The Role Of KLF4 And Mechanism Of KLF4 Actions In Regulating Chromatin Remodeling Of Vascular Smooth Muscle Cells

The vascular smooth muscle cell(SMC) plays a vital role in maintenance of vessel homeostasis, blood pressure, and response to injury. The phenotypic switching of VSMCs plays an important role in cardiovascular diseases, such as hypertension, atherosclerosis and restenosis. Exploring the molecular mechanisms of VSMC phenotype modulation is helpful for therapy of these remodeling cardiovascular diseases.Evidence has been steadily accruing that epigenetic regulation is a important element in the determination of SMC differentiation state, particularly in the areas of histone acetylation and methylation. Previous studies in SMCs have demonstrated that retinoic acid treatment-induced H4 hyperacetylation and H3K4 dimethylation(H3K4dime) of histones within the CAr G-containing regions of the SM marker gene simultaneously increased serum response factor(SRF) binding to CAr G box DNA sequences within the promoters of the SM marker genes and promoted the expression of these genes. CBP and its close homolog, p300, are proteins that contain a catalytic histone acetyltransferase(HAT) domain and that act as transcriptional co-activators for many sequence-specific transcription factors. Several studies have indicated that p300 is necessary for histone acetylation and for SMC differentiation induced by retinoic acid.Chromatin modifications and subsequent remodeling that control cell-specific marker expression are considered key processes in cell differentiation. Understanding the relationship between gene transcriptional activation by transcription factors and chromatin remodeling is extremely important. Krüppel-like factor 4(KLF4), which is a zinc-finger-containing transcription factor, plays a key role in cell proliferation, differentiation, apoptosis, cell cycle and embryonic development. Several reports have demonstrated that the N-terminus of KLF4 contains a strong transactivation domain, which interacts with the transcriptional co-activators p300/CBP. p300/CBP is assumed to be recruited to specific regions of DNA by sequence-specific transcription factors and that this interaction can promote localized histone acetylation. But how KLF4 recruits p300 to the KLF4-regulated gene promoter by altering its cofactor interactions and modification status is not well characterized.The aim of the present study was to investigate whether and how histone acetylation was mediated by KLF4 during VSMC differentiation. Part KLF4 mediates TGFⅠ-β1-induced histone H3 acetylationObjective: To explore whether TGF-β1 increases histone H3 acetylation and the actual relationship between KLF4 and histone acetylation in TGF-β1-stimulated VSMCs.Methods: Western blotting was performed to examine the expression of KLF4, H3 and H4 acetyation. H3 and H4 acetyation in VSMCs induced by TGF-β1 was determined by cell immunofluorescence analysis. Chromatin immunoprecipitation(Ch IP) analysis was applied to detect the occupancy of acetylated H3, p300 and KLF4 at the p21 and TβRI promoters, which were activated by KLF4 in VSMCs.Results:1 TGF-β1 induces KLF4 expression and histone H3 and H4 acetylationHistone acetylation is closely associated with transcriptional activation. TGF-β1 regulates the expression of proliferation and differentiation-related genes in VSMCs. Western blot analysis showed that TGF-β1 increased KLF4 expression and histone H3 and H4 acetylation in a time- and dose-dependent manner. Similar results were obtained in VSMCs by cell immunofluorescence analysis, showing that histone H3 and H4 acetylation markedly increased in VSMC nuclei 24 h after TGF-β1 treatment.2 KLF4 mediates TGF-β1-induced histone H3 acetylationTo validate the roles of KLF4 in TGF-β1-induced histone acetylation, KLF4 overexpression was obtained by an adenovirus vector infection using the vector Ad-GFP-KLF4. KLF4 overexpression further increased histone H3 acetylation by TGF-β1. In contrast, when VSMCs were transfected with KLF4-specific si RNA(si-KLF4) to block endogenous KLF4 expression, TGF-β1-induced H3 acetylation was abrogated. However, only a slight change in the level of acetylated H4 was observed in VSMCs infected with p Ad-GFP-KLF4 or transfected with KLF4-specific si RNA. These results indicate that KLF4 is essential for the induction of H3 acetylation when KLF4-dependent transcription is activated by TGF-β1 signaling.3 KLF4 increases the occupancy of acetylated H3 on the TGF-β1-controlled gene