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Cynthia L. von Zee, Kelly A. Langert, Evan B. Stubbs, Jr.; Transforming Growth Factor-β Promotes Rho G-protein Mediated Synthesis and Secretion of Endothelin-1 in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3239.
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Analysis of aqueous humor from patients with primary open-angle glaucoma reveal marked and potentially pathologic increases in the content of endothelin-1 (ET-1) and transforming growth factor-beta (TGF-β). Here, we determined the consequences of TGF-β signaling on regulation of ET-1 expression and secretion by human trabecular meshwork (TM) cells.
Human TM cell lines (NTM5 and GTM3) cultured in serum-free media were incubated in the absence or presence of TGF-β1 or -β2, and relative changes in preproendothelin (ppET)-1 mRNA content and secreted mature ET-1 peptide were quantified by real-time PCR (qRT-PCR) and ELISA, respectively. In some experiments, TGF-β or ET-1 receptor antagonists, or inhibitors of Rho G-protein activation, were evaluated for effects on TGF-β signaling. Filamentous actin (F-actin) organization was visualized by AlexaFluor488-conjugated phalloidin.
Human TM cells cultured in the presence (5 ng/ml, 24h) of TGF-β1 or TGF-β2 exhibit a marked (>9-fold) increase in ppET-1 mRNA content compared with vehicle-treated controls. Co-incubation with SB-505124, a selective inhibitor of TGFβRI/ALK-5 signaling, prevented TGF-β mediated ppET-1 mRNA expression. In contrast, co-incubation with ETA (BQ-123) or ETB (BQ-788) receptor antagonists had no effect on TGF-β mediated ppET-1 mRNA expression. TGF-β elicited a robust (>7-fold) biphasic secretion of mature ET-1 from TM cells while functionally enhancing F-actin stress fiber organization. Inhibition of Rho signaling significantly attenuated TGF-β mediated increases in ppET-1 mRNA content, secretion of mature ET-1, and F-actin stress fiber assembly.
TGF-β, signaling through the TGFβRI/ALK-5 receptor, elicits marked increases in ET-1 mRNA content and ET-1 secretion from cultured human TM cells. Elevated levels of TGF-β2 present in AH of POAG patients may elevate IOP, in part, by eliciting aberrant Rho G-protein dependent cell contraction, and increasing ET-1 synthesis and secretion, in human TM cells.
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