Hyaluronic acid induces COX-2 expression via CD44 in orbital fibroblasts from patients with thyroid-associated ophthalmopathy.

PURPOSE: The aim of this study was to determine the effect of hyaluronic acid (HA) on cyclooxygenase (COX)-2 expression in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). METHODS: Primary cultured orbital fibroblasts were obtained from patients with TAO and non-TAO subjects. Dermal and conjunctival fibroblasts were cultured from the eyelid skin of subjects undergoing cosmetic lid surgery or cataract surgery, respectively. The cells were treated with HA and the transcriptional and translational levels of COX-2 were measured. The expression of CD44 on each type of cells was determined, and the involvement of CD44 in the HA-induced COX-2 increase in orbital fibroblasts from patients with TAO was evaluated by using CD44 knockdown cells and by pretreatment with neutralizing antibody. The relevance of the mitogen-activated protein kinase (MAPK) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated signaling pathway was assessed by immunoblotting for the phosphorylated form of each MAPK or IκB and by using specific inhibitors to these pathways. RESULTS: Hyaluronic acid increased COX-2 expression in orbital fibroblasts from patients with TAO, which was not observed in the cells from non-TAO subjects and conjunctival or dermal fibroblasts. Orbital fibroblasts from patients with TAO expressed significantly higher level of CD44 than non-TAO cells, and the increased COX-2 expression by HA in these cells was attenuated by knockdown or neutralizing of CD44. Hyaluronic acid induced MAPK and IκB phosphorylation; and cotreatment with specific MAPK or NF-κB inhibitors halted HA-induced transcription of COX-2, suggesting the involvement of these signaling pathways. CONCLUSIONS: Hyaluronic acid induced COX-2 expression in orbital fibroblasts from patients with TAO via CD44 through the MAPK and NF-κB-mediated signaling pathways. These results suggest that HA may have a proinflammatory role in the pathogenesis of TAO by inducing COX-2.

Pirfenidone attenuates the IL-1ß-induced hyaluronic acid increase in orbital fibroblasts from patients with thyroid-associated ophthalmopathy.

PURPOSE: This study aimed to investigate the effect of pirfenidone on the IL-1ß-induced hyaluronic acid (HA) increase in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). METHODS: Primary cultured orbital fibroblasts were obtained from patients with TAO, and the excreted levels of HA from IL-1ß-treated cells with or without pirfenidone were measured. The effect of pirfenidone on IL-1ß-induced hyaluronic acid synthase (HAS) expression was evaluated. The relevance of the mitogen-activated protein kinase (MAPK)-mediated signaling pathway in IL-1ß-induced HAS expression was assessed using specific inhibitors to p38, extracellular signal-regulated kinase (ERK), or c-Jun N-terminal kinase (JNK). The phosphorylation level of each MAPK in IL-1ß-treated cells with or without pirfenidone and the level of AP-1 DNA binding were measured. The inhibitory potency of pirfenidone on HA production was evaluated using dexamethasone as a reference agent. RESULTS: Pirfenidone strongly attenuated the IL-1ß-induced HA release in a dose-dependent manner. The IL-1ß-induced HAS expression was decreased significantly following cotreatment with pirfenidone at the mRNA and protein levels. The production of mRNAs was halted by cotreatment with inhibitors of ERK and p38, but not by inhibitors of JNK. The IL-1ß-induced ERK and p38 phosphorylation, and AP-1 DNA binding were attenuated in the presence of pirfenidone. Pirfenidone showed greater potency than dexamethasone in inhibiting increases in IL-1ß-induced HA. CONCLUSIONS: Pirfenidone attenuates the IL-1ß-induced HA production in orbital fibroblasts from patients with TAO, at least in part, through suppression of the MAPK-mediated HAS expression. These results support the potential use of pirfenidone for treatment of patients with TAO.

Pirfenidone attenuates IL-1ß-induced COX-2 and PGE2 production in orbital fibroblasts through suppression of NF-κB activity.

The aim of this study was to determine the effect of pirfenidone on interleukin (IL)-1ß-induced cyclooxygenase (COX)-2 and prostaglandin (PG)E2 expression in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). Primary cultures of orbital fibroblasts from patients with TAO (n = 4) and non-TAO subjects (n = 4) were prepared. The level of PGE2 in orbital fibroblasts treated with IL-1ß in the presence or absence of pirfenidone was measured using an enzyme-linked immunosorbent assay. The effect of pirfenidone on IL-1ß-induced COX-2 expression in orbital fibroblasts from patients with TAO was evaluated by reverse transcription-polymerase chain reaction (PCR) and quantitative real-time PCR analyses, and verified by Western blot. Activation of nuclear factor-κB (NF-κB) was evaluated by immunoblotting for inhibitor of κB (IκB)α and phosphorylated IκBα, and DNA-binding activity of p50/p65 NF-κB was analyzed by electrophoretic mobility shift assay. In addition, IL-1 receptor type 1 (IL-1R1) expression was assessed by RT-PCR in IL-1ß-treated cells with or without pirfenidone. Pirfenidone significantly attenuated IL-1ß-induced PGE2 release in both TAO and non-TAO cells. IL-1ß-induced COX-2 mRNA and protein expression decreased significantly following co-treatment with pirfenidone. IL-1ß-induced IκBα phosphorylation and degradation decreased in the presence of pirfenidone and led to decreased nuclear translocation and DNA binding of the active NF-κB complex. In our system, neither IL-1ß nor pirfenidone co-treatment influenced IL-1R1 expression. Our results suggest that pirfenidone attenuates the IL-1ß-induced PGE2/COX-2 production in TAO orbital fibroblasts, which is related with suppression of the NF-κB activation.