Redox-Induced Src Kinase and Caveolin-1 Signaling in TGF-β1-Initiated SMAD2/3 Activation and PAI-1 Expression

Plasminogen activator inhibitor-1 (PAI-1), a major regulator of the plasmin-based pericellular proteolytic cascade, is significantly increased in human arterial plaques contributing to vessel fibrosis, arteriosclerosis and thrombosis, particularly in the context of elevated tissue TGF-β1. Identification of molecular events underlying to PAI-1 induction in response to TGF-β1 may yield novel targets for the therapy of cardiovascular disease.Reactive oxygen species are generated within 5 minutes after addition of TGF-β1 to quiescent vascular smooth muscle cells (VSMCs) resulting in pp60(c-src) activation and PAI-1 expression. TGF-β1-stimulated Src kinase signaling sustained the duration (but not the initiation) of SMAD3 phosphorylation in VSMC by reducing the levels of PPM1A, a recently identified C-terminal SMAD2/3 phosphatase, thereby maintaining SMAD2/3 in an active state with retention of PAI-1 transcription. The markedly increased PPM1A levels in triple Src kinase (c-Src, Yes, Fyn)-null fibroblasts are consistent with reductions in both SMAD3 phosphorylation and PAI-1 expression in response to TGF-β1 compared to wild-type cells. Activation of the Rho-ROCK pathway was mediated by Src kinases and required for PAI-1 induction in TGF-β1-stimulated VSMCs. Inhibition of Rho-ROCK signaling blocked the TGF-β1-mediated decrease in nuclear PPM1A content and effectively attenuated PAI-1 expression. TGF-β1-induced PAI-1 expression was undetectable in caveolin-1-null cells, correlating with the reduced Rho-GTP loading and SMAD2/3 phosphorylation evident in TGF-β1-treated caveolin-1-deficient cells relative to their wild-type counterparts. Src kinases, moreover, were critical upstream effectors of caveolin-1(Y14) phosphoryation and initiation of downstream signaling.TGF-β1-initiated Src-dependent caveolin-1(Y14) phosphorylation is a critical event in Rho-ROCK-mediated suppression of nuclear PPM1A levels maintaining, thereby, SMAD2/3-dependent transcription of the PAI-1 gene

PAI-1 induction in response to TGF-β1 involves reactive oxygen species (ROS).

<p>DCF fluorescence measurements (as described in Methods) were used to determine ROS generation (per equivalent number of cells) and expressed relative to unstimulated cultures (set as a.u. = 1). ROS levels increase within 5 minutes after addition of TGF-β1 (1 ng/ml) to serum-deprived quiescent VSMCs (<b>A</b>). ROS generation appears to be important in TGF-β1-stimulated PAI-1 expression since PAI-1 induction is effectively suppressed by even low concentrations of the established inhibitors of free radical generation NAC (<b>B</b>) and DPI (<b>C</b>). NAC pretreatment also attenuates (at 2 mM) and completely eliminates (at concentrations ≥5 mM) TGF-β1-dependent ERK1/2 and SMAD2/3 phosphorylation but has no effect of EGF-stimulated ERK1/2 activation (<b>E</b>). Both NAC (<b>B,F,G</b>) and DPI (<b>C,H</b>) pretreatment (30 mins) served to assess the role of ROS in TGF-β1- and EGF-mediated PAI-1 induction. ERK2 provided a loading control. Data plots (<b>A,D,F</b>) represent the mean ± S.D. of three independent experiments; statistical significance among the indicated groups was calculated by t-test.</p

A model for TGF-β1 stimulated maintenance of SMAD3 phosphorylation and PAI-1 induction via <i>Src</i>/FAK/Caveolin-1 signaling.

<p>TGF-β1 stimulates caveolin-1^Y14 phosphorylation in a reactive oxygen species-FAK/<i>c-Src</i> dependent manner removing repressive influences on EGFR signaling (in red) leading to EGFR transactivation (also by <i>c-Src</i>), thereby, initiating signaling events leading to the MEK-ERK pathway activation necessary for PAI-1 induction. <i>Src</i> kinase phosphorylation of caveolin-1^Y14 also stimulates Rho-GTP loading and ROCK (an established downstream target of Rho) activation is necessary for PAI-1 induction. pCaveolin-1^Y14-Rho-ROCK mediated signaling leads to inhibition of PTEN-PPM1A interactions resulting in a reduction of nuclear PPM1A phosphatase (black pathway), thereby, maintaining the pSMAD2/3 levels (highlighted in blue) required for PAI-1 induction by TGF-β1 (see text). PAI-1 is elevated in atherosclerotic plaques frequently colocalizing with α-smooth muscle actin-expressing cells, presumably VSMCs (insert).</p

RhoA both interacts with caveolin-1^Y14 in response to TGF-β1 and required for PAI-1 induction.

