Role Of KLF4 In ATRA-induced And PDGF-BB-repressed SM22α Expression In Vascular Smooth Muscle Cells

The phenotypic switching of vascular smooth muscle cells (VSMCs) plays a key role in the development of vascular remolding diseases, such as atherosclerosis and restenosis. Normally, VSMCs in mature animal vessels are mainly differentiated cells, also named as contractile phenotype, which express smooth muscle (SM)-specific contractile proteins, such as SMα-actin, SM-myosin heavy chain (SM-MHC), and SM22α. In response to vascular injury, VSMCs can de-differentiate from a contractile to a synthetic phenotype and then proliferate, subsequent repressing the SMC markers'expression. Despite the importance of VSMC phenotypic switching in the development of vascular diseases, the exact mechanisms controlling VSMC phenotype are not completely understood. Therefore, understanding of the molecular mechanisms regulating VSMC phenotype is helpful for the treatment of vascular remolding diseases.Smooth muscle 22 alpha (SM22α) is one of the markers of differentiated SMCs, the molecular weight of which is 22~25 kD. All-trans retinoic acid (ATRA), a derivative of vitamin A, can promote cell differentiation through binding to its intracellular receptor RAR/RXR. Platelet-derived growth factor (PDGF), a potent mitogenic agent, can promote cell proliferation through binding to its specific receptor PDGFR. Krüppel-like factors (KLFs) is a pleiotropic transcription factor characterized by zinc finger motifs, which play a key role in cell proliferation, apoptosis, differentiation, embryonic development and somatic cell reprogramming. Recent studies showed that KLF4, as a transcription factor, can regulate gene expression by directly binding to DNA element. We and Owens demonstrated that ATRA and PDGF-BB could induce KLF4 expression in VSMCs, and KLF4 plays a key role in ATRA-induced and PDGF-BB-repressed expression of SMC marker genes. However, the molecular mechanisms by which KLF4 plays different roles in ATRA- and PDGF-BB-dependent regulation of SMC marker genes are not fully understood.In this study we illustrate the function and molecular mechanism of KLF4 in ATRA-induced VSMC differentiation and PDGF-BB-induced VSMC proliferation.1 KLF4 mediates ATRA-induced VSMC differentiation and PDGF-BB-induced VSMC proliferationIn this study we used the differentiation and dedifferentiation marker genes to decide the phenotype of VSMCs, and examined the effect of ATRA and PDGF-BB on VSMC proliferation and phenotypic modulation. To further verify the role of KLF4 in ATRA-induced VSMC differentiation and PDGF-BB-induced VSMC proliferation, we infected VSMCs with pAd-KLF4 or transfected VSMCs with siRNA targeting KLF4 to overexpress or knockdown KLF4 expression. Results are as follows: 1.1 The effect of ATRA and PDGF-BB on VSMC proliferationThe results of cell number and BrdU incorporation assays showed that treatment of VSMCs with ATRA resulted in a significant inhibition of VSMC proliferation in a dose-dependent (5, 10, 20μM) and time-dependent (12, 24, 48 h) manner. A significant inhibitory effect was observed by treating VSMCs with 10μM ATRA for 48 h. Otherwise, treatment of VSMCs with PDGF-BB resulted in a significant induction of VSMC proliferation in a dose-dependent (5, 10, 20 ng/mL) and time-dependent (12, 24, 48 h) manner. A significant inducing effect was observed by treating VSMCs with 10 ng/mL PDGF-BB for 48 h.1.2 The effect of ATRA and PDGF-BB on the expression of KLF4 and SMC marker genesWestern blot results showed that treatment of VSMCs with ATRA and PDGF-BB significantly increased KLF4 expression in a time-dependent (12, 24, 48 h) and dose-dependent (5, 10, 20μM or 5, 10, 20 ng/mL) manner. SM22αis the differentiated marker gene of VSMCs, while PCNA (proliferating cell nuclear antigen) is the VSMC-dedifferentiated marker gene. Western blot results showed that treatment of VSMCs with ATRA significantly increased the expression of SM22αand decreased the protein level of PCNA. In contrast, PDGF-BB increased the level of PCNA and decreased the level of SM22α.1.3 The effect of knockdown