ABSTRACT
BACKGROUND: Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. Although transforming growth factor (TGF)-ß1-induced connective tissue growth factor (CTGF/CCN2) expression has been presented in SSc fibrosis, the therapeutic potential of targeting CTGF in SSc has not been fully explored. COA-Cl is a novel nucleic acid analog, which is reported to have pleiotropic beneficial biologic effects. OBJECTIVE: We examine the effects of COA-Cl on TGF-ß1-induced CTGF expression in normal human dermal fibroblast (NHDF). We also examined the effects of COA-Cl on CTGF expression in a mouse SSc model of angiotensin II (Ang II)-induced skin fibrosis. METHODS: NHDF was cultured for in vitro experiments. For in vivo experiments, C57BL/6J mice were treated with Ang II for 14 days by subcutaneous osmotic pump. Quantitative real-time polymerase chain reaction, western blot analysis, immunohistochemical staining and immunofluorescence staining were performed to examine the expression levels of CTGF and phosphorylation levels of Smad2/3, ERK1/2 and Akt. RESULTS: COA-Cl attenuated the TGF-ß1-induced expression of both CTGF mRNA and protein in NHDF. Although COA-Cl did not alter the TGF-ß1-induced phosphorylation of Smad2/3 or ERK1/2, it reduced the TGF-ß1-induced phosphorylation levels of Akt in NHDF. Notably, COA-Cl dephosphorylated the Akt of lysates of TGF-ß1-treated NHDF. COA-Cl reduced the levels of CTGF mRNA, CTGF protein, dermal thickness, collagen content and Akt phosphorylation in the skin of mice SSc model. CONCLUSION: These results imply that the inhibition of TGF-ß1-induced CTGF expression by COA-Cl may be a therapeutic approach for SSc.
Subject(s)
Adenosine/analogs & derivatives , Connective Tissue Growth Factor/metabolism , Scleroderma, Systemic/drug therapy , Skin/pathology , Transforming Growth Factor beta1/metabolism , Adenosine/pharmacology , Adenosine/therapeutic use , Angiotensin II/toxicity , Animals , Cell Line , Disease Models, Animal , Drug Evaluation, Preclinical , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibrosis/drug therapy , Fibrosis/etiology , Fibrosis/pathology , Humans , Male , Mice , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Scleroderma, Systemic/pathology , Skin/drug effectsABSTRACT
We previously demonstrated a potent angiogenic effect of a newly developed adenosine-like agent namedCOA-Cl.COA-Cl exerted tube forming activity in human umbilical vein endothelial cells in the presence of normal human dermal fibroblasts (NHDF). We therefore explored whether and howCOA-Cl modulates gene expression and protein secretion ofVEGF, a master regulator of angiogenesis, inNHDFRT-PCRandELISArevealed thatCOA-Cl upregulatedVEGF mRNAexpression and protein secretion inNHDFHIF1α(hypoxia-inducible factor 1α), a transcription factor, andPGC-1α(peroxisome proliferator-activated receptor-γcoactivator-1α), a transcriptional coactivator, are known to positively regulate theVEGFgene. Immunoblot andRT-PCRanalyses revealed thatCOA-Cl markedly upregulated the expression ofPGC-1αprotein andmRNACOA-Cl had no effect on the expression ofHIF1αprotein andmRNAin both hypoxia and normoxia. SilencingPGC-1αgene, but notHIF1αgene, by small interferingRNAattenuated the ability ofCOA-Cl to promoteVEGFsecretion. When an N-terminal fragment ofPGC-1αwas cotransfected with its partner transcription factorERRα(estrogen-related receptor-α) inCOS-7 cells,COA-Cl upregulated the expression of the endogenousVEGF mRNA However,COA-Cl had no effect on the expression ofVEGF, whenHIF1αwas transfected.COA-Cl inducesVEGFgene expression and protein secretion in fibroblasts. The transcriptional coactivatorPGC-1α, in concert withERRα, plays a key role in theCOA-Cl-inducedVEGFproduction.COA-Cl-induced activation ofPGC-1α-ERRα-VEGFpathway has a potential as a novel means for therapeutic angiogenesis.