promoterTo determine whether acetylated H3 was increased on the KLF4-regulated gene promoter, chromatin immunoprecipitation(Ch IP) analysis was applied to detect the occupancy of acetylated H3 at the p21 and TβRI promoters, which were activated by KLF4 in VSMCs. Twelve pairs of primers that covered a 1.5 kb range of the p21 promoter region and a 1.2 kb range of the TβRI promoter region were employed. Ch IP analysis showed that H3 acetylation was highly concentrated in the region between-645 bp and 57 bp of the p21 promoter in TGF-β1-treated VSMCs and peaked at-225 bp/57 bp. KLF4 overexpression significantly increased the acetylation and enrichment of H3 in this region. In another gene regulated by KLF4, H3 acetylation was highly concentrated in the region between-545 bp and +105 bp of the TβRI promoter, suggesting that KLF4-mediated histone H3 acetylation and recruitment to KLF4-regulated promoters is a generalized event, at least in VSMCs. We chose p21 as a target gene to further study the relationship between KLF4-regulated transcription and H3 acetylation. As expected, the knockdown of endogenous KLF4 by si-KLF4 significantly reduced the level of acetylated H3 at the p21 promoter, regardless of TGF-β1 treatment.4 KLF4 increases the occupancy of p300 on the TGF-β1-controlled gene promoterThe effects of KLF4 and TGF-β1 on the enrichment of p300 were also measured by Ch IP analysis with primer pairs covering this region of the p21 promoter. p300 was bound to two positions, at-645 bp/-397 bp and-225 bp/57 bp, of the p21 promoter following stimulation with TGF-β1. These two positions contain both Smad sites and TCE sites, which are responsible for TGF-β1 regulation. Furthermore, we found that p300 binding was also regulated by KLF4. KLF4 overexpression further increased the binding of p300 to-645 bp/-397 bp and to 225 bp/57 bp of the p21 promoter region. The knockdown of KLF4 by si-KLF4 reduced the inducing effect of TGF-β1 on the binding of p300, regardless of TGF-β1 treatment. To determine the role of p300 in the acetylation and recruitment of H3, we transfected VSMCs with si RNA targeting p300(si-p300) before adding TGF-β1 and examined the enrichment of acetylated H3 on the p21 promoter. The results showed that the knockdown of p300 drastically reduced acetylated H3 binding to-645 bp/-397 bp and to-225 bp/57 bp of the p21 promoter. Previous studies have shown that KLF4 binds to the TCE site of its target genes.We speculated that KLF4 might also bind to the TCE site on the p21 promoter. The effect of TGF-β1 on the binding of KLF4 to the p21 promoter was determined to test this hypothesis. Consequently, the binding of KLF4 to-225 bp/57 bp of the p21 promoter increased in VSMCs infected with Ad-KLF4, and TGF-β1 treatment further increased its binding. These results clearly suggest that TGF-β1 promotes the KLF4-mediated recruitment of p300 to the p21 promoter, subsequently leading to increased H3 acetylation at position-225 bp/57 bp of the p21 promoter.Summary:KLF4 can recruit p300 to the KLF4-binding sites of the genes by TGF-β1 regulation during VSMC differentiation, and mediate TGF-β1-induced histone H3 acetylation. Part Ⅱ PTEN and p300 interact with KLF4, which regulates KLF4phosphorylation and acetylationObjective: To examine whether TGF-β1 influences interaction of KLF4 with p300 and PTEN, and biological significance of their interaction.Methods: Co-immunoprecipitation assay was done to examine the interaction between KLF4, p300 and PTEN. The localization of KLF4 and p300 in VSMCs was determined by cell immunofluorescence analysis. A sequential Ch IP analysis was performed to examine whether TGF-β1 enhanced the recruitment of KLF4-p300 complex to p21 promoter. A GST pull-down assay was performed to further reveal whether TGF-β1 directly affected the interaction between KLF4, p300 and PTEN in vitro.Results:1 TGF-β1 increases the interaction of KLF4 with p300 and inhibits the interaction of KLF4 with PTENCoimmunoprecipitation assay and GST pull-down assay showed that TGF-β1 increased the levels of p300 present in anti-KLF4 immunoprecipitates, peaking at 30 min and subsequently decreasing to control levels by 60 min. Unexpectedly, we found that KLF4 formed a complex with PTEN before TGF-β1 treatment, that TGF-β1 decreased this association within 30 min, and that their interaction was restored to the basal level at 60 min after TGF-β1 treatment.The results indicated that TGF-β1 treatment increases the p300-KLF4 interaction and reduces the PTEN-KLF4 interaction.2 KLF4 