or overexpression of KLF4 on VSMC marker gene expression and stress fiber formationVSMCs were transfected with siRNA targeting KLF4 to knock down KLF4 expression and assess its role in ATRA-induced VSMC differentiation and PDGF-BB-induced VSMC proliferation. First, we demonstrated that KLF4-siRNA knocked down about 75~80% of KLF4 expression induced by ATRA and PDGF-BB. For ATRA treatment, western blot and TRITC-phalloidin immunostaining showed that transfection with KLF4-siRNA reduced SM22αprotein level and the formation of stress fibers when compared with control siRNA. For PDGF-BB treatment, transfection with KLF4-siRNA increased SM22αprotein level and the formation of stress fibers.In addition, VSMCs were infected with pAd-KLF4 to observe the effect of KLF4 overexpression on SM22αexpression and stress fiber formation. Results showed that overexpression of KLF4 markedly increased the expression of SM22αand the formation of stress fibers.2 KLF4 regulates the SM22αpromoter activity in an acetylation/deacetylation-dependent mannerRecent studies have shown that the regulation of KLF4 activity can occur on multiple ways, including interaction with partners or post-translational modifications (e.g. acetylation, phosphorylation, sumoylation, et al). In this study we detected the effect of ATRA and PDGF-BB on KLF4 acetylation and studied the function of acetylated KLF4 in regulating SM22αexpression. 2.1 ATRA promotes and PDGF-BB represses the binding of KLF4 to the SM22αpromoter The SM22αpromoter region from position–676 to +1 contains a typical KLF4 binding site (CACCC) (termed'site 1') and its reverse orientation sequence (GTGGG) (termed'site 2'), the chromatin immunoprecipitation (ChIP) and oligonucleotide pull down assay showed that ATRA treatment increased the binding of KLF4 to the site 2, and PDGF-BB decreased the binding of KLF4 to the site 1.2.2 KLF4 regulates the SM22αpromoter activity through different binding sites in an acetylation-dependent mannerTo examine if the different binding of KLF4 to sites 1 and 2 was associated with its acetylation level, we detected the effect of ATRA and PDGF-BB on KLF4 acetylation. Western blot results showed that ATRA increased and PDGF-BB decreased KLF4 acetylation. Oligonucleotide pull down assay showed that KLF4 acetylation induced by ATRA increased its binding to site 2, whereas KLF4 deacetylation caused by PDGF-BB reduced its binding to site 1.2.3 KLF4 is acetylated by histone acetyltransferase p300Reporter gene assays showed that co-expression of KLF4 and p300 markedly activated the SM22αpromoter. The acetylation-deficient mutant K225/229R of KLF4 could not enhance SM22αpromoter activity irrespective of whether or not p300 was present. In vitro acetylation assay further confirmed that recombinant GST-KLF4 could be strongly acetylated by GST-p300-HAT (active domain of p300) in the presence of acetyl-CoA. Oligonucleotide pull down assay using purified GST-KLF4 showed that increased binding of acetylated KLF4 to each of the two sites after KLF4 was acetylated.The above results demonstrated that when VSMCs were treated with ATRA, a certain number of KLF4 molecules were acetylated, and the binding of acetylated KLF4 to site 2 was increased. In contrast, when VSMCs were treated with PDGF-BB, a certain number of KLF4 molecules were deacetylated, and deacetylated KLF4 was dissociated from site 1. 3 ATRA induces KLF4 acetylation via JNK and p38 pathways, whereas PDGF-BB induces KLF4 deacetylation via ERK and PI3K/Akt pathwaysTo further determine the molecular mechanism of KLF4-mediated SM22αexpression in the context of ATRA and PDGF-BB, we further investigated the effect of ATRA and PDGF-BB on JNK, p38, ERK and PI3K/Akt signaling pathways.3.1 ATRA induces KLF4 acetylation via JNK and p38 pathways, whereas PDGF-BB induces KLF4 deacetylation via ERK and PI3K/Akt pathwaysWhen VSMCs were treated with ATRA (10μM) or PDGF-BB (10 ng/mL) for 15, 30 and 60 min, ATRA increased and PDGF-BB decreased KLF4 acetylation in a time-dependent manner. For ATRA treatment, ATRA dramatically increased the level of phospho-JNK and phospho-p38 but