PIAS1 Mediates TGFβ-Induced SM α-Actin Gene Expression Through Inhibition of KLF4 Function-Expression by Protein Sumoylation
Objective— TGFβ and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) α-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFβ activates SM α-actin through PIAS1.
Methods and Results— An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFβ-induced expression of SM α-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM α-actin promoter activity in a TGFβ control element (TCE)-dependent manner. Because the TCE within the SM α-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmunoprecipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM α-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation.
Conclusions— These results provide evidence that PIAS1 promotes TGFβ-induced activation of SM α-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.
- transforming growth factor β
- protein inhibitor of activated STAT1
- vascular smooth muscle cells
- Krüppel-like factor
Smooth muscle cells (SMCs) play pivotal roles in development of blood vessels, as well as in a variety of major diseases in man including atherosclerosis, hypertension, cancer, asthma, and vascular aneurysms.1 A major challenge for this field has been to identify environmental cues, signaling pathways, and molecular mechanisms that normally control differentiation of SMCs and how these are disrupted in disease states.2 A key to understanding differentiation and phenotypic switching of SMCs is to identify the mechanisms that regulate transcription of SMC-specific or -selective genes including those for SM α-actin, SM myosin heavy chain (SM-MHC), SM22a, calponin, and smoothelin.1 These SMC-specific genes are the most abundant proteins in fully differentiated SMCs, and are downregulated in phenotypically modulated SMCs as found at sites of vascular injury and atherosclerotic lesions. As such, elucidation of mechanisms that control both normal differentiation of SMCs and phenotypic switching in disease states is likely to provide key insights toward understanding of the development of vascular disease.
Although a critical component of vascular disease is modulation of the differentiated state of vascular SMCs, the mechanisms governing SMC differentiation are relatively poorly understood. Expression of many SMC marker genes has been shown to be dependent on multiple CC(A/T)6GG (CArG) elements and their binding factor, serum response factor (SRF).3–6 Activation of SMC marker genes in vivo in transgenic mice has also been shown to be dependent on multiple other cis-elements including E-boxes that bind members of the basic helix-loop-helix (bHLH) proteins,7 and a transforming growth factor β (TGFβ) control element (TCE).8 We have previously identified PIAS1 as a novel molecular partner of class I bHLH proteins and suggested that PIAS1 plays a role in regulating cooperative interaction of E-box/class I bHLH binding factors and CArG/SRF in activating SMC-selective gene expression, such as SM α-actin, SM-MHC and SM22α.9 Recently, PIAS family proteins have been proposed to function as a small ubiquitin-related modifier (SUMO) E3-ligase.10 SUMO E3-ligase posttranslationally modifies many proteins and plays a role in diverse processes including regulation of transcription, chromatin structure, and DNA repair.11 Of significance, our previous study has indicated that disruption of the E3-ligase activity of PIAS1 abolished its ability to activate the SM α-actin promoter, suggesting that its activity is dependent on protein sumoylation, although the protein targets for this sumoylation have not been identified.
TGFβ is thought to regulate a number of cell events underlying the development of vascular lesions, including SMC differentiation. TGFβ has also been shown to induce expression of SMC differentiation marker genes in a variety of cell types in vitro.12,13 Several key transcription factors have been identified and shown to be important in regulation of TGFβ-induced SMC-specific gene expression, including SRF,14 δEF1,15 Sp1/Sp3,16 and Smad family.17 Of interest, studies of the SM α-actin promoter revealed that 3 CArG boxes and TCE were required for TGFβ inducibility.18 Adam et al identified Krüppel-like factor 4 (KLF4) as a TCE binding factor based on a yeast one-hybrid screen and electrophoretic gel shift assays.19 However, KLF4 was subsequently shown to potently repress expression of multiple SMC marker genes through a combination of effects including suppression of myocardin expression, inhibition of SRF binding to intact chromatin, recruitment of histone deacetylases, and suppressing myocardin-induced gene activation.19–21 Observations that the repressor KLF4 binds to a TCE which mediates TGFβ-dependent activation of SMC marker genes are paradoxical, and indicate that there are major unresolved questions regarding the mechanisms by which TGFβ, KLF4, and TCE regulate expression of SMC marker genes such as SM α-actin.
The goals of present study were as follows: (1) to determine whether PIAS1 contributes to TGFβ-induced expression of SMC marker genes; (2) to determine cis-elements and transcription factors that contribute to the expression of SM α-actin gene by PIAS1; (3) to determine whether PIAS1 induced sumoylation of KLF4 and its subsequent degradation contributes to TGFβ-induced expression of SMC marker genes.