and p300 translocate to the nuclear area in TGF-β1-treated VSMCs and interact with each other in p21 promoterDual immunofluorescent detection revealed that KLF4 and p300 were distributed across the entire cell before TGF-β1 treatment but translocated to the nuclear area in TGF-β1-treated VSMCs for 30 min and that KLF4 and p300 primarily colocalized in this period. Furthermore, we performed a sequential Ch IP analysis, and showed that TGF-β1 increased the recruitment of the KLF4-p300 complex to-225 bp/57 bp of the p21 promoter compared with TGF-β1-untreated cells, and KLF4 overexpression before the addition of TGF-β1 further enhanced the interaction of KLF4 with p300.Taken together, these results indicate that KLF4 recruits p300 to specific sites of the p21 promoter through physically associating with p300 upon TGF-β1 signaling activation.3 TGF-β1 induces KLF4 phosphorylation and acetylationWe examined the post-translational modifications of KLF4. Among the tested modifications, TGF-β1 stimulated KLF4 phosphorylation on Ser residues within 15 min, reaching a maximum at 30 min, and then returned to the basal level by 60 min. Under these experimental conditions, KLF4 acetylation simultaneously increased in a time-dependent manner. The level of KLF4 expression was not affected over the time course of the study.4 Post-translational modifications of KLF4, including phosphorylation and acetylation, are regulated by PTEN and p300To determine whether KLF4 acetylation was mediated directly by p300, we tested the effect of p300 knockdown on KLF4 acetylation. The results showed that TGF-β1 markedly stimulated KLF4 acetylation, and the knockdown of p300 abrogated TGF-β1-induced KLF4 acetylation. Next, we examined the effect of PTEN knockdown on KLF4 phosphorylation and found that PTEN knockdown by si-PTEN facilitated TGF-β1-induced KLF4 phosphorylation. When VSMCs were transfected with GFP-PTEN, TGF-β1-induced KLF4 phosphorylation was abrogated. In vivo, we found that KLF4 phosphorylation in the aortas of PTEN-/- mice(smooth muscle-specific PTEN knockout mice) was higher than that of WT mice. These results suggest that PTEN can directly dephosphorylate KLF4.Summary:PTEN forms a complex with KLF4 to inhibit the phosphorylation of the latter under basal conditions. Upon TGF-β1 signaling activation, phosphorylated PTEN loses its ability to dephosphorylate KLF4 and switches the cofactor interacting with KLF4 from PTEN to p300. KLF4-p300 complexes were recruited to KLF4-binding sites of the gene promoter of VSMCs to acetylate histone H3. Part ⅢKLF4 phosphorylation promotes p300 acetylation via the p38pathway and is required for its interaction with p300 and H3acetylationObjective: To observe the role of post-translational modifications of KLF4 in mediating TGF-β1 induced H3 acetylation.Methods: Site-directed mutagenesis and plasmid transfection were performed in vitro study. The protein extracts from VSMCs were used to detect phosphorylation of various signaling molecules by Western blotting. si RNA transfection experiments were performed in VSMCs to knock down KLF4. Immunoprecipitation assay was done to examine the phosphorylation and acetylation of KLF4.Results:1 KLF4 phosphorylation, but not acetylation, is required for its interaction with p300 and KLF4 acetylationBecause our previous study demonstrated that KLF4 is phosphorylated at S470 by Smad and p38 signaling in response to TGF-β1, we sought to examine whether KLF4 phosphorylation affected its interaction with p300 and PTEN. To test this possibility, we performed a serine-to-alanine mutation of KLF4 at Ser470. The S470 A substitution of KLF4 markedly reduced the TGF-β1-induced interaction of KLF4 with p300 but did not affect its interaction with PTEN. Correspondingly, KLF4 acetylation significantly decreased in the S470 A mutant compared with wild-type KLF4. By contrast, no obvious difference in the phosphorylation level was observed between wild-type KLF4 and its acetylation-deficient mutants K225 R, K229 R, and K225/229 R.These results suggest that the acetylation modification of KLF4 at Lys225, Lys229, or Lys225/229 does not affect its phosphorylation at Ser470 by TGF-β1, whereas phosphorylation at Ser470 is necessary for its interaction with p300 and for its acetylation by p300.2 KLF4 phosphorylation is required for TGF-β1-induced H3 acetylationNext, the effect of S470 A substitution on H3 acetylation was measured. The results showed that the induction of H3 acetylation by TGF-β1 was decreased by half when Ser470 