The Role And Mechanism Of KLF4 In Transforming Growth Factor-β1-induced Vsmc Differentiation

Proliferation of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of a variety of proliferative vascular diseases, such as atherosclerosis, restenosis and hypertension. Several lines of evidence have shown that the factors controlling VSMC proliferation and growth inhibition, including various growth factors, signal transduction molecules and transcription factors constitute a complex functional network. Krüppel-like factors (KLFs), retinoic acid receptor-alpha (RARα), platelet-derived growth factor receptor (PDGFR), and transforming growth factor-βtype I receptor (TβRI) and type II receptor (TβRII) are expressed in VSMCs and are components of such a network. Transforming growth factor-β1 (TGF-β1) signaling is involved in the regulation of cell growth, differentiation, apoptosis, cellular homeostasis and other cellular functions. Despite numerous signaling studies, however, the specific contribution of TGF-βsignaling to VSMC phenotypic modulation has not been fully elucidated.Krüppel-like factor 4 (KLF4, GKLF) is a member of Sp1 transcription factor family characterized by three C2H2 zinc finger motifs, mainly involved in regulating cell growth, differentiation, proliferation and apoptosis, by controlling the expression of a large number of genes with GC/GT-rich promoters. It has been demonstrated that KLF4 might function as a pleiotropic factor depending on the interaction partner(s) and the cellular context in VSMCs. However, the actual relationship between KLF4 and TβR in the regulation of VSMC proliferation and growth inhibition is not fully understoodTo elucidate whether and how TGF-β-mediated KLF4 expression regulates TGF-βsignaling in VSMCs, we detected the effects of TGF-β1 on VSMC proliferation, growth inhibition and expression of marker genes. We further detected the actual relationship between KLF4 and TβR in the regulation of VSMC proliferation and growth inhibition.1. KLF4 mediates TGF-β1-induced VSMC differentiation via TβRITGF-β1 treatment of VSMCs either stimulates proliferation or induces differentiation, depending on the experimental conditions. Thus, using cultured rat VSMCs, we examined the effect of TGF-β1 on the proliferation, cell cycle and differentiated marker genes of VSMCs and the role of KLF4 in TGF-β-induced VSMC differentiation.1.1 TGF-β1 inhibits proliferation, migration and cell cycle of VSMCsThe results of MTT and migration assays showed that treatment of VSMCs with TGF-β1 resulted in a significant reduction of cell proliferation and migration in a dose-dependent (0.5, 1, 2, 4 ng/ml) and time-dependent (24, 48 h) manner. A significant inhibiting effect was observed by treating VSMCs with 2 ng/ml of TGF-β1 for 48 h.G1-S switch is a key point in cell proliferation, so we examined the alteration of VSMC cell cycle by flow cytometry. Cell cycle analysis showed that the G0/G1 cell population was increased to 42% after TGF-β1 treatment for 48 h, with only 15% of untreated cells being arrested in the G0/G1 phase of the cell cycle. In addition, there was a reduction in cell proportion in S phase and G2/M phase after TGF-β1 treatment.1.2 TGF-β1 enhances the expression of differentiation marker genes, and simultaneously suppresses the expression of dedifferentiation marker geneSM22αand SMα-actin highly expressed in differentiated VSMCs are the differentiated marker genes, while PCNA (proliferating cell nuclear antigen) is the VSMC-dedifferentiated marker. Western blot showed that treated with 2 ng/ml of TGF-β1 from 0 to 48 h increased expression of SM22αand SMα-actin, while decreasing the PCNA levels in a time-dependent manner. In addition, when treatment with a range of TGF-β1 concentrations from 0 to 4 ng/ml for 48 h resulted in increased the level of SM22αand SMα-actin in a dose-dependent manner, and decreased the level of PCNA in VSMCs.1.3 TβRI expression is closely linked to VSMC differentiation by TGF-β1RNA interference was used to silence the expression of TβRI and to assess its effects on TGF-β1-induced VSMC differentiation. Measurements of protein expression showed that the targeted TβRI siRNA knocked down between 75% and 80% of cellular TβRI that was induced by TGF-β1. Transfection with TβRI siRNA reduced SM22αand SMα-actin protein levels while upregulating PCNA expression in TGF-β1-treated cells.1.4 KLF4 mediates TGF-β1-induced TβRI expression and VSMC differentiationTo confirm that KLF4 is involved in the regulation of VSMC differentiation we examined the effect of TGF-β1 on KLF4 protein expression. The results of Western blot analysis showed that TGF-β1 (2 ng/ml) time-dependently induced KLF4 levels, reaching a maximum after 6 h before returning to basal levels by 48 h of treatment with TGF-β1. A similar pattern