Attenuation of collagen production with small interfering RNA of SPARC in cultured fibroblasts from the skin of patients with scleroderma.
OBJECTIVE
Recently, it was observed that SPARC (secreted protein, acidic and rich in cysteine) is overexpressed in the fibroblasts of skin biopsy specimens obtained from patients with systemic sclerosis (SSc; scleroderma), and that specific inhibition of SPARC expression in normal human fibroblasts attenuated the profibrotic effect of transforming growth factor beta (TGFbeta). The purpose of this study was to examine whether inhibition of SPARC with small interfering RNA (siRNA) can be used to ameliorate the overproduction of major extracellular matrix components in SSc fibroblasts.


METHODS
Fibroblasts obtained from biopsy specimens of the unaffected skin of 3 patients with diffuse SSc and 3 age- and sex-matched healthy controls were cultured and transfected with SPARC siRNA. Before and after transfection, real-time quantitative reverse transcription-polymerase chain reaction, immunostaining, and Western blotting were used to examine the transcription and/or protein levels of SPARC, connective tissue growth factor (CTGF), type I collagen, type II collagen, TGFbeta receptor 1 (TGFbetaR1), and Smad3. Student's paired t-tests were used to determine the significance of the results.


RESULTS
SSc fibroblasts showed overexpression of SPARC, COL1A2, COL3A1, and CTGF, which is consistent with an activated fibroblast phenotype. Upon inhibition of SPARC with siRNA, these activated SSc fibroblasts showed decreased gene expression of COL1A2, COL3A1, and CTGF (43%, 54%, and 58%, respectively). In addition, the overproduction of type I collagen protein in SSc fibroblasts was inhibited and was shown to parallel levels of SPARC expression. In contrast, the expression of TGFBR1 and Smad3 did not change significantly nor was the level of phosphorylation of Smad3 reduced upon SPARC siRNA silencing in unstimulated cultured SSc fibroblasts.


CONCLUSIONS
Overproduction of collagens in SSc skin fibroblasts can be attenuated through SPARC silencing. This inhibition may be associated either with direct interaction between SPARC and collagens or with inhibition of CTGF, which is a downstream effector of TGFbeta signaling. However, SPARC silencing in SSc fibroblasts appears not to be associated with TGFBR1- and Smad3-dependent processes. Application of SPARC silencing represents a potential therapeutic approach to SSc.
Zhou
Xiaodong
X
University of Texas-Houston Health Science Center, Houston, TX 77030, USA. xiaodong@uth.tmc.edu
Tan
Filemon K
FK
Guo
Xinjian
X
Arnett
Frank C
FC
D030201:Activin Receptors, Type I / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D024043:Collagen Type II / Q000378:metabolism*; D055513:Connective Tissue Growth Factor; D005260:Female; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D015676:Osteonectin / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering / Q000235:genetics*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000473:pathology; D012867:Skin; D014162:Transfection
D015415:Biomarkers; C526317:CCN2 protein, human; D024042:Collagen Type I; D024043:Collagen Type II; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D015676:Osteonectin; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human