Materials and Methods
Cell Culture and Transient Transfection and Luciferase Assays
BALBc3T3 cells, rat aortic SMCs, and COS cells were cultured as previously described.2 Cells were transfected using Fugene (Roche) according to the manufacturer’s protocol. The cells were incubated 48 hours before being harvested with Passive Lysis buffer (Promega). Luciferase activity was measured with luciferase assay substrate (Promega), and was normalized to total protein (Coomassie Plus Protein Assay Reagent, Pierce) or Renilla luciferase activities using Dual-luciferase reporter assay system (Promega). Each experiment was used three samples and performed three times, respectively.
Construction of siRNA Plasmid, siRNA Oligonucleotide, and Transfection
A plasmid-based system for production of shRNA was previously described.22 Transfection of siRNA plasmid was carried out using Fugene (Roche) according to the manufacturer’s protocol. siRNA oligonucleotides were purchased from Hayashi Kasei Co Ltd and transient transfection of siRNA oligonucleotide was carried out using lipofectamine2000 (Invitrogen) according to the manufacturer’s protocol.
RNA Extraction and Real-Time RT-PCR
Total RNA was prepared from the cultured SMCs using Isogen (Wako) according to the manufacturer’s protocol. One microgram of RNA was used for reverse transcription with RNA LA PCR Kit (Takara) followed by removing DNA with DNA-free kit (Ambion), and real-time RT-PCR analysis was performed using SYBR green (TOYOBO) according to the manufacturer’s protocol. Each experiment was used three samples and performed 3 times, respectively.
In Vivo Sumoylation Assays
COS7 cells were transfected using FuGENE 6 according to the manufacturer’s instructions. After incubation, cells were lysed in 1 mL of RIPA buffer for 30 minutes on ice. Lysates were first cleared with protein G beads for 30 minutes, followed by incubation with antibodies for 1 hour at 4°C. The antibody complexes were captured with protein G beads for 1 hour. Beads were washed 4 times with the same buffer, and immunoprecipitates were eluted and analyzed by Western blot as described previously.23 Each experiment was performed three times and representative data shown.
Statistical analyses were performed using 1-way ANOVA or Student t test when appropriate. Probability values of less than 0.05 were considered statistically significant.
An siRNA Specific for PIAS1 Inhibited TGFβ-Induced Expression of the SM α-Actin Gene in Cultured SMCs
TGFβ plays a major role in the expression of multiple SMC marker genes in a variety of cell types in vitro.12–14 Results of our previous studies showed that PIAS1 activated the expression of SMC differentiation marker genes in cultured SMCs.9 To determine whether endogenous PIAS1 regulates TGFβ-induced SM α-actin gene expression, the effect of shRNA-induced knockdown of PIAS1 proteins was examined using pMighty plasmid-based shRNA expression system.22 Rat aortic SMCs with or without TGFβ treatment were transfected with SM α-actin promoter-enhancer reporter constructs along with the PIAS1 shRNA vector (pMighty-aPIAS1; Figure 1A), and luciferase activities were measured. The shRNA specific for PIAS1 significantly inhibited TGFβ-induced SM α-actin promoter activities (Figure 1B). To investigate whether an siRNA specific for PIAS1 also inhibits TGFβ-induced expression of endogenous SM α-actin gene, we transfected an siRNA oligonucleotide specific for PIAS1 (Figure 1C) into cultured rat aortic SMCs with or without TGFβ and performed real-time RT-PCR of SM α-actin after DNase treatment. The PIAS1 siRNA oligonucleotide nearly completely inhibited TGFβ induced increases in SM α-actin mRNA levels (Figure 1D). These results indicate that PIAS1 is required for TGFβ-induced expression of the SM α-actin gene. Next, to test whether TGFβ induces PIAS1 expression, we performed real-time RT-PCR by using mRNA from TGFβ-stimulated rat aortic SMCs (supplemental Figure I, available online at http://atvb.ahajournals.org). TGFβ induced SM α-actin gene expression by approximately 2.2-fold within 24 hours, but did not affect PIAS1 mRNA levels.