of TβRI expression following TGF-β1 stimulation was observed. Measurements of mRNA levels by semi-quantitative RT-PCR showed comparable changes in KLF4 and TβRI expression in response to TGF-β1.To assess that KLF4 mediates TGF-β1-induced TβRI expression in VSMCs, KLF4 siRNA or Ad-KLF4 was transfected into VSMCs. Transfection of VSMCs with KLF4 siRNA resulted in the suppression of TβRI mRNA and protein levels. Correspondingly, a reduction was observed in TGF-β1-induced expression of SM22αand SMα-actin proteins in response to KLF4 silencing. Conversely, overexpression of KLF4 markedly induced expression of TβRI, SM22αand SMα-actin.2. TGF-β1 induces KLF4 phosphorylation and its interaction with Smad2/3 via Smad and p38 MAPK signaling in VSMCsThe role of KLF family in the regulation of TGF-βsignaling has been the subject of considerable investigation. However, the precise mechanism whereby KLF4 regulates TGF-βsignaling in VSMCs is still poorly understood. Having identifed TGF-βregulates KLF4, we investigated whether there exists an interesting possibility that one of the TGF-β-activated pathways regulates KLF4.2.1 TGF-β1 induces KLF4 phosphorylation via Smad and p38 MAPK signaling in VSMCsThe potential role of KLF4 in TGF-β1 signaling and function was investigated by determining KLF4 phosphorylation in VSMCs in response to TGF-β1. This experiment showed that TGF-β1 increased phosphorylation of KLF4 within 5 min, reaching a maximum between 20 and 40 min. Under these experimental conditions, the phosphorylation of TβRI, Smad2, Smad3 and p38 MAPK increased concurrently in a time-dependent manner, whereas ERK phosphorylation was negatively affected by TGF-β1.To assess the importance of Smad and p38 MAPK signaling in promoting KLF4 phosphorylation by TGF-β1, the effects of SB431542 and SB203580 on KLF4 phosphorylation induced by TGF-β1 were detected. Pharmacological inhibition of TβRI or p38 MAPK blocked TGF-β1-induced KLF4 phosphorylation. Moreover, silencing Smad2, Smad3 or p38αMAPK blocked TGF-β1-induced KLF4 phosphorylation.2.2 KLF4 phosphorylation by TGF-β1 increases the interaction between KLF4 and Smad2/3Because TGF-β1 induced KLF4 phosphorylation, experiments were conducted to determine whether KLF4 phosphorylation affected its ability to interact with Smad2 or Smad3 in VSMCs. Co-immunoprecipitation experiments confirmed that TGF-β1 time-dependently increased the interaction of Smad2 and Smad3 with KLF4. SB431542 or SB203580 suppressed TGF-β1-dependent recruitment of KLF4 with Smad2 and Smad3. Correspondingly, siRNA-mediated knockdown of p38αMAPK, Smad2 or Smad3 also decreased the interaction of KLF4 with Smad2/3 in TGF-β1-treated cells.To verify that the KLF4-Smad interaction is elicited by KLF4 phosphorylation, point-mutation experiments were conducted. Mutation within the DNA-binding domain at 470 showed that the Ser470Ala substitution markedly reduced TGF-β1-dependent KLF4 phosphorylation; this was accompanied by impaired co-sedimentation with Smad2/3.2.3 KLF4 and Smad2 cooperatively activate the TβRI promoterAfter having demonstrated that KLF4 mediates TGF-β1-induced TβRI expression through interaction with Smad2/3, we then examined whether KLF4-Smad2 association plays a role in the activation of the TβRI promoter. Gene reporter assay showed that co-expression KLF4 and Smad2 induced an ~19-fold increase in luciferase activity.A further induction in TβRI promoter activity was observed when the cells cotransfected with KLF4 and Smad2 expression plasmids were stimulated with TGF-β1. While, SB431542, SB203580 or a combination of both, significantly reduced the reporter gene activity in the TGF-β1-treated 293A cotransfected cells.3. Synergistic cooperation of KLF4 with Smad2 in TGF-β1-induced TβRI gene activation in VSMCsSince KLF4 mediates TβRI expression, we hypothesized that KLF4 enhances TGF-βsignaling via TβRI. Since it has been demonstrated that KLF4 interaction with Smad2 cooperatively activates TβRI promoter, we hypothesized that KLF4 could form a stable complex with Smad2 either at the KLF4-binding region, Smad-responsive element or both of these two regions. Using the TESS computational program, we established that the -472/-339 bp region of the rat TβRI promoter contains KLF4-binding sites (CACCC) and the -806/-561 bp region contains Smad-responsive region (CAGAC).3.1 KLF4 binds directly to the KLF4-binding sites 2 and 3 of the TβRI promoter HEK293A cells were transfected with a TβRI promoter-reporter plasmid and increasing amounts of KLF4 expression plasmids. Measurements of luciferase activity showed that overexpression of KLF4 dose-dependently increased by several fold the transcriptional activity of TβRI when compared with control plasmids.Using the TESS computational program, we established that the -472/-339 bp region of the