p38 MAP Kinase Pathway Mediation of Stromelysin Production in Trabecular Meshwork Cells with TNF or IL–1 Treatment

Abstract

Abstract: : Purpose: The regulated expression of stromelysin–1, matrix metalloproteinase–3 (MMP–3), by the trabecular meshwork (TM) affects extracellular matrix turnover and aqueous outflow facility. IL–1α and TNFα regulate MMP–3 production in TM cells. We investigated the p38 MAP kinase intracellular signaling pathway in this process. Methods: Cultured porcine TM cells were treated with TNFα or IL–1α. Phosphorylation of p38 on Thr180/Tyr182 and of the transcription factor, ATF–2 on Thr69/71 was analyzed by western immunoblot. The effect of a p38 inhibitor, SB202190, on MMP–3 production following IL–1α or TNFα addition was determined. To verify the role of p38 in this process, a dominant negative p38 construct and a secreted alkaline phosphatase (SEAP) reporter construct containing the 2.3 kb MMP–3 promoter were co–transfected into TM cells. Expression of SEAP in the media was determined by a chemiluminenscent assay. Results: Phosphorylation of p38 MAP kinase increases at 5 and 15 minutes after treatment with TNFα. ATF–2 phosphorylation increases 15 minutes after treatment with either TNFα or IL–1α. MMP–3 levels increase after 48 or 72 hours of treatment with either TNFα or IL–1α. SB202190 blocks the TNFα stimulation of MMP–3, but the inhibitor's effects on IL–1α stimulation of MMP–3 levels vary with the activation state. The dominant negative p38 blocks both the TNFα and the IL–1α increases of MMP–3 promoter activity. Conclusions: Increased phosphorylation of p38 at 5 and 15 min after IL–1α or TNFα treatment suggests p38 MAP kinase pathway involvement in MMP–3 expression. The effects of the p38 inhibitor and the dominant negative construct provide strong support for this conclusion.

Keywords: signal transduction • outflow: trabecular meshwork • phosphorylation