Connective tissue growth factor expression and induction by transforming growth factor-β is abrogated by simvastatin via a Rho signaling mechanism

Abstract

Connective tissue growth factor (CTGF), a potent profibrotic mediator, acts downstream and in concert with transforming growth factor (TGF)-β to drive fibrogenesis. Significant upregulation of CTGF has been reported in fibrogenic diseases, including idiopathic pulmonary fibrosis (IPF), and is partly responsible for associated excessive fibroblast proliferation and extracellular matrix deposition, but no effective therapy exists for averting such fibrogeneic events. Simvastatin has reported putative antifibrotic actions in renal fibroblasts; this study explores such actions on human IPF-derived and normal lung fibroblasts and examines associated driving mechanisms. Simvastatin reduces basal CTGF gene and protein expression in all fibroblast lines, overriding TGF-β induction through inhibition of the cholesterol synthesis pathway. Signaling pathways driving simvastatin's effects on CTGF/TGF-β interaction were evaluated using transient reporter transfection of a CTGF promoter construct. Inhibition of CTGF promoter activity by simvastatin was most marked at 10 μM concentration, reducing activity by 76.2 and 51.8% over TGF-β-stimulated cultures in IPF and normal fibroblasts, respectively. We also show that geranylgeranylpyrophosphate (GGPP), but not farnesylpyrophosphate, induces CTGF promoter activity following simvastatin inhibition by 55.3 and 31.1% over GGPP-negative cultures in IMR90 and IPF-derived fibroblasts, respectively, implicating small GTPase Rho involvement rather than Ras in these effects. Indeed, the specific Rho inhibitor C3 exotoxin significantly ( P < 0.05) suppressed TGF-β-induced CTGF promoter activity in transfected lung fibroblasts, a finding further supported by transfection of dominant-negative and constitutively active RhoA constructs, thus demonstrating that simvastatin through a Rho signaling mechanism in lung fibroblasts can modulate CTGF expression and interaction with TGF-β.

RhoA Modulates Smad Signaling during Transforming Growth Factor-β-induced Smooth Muscle Differentiation

“…6A). To confirm the role of RhoA in regulating functional activity of Smads, we transfected the SBE reporter into C3H10T1/2 cells, and the cells were then treated with a RhoA specific inhibitor C3 exotoxin followed by TGF-␤ induction (26). Luciferase assay showed that C3 exotoxin also significantly inhibited TGF-␤-induced SBE promoter activity (Fig.…”

Simvastatin Ameliorates Radiation Enteropathy Development After Localized, Fractionated Irradiation by a Protein C-Independent Mechanism

“…Other pleiotropic statin effects that may confer an enteroprotective effect include a reduction of oxidative stress, upregulation of tPA, or downregulation of TF, PAI-1, and connective tissue growth factor (CTGF). CTGF has been implicated in the development of intestinal radiation fibrosis (52)(53)(54)). An effect mediated through CTGF downregulation might explain why the protective effect of statins against delayed toxicity was greater than protection against early toxicity .…”

Repression of IP-10 by Interactions between Histone Deacetylation and Hypermethylation in Idiopathic Pulmonary Fibrosis

“…The internal control Renilla luciferase reporter construct pRL-SV40 was obtained from Promega (Southampton, United Kingdom). Transfections were performed in cells at 75% confluence as performed previously (42). Briefly, 0.25 g of the testing plasmid DNA and 0.25 ng of the internal control Renilla luciferase DNA were added to each well (24-well plate) using a 1:1 ratio of DNA/Transfast in serum-free medium.…”