<p>A Rho-GTPase assay (as described in Methods) was used to assess relative RhoA activation by TGF-β1 in fibroblasts. RhoA-GTP loading increased within 2–4 hours of TGF-β1 addition (0.1 ng/ml) to 1-day serum-deprived wild-type MEFs. In contrast, the level and duration of RhoA activation during this 4 hour window is markedly reduced in caveolin-1-null fibroblasts compared to caveolin-1^+/+ cells (<b>A</b>). Immunoprecipitation (IP) of RhoA followed by phospho-caveolin-1^Y14 western analysis disclosed a time-dependent association between phospho-caveolin-1^Y14 and endogenous RhoA in response to TGF-β1 while total levels of caveolin-1 remain unchanged (<b>B</b>). IP of caveolin-1 followed by western blotting for RhoA similarly confirmed increased interaction between both proteins in wild-type (WT) MEFs upon a 2 to 4 hr stimulation with TGF-β1 but not in caveolin-null cells (<b>C</b>). Transfection of a dominant-negative RhoA construct prior to addition of TGF-β1 effectively inhibited PAI-1 expression while introduction of a GFP control vector was without effect (<b>D</b>) indicating that RhoA is required for TGF-β1-induced PAI-1 expression.</p

Inhibition of ROS generation attenuates TGF-β1 signaling in VSMC.

<p>Quiescent VSMCs were stimulated with TGF-β1 (1 ng/ml) for the times indicated with or without NAC (5 mM) pretreatment for 1 hour. Increases in p<i>Src</i>^Y416, pFAK^Y577 and pCaveolin^Y14 (targets of c-<i>Src</i> kinases) in response to TGF-β1 is completely inhibited by NAC, suggesting an upstream role for ROS generation in activation of Src/FAK/caveolin-1 signaling pathways (<b>A</b>). FAK^Y397 phosphorylation by TGF-β1 (at least within the time frame of 2 hours) is relatively unaffected by NAC blockade of ROS generation. Total levels of <i>c-Src</i>, FAK and caveolin-1 are largely unchanged over the time course of TGF-β1 exposure serving as loading controls (<b>A</b>). To assess the role of ROS generation in SMAD3 activation, TGF-β1-stimulated SMAD3 phosphorylation over time was compared to an identical window with NAC pretreatment. Blots were probed with antibodies to determine both pSMAD3 and total SMAD3 levels (<b>B</b>).</p

c-<i>Src</i> is an upstream regulator of caveolin-1^Y14 phosphorylation.

<p>MEFs were serum-deprived for 1 day prior to incubation with TGF-β1 (0.1 ng/ml) for the times indicated. Western analysis indicated that TGF-β1 stimulated caveolin-1 phosphorylation at the Y14 c-<i>Src</i> kinase target site in caveolin-1^+/+ fibroblasts but not, as expected, in caveolin-1^−/− cells (<b>A,B</b>). Caveolin^Y14 phosphorylation is similarly evident extracts of SYF^+/+/+ but not in SYF^−/−/− MEFs (<b>C</b>). Stable expression of a pp60^c-<i>src</i> construct (+WT <i>Src</i>) in SYF^−/−/− fibroblasts is sufficient to rescue caveolin^Y14 phosphorylation in response to TGF-β1 (but not in empty vector expressing SYF^−/−/− cells) despite comparable caveolin-1 expression in both cell types (<b>D</b>). Pretreatment of serum-deprived VSMC with the <i>Src</i> kinase inhibitor SU6656 (2 µM) prior to addition of TGF-β1 (1 ng/ml) eliminated TGF-β1-induced <i>Src</i>^Y416 activation, caveolin^Y14 phosphorylation and PAI-1 expression (<b>E</b>). Total ERK2 (<b>A,B</b>), caveolin-1 (<b>C,D,E</b>) and c-<i>Src</i> (<b>E</b>) were approximately constant under all culture conditions providing internal loading controls. Data plotted in (<b>B</b>) represent the mean ± S.D. of three independent experiments. To assess potential growth factor-associated changes in caveolin-1 localization, subconfluent serum-deprived MEFs were stimulated with TGF-β1 (0.1 ng/ml) for 2 hrs and the distribution of phospho-caveolin-1^Y14 and total caveolin-1 assessed by immunocytochemistry; control cells remained untreated (<b>F</b>).</p

FAK is a downstream target of <i>Src</i> kinases and is required for PAI-1 induction by TGF-β1.

