Krüppel-like Factor 4 Mediates Mitofusin-2 Gene Expression Induced By All-trans Retinoic Acid In Vascular Smooth Muscle Cells

Proliferative vascular diseases are multiple gene disease caused by interaction of environmental and genetic factors. Vasoactive peptides and their receptors, growth factors and cytokines as well as their receptors, cell signal transduction proteins and cyclins are all involved in the occurrence of these diseases. All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to regulate growth induce differentiation in multiple cells in direct or indirect manner.The Krüppel-like factor 4 (KLF4/GKLF) is a pleiotropic zinc finger transcription factor that regulates genes being involved in both the regulation of cell growth, proliferation, differentiation and apoptosis in several tissues. In vascular smooth muscle cells (VSMCs), KLF4 can function as an anti-proliferation factor or a prodifferentiation factor depending on the interaction partner. We have recently demonstrated that ATRA induced the expression of KLF4 and mitofusin 2 (mfn-2), a member of the mitofusin family, in VSMCs. However, the role of KLF4 in the regulation of mfn-2 expression has not been well characterized.Mfn-2 (also named hyperplasia suppressor gene [HSG]) is a mitochondrial fusion protein that is expressed mainly in tissues with high energetic requirements, such as skeletal muscle and heart. Several lines of evidence indicate that mfn-2 modulates mitochondrial metabolism by regulating mitochondrial membrane potential, fuel oxidation, and the oxidative phosphorylation system. In addition, recent study has shown that mfn-2 exhibits a profound anti-proliferative effect on VSMCs in vitro and in vivo, and promotes VSMC and cardiomyocyte apoptosis. Recent evidence also shows that mfn-2 inhibits tumor cell proliferation and increases their sensitivity to chemotherapy and radiotherapy even above p53. Thus, it is important to identify the molecular mechanisms underlying the activation or repression of mfn-2 expression in order to have a better understanding of its biological functions. Based on the experimental facts that mfn-2 and KLF4 showed a similar expression pattern and that KLF4 is a pleiotropic transcription factor that participates in a wide range of biological functions, we speculated that KLF4 might be a key effector of ATRA-induced mfn-2 expression in VSMCs.Here, we studied the mechanism by which KLF4 mediates ATRA-induced mfn-2 expression in VSMCs. We show that KLF4 binds directly to the mfn-2 promoter and activates its transcription. Further, we demonstrate that ATRA increases the interaction of KLF4 with p300 by inducing KLF4 phosphorylation, and that the activation of JNK and p38 MAPK signaling by ATRA is essential for KLF4 phosphorylation and its interaction with p300. KLF4 acetylation by p300 increases its activity to transactivate the mfn-2 promoter. In further study, we also demonstrate that RARαis involved in KLF4-mediated transactivation of mfn-2 promoter by binding to KLF4 in ATRA-induced VSMCs.1. KLF4 mediates ATRA-induced mfn-2 gene expression in VSMCsATRA is known to induce the expression of KLF4 and mfn-2, however, direct relationship between KLF4 and mfn-2 has not yet been characterized. We sought to determine whether KLF4 is responsible for the mfn-2 expression and function in VSMCs treated by ATRA. The results were as follows:1.1 KLF4 mediates ATRA-induced mfn-2 expression in VSMCsThe results of Western blot and RT-PCR assays showed that treatment of VSMCs with ATRA resulted in upregulation of KLF4 and mfn-2 in a time-dependent manner. A significant change was observed at concentration of 10μM for 24 h.To further determine whether ATRA-induced mfn-2 gene expression was due to the increased expression of KLF4, we transfected VSMCs with KLF4 expression vector (pAd-KLF4) and KLF4-siRNA, the result of Western blotting and RT-PCR assays showed that overexpression of KLF4 resulted in a time-dependent increase in protein and mRNA levels of mfn-2. In contrast, when VSMCs were transfected with rat KLF4-specific siRNA (KLF4-siRNA) to block the endogenous KLF4 expression induced by ATRA, ATRA-elicited increase in mfn-2 expression significantly reduced. 