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

Background 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. Principal Findings Reactive oxygen species are generated within 5 minutes after addition of TGF-β1 to quiescent vascular smooth muscle cells (VSMCs) resulting in pp60c-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-1Y14 phosphoryation and initiation of downstream signaling. Conclusions TGF-β1-initiated Src-dependent caveolin-1Y14 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.

This paper provides novel evidence that TGF-b1 stimulation of VSMC leads to a reduction in nuclear levels of PPM1A, a recently identified C-terminal SMAD2/3 phosphatase capable of attenuating TGF-b1-mediated transcriptional responses including PAI-1 expression [19]. Inhibition of Rho-ROCK signaling prior to addition of TGF-b1 rescues PPM1A expression with correlative decreases in nuclear pSMAD2/3 content implicating the Rho-ROCK pathway as an upstream negative regulator of this serine phosphatase. SMAD2/3 phosphorylation and subsequent PAI-1 induction by TGF-b1 was suppressed by genetic deficiency of caveolin-1 implicating caveolin-1 as an activator of Rho-ROCK-SMAD2/3 signaling. Src kinase activity, moreover, was critical for caveolin-1 Y14 phosphorylation as assessed using mouse embryo fibroblasts deficient in Src, Yes, Fyn kinases (SYF 2/2/2 ), by introduction of a wild-type pp60 c-Src construct in SYF 2/2/2 cells and use of src kinase inhibitors. Significantly reduced SMAD3 phosphorylation and increased PPM1A expression in SYF 2/2/2 cells, relative to wild-type fibroblasts correlated with reduced PAI-1 levels. Src kinase-dependent FAK phosphorylation at Y577 and Y861, moreover, is stimulated by TGF-b1 while TGF-b1-initiated FAK Y397 autophosphorylation was Src-independent. FAK is required for caveolin-1 Y14 phosphorylation, pSMAD3 activation and PAI-1 induction. Finally, stimulation of the Src-FAK-caveolin-1-SMAD3 signaling axis and subsequent PAI-1 expression in response to TGF-b1 requires generation of reactive oxygen species (ROS) linking alterations in cellular redox state to gene reprogramming. TGF-b1 increases the production of ROS likely through several NADPH oxidases (NOXs) of which Nox4 has been linked to PAI-1 expression through mitogen-activated protein kinase phosphatase-1 inhibition [20]. While JNK and p38 appear implicated in the TGF-b1RROS pathway of PAI-1 gene control, integration of other non-canonical SMAD-dependent events are less clear and are the subject of this study.

Transient Transfection of siRNA or Dominant-Negative (DN) Constructs
Semi-confluent (70%) primary VSMC cultures were washed in PBS prior to addition of siRNA constructs to GFP (control), SMAD3, caveolin-1 or PPM1A (Dharmacon; final concentration 1 mM), in Accell siRNA delivery medium (1 ml) for 72-96 hours. Following a brief incubation in serum-free DMEM, VSMCs were stimulated with TGF-b1 for 4 hrs prior to harvesting for extraction. Subconfluent 35-mm cultures of R22 cells were transfected with DN-pp60 c-src , DN-RhoA N17 or control GFP expression constructs as described [13][14][15]. Following transfection, cells were serum-deprived for 2 days prior to TGF-b1 stimulation. Transfection efficiency was 50-70% (assessed by GFP fluorescence microscopy). . 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-b1 (1 ng/ml) to serum-deprived quiescent VSMCs (A). ROS generation appears to be important in TGF-b1-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) and DPI (C). NAC pretreatment also attenuates (at 2 mM) and completely eliminates (at concentrations $5 mM) TGF-b1-dependent ERK1/2 and SMAD2/3 phosphorylation but has no effect of EGF-stimulated ERK1/2 activation (E). Both NAC (B,F,G) and DPI (C,H) pretreatment (30 mins) served to assess the role of ROS in TGF-b1-and EGF-mediated PAI-1 induction. ERK2 provided a loading control. Data plots (A,D,F) represent the mean 6 S.D. of three independent experiments; statistical significance among the indicated groups was calculated by t-test. doi:10.1371/journal.pone.0022896.g001

Immunoprecipitation
Cells were disrupted for 30 min (in cold 50 mM HEPES, pH 7.5, 1% Triton X-100, 1% NP-40, 0.5% deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mM Na-orthovanadate, 0.1% SDS, protease cocktail inhibitor) and extracts clarified at 14,000 g for 15 min. Lysate protein (500 mg) from control and TGF-b1treated cells were incubated with antibodies to RhoA (2 mg, RhoA; Santa Cruz Biotechnology) for 2 h in a total volume of 500 ml. Immune complexes were collected with Protein A/G Plus-agarose, washed three times with lysis buffer without SDS and boiled in sample buffer.

