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P.B. Liton, C. Luna, P. Gonzalez, D.L. Epstein; Involvement of TGF–Beta1 and MAPKs in the Stretch–Induced Expression of IL–6 in Human Trabecular Meshwork (HTM) Cells: A Potential Role in Outflow Homeostasis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4766.
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We have previously demonstrated the induction of both TGF–ß1 and IL–6 in HTM cells subjected to cyclic mechanical stress (CMS). Since TGF–ß1 has been shown to be a potent regulator of IL–6 in several cell types, we investigated the effect of TGF–ß1 on IL–6 expression in HTM cells, as well as the mechanisms underlying the stretch–induced expression of IL–6 in HTM primary cultures. We hypothesize that TGF–ß1 activates IL–6 expression in HTM cells and that this activation is mediated through the MAPK pathways.
All experiments were conducted in confluent monolayers of HTM cells. Concentration of secreted IL–6 was quantified by ELISA. Activation of the IL–6 promoter was quantified by measuring the secreted alkaline phosphatase protein (SEAP) released into the culture medium by HTM cells infected with an adenovirus expressing the SEAP reporter gene controlled by the IL–6 promoter (AdIL6–SEAP). CMS (5% elongation, 1 cycle per second) was applied using the Flexcell System. Reagents used in this study included TGF–ß1, anti–human TGF–ß1 antibody, and the inhibitors for the p38 MAPK (SB202190), JNK (SP600125) and ERK (PD98059).
Incubation of HTM cells with increasing concentrations of TGF–ß1 (1, 2, and 5 ng/ml) for 20 hours significantly increased the levels of secreted IL–6 (2.5 ± 0.05 fold) and the transcription of the IL–6 promoter (0.73 ± 0.03 fold). The TGF–ß1–induced expression of IL–6 was completely abolished when HTM primary cultures were pre–treated for 2 hours with inhibitors for p38 MAPK or JNK prior to TGF–ß1 treatment. In addition, the presence of any of the three MAPK pathway inhibitors (10 µM) or an anti–human TGF–ß1 antibody (2 µg/ml) completely blocked or significantly diminished (22.5% ± 6.2), respectively, the induction of IL–6 expression by CMS.
Our results suggest that exposure of HTM cells to CMS triggers a molecular mechanism involving an autocrine amplification loop between TGF–ß1 and MAPKs, which leads to increased expression of IL–6. Since IL–6 has been shown to increase both outflow facility in perfused anterior segments as well as the permeability of SC cell monolayers, such a mechanism involving TGF and MAPKs might represent a potential homeostatic response in the TM by which mechanical stress, associated with elevated IOP, could promote the release of factors, like IL–6, capable of increasing outflow facility and restoring normal IOP levels.
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