1.2 KLF4 binds directly to the mfn-2 promoter and regulates its transcriptionTo identify whether KLF4 activates the mfn-2 promoter, A293 cells were transiently co-transfected with the mfn-2 promoter (pGL3-mfn-2-luc) and KLF4 expression plasmid (GFP-KLF4). Reporter gene assays showed that KLF4 overexpression significantly increased the mfn-2 promoter activity in a dose-dependent fashion.Using the TESS computational program, we established that the -1333/+15 bp region of the mfn-2 promoter contains four typical KLF4-binding sites (CACCC/GTGGG). To identify the KLF4-responsive elements involved in KLF4-induced mfn-2 expression, progressive 5' deletion constructs of the mfn-2 promoter fused to the luciferase reporter gene were generated and transiently transfected into A293 cells with KLF4. Measurements of luciferase activity suggested that the region between -441 and -273 is critical for conferring the capacity to respond to KLF4.To further evaluate which site binds KLF4 and activates the promoter, the four boxes (–1152GTGGG–1148,–840CACCC–836,–356CACCC–352, and–122GTGGG–118) were mutated, respectively, and functional analysis was performed in A293 cells. The result showed that the inactivation of the box 3 completely blocked the response to KLF4, indicating that box 3 element plays a critical role in the KLF4-mediated transcriptional activation of mfn-2.To investigate whether KLF4 binds to the mfn-2 promoter within intact chromatin, ChIP assays were performed. An increased binding of KLF4 to the mfn-2 promoter was observed in KLF4-overexpressing VSMCs.1.3 KLF4 mediates ATRA-induced perinuclear mitochondrial clustering and mitochondrial depolarizationTo examine the effect of upregulation of KLF4 and mfn-2 expression induced by ATRA on mitochondrial dynamics in VSMCs, fluorescent microscopy and electron microscopy were done. The perinuclear mitochondrial clustering was observed in KLF4-overexpressing and ATRA-treated VSMCs. In fluorescent microscopy, when VSMCs were transfected with KLF4-specific siRNA (siRNA-KLF4) to block the endogenous KLF4 expression induced by ATRA, we could not observe perinuclear mitochondrial clustering anymore. To further evaluate whether mitochondrial membrane potential (MMP) was also altered in KLF4-overexpressing cells or in ATRA-treated cells and whether some of the effects of KLF4 on the membrane potential depended on mfn-2, we stained the VSMCs with JC-1. The results suggested that KLF4 overexpression induced mitochondrial perinuclear clustering, and decreased the mitochondrial membrane potential, while transfecting cells with siRNA-mfn-2 abrogated the effect of KLF4 on mitochondrial membrane potential. As expected, these changes were also reflected in the MMP as measured by flow cytometer. Thus, we speculate that KLF4-induced decrease in the mitochondrial membrane potential of VSMCs is at least in part through the up-regulation of mfn-2.These results suggest that the mfn-2 promoter is a direct transcriptional target for KLF4, while KLF4 mediates ATRA-induced mfn-2 expression and plays a role in ATRA signaling and function in VSMCs.2. ATRA promotes the activation of the mfn-2 promoter by KLF4 via inducing KLF4 acetylationLike other zinc finger-containing proteins, the KLF activity may be regulated not only by de novo synthesis but also by protein-protein interactions and posttranslational modification such as phosphorylation, acetylation, ubiquitination or SUMOylation. In this part, we investigated the modification of KLF4 in ATRA-induced VSMCs and the roles of KLF4 modification in activation of the mfn-2 promoter.2.1 ATRA induces acetylation of KLF4Because KLF4 overexpression and ATRA treatment induce the mfn-2 expression, we sought to determine the mechanism whereby KLF4 regulates mfn-2 gene expression in ATRA-treated VSMCs. Western blot assays showed that KLF4 overexpression increased mfn-2 expression, while ATRA treatment increased the inductive effect of KLF4 on mfn-2 expression. To test whether KLF4 binds to the mfn-2 promoter after ATRA stimulation, we performed ChIP assays using primers specific for the mfn-2 promoter containing the putative KLF4-binding site 3. Upon ATRA stimulation, KLF4 binding to DNA was increased.To further demonstrate the transcriptional activation of mfn-2 by KLF4, we overexpressed or knocked down KLF4 and detected the effect of ATRA on the mfn-2 promoter. Reporter gene assays showed that knockdown of KLF4 abrogated the effect of ATRA on the