Reactive Oxygen Species (ROS) Assay
The carboxy derivative of fluorescein, 29,79-dichlorofluorescein (carboxy-H2DCFDA) (Molecular probes; C400) was used to determine ROS generation in response to TGF-b1 according to manufacturer's recommendations. Briefly, cells were stimulated with TGF-b1 for the times indicated, medium removed and cells incubated with 5 mM DCFDA in PBS for 15 minutes prior to scrape harvest. Equivalent number of cells were used to assess baseline fluorescence (unstimulated) and response to TGF-b1 stimulation with a multi-detection microplate reader (Synergy HT; Bio-Tek) at an excitation wavelength of 495 nm.
Since changes in redox state by TGF-b1 affects both the canonical SMAD and non-canonical pathways (e.g., Figure 1E), it was important to clarify the impact on downstream TGF-b1 effectors (e.g., src, EGFR, FAK, caveolin-1, SMADs). NAC effectively suppressed TGF-b1-induced c-Src Y416 as well as FAK Y577 (a target of activated c-Src kinases) phosphorylation (Figure 2A), positioning ROS upstream of c-Src -and FAKmediated signaling. While TGF-b1-stimulated caveolin-1 Y14 phosphorylation is also NAC sensitive, FAK Y397 autophosphorylation is only marginally affected by NAC pretreatment suggesting the participation of non-ROS-dependent mechanisms in FAK auto-activation ( Figure 2A). Time-course assessments indicated, moreover, that NAC preincubation suppressed both the amplitude and duration of SMAD3 phosphorylation as well as the inhibition of PAI-1 induction (cf., Figures 1E,2B). Consistent with suppression of SMAD3 phosphorylation, PAI-1 induction by TGF-b1 is also effectively attentuated by NAC preincubation ( Figure 1D,2B).
Given the importance of Src kinases as downstream effectors of ROS-sensitive pathways [21], the Src-dependency of TGF-b1initiated signaling was further assessed using Src, Yes, Fyn triple-null (SYF 2/2/2 ) and wild-type (SYF +/+/+ ) MEFs. c-Src protein as well as c-Src Y416 phosphorylation was evident, as expected, in TGF-b1stimulated wild-type but not SYF-null cells ( Figure 3A). EGFR activation in response to TGF-b1, moreover, is significantly diminished in SYF +/+/+ compared to SYF 2/2/2 fibroblasts consistent with involvement of Src kinases in TGF-b1-mediated EGFR transactivation in VSMCs [13][14][15]. SMAD3 phosphorylation (both extent and duration) is also significantly reduced in SYF 2/2/2 cells compared to their wild-type counterparts over the (1 ng/ml) for the times indicated with or without NAC (5 mM) pretreatment for 1 hour. Increases in pSrc Y416 , pFAK Y577 and pCaveolin Y14 (targets of c-Src kinases) in response to TGF-b1 is completely inhibited by NAC, suggesting an upstream role for ROS generation in activation of Src/FAK/caveolin-1 signaling pathways (A). FAK Y397 phosphorylation by TGF-b1 (at least within the time frame of 2 hours) is relatively unaffected by NAC blockade of ROS generation. Total levels of c-Src, FAK and caveolin-1 are largely unchanged over the time course of TGF-b1 exposure serving as loading controls (A). To assess the role of ROS generation in SMAD3 activation, TGF-b1-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). doi:10.1371/journal.pone.0022896.g002 time course of TGF-b1-stimulation and PAI-1 induction is completely eliminated in Src kinase-deficient MEFs ( Figure 3B). This is in keeping with the higher levels of PPM1A evident in SYF 2/2/2 relative to wild-type fibroblasts. VSMC pretreatment with the src kinase-specific inhibitor SU6656, as expected, prevented the TGF-b1-dependent increase in c-Src Y416 phosphorylation ( Figure 3C). SU6656, however, did not impact TGF-b1initiated SMAD2/3 activation at early time points (e.g., 1 hour) but completely eliminated later-stage (e.g., 4 hrs) SMAD2/3 phosphorylation ( Figure 3C). Transient transfection of VSMCs . Downstream