decreased the level of phospho-ERK, and the level of phospho-Akt had no detectable change. For PDGF-BB treatment, PDGF-BB dramatically increased the level of phospho-JNK, phospho-p38, phospho-ERK and phospho-Akt.VSMCs were incubated with the JNK inhibitor SP600125, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, the ERK inhibitor PD98059, or the PI3K/Akt inhibitor LY294002 for 2 h before exposure to ATRA or PDGF-BB. Inhibition of JNK or p38 blocked the ATRA-induced KLF4 acetylation. Inhibition of ERK and PI3K/Akt blocked the PDGF-BB-induced KLF4 deacetylation, whereas JNK and p38 inhibition had no effect on the KLF4 deacetylation induced by PDGF-BB. To independently confirm these observations, cells were transfected with siRNA targeting JNK, p38, ERK, or Akt and then stimulated with ATRA or PDGF-BB. Under these conditions, acetylation of KLF4 by ATRA and KLF4 deacetylation by PDGF-BB were blocked, consistent with a decrease in the levels of phosphoactive forms of JNK, p38, ERK, and Akt.We carried out an oligonucleotide pull down assay to further confirm whether the acetylation of KLF4 affects its binding to the KLF4 binding sites 1 and 2 on the SM22αpromoter. Inhibition of the JNK or p38 by pharmacological inhibitors or the specific siRNA significantly decreased ATRA-induced KLF4 binding to site 2, but did not markedly affect KLF4 binding to site 1. Conversely, inhibition of the ERK or PI3K/Akt by pharmacological inhibitors or the specific siRNA significantly increased the binding of KLF4 to site 1, but had little effect on KLF4 binding to site 2.3.2 ATRA induces KLF4 phosphorylation and its interaction with p300 viaJNK and p38 pathways, PDGF-BB induces KLF4 dephosphorylation and its interaction with HDAC2 via ERK and PI3K/Akt pathways To understand whether ATRA- and PDGF-BB-induced KLF4 acetylation changes depend on KLF4 phosphorylation changes, we first identified that ATRA increased and PDGF-BB decreased KLF4 phosphorylation in a time-dependent manner. To further determine the signaling pathways leading to changes in the phosphorylation and acetylation of KLF4, we blocked the different signaling pathways by pharmacological inhibitors and RNA interference. Results showed that ATRA induced KLF4 phosphorylation via JNK and p38 pathways, and PDGF-BB induced KLF4 dephosphorylation via ERK and PI3K/Akt pathways.To understand whether KLF4 phosphorylation affects its interaction with p300 and HDAC2, we conducted co-immunoprecipitation assays. Results showed that inhibition of JNK and p38 abrogated ATRA-induced interaction of KLF4 with p300 and ATRA-repressed interaction of KLF4 with HDAC2, whereas inhibition of ERK and PI3K/Akt reversed PDGF-BB-repressed interaction of KLF4 with p300 and abolished PDGF-BB-induced interaction of KLF4 with HDAC2. Likewise, reciprocal immunoprecipitation with anti-p300 showed comparable changes in KLF4 co-sedimentation in response to ATRA or PDGF-BB. Correspondingly, siRNA-mediated knockdown of JNK, p38, ERK, or Akt was accompanied by marked changes in p300 or HDAC2 complexed with KLF4 when compared with the NS-siRNA.The above results suggest that ATRA promotes KLF4 interaction with p300 by inducing KLF4 phosphorylation via JNK and p38 pathways, whereas PDGF-BB promotes KLF4 interaction with HDAC2 by inducing KLF4 dephosphorylation via ERK and PI3K/Akt pathways. CONCLUSIONS1 KLF4 plays an important role in ATRA-induced and PDGF-BB-repressed SM22αexpression.2 KLF4 regulates SM22αpromoter activity in the context of ATRA and PDGF-BB through different binding sites in an acetylation-/deacetylation-dependent manner.3 ATRA induces KLF4 acetylation via JNK and p38 pathways, and promotes the binding of KLF4 to site 2 (-136~-132, GTGGG), subsequently activating the expression of SM22α; PDGF-BB induces KLF4 deacetylation via ERK and PI3K/Akt pathways, and inhibits the binding of KLF4 to site 1 (-263~-259, CACCC), subsequently repressing the expression of SM22α.4 ATRA induces KLF4 phosphorylation and its interaction with p300 via JNK and p38 pathways, PDGF-BB induces KLF4 dephosphorylation and its interaction with HDAC2 via ERK and PI3K/Akt pathways.