimental urethral obstruction. The activity of CCN2 promoter constructs, transiently transfected into cultured SMCs, was increased (up to 6-fold) by continuous cyclic stretching. Molecular analyses of the CCN2 promoter by serial construct deletions, cis-element mutagenesis, and electrophoretic mobility shift assays revealed that a highly conserved NF-B binding site located within the CCN2 proximal promoter region is responsible for the activation of the promoter by stretch. Chromatin immunoprecipitation assays showed that NF-B binds to the endogenous CCN2 promoter in both stretched cells and mechanically overloaded bladder tissues. Furthermore, stretch-dependent CCN2 promoter activity was significantly reduced upon inhibition of either phosphatidylinositol 3-kinase, p38 stress-activated kinase, or RhoA GTPase and was completely abolished upon inhibition of actin polymerization. Concordantly, actin polymerization was increased in either mechanically stretched cells or overloaded bladder tissues. Incubation of cultured SMCs with a cell-penetrating peptide containing the N-terminal sequence, Ac-EEED, of smooth muscle ␣-actin, altered both actin cytoskeleton organization and stretch-mediated nuclear relocation of NF-B, and subsequently, it reduced CCN2 promoter activity. Thus, mechanical stretch-induced changes in actin dynamics mediate NF-B activation and induce CCN2 gene expression, which probably initiates the fibrotic reactions observed in mechanical overloadassociated pathologies.

CCN2 Suppresses Catabolic Effects of Interleukin-1β through α5β1 and αVβ3 Integrins in Nucleus Pulposus Cells

“…Our results are in agreement with studies showing CCN2 suppression by inflammatory cytokines in other cell types, including chondrocytes, where prostaglandin E 2 , IL-1␤, and TNF-␣ are inhibitory (27)(28)(29)(30). Unlike in NP cells, some studies have reported the involvement of NF-B signaling in the induction of CCN2 in response to LPS, mechanical stretch, and hepatocyte growth factor (25,31,32). Thus, our results suggest that although p65-mediated gene repression is less common, it is context-and cell type-dependent (33).…”

“…There are two conserved NF-B binding sites at Ϫ93/Ϫ86 and Ϫ546/Ϫ537 bp on the human CCN2 promoter (24,25). We used the Multiz alignment in the UCSC Genome Browser to compare conservation of these sites between species.…”

The G Protein-coupled Receptor 30 Is Up-regulated by Hypoxia-inducible Factor-1α (HIF-1α) in Breast Cancer Cells and Cardiomyocytes

“…The CTGF luciferase reporter plasmid pCTGF (Ϫ1999/ϩ36)-luc (30), which is based on the backbone of vector pGL3-basic (Promega), was a gift from Dr. B. Chaqour.…”

Connective Tissue Growth Factor Regulates Retinal Neovascularization through p53 Protein-dependent Transactivation of the Matrix Metalloproteinase (MMP)-2 Gene

“…Chromatin Immunoprecipitation (ChIP) Assay-ChIP was performed as described previously with modifications allowing for a quantitative analysis of protein-DNA interaction (9). DNA-protein complexes were fixed by directly adding formaldehyde (1%) to freshly dissected retinas or cultured cells.…”