Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-β1-stimulated porcine skin fibroblasts

Previous studies in a pig model of skin wound healing showed a coordinate expression of transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF), and exposure of porcine skin fibroblasts in vitro to recombinant human CTGF significantly up-regulated mRNA levels for a number of molecules. Therefore, based on recent reports that small interfering RNA (siRNA; double-stranded RNA) can effect silencing of the expression of gene(s), this approach has now been used with CTGF-specific siRNA to better understand the function of this growth factor in regulating matrix homeostasis and repair. Normal skin fibroblasts from Yorkshire pigs were treated with 0.1-0.8 muM CTGF siRNA, TGF-beta, or TGF-beta plus CTGF siRNA for 12-48 hours. Total RNA was isolated and quantified, and then mRNA levels for specific molecules were analyzed by reverse transcription-polymerase chain reaction. Protein levels for CTGF and HSP47 were assessed by Western-blot analysis. CTGF siRNA transfection led to significant decreases in mRNA and protein levels for CTGF in both a dose- and time-dependent manner. mRNA levels for types I and III procollagen, decorin, HSP47, tissue inhibitor of metalloproteinase -1, -2, -3, and basic fibroblast growth factor were also significantly and uniquely decreased following exposure of cells to CTGF siRNA. Addition of TGF-beta to the cells led to increases in CTGF mRNA levels that were blocked by CTGF siRNA. CTGF siRNA exposure also significantly and selectively down-regulated TGF-beta-mediated increases in mRNA levels for types I and III procollagen. The results indicate that CTGF can regulate extracellular matrix molecule, growth factor, and proteinase inhibitor gene expression, and that some of the TGF-beta effects on skin fibroblasts are via a CTGF-dependent pathway.