TGFβ inhibits ezrin phosphorylation at Thr-567, resulting in PKA activation and inhibition of XIAP and survivin expression.
A, TGFβ1 treatment of FET cells decreased ezrin phosphorylation at Thr-567 in a time-dependent manner (left panel). Relative levels of p-ezrin Thr-567 to total ezrin were analyzed, and the results are shown on the right. ***, p < 0.001 (n = 3). B, expression of Smad2 and Smad3 was knocked down in FET cells (left panel). Quantification of Western blots is shown on the right. ***, p < 0.001 (n = 2). C, Smad3 KD, but not Smad2 KD, prevented TGFβ1-mediated inhibition of ezrin phosphorylation (left panel). Relative levels of p-ezrin Thr-567 to total ezrin were determined and are shown on the right. **, p < 0.01. NS, not statistically significant (n = 3). D, TGFβ1 treatment increased PKA activation to a much lesser degree in ezrin KD cells than in NT sh control cells. **, p < 0.01; ***, p < 0.001 (n = 3). E, NSC attenuated TGFβ-induced PKA activation. ***, p < 0.001 (n = 3). F, ezrin KD reduces TGFβ1-induced apoptosis. **, p < 0.01 (n = 2). G and H, ezrin KD has no effect on TGFβRI or TGFβRII expression (G) or on Smad3 expression or TGFβ-mediated Smad3 phosphorylation (H, left). Quantification of Western blots is shown on the right. **, p < 0.01; ***, p < 0.001. NS, not statistically significant (n = 3).