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Cynthia Lynn Pervan, Jonathan Lautz, Evan B Stubbs; Rho GTPase-mediated activation of the MAP kinase pathway facilitates TGF-β2 dependent ET-1 expression. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4687.
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© ARVO (1962-2015); The Authors (2016-present)
Elevated content of transforming growth factor (TGF)-β2 and endothelin-1 (ET-1) within the aqueous humor of affected patients is associated with the development of primary open-angle glaucoma (POAG). We have previously demonstrated that TGF-β2 markedly enhances ET-1 expression and secretion in human trabecular meshwork (TM) cells through concurrent activation of both canonical (Smad3) and non-canonical (Rho GTPase) signaling pathways. Here, we elucidate the mechanism by which Rho GTPases promote TGF-β2 mediated induction of ET-1 expression in human TM cells.
Confluent human TM cells were cultured in serum-free media and incubated x 24h in the absence (vehicle) or presence of TGF-β2 (5 ng/ml). In some experiments, Rho GTPase dependent signaling was blocked by pre-treatment with exoenzyme C3 transferase or siRNA-targeted knockdown of RhoA or RhoB. p38 dependent MAP kinase signaling was inhibited by pre-treatment with SB-203580. Content of RhoA, RhoB, or total and phosphorylated p38 proteins were determined by Western immunoblot. Relative changes in preproendothelin (ppET)-1 mRNA content and secreted ET-1 peptide were quantified by real-time PCR and ELISA, respectively.
Human TM cells pretreated (1h) with SB-203580 exhibit a significant attenuation of TGF-β2 mediated ET-1 induction. Pretreating TM cells with exoenzyme C3 transferase (1h), or with siRNA targeting RhoA, similarly attenuates TGF-β2 mediated ET-1 induction. Consistent with these findings, TGF-β2 was found to elicit a marked increase in acute (30 min) phosphorylation of p38 MAP kinase. By comparison, pre-treatment with exoenzyme C3 transferase (1h) modestly attenuates TGF-β2 mediated acute phosphorylation of p38 MAP kinase.
TGF-β2 induction of ET-1 expression in human TM cells is mediated by RhoA GTPase dependent activation of p38 MAP kinase signaling.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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