<p>MEFs were serum-deprived for 1 day prior to addition of TGF-β1 (0.1 ng/ml). TGF-β1 stimulates FAK phosphorylation at the Y577 and Y861 sites in SYF^+/+/+ but not SYF^−/−/−cells consistent with an upstream role of <i>Src</i> kinases in FAK activation. TGF-β1-induced FAK^Y397 autophosphorylation, in contrast, is unaffected by genetic ablation of <i>src</i> family kinases (<b>A</b>). To assess the role of FAK in TGF-β1-induced PAI-1 and CTGF expression, serum-deprived FAK^+/+ and FAK^−/− MEFs were stimulated with TGF-β1 and blots probed with antibodies to PAI-1 and CTGF (<b>B</b>). TGF-β1 stimulates FAK phosphorylation at Y397, Y561 and Y861 only in wild-type but not, as anticipated, in FAK-null fibroblasts (<b>C</b>) providing antibody specificity controls for panels <b>A–C</b>. TGF-β1-stimulated c-<i>Src</i> and EGFR activation is significantly attenuated in FAK^−/− cells relative to FAK^+/+ MEFs (<b>C</b>). SMAD3 C-terminal phosphorylation in response to TGF-β1 is reduced in FAK^−/− as compared to FAK^+/+ cells; total SMAD2/3 levels were unchanged regardless of FAK genetic status (<b>D</b>). Western analysis was used to evaluate the effect of FAK genetic status (FAK^−/− vs. FAK^+/+) on TGF-β1-induced caveolin-1^Y14 phosphorylation (<b>D</b>). Consistent with previous observations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022896#pone.0022896-Bailey1" target="_blank">[40]</a>, total caveolin-1 is lower in FAK^−/− MEFs compared to wild-type cultures (<b>D</b>). Assessment of total FAK (<b>A,B</b>), ERK2 (<b>B,C</b>) and SMAD3 (<b>D</b>) provided loading controls.</p

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.</p

Downstream signaling events initiated by TGF-β1-activated S<i>rc</i> kinase.

<p>SYF^+/+/+ and SYF^−/−/− fibroblasts were serum-deprived for 1 day prior to stimulation with TGF-β1 (0.1 ng/ml) for the times indicated and lysates subject to western analysis. <i>Src</i> activation (assessed using phospho- <i>Src</i>^Y416 antibodies) and increased EGFR phosphorylation at the <i>Src</i> kinase target Y845 site, are both evident in TGF-β1-stimulated wild-type (SYF^+/+/+) MEFs but not <i>Src</i>, <i>Fyn</i>, <i>Yes</i> triple-null (SYF^−/−/−) cells (<b>A</b>). The level (at 15 and 30 minutes) and maintenance (at 4 hrs) of SMAD3 phosphorylation is significantly reduced in SYF^−/−/− fibroblasts compared to their wild-type counterparts (<b>B</b>). In contrast to the typical time course-dependency of PAI-1 induction in response to TGF-β1 in SYF^+/+/+ cells, PAI-1 was not detectable in <i>Src</i>-deficient MEFs regardless of the duration of TGF-β1 exposure. The absence of PAI-1 expression and attenuated SMAD3 phosphorylation reflected increased PPM1A levels in SYF^−/−/− as compared to SYF^+/+/+ fibroblasts (<b>B</b>). Pretreatment of VSMCs with the <i>Src</i> kinase inhibitor SU6656 (2 µM) blocked the long-term maintenance (but not the initiation) of SMAD2/3 phosphorylation in response to TGF-β1 while total SMAD levels remain unchanged (<b>C</b>). <i>Src</i>^Y416 phosphorylation by TGF-β1 was completely eliminated by SU6656 confirming the effectiveness of this inhibitor (<b>C</b>). Transient transfection of VSMCs with a dominant-negative pp60^c-<i>src</i> (DN-<i>Src</i>) expression construct (or a GFP control vector) 72 hours prior to incubation with TGF-β1 for 6 hours was followed by western analysis for PAI-1. TGF-β1-stimulated PAI-1 induction was effectively suppressed by the DN-<i>Src</i> but not the GFP construct (<b>D</b>). SYF^−/−/− cells genetically-engineered to express wild-type pp60^c-<i>src</i> (SYF^−/−/−+WT <i>Src</i>) rescued PAI-1 inducibility in response to TGF-β1 (<b>E</b>). ERK2 (<b>A,D,E</b>) and SMAD3 (<b>C</b>) serve as a loading controls.</p