Systemic sclerosis (SSc) or scleroderma is a multi-systemic disease of unknown etiology. Skin is the most obvious organ involved. Skin fibroblasts from SSc patients demonstrate an activated phenotype that includes increased synthesis of extracellular matrix (ECM) components. Abnormal interactions among fibroblasts, ECM and cytokines may contribute to the production of excessive amount of ECM components. Increased activities of fibrogenic cytokines, such as transforming growth factor-beta (TGF-beta), have been implicated in some of the pathogenic lesion of SSc. SPARC (secreted protein, acidic and rich in cysteine), a matricellular protein, is an important regulator of cell-matrix interaction and modulator of growth factors. Our recent studies indicated that gene and protein expression of SPARC is significantly increased in SSc dermal fibroblasts. Inhibition of the SPARC gene in normal fibroblasts induced a decreased expression of collagen type I and diminished response of both SPARC and collagen type I to TGF-beta1 stimulation. Therefore our hypothesis are: 1) increased expression of SPARC in SSc fibroblasts may alter the regulation of cell-matrix interaction, and selective suppression of SPARC may restore normal cell-matrix interaction, and perhaps attenuate the fibrotic phenotype in SSc dermal fibroblasts; 2) TGF-beta1 and SPARC may participate in a reciprocal feedback loop in the regulation of cell-matrix interactions, and suppression of the SPARC gene may diminish fibrogenic effects of TGF-beta1 in SSc and normal fibroblasts. To examine our hypothesis, we propose the following specific aims:
Specific aim 1 : To investigate whether specific inhibition of SPARC can abrogate fibrotic activity in SSc dermal fibroblasts, we will use specific siRNA to inhibit gene expression of SPARC in SSc dermal fibroblasts, and then examine these fibroblasts for: 1). Alterations in gene and protein expressions of ECM constituents (e.g., collagens, fibronectin, MMPs and TIMPs) and fibrogenic cytokines/growth factors that are dysregulated in SSc (e.g., TGF-beta, CTGF, IL-ICT, IL-6, IL-8, MCP-1; 2). Alterations in collagen synthesis and degradation; 3). Alterations in fibroblast proliferation and functional contraction. In addition, the ECM will be examined for protein deposition (e.g. collagen and fibronectin) and enzyme activity (e.g. MMPs).
Specific aim 2 : To investigate whether specific inhibition of SPARC in SSc and normal dermal fibroblasts can attenuate fibrogenic effects of TGF-beta, we will compare the effects of exogenous TGF-beta1 on cultured fibroblasts with or without suppression of the SPARC gene, by examining these fibroblasts for alterations in gene and protein expressions of ECM constituents (e.g., collagens, fibronectin, MMPs and TIMPs) and factors involving in the regulatory pathway of TGF-beta, as well as fibroblast proliferation and functional contraction, and protein deposition and enzyme activity in the ECM. In addition, we will investigate whether exogenous SPARC can restore or increase fibrogenic effects of TGF-beta on cultured fibroblasts with or without suppression of the SPARC gene using recombinent SPARC protein.
