Rho-ROCK pathway regulates nuclear levels of PPM1A and maintains SMAD3 activation.

<p>VSMCs maintained under serum-deprived conditions for 1 day were TGF-β1-stimulated (1 ng/ml) with or without the ROCK inhibitor, Y-27632 (10 µm) and cellular lysates probed for pSMAD2, total SMAD2/3, PAI-1 and ERK2 (<b>A</b>). Late-stage (4 hour) pSMAD2 levels were markedly attenuated and PAI-1 expression completely inhibited by ROCK blockade (<b>A</b>). TGF-β1-induced SMAD phosphorylation at the late time points (4 hours) is significantly reduced by inhibition of ROCK signaling. Serum-deprived VSMCs were pretreated for 30 minutes with Y-27632 (at indicated concentrations) prior to exposure to TGF-β1 for 4 hours. Cell lysates were probed for PAI-1, SMAD2/3, pSMAD2/3 and PPM1A (<b>C</b>). PAI-1 expression in response to TGF-β1 was completely blocked by Y-27632 pre-exposure (10 µM final concentration) despite the initial increase in SMAD2 phosphorylation in Y-27632-treated cells. Concentrations of Y-27632 that effectively inhibit PAI-1 induction and suppress SMAD2 phosphorylation also increase PPMIA levels (<b>C</b>). Transient knock-down of SMAD3 with siRNA constructs (as detailed in Methods) (<b>D,E</b>) or pre-incubation with the small molecule inhibitor of SMAD3 phosphorylation SIS3 (5 µM) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022896#pone.0022896-Jinnin1" target="_blank">[41]</a> (<b>D,F,G</b>) eliminates TGF-β1-induced PAI-1 expression in VSMCs (<b>D,E</b>) and MEFs (<b>F,G</b>). Cell fractionation studies confirmed that nuclear accumulation of pSMAD3 in response to TGF-β1 is blocked while nuclear PPM1A content increased upon pre-incubation with Y-27632 (<b>H</b>). TGF-β1 stimulation for 4 hours actually reduced nuclear PPM1A levels, which was restored by Y-27632 pretreatment (<b>H</b>). siRNA-mediated PPM1A knockdown in VSMCs resulted in a significantly increased TGF-β1-induced PAI-1 response compared to cells transfected with control siRNA constructs (<b>I</b>). ERK2 (<b>A,B,D–G</b>), SMAD2/3 (<b>A,C,F</b>), tubulin (<b>D</b>), lamin (<b>H</b>) and actin (<b>I</b>) provide loading controls. Data plotted in (<b>B,E,G</b>) is the mean ± S.D. of three independent experiments.