promoter, while ATRA further induced the mfn-2 promoter activity by KLF4 overexpression in A293 cells.To explore the molecular mechanism of ATRA-increased inductive effect of KLF4 on mfn-2 gene expression, the level of acetylated-KLF4 was detected firstly. CoIP assay showed that ATRA time-dependently enhanced the acetylation level of KLF4 within 24 h. To further understand whether acetylation of KLF4 is related to p300, effect of ATRA on p300 subcellular localization in VSMCs was observed. p300 translocated from the cytoplasm into the nucleus after ATRA stimulation for 1 hour. Effects on the interaction between KLF4 and p300 after p300 nuclear import were further investigated by CoIP assay, suggesting that ATRA promoted the interaction of KLF4 with p300.To further determine the effect of KLF4 acetylation on the mfn-2 promoter activation, the luciferase activity was detected. The results showed that the coexpression of KLF4 with p300 increased the mfn-2 promoter activity than overexpression of KLF4 alone. When A293 cells were co-transfected with KLF4 and HDAC2, the mfn-2 promoter activity was reduced to 10% of that transfected with KLF4 alone. Meanwhile, when acetylation-deficient mutant of KLF4 (K225/229R) and p300 were co-transfected, the promoter activity could not be enhanced, indicating that KLF4 acetylation by p300 is required for the activation of the mfn-2 promoter.2.2 ATRA promotes the interaction between KLF4 and p300 by inducing KLF4 phosphorylation via JNK and p38 signalingTo understand whether ATRA-induced interaction between KLF4 and p300 depends on KLF4 phosphorylation, we first detected the levels of phospho-KLF4 in VSMCs treated with ATRA. CoIP assay showed that ATRA stimulation rapidly induced the phosphorylation of KLF4 within 0.5 h. KLF4 phosphorylation reached a maximum at 1 h and remained constant for at least 2 h. Under these experimental conditions, the phosphorylation of JNK and p38 MAPK increased concurrently in a time-dependent manner, and the phosphorylation of Akt and ERK was not changed following ATRA treatment.To further determine whether JNK and p38 MAPK signaling mediate phosphorylation of KLF4 induced by ATRA, we performed the inhibitor studies on KLF4 phosphorylation. VSMCs treated with either JNK inhibitor SP600125 or p38 MAPK inhibitor SB203580 blocked ATRA-induced KLF4 phosphorylation, whereas pretreatment with ERK inhibitor PD98059 and Akt inhibitor LY294002, did not affect the KLF4 phosphorylation.Next, we investigated whether KLF4 phosphorylation affects its interaction with p300. The effects of JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 on ATRA-induced KLF4 phosphorylation and ATRA-increased interaction between KLF4 and p300 were detected. The results suggest that ATRA-increased interaction between KLF4 and p300 was reduced to the level of the control by treating VSMCs with SP600125 or SB203580 rather than PD98059 or LY294002. To further demonstrate that the phosphorylation of KLF4 is important for its interaction with p300, we mutated three phosphorylation sites (S415A, S444A, and S470A), and found that both KLF4 phosphorylation and the interaction of KLF4 with p300 induced by ATRA was reduced when the Ser470 was mutated to alanine.To further understand whether KLF4 acetylation depends on its phosphorylation and its interaction with p300, ATRA-induced KLF4 acetylation in VSMCs treated with SP600125 and SB203580 or in VSMCs transfected with the mutant of phosphorylation site (Ser470) was detected. The results showed that inhibiting JNK and p38 activation by their respective inhibitors or transfecting with the mutant of phosphorylation site (Ser470) abrogated ATRA-induced KLF4 acetylation.To evaluate the effect of the KLF4 phosphorylation on ATRA-induced mfn-2 promoter activity, luciferase activity was detected. The results showed that the coexpression of the S470A mutant of KLF4 with p300 could not activate the mfn-2 promoter as compared with the coexpression of the wild-type KLF4 with p300. Similarly, ATRA-elicited increase in the mfn-2 promoter activity was abrogated by treatment with SP600125 and SB203580 or by transfecting the S470A mutant.These results further suggest that ATRA stimulates the interaction of KLF4 with p300 by inducing KLF4 phosphorylation, which in turn increases KLF4 acetylation and its transactivation to the