was substituted with alanine. K225 R, K229 R, or K225/229 R substitution in the KLF4 molecule did not affect TGF-β1-induced H3 acetylation compared with the wild-type KLF4. Next, we determined whether garcinol, which is a potent p300 histone acetyltransferase inhibitor, could inhibit H3 acetylation. As expected, garcinol clearly decreased the level of acetylated H3, regardless of TGF-β1 treatment. Altogether, these findings suggest that KLF4 phosphorylation at Ser470 is necessary for its interaction with p300 and for H3 acetylation by p300 in response to TGF-β1.3 KLF4 phosphorylation promotes p300 acetylation induced by TGF-β1Western blot analysis showed that TGF-β1 rapidly stimulated PTEN phosphorylation on Ser residues within 15 min, reaching a maximum at 30 min, and then returned to the basal level by 60 min. Under these experimental conditions, p300 acetylation simultaneously increased in a time-dependent manner, which is consistent with the phosphorylation and acetylation modifications of KLF4. Next, we determined the relationship between KLF4 and p300 acetylation. p300 acetylation markedly decreased when Ser470 of KLF4 was substituted with alanine; however, S470 A substitution did not affect PTEN phosphorylation. When Lys225, Lys229, or Lys225/229 of KLF4 were substituted with arginine, p300 acetylation was not affected, suggesting that KLF4 phosphorylation precedes p300 acetylation in response to TGF-β1 and that KLF4 phosphorylation is required for p300 acetylation.4 TGF-β1 induces post-translational modifications of KLF4 and p300 acetylation via Smad and p38 pathwayFurther, we examined which signaling pathway mediated the post-translational modifications of KLF4, p300 and PTEN in response to TGF-β1. Western blot analysis showed that upon stimulation with TGF-β1, the phosphorylation of p38, JNK, Smad2, and Akt simultaneously increased in a time-dependent manner, whereas ERK phosphorylation was negatively regulated by TGF-β1. The expression levels of these signal molecules did not significantly change during the course of stimulation with TGF-β1. Then, VSMCs were incubated with the p38 inhibitor SB203580, the JNK inhibitor SP600125, the ERK inhibitor PD98059, the Smad inhibitor SB431542, or the PI3K/Akt inhibitor LY294002 for 2 h before exposure to TGF-β1. The inhibition of Smad or p38 MAPK blocked TGF-β1-induced KLF4 acetylation and phosphorylation. Interestingly, the pharmacological inhibition of Smad or p38 MAPK also blocked TGF-β1-induced p300 acetylation, again suggesting that Smad- and p38 MAPK-mediated KLF4 phosphorylation by TGF-β1 is necessary for p300 acetylation. In addition, the inhibition of p38 MAPK or PI3K/Akt signaling significantly decreased TGF-β1-induced PTEN phosphorylation. Together, these results clearly suggest that Smad and p38 MAPK pathways mediate TGF-β1-induced KLF4 phosphorylation, followed by KLF4 and p300 acetylation in VSMCs, whereas p38 MAPK or PI3K/Akt signaling pathways mediate PTEN phosphorylation.5 KLF4 promotes p300 acetylation via the p38 pathwayThen, we tested whether KLF4 could influence Smad or p38 MAPK signaling pathways. This experiment showed that Smad2 phosphorylation was dramatically increased by TGF-β1 and was not affected by KLF4 overexpression or by KLF4 knockdown. Likewise, both KLF4 overexpression and TGF-β1 treatment promoted p38 MAPK phosphorylation and KLF4 knockdown reduced the TGF-β1-induced p38 MAPK phosphorylation, suggesting that KLF4 promotes p300 acetylation via the p38 MAPK signaling pathway.Summary:TGF-β1 treatment promotes KLF4 phosphorylation via p38 MAPK or Smad signaling, thus leading to KLF4-p300 complex formation. Phosphorylated KLF4 can enhance p300 HAT activity via the p38 MAPK pathway.Conclusions:1 KLF4 mediates TGF-β1-induced histone H3 acetylation.2 PTEN forms a complex with KLF4 to inhibit the phosphorylation of the latter under basal conditions. Upon TGF-β1 signaling activation, phosphorylated PTEN loses its ability to dephosphorylate KLF4 and switches the cofactor interacting with KLF4 from PTEN to p300.3 TGF-β1 treatment promotes KLF4 phosphorylation via p38 MAPK or Smad signaling, thus leading to KLF4-p300 complex formation, which recruits p300 to the KLF4-binding sites of the p21 promoter in VSMCs, and the p21 promoter chromatin-localized p300 acetylates histone H3, leading to chromatin remodeling and p21 promoter activation by KLF4.4 Phosphorylated KLF4 can enhance p300 HAT activity via the p38 MAPK pathway and increases H3 acetylation, which leads to p21 promoter activation.