Tissue fibrosis occurs in a wide range of disease conditions resulting from excessive deposition of extracellular matrix (ECM). The ECM contains 3 major components: structural proteins and proteoglycans, matricellular proteins, and growth factors (1). Although structural proteins such as collagens are most prominently increased in fibrotic tissues, matricellular proteins and growth factors are believed to be the major players in the maintenance of homeostasis in the ECM (1).
SPARC (secreted protein, acidic and rich in cysteine), which is also referred to as osteonectin or BM-40, is a matricellular protein in the ECM that has multiple biologic functions. It participates in the modulation of cell–matrix interactions, cell adhesion, wound repair, and angiogenesis (1–3). Recently, accumulating evidence has suggested that SPARC may play an important role in fibrosis. Previous studies have shown increased expression of SPARC in many fibrotic disorders, including scleroderma (systemic sclerosis [SSc]), pulmonary fibrosis, renal interstitial fibrosis, hepatic cirrhosis, and atherosclerotic vascular lesions (4–8). Although the physiologic and pathologic significance of these observations is not clear, SPARC has shown the ability to stimulate the transforming growth factor β (TGFβ) signaling system through a TGFβ receptor– and Smad2/3-dependent pathway (9).
In animal models, SPARC-null mice displayed a diminished amount of pulmonary fibrosis compared with control mice after exposure to the profibrotic drug bleomycin (10). SPARC-null mesangial cells displayed reduced expression of both TGFβ1 and type I collagen, while the addition of SPARC to these cells induced early production of TGFβ1 and later augmentation of type I collagen (11). These observations suggest that SPARC regulates collagen expression that may be associated with its influence on TGFβ1 expression in mesangial cells. Type I collagen genes are stimulated by TGFβ1 through their promoters (12, 13), while any direct interactions between type I collagen and SPARC have not been defined.
In studies of SSc, a devastating human fibrotic disease that demonstrates overexpression of SPARC in dermal fibroblasts, our previously reported data suggested that a specific single-nucleotide polymorphism at the 3′-untranslated region of the SPARC gene is associated with quantitative expression of SPARC, as well as genetic susceptibility to this fibrosing disease (8). Therefore, increased amounts of SPARC may be harmful to the local environment in living tissues, presumably through dysregulation of the ECM. All of these factors make SPARC an attractive candidate to be targeted in the treatment of fibrotic conditions.
TGFβ is a multifunctional cytokine that controls proliferation, differentiation, and other functions in many types of cells. TGFβ has been widely accepted as a profibrotic cytokine that not only is found in fibrotic tissues of certain disease states but that also can convert normal tissues into a fibrotic phenotype (14, 15). TGFβ1 mediates the formation of ECM by stimulation of the synthesis of components such as collagen, suppression of matrix metalloproteinases, and induction of tissue inhibitors of these enzymes (16).
Considering the regulatory roles of both SPARC and TGFβ in the ECM, altered expression of one may affect the expression and functions of the other and may ultimately influence the synthesis of structural components of the ECM, such as type I collagen. This mutually regulatory relationship could provide the basis for exploring therapeutic strategies in the treatment of fibrotic diseases. The purpose of the in vitro studies described here was to examine whether specific inhibition of SPARC with small interfering RNA (siRNA) can influence the expression of type I collagen and ameliorate the profibrotic activity of TGFβ1 on normal skin fibroblasts.
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- MATERIALS AND METHODS
In fibrotic diseases such as SSc, fibroblasts are activated to produce excessive amounts of ECM components such as collagen (19). Numerous studies have shown that ECM biosynthesis and deposition are regulated by matricellular proteins and growth factors and by alterations in cell–ECM interactions that are accompanied by reorganization of the cytoskeletal network (20). SPARC and TGFβ represent a matricellular protein and a growth factor, respectively, that are 2 major regulators of the ECM.
In studies aimed at finding ways to ameliorate the fibrotic process, many profibrotic stimuli, such as TGFβ and other cytokines, have been intensely investigated. Few studies, however, have investigated how matricellular proteins, another group of important ECM regulators, influence the effects of these cytokines, modulate cellular functions, and ultimately prevent fibrosis. SPARC is a matricellular glycoprotein with multiple cellular and extracellular functions. SPARC and TGFβ have been shown to maintain homeostasis of the ECM in a cooperative and mutually regulatory manner. Results of recent studies suggest that SPARC may regulate the TGFβ signaling system through a TGFβ receptor– and Smad2/3-dependent pathway (9).
Currently, application of double-stranded siRNA to induce RNA silencing in cells has been widely accepted in many studies of gene functions (21). Gene-specific siRNA has been demonstrated to be able to facilitate the degradation of homologous RNA, resulting in corresponding gene silencing (22). Using siRNA to disrupt clinically important genes, such as p53, Ras (V12), CD4, and CD25, has exemplified potential therapeutic applications of siRNA (21).
In this study, we designed a SPARC sequence–specific siRNA that showed inhibition of SPARC gene expression in normal cultured human fibroblasts. Interestingly, SPARC “knock-down” fibroblasts with transient transfection of siRNA also showed reduction of type I collagen expression. This indicates that an important relationship between SPARC and type I collagen may be present in the cells and also supports the previous observation in mouse mesangial cells that SPARC-null mice displayed diminished expression of type I collagen (11). The finding that the addition of exogenous TGFβ1 to normal fibroblast cultures induced increased expression of both SPARC and type I collagen indicates that a fibrogenic stimulus impairs cellular and/or ECM homeostasis involving both matricellular and structural proteins. In contrast to the normal fibroblasts, the SPARC “knock-down” fibroblasts showed a diminished response of both SPARC and type I collagen to TGFβ1 stimulation. These observations indicate that controlling SPARC expression in cultured fibroblasts may influence the expression of collagen and attenuate the fibrotic activity of TGFβ1. This finding is of potential interest, especially given the knowledge that TGFβ is a major player in many forms of tissue fibrosis. Regulation of SPARC expression in tissue may optimize cellular and/or extracellular conditions to prevent the stimulation of fibrosis. Therefore, it will be interesting to pursue the studies of SPARC siRNA in certain fibrotic tissues, such as skin from patients with scleroderma, in the future.
Although this study represents SPARC inhibition in cultured fibroblasts only, and the precise mechanisms involved in the process need to be further explored, it represents a first attempt to use SPARC siRNA in the regulation of a major ECM component in culture conditions. Application of SPARC siRNA in controlling cellular and/or extracellular response to fibrotic stimulus may provide insight into therapeutic strategies for treating fibrotic diseases.