COX-2, PPAR-γ, TGF-β及各種細胞激素在甲狀腺眼外肌病變所扮演的角色

Abstract

Purpose: Graves’ophthalmopathy (GO) is an autoimmune inflammatory disorder.Recent evidence implicates the TSH receptor (TSH-R) as an important orbital autoantigen in GO. The characteristic clinical manifestations of GO, including proptosis, chemosis, periorbital edema, and extraocular muscle dysfunction, result from the intraorbital inflammatory process and enlargement of the adipose tissue and muscles within the orbit. After antibody binding with antigen, a cascade of inflammation starts, including monocytes and macrophages releasing cytokines, eg. IL-1β. IL-1β can induce COX-2, which can produce prostaglandin H2. After the conversion of prostaglandin H2 into prostaglandin E2, inflammation is induced. On the other hand, prostaglandin H2 can be converted to prostaglandin D2. Prostaglandin J2 is formed after dehydration of cyclopentane ring and is further metabolized to yield 15d-PGJ2, a natural ligand of PPAR-γ. After the binding of 15d-PGJ2, PPAR-γ will be activated and induce anti-inflammatory activity, combined with adipogenesis. A distinctive histopathological feature associated with GO is the accumulation of the glycosaminoglycan, mainly hyaluronan, in orbital connective tissues. In recent study, hyaluronan synthesis in orbital fibroblasts has been shown to be up-regulated by TGF-β. Overproduction of TGF-β will result in excessive scar formation and fibrosis. Through action of PGE2, COX-2 induces TGF-β and produces fibrosis, and activates HAS, starting accumulation of glycosaminoglycan. TGF-β can increase the formation of hyaluronan. In turn, hyaluronan and TGF-β can stimulate the production of COX-2 and prostaglandin. Previous study found that mRNA expression of IGF-1α, IGF-1R, IL-1β, IFN-γ, PPAR-γ, TSH-R, COX-2, HAS in GO orbital fat tissues by real time PCR were up regulated as compared to normal orbital tissues. PPAR-γ disrupts TGF-β signal transduction and blocks profibrotic responses. Some of these responses may take part in the pathogenesis of GO. Synergistic and antagonistic interactions can occur between the pathways producing prostaglandin (PGE2) and hyaluronan or those regulating glycosaminoglycan production and adipogenesis. In this study, we will investigate the effect of different cytokines on the expression of PPAR-γ, COX-2, TGF-β, Adiponectin, CXCL10, TSH-R mRNA and production of protein by myoblasts cultured from the extraocular muscles. We will also try to evaluate the effect of PPAR-γ agonist, antagonist and COX-2 inhibitor on the expression of TGF-β, hyaluronan synthase (HAS1, HAS2, HAS3) and hyaluronan. Methods: To determine whether cytokines induce these genes expression, we treated orbital myoblasts with or without IFN-γ, IGF-1, IL-1β , TNF-α. Data were analyzed by real time PCR for PPAR-γ, COX-2, TGF-β, CXCL10, Adiponectin, TSH-R mRNA. ELISA and Western blot were used to confirm the TGF-β, CXCL10, COX-2, TSH-R protein expression in cytokines treated orbital myoblasts. We cultured orbital extraocular muscles and treated them with TNF-α in the presence of the PPAR-γ agonist, antagonist and COX-2 inhibitor. ELISA and Western blot were used to determine their effect on TGF-β, hyaluronan synthase (HAS1, HAS2, HAS3) and hyaluronan protein synthesis. Results: After treating patients with Graves' ophthalmopathy group myoblasts especially in IL-1β or TNF-α for 24 hours, gene expression of the mRNA of PPAR-γ, COX-2, TGF-β, CXCL10, Adiponectin, TSH-R show significantly increase. ELISA results of TGF-β, CXCL10 and Western blot results of COX-2, TSH-R were compatable with RT-PCR. There was no significant gene expression in normal group. All myoblasts gene expressions show differences between the disease and control groups. In this in vitro experiment of cytokine treated GO myoblasts, there is a trend that the TGF-β and hyaluronan gene expression was under inhibition with COX-2 inhibitor (Celecoxib) or PPAR-γ agonist (Pioglitazone), though not reach significant level. We hypothesis that TGF-β activate hyaluronan synthesis via HAS signals. Both Celecoxib and Pioglitazone treatment inhibited cytokine induced HAS3 expression, but did not afftect HAS1 and HAS2 expression. The signaling pathways by which TGF-β regulates HAS3 are still poorly understood. HAS3 can induce hyaluronan production which may be important pathogenesis to Graves' ophthalmopathy. Conclusion: To our knowledge, this is the first in vitro experiment demonstrates PPAR-γ, COX-2, TGF-β, CXCL10, Adiponectin, TSH-R production stimulated by cytokines in GO extraocular muscles. These results suggest that the extraocular muscles indeed play an important role in GO pathogenesis besides orbital fibroblasts. Taking together, the present data did partly support our working hypothesis that COX-2 dependent PGs induce TGF-β signaling, stimulate hyaluronan production via HAS activation in GO myoblasts. In our study, we observed COX2 inhibitor and PPAR-γ agonist suppress TGF-β, HAS and hyaluronan protein production. In the future, more studies will be helpful to get useful information in the treatment of Graves’ophthalmopathy with restrictive myopathy and adipogenesis.