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Cynthia Von Zee, Jonathan Lautz, Kelly Langert, Evan Stubbs; Smad3 phosphorylation promotes Transforming Growth Factor-β2 mediated expression of endothelin-1 in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3558. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In human trabecular meshwork (TM) cells, Transforming Growth Factor (TGF)-β2 markedly enhances synthesis and secretion of the vasoactive peptide endothelin-1 (ET-1), in part, through activation of the canonical (Smad) signaling pathway. Here, we determined the role of Smad3 signaling in facilitating TGF-β2 mediated enhancement of ET-1 expression in human TM cells.
Transformed human TM cells (GTM3) cultured in serum-free media were incubated in the absence or presence of Specific Inhibitor of Smad3 (SIS3; 10 µM) followed by treatment with TGF-β2 (5 ng/ml) for 30 min, 1h, 6h, or 24h. Relative changes in preproendothelin (ppET)-1 mRNA content and Smad3 phosphorylation were quantified by quantitative real-time PCR (qRT-PCR) and Western blot, respectively.
Human TM cells cultured in the presence (5 ng/ml, 24h) of TGF-β2 exhibit a marked, time-dependent, and sustained induction of Smad3 phosphorylation, compared to vehicle controls. Pre-treatment with SIS3 (30 min) attenuated Smad3 phosphorylation induced by TGF-β2 (30 min). In marked contrast, Smad3 phosphorylation was enhanced in GTM3 cells treated (1h) with SIS3 prior to incubation with TGF-β2 (6h, 24h). GTM3 cells treated with TGF-β2 alone exhibited a marked (>9-fold), time-dependent significant increase in ppET-1 mRNA content by 6h. By comparison, GTM3 cells pre-treated (1h) with SIS3 followed by TGF-β2 (6h, 24h) exhibited further enhancement of ppET-1 mRNA content compared to TGF-β2 treated controls.
The selective inhibitor SIS3 effectively attenuates short-term (30 min) Smad3 phosphorylation. However, SIS3 enhances chronic, long-term (6h, 24h) Smad3 phosphorylation by TGF-β2. Enhanced Smad3 phosphorylation markedly augments TGF-β2 mediated increases in ppET-1 mRNA content in human TM cells. Elevated levels of TGF-β2 present in AH of POAG patients may increase ET-1 synthesis in human TM cells by promoting activation of Smad3 signaling.
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