Abstract: |
PURPOSE: Analysis of aqueous humor from patients with primary open-angle glaucoma (POAG) revealed marked increases in the content of endothelin-1 (ET-1) and transforming growth factor-beta (TGF-beta). We determined the consequences of TGF-beta signaling on ET-1 expression and secretion by human trabecular meshwork (TM) cells. METHODS: Primary or transformed (NTM5 and GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-beta1 or -beta2. Relative changes in preproendothelin (ppET)-1 mRNA content and secreted ET-1 peptide were quantified by real-time PCR and ELISA, respectively. In some experiments, TGF-beta or ET-1 receptor antagonists, or Rho G-protein inhibitors, were evaluated for effects on TGF-beta signaling. Filamentous actin organization was visualized by phalloidin. RESULTS: Primary or transformed human TM cells cultured in the presence of TGF-beta1 or -beta2 exhibit a marked (>8-fold) increase in ppET-1 mRNA content compared to vehicle controls. Coincubation with SB-505124, an inhibitor of TGFbetaRI/ALK-5 signaling, prevented TGF-beta-mediated ppET-1 mRNA expression. In contrast, coincubation with ET(A) (BQ-123) or ET(B) (BQ-788) receptor antagonists had no effect on TGF-beta-mediated ppET-1 mRNA expression. TGF-beta1 and -beta2 each elicited a robust (>7-fold) secretion of ET-1 while enhancing stress fiber organization. Inhibition of Rho signaling attenuated TGF-beta-mediated increases in ppET-1 mRNA content, ET-1 secretion, and stress fiber organization. CONCLUSIONS: TGF-beta, signaling through the TGFbetaRI/ALK-5 receptor, elicits marked increases in ET-1 mRNA content and ET-1 secretion from cultured primary or transformed human TM cells. Elevated levels of TGF-beta2 present in AH of POAG patients may elevate intraocular pressure, in part, by eliciting aberrant Rho G-protein dependent cell contraction, and increasing ET-1 synthesis and secretion, in human TM cells. |