An siRNA Specific for ubc9, an E2-Ligase for Sumoylation, Inhibited TGFβ-Induced SM α-Actin Gene in Cultured SMCs
PIAS family members possess E3-ligase activity for SUMO. Of interest, our previous study showed mutation of the E3-ligase region of PIAS1 abolished its ability to activate the SM α-actin promoter, suggesting that sumoylation plays an important role in PIAS1-induced SM α-actin gene transactivation.9 To test whether sumoylation contributes to TGFβ-induced expression of SM α-actin gene, the effect of an siRNA specific for ubc9, an E2-ligase for SUMO (Figure 2A), on the expression of SM α-actin gene was examined in cultured SMCs with or without TGFβ (Figure 2B). Suppression of ubc9 expression reduced the induction of SM α-actin gene expression by TGFβ. These results indicate that ubc9 is required for TGFβ-induced SM α-actin gene expression.
TCE Was Required for the PIAS1-Mediated Increase in SMα-Actin Promoter Activity
To determine the mechanisms by which PIAS1 regulates SM α-actin promoter activity, a series of mutation constructs of SM α-actin promoter and pCMV-flag-PIAS1 were transfected into BALBc3T3 cells (Figure 3A). PIAS1 significantly activated wild-type, double E-box mutant, and triple CArG mutant constructs by approximately 39-fold, 28-fold, and 29-fold, respectively. The activation of the triple CArG mutant is quite surprising in that all 3 CArG elements are required for expression of SM α-actin in vivo,4 and this construct is also unresponsive to the highly potent SRF coactivator myocardin.22 In contrast, mutation of the SM α-actin TCE completely abolished the responsiveness to PIAS1. KLF4 has been shown to bind the TCE and potently repress expression of multiple SMC marker genes through a combination of effects including suppression of myocardin expression, inhibiting SRF binding to intact chromatin, and suppressing myocardin-induced gene activation.19–21 Thus, the following experiments were performed to examine the possible involvement of KLF4 in this process. First, we determined whether PIAS1 and KLF4 proteins interact by performing mammalian two-hybrid assays in BALBc3T3 cells transiently transfected with GAL4BD-KLF4 (full-length KLF4 fused to GAL4 BD), VP16-PIAS1 (full-length PIAS1 fused to VP16), and pG5Luc reporter plasmids (Figure 3B). Cotransfection of only GAL4BD-KLF4 or VP16-PIAS1 stimulated the pG5Luc reporter activity by threefold and sevenfold, respectively, whereas expression of both GAL4BD-KLF4 and VP16-PIAS1 activated the reporter plasmid by approximately 20-fold. The interaction between PIAS1 and KLF4 was further tested using coimmunoprecipitation assays in 293T cells transfected with GAL4BD-KLF4 and flag-tagged PIAS1 (supplemental Figure II). Results showed the presence of PIAS1 in anti-GAL4 immunoprecipitates but not control IgG precipitates based on Western blotting with the anti-flag antibody. Taken together, these results suggest that KLF4 can interact with PIAS1. Next, to determine the effect of PIAS1 on KLF4-induced repression of SMC marker gene expression, expression plasmids for KLF4 or PIAS1 and SM α-actin promoter reporter gene were transiently transfected into BALBc3T3 cells (Figure 3C). KLF4 reduced SM α-actin promoter activity by 32%, whereas PIAS1 dramatically attenuated repression of SM α-actin promoter activity by KLF4 in a dose-dependent manner in BALBc3T3 cells.
KLF4 Was Modified by SUMO-1
To determine whether KLF4 is modified by SUMO-1, in vivo sumoylation assays using COS cells transiently expressing flag-tagged KLF4 and HA-tagged SUMO-1 were performed (Figure 4). Western blot analysis using antiflag antibody revealed the presence of flag-tagged KLF4 in all cells transfected with the plasmid expressing flag-KLF4. When HA-SUMO-1 was coexpressed, 2 additional slower migrating bands were detected by the flag antibody, and HA antibody identified the slower migrating forms of KLF4. Results suggest that SUMO-1 was conjugated to KLF4.
PIAS1 Promoted Degradation of KLF4
PIAS family members affect protein stability and its function.24 To determine whether PIAS1 induces degradation of KLF4, we overexpressed GAL4-KLF4 with increasing amounts of PIAS1. As shown in Figure 5A, increasing amounts of PIAS1 resulted in decreasing levels of KLF4. The half-life of KLF4, measured by cycloheximide (20 μg/mL, a nonspecific inhibitor for protein synthesis) blockade experiments, showed a decrease in the presence of PIAS1 (Figure 5B). To test whether KLF4 degradation requires the E3-ligase for sumoylation activity of the PIAS1 protein, we performed Western blot analyses using a mutant of PIAS1 defective in E3-ligase activity (C351S). As shown in Figure 5B, the PIAS1 (C351S) mutant did not decrease KLF4 protein levels. These results indicated that the PIAS1 promotes degradation of KLF4 and that E3-ligase activity for sumoylation is required to exert this effect. To test whether PIAS1 might influence the stability of SRF, the half-life of SRF was measured in the presence or absence of cycloheximide (supplemental Figure III). PIAS1 did not affect the half-life of SRF, statistically.