signaling events initiated by TGF-b1-activated Src kinase. SYF +/+/+ and SYF 2/2/2 fibroblasts were serum-deprived for 1 day prior to stimulation with TGF-b1 (0.1 ng/ml) for the times indicated and lysates subject to western analysis. Src activation (assessed using phospho-Src Y416 antibodies) and increased EGFR phosphorylation at the Src kinase target Y845 site, are both evident in TGF-b1-stimulated wild-type (SYF +/+/+ ) MEFs but not Src, Fyn, Yes triple-null (SYF 2/2/2 ) cells (A). The level (at 15 and 30 minutes) and maintenance (at 4 hrs) of SMAD3 phosphorylation is significantly reduced in SYF 2/2/2 fibroblasts compared to their wild-type counterparts (B). In contrast to the typical time coursedependency of PAI-1 induction in response to TGF-b1 in SYF +/+/+ cells, PAI-1 was not detectable in Src-deficient MEFs regardless of the duration of TGF-b1 exposure. The absence of PAI-1 expression and attenuated SMAD3 phosphorylation reflected increased PPM1A levels in SYF 2/2/2 as compared to SYF +/+/+ fibroblasts (B). Pretreatment of VSMCs with the Src kinase inhibitor SU6656 (2 mM) blocked the long-term maintenance (but not the initiation) of SMAD2/3 phosphorylation in response to TGF-b1 while total SMAD levels remain unchanged (C). Src Y416 phosphorylation by TGF-b1 was completely eliminated by SU6656 confirming the effectiveness of this inhibitor (C). Transient transfection of VSMCs with a dominant-negative pp60 c-src (DN-Src) expression construct (or a GFP control vector) 72 hours prior to incubation with TGF-b1 for 6 hours was followed by western analysis for PAI-1. TGF-b1-stimulated PAI-1 induction was effectively suppressed by the DN-Src but not the GFP construct (D). SYF 2/2/2 cells genetically-engineered to express wild-type pp60 c-src (SYF 2/2/2 +WT Src) rescued PAI-1 inducibility in response to TGF-b1 (E). ERK2 (A,D,E) and SMAD3 (C) serve as a loading controls. doi:10.1371/journal.pone.0022896.g003 . FAK is a downstream target of Src kinases and is required for PAI-1 induction by TGF-b1. MEFs were serum-deprived for 1 day prior to addition of TGF-b1 (0.1 ng/ml). TGF-b1 stimulates FAK phosphorylation at the Y577 and Y861 sites in SYF +/+/+ but not SYF 2/2/2 cells consistent with an upstream role of Src kinases in FAK activation. TGF-b1-induced FAK Y397 autophosphorylation, in contrast, is unaffected by genetic ablation of src family kinases (A). To assess the role of FAK in TGF-b1-induced PAI-1 and CTGF expression, serum-deprived FAK +/+ and FAK 2/2 MEFs were stimulated with TGF-b1 and blots probed with antibodies to PAI-1 and CTGF (B). TGF-b1 stimulates FAK phosphorylation at Y397, Y561 and Y861 only in wild-type but not, as anticipated, in FAK-null fibroblasts (C) providing antibody specificity controls for panels A-C. TGF-b1-stimulated c-Src and EGFR activation is significantly attenuated in FAK 2/2 cells relative to FAK +/+ MEFs (C). SMAD3 C-terminal phosphorylation in response to TGF-b1 is reduced in FAK 2/2 as compared to FAK +/+ cells; total SMAD2/3 levels were unchanged regardless of FAK genetic status (D). Western analysis was used to evaluate the effect of FAK genetic status (FAK 2/2 vs. FAK +/+ ) on TGF-b1-induced caveolin-1 Y14 phosphorylation (D). Consistent with previous observations [40], total caveolin-1 is lower in FAK 2/2 MEFs compared to wild-type cultures (D). Assessment of total FAK (A,B), ERK2 (B,C) and SMAD3 (D) provided loading controls. doi:10.1371/journal.pone.0022896.g004 with a dominant-negative c-Src construct, furthermore, effectively inhibited PAI-1 expression upon TGF-b1 addition ( Figure 3D). Stable reconstitution of wild-type pp60 c-src in SYF 2/2/2 cells (SYF 2/2/+WT-Src ) was sufficient to ''rescue'' TGF-b1-mediated PAI-1 inducibility ( Figure 3E) confirming participation of pp60 c-src in PAI-1 gene control.
