Small interfering RNA inhibition of SPARC attenuates the profibrotic effect of transforming growth factor β1 in cultured normal human fibroblasts

Abstract

Objective. SPARC (secreted protein, acidic and rich in cysteine), or osteonectin, is a matricellular protein. Recently, it was observed to be overexpressed in fibroblasts obtained from the skin of patients with scleroderma, as well as in different tissues from patients with several other fibrotic disorders. Moreover, a genetic polymorphism in SPARC has been associated with susceptibility to scleroderma. Transforming growth factor ␤1 (TGF␤1) is a profibrotic cytokine that stimulates excessive collagen production in patients with scleroderma or other fibrotic diseases. The purpose of this study was to examine whether specific inhibition of SPARC can influence the expression of type I collagen and ameliorate the profibrotic activity of TGF␤1 on normal human fibroblasts.Methods. Fibroblasts obtained from the skin of 4 healthy individuals were cultured and transfected with SPARC small interfering RNA (siRNA). TGF␤ was used as a fibrosis stimulus in cultured fibroblasts. Real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting were used to measure transcription and protein levels of SPARC and type I collagen, respectively.Results. The fibroblasts transfected with SPARC siRNA showed decreased expression of both SPARC and type I collagen. Exogenous TGF␤1 induced increased expression of both SPARC and type I collagen in cultured normal human fibroblasts, but this response was significantly blunted in the fibroblasts transfected with SPARC siRNA. Conclusion. TGF␤1 can induce increased expression of both SPARC and type I collagen. Specific inhibition of SPARC led to decreased expression of type I collagen and attenuated the profibrotic effect of TGF␤1 in cultured normal human fibroblasts. Use of siRNA to silence SPARC represents a potential therapeutic approach to fibrotic disorders such as scleroderma.

SPARC downregulation attenuates the profibrogenic response of hepatic stellate cells induced by TGF-β1and PDGF

“…As previously published, siSPARC treatment significantly reduced TGF-␤ 1 mRNA and protein levels in HSCs (10). As expected, SPARC knockdown also led to downregulation of collagen type I expression in both LX-2 and rat primary cultured HSCs (34,39). Since only a 50% reduction in SPARC levels was observed in SPARC siRNA-treated CFSC-2G cells, downregulation of collagen type I was not significant in this cell line; however, the expression of this ECM protein was decreased when SPARCdeficient CFSC-2G cells were treated with TGF-␤ 1 .…”

SPARC Suppresses Apoptosis of Idiopathic Pulmonary Fibrosis Fibroblasts through Constitutive Activation of β-Catenin

“…In an intratracheal bleomycin model, Strandjord et al (37) showed that collagen accumulation is decreased in SPARC-null mice. Strandjord et al showed a decrease in collagen accumulation in the lungs of SPARC-null mice following bleomycin injury, a finding supported by recent studies such as those of Zhou et al (38) showing that SPARC regulates collagen expression. The bleomycin model in mice clearly does not completely mirror IPF, especially in regard to chronicity and the presence of fibroblastic foci in IPF but not in the bleomycin model.…”

Lack of host SPARC enhances vascular function and tumor spread in an orthotopic murine model of pancreatic carcinoma

“…Similar to SPARC, transforming growth factor- (TGF-) isoforms have been shown to have increased expression in pancreatic cancer; to correlate with increased invasion and decreased survival in late-stage disease; and to have paradoxical effects on pancreatic tumor progression (Pardali and Moustakas, 2007;Truty and Urrutia, 2007). Moreover, TGF- isoforms activate pancreatic stellate cells and increase SPARC expression to induce a desmoplastic response that is characteristic of pancreatic ductal adenocarcinoma (Mahadevan and Von Hoff, 2007;Podhajcer et al, 2008;Zhou et al, 2005). A common occurrence in the metastatic progression of pancreatic carcinoma is epithelial-to-mesenchymal transition (EMT).…”