Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-beta 1-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 microM 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.
Wang
Jian Fei
JF
McCaig Center for Joint Injury and Arthritis Research, Department of Microbiology and Infectious Disease, Faculty of Medicine, University of Calgary,Calgary, Alberta, Canada.
Olson
Merle E
ME
Ma
Lingling
L
Brigstock
David R
DR
Hart
David A
DA
D000818:Animals; D018929:Cell Culture Techniques; D055513:Connective Tissue Growth Factor; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000502:physiology*; D012867:Skin / Q000378:metabolism / Q000473:pathology*; D013552:Swine; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1
C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor