A9.3 Hydrogen sulfide and MEK1 inhibition reduce IL-1[beta]-induced activation of fibroblast-like synoviocytes

Background and Objectives In the pathophysiology of osteoarthritis (OA) the secretion of the pro-inflammatory cytokine interleukin (IL)-1β is among the critical steps mediating the aberrant degenerative processes in which activated fibroblast-like synoviocytes (FLS) play a pivotal role. The major goal of this study was to investigate the molecular mechanisms underlying this activation and how treatment with hydrogen sulfide (which is applied for treatment of OA in the form of sulfur spa therapy), or various mitogen-activated protein kinase (MAPK) inhibitors influences them. Methods FLS derived from OA patients were used throughout this study. The effect of IL-1β and NaHS treatment on the phosphorylation of 26 MAPkinases and other serine / threonine kinases was analysed by proteome profiler array. The secretion of IL-6 was monitored by ELISA in cell culture supernatants from conventional cultured FLS treated with NaHS or MAPK inhibitors. In addition, FLS were grown in 3-dimensional extracellular matrix micromass cultures, stimulated with IL-1β and treated with NaHS or the MAPK inhibitors PD98059 (MEK1), SB203580 (p38) and SP600125 (JNK). Results Data gained from human phospho-MAPK proteome profiler array revealed that IL-1β as well as NaHS increased the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 (68 ± 16% and 90 ± 27%), mitogen- and stress activated protein kinase (MSK)2 (37 ± 5%), p38 subunits α (232 ± 51%), β (125 ± 38%) and [GAMMA] (417 ± 216%) as well as c-Jun-N-terminal (JNK) 1/2 (33 ± 7% and 89 ± 3%). Interestingly, NaHS treatment inhibited the IL-1β-induced activation of ERK1/2, MSK2, p38δ and JNK2 between 10 up to 30%. Furthermore, treatment with NaHS or MEK1 inhibitor completely abolished the IL-1β-induced secretion of IL-6, whereas inhibitors of p38 and JNK did not show this effect. FLS micromass cultures stimulated with IL-1β developed a hyperplastic lining layer structure and treatment with NaHS or the MEK1 inhibitor was found to almost completely inhibit this process. Conclusion These experiments demonstrate that hydrogen sulfide decreases the IL-1β-induced phosphorylation of ERK1/2 and MSK2, which signal downstream of MEK1/2. This suggests a direct effect of hydrogen sulfide on MEK1/2 activation leading to decreased IL-6 secretion and inhibition of formation of hyperplastic lining layer structures, which might explain some of the beneficial effects of sulfur spa therapy. Gene expression studies will help to elucidate the