Expression of PIAS1 Was Regulated in Human Aorta
To determine whether the expression of PIAS1 is regulated in human atherosclerotic lesions, we performed real-time RT-PCR using human autopsy specimens. As shown in Figure 6A, immunostaining of SM α-actin revealed the downregulation of SM α-actin in the advanced atherosclerotic lesion. PIAS1, SM α-actin, and TGFβ were expressed at higher levels in diffuse intimal thickening (DIT) than in atherosclerotic lesions (Figure 6B). In contrast, KLF4 and BMP2, which have been implicated in vascular calcification that accompanies the loss of SMC marker gene expression,25 were expressed less prominently in DIT than in atherosclerotic lesions. These results are consistent with the possibility that PIAS1 is involved in regulating SMC gene expression within atherosclerotic lesions through KLF4-dependent mechanisms. That is, increased PIAS1 levels appear to be associated with reduced KLF4 expression and increased SM α-actin expression.
In this report, we provide several lines of evidence in support of the hypothesis that TGFβ-induced activation of SM α-actin in cultured SMCs is mediated at least in part by PIAS1 dependent sumoylation and degradation of KLF4, a TCE binding protein that potently represses SMC gene expression. First, siRNA-induced suppression of endogenous PIAS1 or ubc9 expression markedly reduced TGFβ-induced expression of the SM α-actin gene. Second, PIAS1 failed to activate a SM α-actin promoter containing a mutated TCE. Third, PIAS1 interacted with the TCE binding factor KLF4 based on coimmunoprecipitation and mammalian two-hybrid assays. Fourth, KLF4 was modified by SUMO-1, and overexpression of PIAS1 promoted degradation of KLF4 which was dependent on its E3-ligase activity for sumoylation. Fifth, real-time RT-PCR using human samples indicated that the expression of PIAS1 and TGFβ were significantly attenuated in advanced atherosclerotic lesions which exhibit reduced SMC marker gene expression. These results suggest that PIAS1 contributes to TGFβ-induction of SM α-actin gene expression by binding the TCE repressor protein KLF4 and targeting it for sumoylation and degradation.
Intense effort has been devoted toward understanding the molecular mechanisms underlying the effects of TGFβ on differentiation of vascular SMCs. Previous studies showed that TGFβ-induced expression of SMC marker genes was mediated through a combination of mechanisms including upregulation of SRF expression,14,26 Smad expression or translocation,17,27 δEF1 expression,15 increased Sp1/3,16 and the downregulation of KLF4.19 The results of the present studies indicate that this downregulation of KLF4 is PIAS1 dependent and likely involves sumoylation of KLF4. Sumoylation of transcription factors has been shown to affect their stability, localization, and activity as activators or repressors, by altering protein-protein interactions including facilitating favor recruitment of corepressors, by regulating their subnuclear localization, or by inducing conformational changes in the structure of the transcriptional factor.11 Results of the present study also showed that siRNA-induced knockdown of ubc9, an E2-ligase for sumoylation, attenuated TGFβ-mediated activation of SM α-actin gene expression. To our knowledge, this report provides the first evidence indicating that PIAS1 and sumoylation contribute to TGFβ-induced SMC gene expression.