To investigate downstream targets of caveolin-1 in transducing TGF-b1 signals, focus centered on RhoA as TGF-b1 stimulates Rho GTP loading ( Figure 7A). Caveolin-1 interacts with RhoA in response to TGF-b1 ( Figure 7B,C) and active RhoA (2-4 hrs post TGF-b1 stimulation) is markedly reduced in caveolin-1 2/2 compared to wild-type fibroblasts despite equivalent RhoA levels ( Figure 7A). Transient expression of a DN-RhoA construct or preincubation with the ROCK inhibitor Y-27632 eliminated PAI-1 induction by TGF-b1 establishing the signaling relevance of an intact RhoA-ROCK pathway in PAI-1 gene control ( Figures 7D,  8A). Time-course and dose-response assessments indicated, furthermore, that ROCK inhibition only marginally affected TGF-b1induced pSMAD2/3 levels at one hour but completely blocked SMAD2/3 phosphorylation and nuclear accumulation at 4 hours ( Figure 8A-C) suggesting that the Rho-ROCK pathway impacts not the initiation but the maintenance of SMAD2/3 phosphorylation. SMAD3 is, in fact, a critical downstream effector of TGF-b1dependent PAI-1 expression as SMAD3 knockdown ( Figure 8D,E) or pre-treatment with SIS3 (a selective inhibitor of SMAD3 phosphorylation) ( Figure 8D,E) completely suppressed PAI-1 induction in VSMCs ( Figure 8D; not shown) as well as in MEFs ( Figure 8F,G). Nuclear levels of pSMAD3 increase over the 4 hour time course response to TGF-b1 stimulation as expected; Y-27632 preincubation virtually eliminated pSMAD3 nuclear accumulation coincident with elevations in the nuclear content of the C-terminal pSMAD phosphatase PPM1A ( Figure 8H). Addition of Y-27632 prior to TGF-b1 stimulation rescued nuclear levels of PPM1 to that approximating control conditions with the corresponding characteristic decrease in nuclear pSMAD3 evident at 4 hours post-TGF-b1 addition ( Figure 8C,H). These data suggest that ROCK regulates PPM1A levels modulating, thereby, pSMAD3 nuclear abundance. Consistent with the concept that PPM1A is a negative Figure 7. RhoA both interacts with caveolin-1 Y14 in response to TGF-b1 and required for PAI-1 induction. A Rho-GTPase assay (as described in Methods) was used to assess relative RhoA activation by TGF-b1 in fibroblasts. RhoA-GTP loading increased within 2-4 hours of TGF-b1 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 (A). 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-b1 while total levels of caveolin-1 remain unchanged (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-b1 but not in caveolinnull cells (C). Transfection of a dominant-negative RhoA construct prior to addition of TGF-b1 effectively inhibited PAI-1 expression while introduction of a GFP control vector was without effect (D) indicating that RhoA is required for TGF-b1-induced PAI-1 expression. doi:10.1371/journal.pone.0022896.g007 regulator of TGF-b1/SMAD2/3 signaling, suppression of endogenous PPM1A in VSMCs with siRNA constructs further augments TGF-b1-induced PAI-1 expression compared to identically-stimu-lated control siRNA transfectants( Figure 8I). Collectively, these findings implicate PPM1A in TGF-b1 signaling pathways in VSMCs. Figure 8. Rho-ROCK pathway regulates nuclear levels of PPM1A and maintains SMAD3 activation. VSMCs maintained under serumdeprived conditions for 1 day were TGF-b1-stimulated (1 ng/ml) with or without the ROCK inhibitor, Y-27632 (10 mm) and cellular lysates probed for pSMAD2, total SMAD2/3, PAI-1 and ERK2 (A). Late-stage (4 hour) pSMAD2 levels were markedly attenuated and PAI-1 expression completely inhibited by ROCK blockade (A). TGF-b1-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-b1 for 4 hours. Cell lysates were probed for PAI-1, SMAD2/3, pSMAD2/3 and PPM1A (C). PAI-1 expression in response to TGF-b1 was completely blocked by Y-27632 pre-exposure (10 mM 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 (C). Transient knock-down of SMAD3 with siRNA constructs (as detailed in Methods) (D,E) or pre-incubation with the small molecule inhibitor of SMAD3 phosphorylation SIS3 (5 mM) [41] (D,F,G) eliminates TGF-b1induced PAI-1 expression in VSMCs (D,E) and MEFs (F,G). Cell fractionation studies confirmed that nuclear accumulation of pSMAD3 in response to TGF-b1 is blocked while nuclear PPM1A content increased upon pre-incubation with Y-27632 (H). TGF-b1 stimulation for 4 hours actually reduced nuclear PPM1A levels, which was restored by Y-27632 pretreatment (H). siRNA-mediated PPM1A knockdown in VSMCs resulted in a significantly increased TGF-b1-induced PAI-1 response compared to cells transfected with control siRNA constructs (I). ERK2 (A,B,D-G  TGF-b1 stimulates caveolin-1 Y14 phosphorylation in a reactive oxygen species-FAK/c-Src dependent manner removing repressive influences on EGFR signaling (in red) leading to EGFR transactivation (also by c-Src), thereby, initiating signaling events leading to the MEK-ERK pathway activation necessary for PAI-1 induction. Src 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-b1 (see text). PAI-1 is elevated in atherosclerotic plaques frequently colocalizing with a-smooth muscle actin-expressing cells, presumably VSMCs (insert). doi:10.1371/journal.pone.0022896.g009
Src kinases are upstream effectors of both FAK and caveolin-1 activation as FAK Y577 and Y861 and caveolin-1 Y14 phosphorylation upon TGF-b1 stimulation is not detected in triple-deficient SYF 2/2/2 cells. Stable reconstitution of pp60 c-Src expression in SYF-null cells rescued caveolin-1 Y14 phosphorylation and PAI-1 induction in response to TGF-b1. Moreover, FAK also impacts caveolin-1 Y14 site phosphorylation in the TGF-b1 signaling cascade since phospho-caveolin Y14 is undetectable in FAK 2/2 cells. TGF-b1 fails to induce PAI-1 in caveolin-1 2/2 fibroblasts while re-expression of a wild-type caveolin-1 construct in caveolin-1-deficient cells effectively rescued TGF-b1 inducibility of this serine protease inhibitor. Although gene-specific pathways downstream of caveolin-1 are only beginning to be defined, RhoA interacts with caveolin-1 in response to TGF-b1 and its activation is regulated by caveolin-1 as this response is attenuated in caveolin-1-null fibroblasts. These observations are also consistent with the requirements for fibronectin induction by TGF-b1 in mesangial cells which also involves src-caveolin-1-RhoA signaling [18]. Moreover, TGF-b1-stimulated PAI-1 expression in hepato-cytes similarly requires caveolin-1-dependent signaling and SMAD2/3 activity [25].
Negative regulators of SMAD signaling also impact transcriptional and biological outcomes [16]. Src-deficient fibroblasts exhibit elevated expression PPM1A (a SMAD phosphatase) which accounts, at least in part, for reduced pSMAD levels as well as attenuated PAI-1 induction in response to TGF-b1, Indeed, ectopic overexpression of PPM1A in HaCaT keratinocytes suppressed, while shRNA depletion of PPM1A enhanced, PAI-1 transcription in response to TGF-b1 [19]. How PPM1A is regulated and its specific role in TGF-b1-driven pathophysiologic disorders (e.g., cardiovascular disease, tissue fibrosis, cancer progression/invasion) is not known. Long-term (4 hour) TGF-b1-stimulation reduced nuclear levels of PPM1A in VSMCs, consistent with observations that TGF-b1-induced proteosomal degradation of PPM1A involves attenuation of PPM1A-PTEN (phosphatase and tensin homologue) interactions [35]. Inhibition of Rho/ROCK signaling, moreover, prevented the TGF-b1induced reduction in nuclear PPM1A levels, suggesting that the Rho-ROCK pathway positively mediates PPM1A degradation likely accounting for maintenance of nuclear pSMAD3 necessary for PAI-1 induction. PTEN activity and cellular location is also regulated by Rho kinases and ROCK can directly phosphorylate PTEN facilitating PTEN-Rho-ROCK interactions [36]. Complex formation may destabilize PTEN-PPM1A interactions. One model consistent with current data suggests that Rho phosphorylates PTEN causing dissociation of PTEN-PPM1A complexes resulting in PPM1A degradation, thereby, retaining SMAD transcriptional activity (Figure 9). PTEN knockdown, moreover, results in hyper-induction of PAI-1 expression in response to TGF-b1 [37] and PTEN deletion in fibroblasts is sufficient to induce PAI-1 and cellular senescence [38,39]. Recent findings suggest that TGF-b1 induces a ''senescence-like'' growth arrest, at least in primary VSMCs, with accompanying increases in p21, PAI-1 and CTGF expression (unpublished). Current studies focus on evaluation of the role of PAI-1, induced via ROS/caveolin-1/ SMAD-dependent signaling, in this response.

Author Contributions
Conceived and designed the experiments: RS PJH. Performed the experiments: RS SSC SPH CEH. Analyzed the data: RS PJH. Contributed reagents/materials/analysis tools: RS SSC SPH CEH JCK PJH. Wrote the paper: RS JCK PJH.