rat TβRI promoter contains two typical KLF4-binding sites (CACCC) and one with a reverse orientation sequence (GGGTG). Chromatin immunoprecipitation studies showed that KLF4 specifically interacts with TGF-β1-responsive sites of TβRI promoter. To determine which DNA elements are involved in the KLF4 induction of TβRI, a series of 5'-deletion mutants and KLF4-binding site mutants were tested in 293A cells co-transfected with pEGFP-KLF4. Measurements of luciferase activity suggested that the KLF4-responsive elements are located in the -453/+21 region in the TβRI promoter.To test the role of these putative KLF4-binding sites in TβRI induction by KLF4, reporter plasmids containing TβRI promoter with site-specific mutations were analyzed. Disruption of KLF4-binding site 1 (-472/-468 bp region) did not affect the transcriptional induction by KLF4, whereas disruption of site 2 (-402/-398 bp region) or 3 (-343/-339 bp region) reduced significantly the KLF4's transcriptional response. To verify the binding activities of the KLF4-binding sites 2 and 3, Oligonucleotide pull-down assays were performed. The same pattern of KLF4 protein bands was observed with both oligonucleotide probes. Extracts from cells treated with TGF-β1 showed stronger KLF4 binding to the two probes by ~2-fold.3.2 Effect of Smad2 on TGF-β1-induced TβRI expressionSmads are central mediators of TβR signals, so we determined whether TGF-β1-dependent induction of TβRI levels is influenced by inhibition of Smad2 and/or Smad3. Silencing of either Smad2 or Smad3 markedly suppressed TβRI up-regulation by TGF-β1. Conversely, overexpression of Smad2 markedly induced expression of TβRI.3.3 The Smad-responsive element is responsible for Smad2 transactivation on the TβRI promoterWe examined whether Smad2 plays a role in the activation of the TβRI promoter by luciferase reporter gene assay. Measurements of luciferase activity showed that overexpression of Smad2 dose-dependently increased by several fold the transcriptional activity of TβRI when compared with control plasmids.Because Smad2 could activate the TβRI promoter, we want to determine whether Smad2 binds directly to this promoter. ChIP assays showed that constitutive Smad2 binds to the TβRI promoter. The TGF-β1 treatment increased Smad2 binding by ~2.2-fold to the putative Smad-binding region as compared to untreated cells.To define which DNA elements are involved in the Smad2 induction of TβRI, a series of 5'-deletion mutants were tested in 293 cells co-transfected with pcDNA-Smad2. pTβRI/-993 elicited a strong transcriptional response by Smad2, whereas pTβRI/-453 and pTβRI/-386 were unresponsive to Smad2.3.4 Interaction of KLF4 with Smad2 bound to the Smad-binding element provides cooperative activation of the TβRI promoterSince KLF4 interaction with Smad2 cooperatively activates TβRI promoter, we hypothesized that KLF4 could form a stable complex with Smad2 either at the KLF4-binding region, Smad-responsive element or both of these two regions. ChIP and two-step ChIP assay showed that KLF4 forms a stable complex with Smad2 that is bound to the Smad-binding region of the TβRI promoter.To further determine the role of the Smad-binding region in the cooperative transcriptional induction of TβRI by KLF4 and Smad2, 293A cells were cotransfected with pTβRI/-993, pTβRI/-993(?-483/-241) or pTβRI/-993(?-806/-561) together with empty vector or the expression vectors encoding KLF4 and Smad2. Measurements of luciferase showed that deletion of the -483/-241 KLF4-binding region abolished KLF4's transcriptional response while maintaining Smad2 responsiveness and the cooperative activation by Smad2 and KLF4. In contrast, deletion of the -806/-561 Smad2-binding motif had no inhibitory effect on the transcriptional induction of TβRI promoter by KLF4; however, both the Smad2 responsiveness and cooperative activation by Smad2 and KLF4 were lost. Moreover, Knockdown of Smad2 with siRNA abolished the additive effect in cells cotransfected with Smad2 and KLF4 expression plasmids.CONCLUSIONS1 TGF-β1 inhibits cell cycle progression and induces differentiation in cultured rat VSMCs. This activity of TGF-β1 is accompanied by upregulation of KLF4, with concomitant increase in TβRI expression.2 KLF4 is found to transduce TGF-β1 signals via phosphorylation-mediated activation of Smad2, Smad3 and p38 MAP kinase. The activation of both pathways, in turn, increases the phosphorylation of KLF4, which enable the formation of KLF4-Smad2 complex in response to TGF-β1.3 KLF4 binds directly to the KLF4-binding sites 2 and 3 of the TβRI promoter and Smad2 recruits to the Smad-responsive region to activate TβRI promoter.4 Formation of a stable KLF4-Smad2 complex to the promoter's Smad-responsive region mediates cooperative TβRI promoter transcription in response to TGF-β1.