Action Mechanisms Of HDAC2 In Regulating VSMC Differentiation Mediated By Krüppel-like Factor 4

In response to pathogenic vascular injuries, vascular smooth muscle cells (VSMC) can revert from differentiated phenotype to dedifferentiated phenotype, and then proliferate. It is well established that phenotypic change of VSMC from the differentiated to the dedifferentiated state accompanies their migration and proliferation, which is the important pathological basis of some vascular proliferative diseases, such as hypertension, atherosclerosis and postangioplasty restenosis. Therefore, exploring the regulatory mechanisms of VSMC phenotype is helpful for therapy of these remodeling cardiovascular diseases.All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to inhibit VSMC proliferation and induce VSMC differentiation through regulating numerous differentiation-related genes. The gut enriched krüppel-like factor (GKLF/KLF4) is a member of KLF family which plays important roles in cell growth, proliferation, differentiation, and embryogenesis as well as carcinogenesis. ATRA has been shown to induce expression of KLF4 and promote VSMC differentiation, but the action mechanisms of KLF4 in VSMC phenotype modulation induced by ATRA are unclear.Histone deacetylases (HDACs) play an important role in the regulation of transcription via triggering deacetylation of histones and some transcription factors. HDAC2 is one of the most thoroughly studied HDACs, and can undergo different posttranslational modifications such as nitration, phosphorylation and ubiquitination. However, little is known of the role of HDAC2 in the regulation of KLF4 activity during ATRA-induced VSMC differentiation. In the present studies, we investigated crucial mechanisms of the interaction between KLF4 and HDAC2 induced by ATRA, which will contribute to penetrate VSMC differentiation.1 The effect of ATRA on the interaction between KLF4 and HDAC2We studied the effect of ATRA on the interaction between KLF4 and HDAC2 by co-IP and GST pull down assay. The results were as follows:1.1 ATRA inhibits the interaction between KLF4 and HDAC2Co-immunoprecipitation assay showed that ATRA inhibited the interaction between KLF4 and HDAC2. Treatment of VSMC with ATRA resulted in a time-dependent (0, 30, 60 min) reduction in the interaction of KLF4 with HDAC2. GST pull down assay further identified that the interaction between KLF4 and HDAC2 was significantly inhibited in VSMC treated with ATRA in a time-dependent manner. The above findings suggested that ATRA inhibits the interaction between KLF4 and HDAC2 in a time-dependent manner.1.2 ATRA induces HDAC2 phosphorylation via JNK-dependent signal pathway in VSMCThe lysates of VSMC treated with ATRA for 15, 30 and 60 min were immunoprecipitated with anti-phosphoserine antibody, and then phospho-HDAC2 was detected by Western blotting with anti-HDAC2 antibody. The results showed that ATRA treatment time-dependently increased the levels of phospho-HDAC2 within 60 min in VSMC. To determine which signal pathway mediates HDAC2 phosphorylation in VSMC treated with ATRA, we examined the phosphorylation of ERK1/2, JNK and Akt by Western blotting with phosphospecific antibodies. The results showed that ATRA markedly increased levels of phospho-JNK at 15 min after ATRA treatment. In contrast, ATRA did not affect the phosphorylation of ERK and Akt. To further identify whether JNK activation mediates HDAC2 phosphorylation induced by ATRA, VSMC were pretreated for 2 h with 20μM PD98059 (a specific inhibitor of ERK), 20μM LY294002 (a specific inhibitor of Akt) or 20μM SP600125 (a specific inhibitor of JNK), and then treated with 10μM ATRA for 1 h. Inhibition of JNK by SP600125 blocked the ATRA-induced phosphorylation of HDAC2, whereas PD98059 and LY294002 had no significant effect on HDAC2 phosphorylation induced by ATRA. After cells were transfected with JNK-specific siRNA, HDAC2 phosphorylation was no significant change even by ATRA stimulation. Together, these results suggest that JNK-, but not ERK- and Akt-dependent signal pathway, is involved in the ATRA-induced HDAC2 phosphorylation in VSMC, subsequently leading to the reduction in the interaction between KLF4 and HDAC2 in VSMC.2 The effects of HDAC2 phosphorylation on the deacetylation of KLF4 and DNA binding activityThe above results suggested that ATRA reduces the interaction between KLF4 and HDAC2 by inducing HDAC2 phosphorylation. To identify whether HDAC2 phosphorylation can regulate KLF4 modification, we detected the phosphorylation of HDAC2, acetylation of KLF4 and its DNA binding activity.2.1 ATRA induces KLF4 acetylationImmunoprecipitation assay and Western blotting assays showed that the acetylation of KLF4 was induced by ATRA in a time-dependent (0, 30, 60 min) manner. After cells were transfected with HDAC2-siRNA, ATRA did not acetylate KLF4 in VSMC. The results suggested that KLF4 acetylation induced by ATRA is dependent on HDAC2 phosphorylation.2.2 Phosphorylated HDAC2 dissociates from KLF4Compared with wild HDAC2, the interaction of HDAC2S424A with KLF4 in ATRA-induced VSMC is significantly increased. Dissociation of phosphorylated HDAC2 from