Abstract
Connective tissue growth factor (CTGF) associated with transforming growth factor-β (TGF-β) play a pivotal role in the pathophysiology of many fibrotic disorders. However, it is not clear whether this interaction also takes place in GO. In this study, we investigated the role of CTGF in TGF-β-induced extracellular matrix production and myofibroblast transdifferentiation in Graves’ orbital fibroblasts. By Western blot analysis, we demonstrated that TGF-β1 induced the expression of CTGF, fibronectin, and alpha-smooth muscle actin (α-SMA) in Graves’ orbital fibroblasts. In addition, the protein levels of fibronectin and α-SMA in Graves’ orbital fibroblasts were also increased after treatment with a recombinant human protein CTGF (rhCTGF). Moreover, we transfected the orbital fibroblasts with a small hairpin RNA of CTGF gene (shCTGF) to knockdown the expression levels of CTGF, which showed that knockdown of CTGF significantly diminished TGF-β1-induced expression of CTGF, fibronectin and α-SMA proteins in Graves’ orbital fibroblasts. Furthermore, the addition of rhCTGF to the shCTGF-transfected orbital fibroblasts could restore TGF-β1-induced expression of fibronectin and α-SMA proteins. Our findings demonstrate that CTGF is an essential downstream mediator for TGF-β1-induced extracellular matrix production and myofibroblast transdifferentiation in Graves’ orbital fibroblasts and thus may provide with a potential therapeutic target for treatment of GO.
Highlights
Graves’ ophthalmopathy (GO) is a cosmetically disfiguring and vision-threatening disease
We recently demonstrated that mRNA and protein expression levels of Connective tissue growth factor (CTGF) were significantly increased in GO orbital fibroblasts compared with normal fibroblasts[15]
By Western blot analysis, we found that the fibrosis-related proteins including CTGF, fibronectin and α-SMA were significantly increased in the primary cultures of GO orbital fibroblasts (GO1-GO4) as compared to those of normal subjects (N1-N4) (p = 0.0232, 0.0083, and 0.0047, respectively) (Fig. 1)
Summary
Graves’ ophthalmopathy (GO) is a cosmetically disfiguring and vision-threatening disease. Multiple studies have established that transforming growth factor-β (TGF-β) play important roles in tissue remodeling and fibrosis via myofibroblast activation in various cell types[5,6,7,8]. The protein and mRNA expression levels of TGF-β have been found to be significantly higher in GO orbital tissues and orbital fibroblasts compared with those of normal controls[9,10,11]. Connective tissue growth factor (CTGF), a secreted protein, is thought to be involved in extracellular matrix remodeling during development and pathologic conditions, and can mediate several downstream actions of TGF-β13,14. We recently demonstrated that mRNA and protein expression levels of CTGF were significantly increased in GO orbital fibroblasts compared with normal fibroblasts[15]. We investigated the role of CTGF in TGF-β-induced extracellular matrix production and myofibroblast transdifferentiation in Graves’ orbital fibroblasts
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