Extracellular SPARC cooperates with TGF-β signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts

Abstract

Objectives: SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc.

Methods: Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-β1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot.

Results: Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-β and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-β signalling.

Conclusion: These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-β dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

Keywords: SPARC; SSc; TGF-β; dermal fibroblasts; fibrosis; signalling.

Fig. 1

SPARC induces the expression of ECM components and fibrotic mediators in dermal fibroblast of SSc patients (A and B) mRNA expression of ECM components and fibrotic genes by dermal fibroblast from breast (open squares), abdomen (filled squares) or forearm (open circles) of HC (A) and from forearm of lcSSc (open circles) and dcSSc patients (filled squares) (B) stimulated with SPARC (1 µg/ml) for 24 h. (C and D) Representative immunoblots (C) and densitometric analysis (D) of Col I, Col IV, fibronectin, TGF-β and SPARC protein expression in dermal fibroblasts of HC and SSc patients stimulated with SPARC (1 µg/ml) for 48 h. Data is shown as relative expression with respect to H3 expression. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with medium. HC: healthy controls; SPARC: secreted protein acidic and rich in cysteine; ECM: extracellular matrix.

Fig. 2

SPARC induces the expression of ECM components and fibrotic mediators in a TGF-β signalling dependent manner (A) Representative immunoblots of SMAD2 activation in dermal fibroblasts of HC and SSc patients stimulated with SPARC (1 µg/ml) for 1 and 6 h. (B) Representative immunoblots of Col I, fibronectin and TGF-β protein expression in skin fibroblasts of SSc patients pre-incubated 1 h with a TGB-R1 inhibitor (10 nM) and further stimulated with SPARC (1 µg/ml) for 48 h. (C) mRNA expression of ECM components by skin fibroblast of HC stimulated with SPARC (1 µg/ml) alone or in combination with TGF-β1 (10 ng/ml) for 24 h. Data is shown as connected dots. (D) Representative immunoblots of Col I and fibronectin protein expression in skin fibroblasts of HC stimulated with SPARC (1 µg/ml) alone or in combination with TGF-β1 (10 ng/ml) for 48 h. #P < 0.05 and ## P < 0.01 compared with medium. *P < 0.05 and **P < 0.01. HC: healthy controls; SPARC: secreted protein acidic and rich in cysteine; ECM: extracellular matrix.