KLF4 made its catalytic activity lose. Sequentially, acetylation of KLF4 increased.2.3 ATRA promotes nuclear export of phospho-HDAC2 by activating JNK-signaling pathwayImmunocytochemistry and cell immunofluorescence analysis indicated that majority of HDAC2 proteins were located in the nucleus, but located mostly in the cytoplasm in VSMC treated with ATRA for 40 min.The results suggested that HDAC2 phosphorylation may lead to HDAC2 nuclear export.2.4 KLF4 acetylation increases the binding of KLF4 to the promoter of VSMC differentiation marker genesTo identify that the expression of VSMC differentiation marker genes SM22αand SMα-actin were related with KLF4 acetylation, ChIP assays were performed. The results showed that when the acetylation of KLF4 reached a maximum after ATRA stimulation, DNA binding activity of KLF4 increased significantly; the interaction of KLF4 with SM22αand SMα-actin promoter was significantly inhibited by SP600125 pretreatment. The results suggested that KLF4 acetylation increases its DNA binding activity.3 The effects of KLF4 acetylation on espression of VSMC differentiation marker genesIn this part, we detected the effect of long-time stimulation of ATRA on HDAC2 phosphorylation, KLF4 acetylation and the expression of VSMC differentiation marker genes. The results were as follows:3.1 ATRA induces VSMC marker gene expressionAfter treatment by ATRA from 0 to 48 h, SM22αand SMα-actin (α-SMA) which are highly expressed in differentiated VSMC increased in a time-dependent manner, suggesting that long-time stimulation of ATRA can induce VSMC phenotypic switching by promoting VSMC marker gene expression.3.2 ATRA induces KLF4 and HDAC2 expression in VSMCWestern blot analysis showed that expression of KLF4 and HDAC2 was induced by ATRA in a time-dependent manner, especially 12 h and 48 h after ATRA treatment, suggesting that long-time stimulation of ATRA can induce VSMC differentiation marker gene expression by inducing the acetylation of KLF4.3.4 ATRA induces HDAC2 phosphorylation and KLF4 acetylationConsistent results were obtained in the model rats with the data in ATRA-induced VSMC.These evidences prove that ATRA can keep JNK pathway persistent activating and long-timely induce HDAC2 phosphorylation. On the other hand, ATRA could induce KLF4 expression in VSMC. Therefore, ATRA-induced VSMC marker gene expression is involved in KLF4 gene expression and its post-translational modification.4 ATRA inhibits neointimal formation induced by balloon injuryThe animal model was established through de-endothelializing by a Fogarty's tube. The effects of ATRA on neointimal formation, HDAC2 phosphorylation and KLF4 acetylation were evaluated in this part.4.1 ATRA inhibits neointimal formation induced by balloon injuryTo investigate the effect of ATRA on neointimal formation, the intima to media area (I/M) ratio was evaluated by morphometric analysis in each cross-section of arteries at 14 days after balloon injuries. The I/M ratio was 1.23±0.11 in ATRA group and 4.30±0.26 in injured group.The expression of PCNA was detected by immunohistochemical staining,and the results demonstrated that the level of PCNA in the ATRA group was lower than that in the injured group. Immunohistochemical staining showed that the number of PCNA-positive cells in the ATRA group was significantly decreased compared to the injured group. The results indicated that ATRA significantly inhibits neointimal hyperplasia.4.2 ATRA induce VSMC marker gene expression during the progress of neointimal formationWestern blot analysis showed that the level of SM22αand SMα-actin in the carotid arteries of ATRA group was much higher than that in the injured group. Immunohistochemical staining also showed that the number of SM22αand SMα-actin positive cells in ATRA group increased significantly, compared to the sham operated group. The results of immunohistochemistry staining were similar to that of Western blot, suggesting that ATRA induces the expression of differentiation marker genes in neointimal formation.4.3 ATRA induces HDAC2 phosphorylation by JNK-dependent pathway in neointimal formation induced by balloon injuryWestern blot analysis showed that compared with the sham operated group and the injured group, phospho-JNK in the ATRA group was significantly increased, whereas phospho-ERK or phospho-Akt had no valuable change. The results from animal model were similar to those from cultured VSMC in vitro.4.4 Dissociation of HDAC2 from KLF4 inhibits KLF4 deacetylationHDAC2 phosphorylation was induced in the ATRA group compared with the injured group. Simultaneously, the interaction between KLF4 and HDAC2 was reduced, leading to KLF4 acetylation at a relative high level.Conclusion:1 ATRA induces HDAC2 phosphorylation by activating JNK-dependent pathway. Dissociation of phospho-HDAC2 from KLF4 prevents KLF4 deacetylation.2 Phosphorylation of HDAC2 at serine residue leads to its nuclear export.3 Acetylation of KLF4 increases its binding activity to the promoter of VSMC differentiation marker genes.4 ATRA induces KLF4 expression in VSMC.5 ATRA inhibits neointimal formation inducing by balloon injury in rats via the JNK signaling.