Previously we have shown that PIAS1 interacted with SRF and class I bHLH proteins. Recently, several studies have demonstrated that SRF, Sp3, Smad family, or myocardin, which have been reported to modulate the induction of SMC marker genes by TGFβ, have been shown to interact with PIAS1 and to be posttranslationally modified by SUMO-1.9,28–32 Results of the present studies provide novel evidence that PIAS1 induced activation of SM α-actin is also mediated by sumoylation of KLF4. Surprisingly, this activation occurred in an E-box- and CArG-element-independent manner. However, in light of results or previous studies showing that the SM α-actin E-boxes and CArG-elements are required for expression in vivo in transgenic mice,4,7 these results may be a function of the unique experimental conditions used or cellular context and may or may not be applicable to normal regulation of these genes in SMCs in vivo. Nevertheless, results raise the possibility that CArG-elements/E-box independent activation of SMC marker genes may occur under some circumstances in vivo. In addition they provide evidence that the TCE element may be capable of recruiting higher order PIAS1 containing protein complexes that are sufficient to activate SM α-actin transcription. These latter observations are analogous to previous studies by Molkentin et al showing that MEF2 and MyoD can transactivate skeletal muscle promoters in cultured myoblast systems as long as at least one of the cognate cis-elements for these factors was present within the promoter.33
KLF4 has been identified as a negative regulatory factor which binds to the SM α-actin TCE and represses the expressions of the SM α-actin, SM-MHC, and SM22α.19 Overexpression of KLF4 markedly suppressed expression of myocardin, and significantly reduced SRF binding to CArG-containing regions of SMC marker genes in the context of intact chromatin, and induced hypoacetylation of histone H4 at SMC CArG regions.20,21 However, the precise molecular mechanisms by which the transcriptional silencer KLF4 mediates TGFβ induction of SM α-actin remains to be determined. Results of the present study showed that PIAS1 and TGFβ were downregulated in human advanced atherosclerotic lesions in which SMC marker genes are repressed, whereas KLF4 gene was expressed. In addition, Yoshida et al recently showed that conditional knockout of KLF4 in mice resulted in a transient delay in suppression of SMC marker genes following vascular injury, but subsequently to enhanced neointima formation through loss of KLF4 dependent activation of the growth suppressor gene p21waf.34 Results indicate that KLF4 plays a key role in regulation of SMC growth and phenotypic switching in vivo, and further highlight the potential importance of understanding mechanisms by which PIAS1, TGFβ regulate KLF expression or functional activity. Of interest, our results showed that sumoylation is likely to lead KLF4 to degradation. Previous studies showed that SUMO modification can serve as a targeting signal in the ubiquitin/proteasome system. Substrates marked by sumoylation are recognized and ubiquitinated by ubiquitin ligases for SUMO conjugates.35 Moreover, SUMO-targeted Ubiquitin Ligases (STUbLs) are recruited to sumoylated target proteins or those containing SUMO-like domains to catalyze their ubiquitination and desumoylation or degradation.36 Taken together, these and our results indicate that PIAS1 may induce sumoylation of KLF4 followed by ubiquitin-dependent degradation.
A critical question is how TGFβ changes the activity of PIAS1. Our data showed that TGFβ had no apparent effect on the expression of PIAS1. Moreover, we found no evidence that TGFβ or knock-down of ubc9 or PIAS1 influenced nuclear localization of PIAS1 and KLF4 (unpublished data K. Kawai-Kowase and M. Kurabayashi, 2007). An alternative possibility is that TGFβ induces posttranslational modifications of PIAS1 that increases its activity. Consistent with this possibility, recent studies provided evidence that phosphorylation of PIAS1 was required for tumor necrosis factor (TNF) induced transcriptional repression.37 Although we found no evidence that TGFβ induced phosphorylation of PIAS1 in cultured SMCs, it is possible this may be a very transient event and difficult to detect (unpublished data K. Kawai-Kowase and M. Kurabayashi, 2007). Finally, it is worth noting that because PIAS1 acts at least in part through enzymatic mechanisms, very little activated PIAS1 may be required to elicit a large biological effect. In any case, it is clear that further studies of the mechanisms that regulate the activity of PIAS1 are required to resolve the preceding issues.
In summary, results of the present studies provide novel evidence showing that PIAS1 and protein sumoylation contribute to TGFβ-induced expression of SM α-actin gene in cultured SMCs. Moreover, we showed that PIAS1 interacted with KLF4 and inhibited KLF4-repressed SM α-actin gene expression, at least in part, through promoting protein sumoylation and degradation. Because PIAS1 has been shown to interact with multiple transcription factors and modify their transcriptional activities, a major the challenge for the future studies is to further elucidate the environmental cues and signaling pathways whereby TGFβ regulates the interactions between PIAS1, KLF4, SRF, Smad family, and other transcription factors, as well as how these regulatory mechanisms are altered within human atherosclerotic lesions.
We are grateful to Miki Matsui and Yukiyo Tosaka for excellent technical assistance.
Sources of Funding
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sport, and Culture of Japan, and a grant from the Japan Cardiovascular Foundation to M.K., NIH grants P01 HL19242, and R01 HL38854 to G.K.O., and Japan Heart Foundation/Novartis Grant, Japan Heart Foundation Grant for Research on Atherosclerosis Update, and a grant from Kowa pharmaceutical to K.-K.K.
Original received October 9, 2007; final version accepted October 3, 2008.
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