TGF-β2 Mediated Canonical Induction of ET-1 Requires Functional Rho GTPase Signaling In Human Trabecular Meshwork Cells

Abstract

Purpose: 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 activation of both canonical (Smad) and non-canonical (Rho GTPase) signaling pathways. Here, we examined whether functional cross-talk between Smads and Rho GTPases promotes TGF-β2 mediated induction of ET-1 expression.

Methods: Primary human TM cells were conditioned in serum-free media and incubated in the absence (vehicle) or presence of TGF-β2 (5 ng/ml). TGF-β2 signaling was blocked using SB-431542 (1 µM) or targeted siRNA knockdown of Smad2 or Smad3. Activation of Rho GTPases was blocked by pre-treatment with exoenzyme C3 transferase (10 µM). Relative changes in preproendothelin (ppET)-1 mRNA content and secreted ET-1 peptide were quantified by real-time PCR and ELISA, respectively. Content of phosphorylated or total Smad2 and Smad3 proteins was determined by Western immunoblot.

Results: Primary human TM cells incubated with TGF-β2 exhibit markedly enhanced ppET-1 mRNA expression and ET-1 peptide secretion, with a concurrent increase in the content of phosphorylated Smad2/3, compared with vehicle-treated controls. TGF-β2 mediated increases in ET-1 were prevented by co-incubation with SB-431542 as well as targeted siRNA knockdown of Smad3, but not Smad2. Pre-treatment with C3 also prevented TGF-β2 mediated increases of ET-1 expression with no observed changes in the content of phosphorylated Smad3.

Conclusions: This study demonstrates that TGF-β2 mediated activation of Smad3, but not Smad2, facilitates ET-1 expression and secretion in human TM cells. Blocking Rho GTPase signaling prevents TGF-β2 mediated enhancement of ET-1 expression, with no effect on Smad3 phosphorylation. These findings suggest that TGF-β2 mediated activation of Rho GTPase signaling is required for Smad3-mediated transcription of ppET-1. This study demonstrates for the first time a functional cross-talk between the canonical Smad and non-canonical Rho GTPase signaling pathways in human TM cells.

Keywords: 735 trabecular meshwork • 490 cytokines/chemokines • 714 signal transduction