mfn-2 promoter.3. RARαenhances activation of the mfn-2 promoter by KLF4 via binding to KLF4 in ATRA-induced VSMCsThe above study shows that KLF4 mediates mfn-2 expression in ATRA-stimulated VSMCs. However, it remains unclear whether RARs are involved in KLF4-mediated mfn-2 expression in ATRA-stimulated VSMCs. In this part, we determined the role of RARs in activation of the mfn-2 promoter by KLF4.3.1 ATRA induces the expression of RARαand RARβin VSMCsIt is not yet clear whether retinoid receptors (RARs) are expressed in VSMCs. Thus, as a first step toward understanding the potential role of RARs in KLF4-mediated mfn-2 expression, we examined the effect of ATRA on expression of RARs. Western blot and RT-PCR analysis showed that ATRA time- and dose- dependently induced the expression of RARα, and slightly up-regulated the expression of RARβ, and had little or no effect on RARγexpression.3.2 RARαis involved in ATRA-induced KLF4 acetylationThere is no RAR-binding site in the mfn-2 promoter region. Thus, we think that mfn-2 expression is indirectly regulated by RARs. To dissect the contribution of RARs to KLF4 activity regulation in VSMCs, we treated VSMCs with RARαantagonist (Ro 41-5253), RARβantagonist (LE135) or transfected VSMCs with RARs-specific siRNA and determined the effects of RARs on KLF4 phosphorylation in ATRA-stimulated VSMCs. The results indicated that knockdown of RARs did not affect the KLF4 phosphorylation. To further understand whether the levels of acetylated-KLF4 were changed when RARs were knocked down with their siRNA, Western blot was performed, and the result revealed that silencing of RARα, but not RARβand RARγ, inhibited the KLF4 acetylation.To further investigate whether RARs affect KLF4 acetylation in ATRA-stimulated VSMCs, we performed a co-immunoprecipitation (Co-IP) assay. The results showed that the precipitates pulled down with KLF4 antibody, RARαwas increased, RARβwas unchanged, and RARγwas undetectable.3.3 RARαpromotes activation of the mfn-2 promoter by KLF4The functional interaction between KLF4 and RARαin the activation of the mfn-2 promoter by KLF4 was further determined by reporter gene assays. The results showed that the mfn-2 promoter activity induced by KLF4 overexpression was further enhanced after cotransfection of KLF4 with RARα. Upon ATRA treatment, the mfn-2 promoter activity was further induced. However, the mfn-2 promoter activity kept at the baseline when cells were transfected with RARαalone, regardless of with or without ATRA treatment.3.4 Interaction of KLF4 with RARαis dependent on KLF4 phosphorylationBecause we have found that the phosphorylation of KLF4 induced by ATRA was independent on RARαexpression, thus we wanted to demonstrate whether KLF4 phosphorylation affects its interaction with RARα. Crossing Co-IP assay indicated that ATRA-increased interaction between KLF4 and RARαwas reduced to the level of the control by treating VSMCs with SP600125 or SB203580 and by transfecting the mutant of KLF4 phosphorylation site (Ser470).3.5 Knockdown of RARαdecreases the interaction of KLF4 with p300Since ATRA increased KLF4 acetylation by increasing the interaction of KLF4 with p300, the role of RARαin the interaction of KLF4 with p300 needs to be further detected. The result showed that the knockdown of RARαresulted in an obvious reduction in the interaction of KLF4 with p300.These results showed that the acetylation rather than the phosphorylation of KLF4 is dependent on the RARαexpression. Moreover, ATRA increases the interaction of phospho-KLF4 with RARαand their interaction promotes the binding of KLF4 to the mfn-2 promoter.CONCLUSIONS1 KLF4 mediates not only ATRA-induced mfn-2 expression in VSMCs by binding directly to the mfn-2 promoter but also ATRA-induced perinuclear mitochondrial clustering and mitochondrial depolarization.2 ATRA promotes the activation of the mfn-2 promoter by KLF4 via inducing acetylation of KLF4 by p300. KLF4 acetylation by p300 increases its activity to transactivate the mfn-2 promoter.3 ATRA increases the interaction of KLF4 with p300 by inducing KLF4 phosphorylation, and the activation of JNK and p38 MAPK signaling by ATRA is essential for KLF4 phosphorylation and its interaction with p300.4 RARαpromotes the activation of the mfn-2 promoter by KLF4 via increasing the interaction of phospho-KLF4 with p300.