A potential role for nuclear factor of activated T-cells in receptor tyrosine kinase and G-protein-coupled receptor agonist-induced cell proliferation.
ABSTRACT: We have studied the role of nuclear factor of activated T-cells (NFAT) transcription factors in the induction of vascular smooth muscle cell (VSMC) growth by platelet-derived growth factor-BB (PDGF-BB) and thrombin, the receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) agonists, respectively. NFATc1 but not NFATc2 or NFATc3 was translocated from the cytoplasm to the nucleus upon treatment of VSMCs with PDGF-BB or thrombin. Translocation of NFATc1 was followed by an increase in NFAT-DNA binding activity and NFAT-dependent reporter gene expression. Cyclosporin A (CsA), a potent and specific inhibitor of calcineurin, a calcium/calmodulin-dependent serine phosphatase involved in the dephosphorylation and activation of NFATs, blocked NFAT-DNA binding activity and NFAT-dependent reporter gene expression induced by PDGF-BB and thrombin. CsA also completely inhibited PDGF-BB- and thrombin-induced VSMC growth, as measured by DNA synthesis and cell number. In addition, forced expression of the NFAT-competing peptide VIVIT for calcineurin binding significantly attenuated the DNA synthesis induced by PDGF-BB and thrombin in VSMCs. Together, these findings for the first time demonstrate a role for NFATs in RTK and GPCR agonist-induced growth in VSMCs.
Project description:Platelet-derived growth factor BB (PDGF-BB) induced cyclin A expression and CDK2 activity in vascular smooth muscle cells (VSMC). Inhibition of nuclear factors of activated T cell (NFAT) activation by cyclosporin A (CsA) and VIVIT suppressed PDGF-BB-induced cyclin A expression and CDK2 activity, resulting in blockade of VSMC in the G(1) phase. In addition, CsA- and VIVIT-mediated inhibition of NFATs and small interfering RNA-targeted down-regulation of cyclin A levels suppressed PDGF-BB-induced VSMC DNA synthesis. PDGF-BB also induced cyclin A mRNA levels in VSMC in an NFAT-dependent manner. Cloning and bioinformatic analysis of rat cyclin A promoter revealed the presence of NFAT-binding elements, and PDGF-BB induced the binding of NFATs to these regulatory sequences in a CsA- and VIVIT-sensitive manner. Chromatin immunoprecipitation analysis showed that NFATc1 binds to the cyclin A promoter in response to PDGF-BB in a VIVIT-sensitive manner. Furthermore, PDGF-BB induced cyclin A promoter-luciferase reporter gene activity in VSMC, and it was inhibited by both CsA and VIVIT. Balloon injury induced cyclin A expression and CDK2 activity in rat carotid arteries, and these responses were also blocked by VIVIT. In addition, VIVIT attenuated balloon injury-induced SMC proliferation, resulting in reduced restenosis. Down-regulation of NFATc1 by its small interfering RNA inhibited PDGF-BB-induced cyclin A expression and DNA synthesis both in rat and human VSMC. Together, these findings demonstrate that the cyclin A-CDK2 complex may be a potential effector of NFATs, specifically NFATc1, in mediating SMC multiplication leading to neointima formation. Therefore, NFATs may be used as target molecules for the development of therapeutic agents against vascular diseases such as restenosis.
Project description:AIM: Catecholamine-induced vascular smooth muscle cell (VSMC) proliferation is one of the major events in the pathogenesis of atherosclerosis and vascular remodeling. The calcineurin-NFAT pathway plays a role in regulating growth and differentiation in various cell types. We investigated whether the calcineurin-NFAT pathway was involved in the regulation of phenylephrine-induced VSMC proliferation. METHODS: Proliferation of VSMC was measured using an MTT assay and cell counts. Localization of NFATc1 was detected by immunofluorescence staining. NFATc1-DNA binding was determined by EMSA and luciferase activity analyses. NFATc1 and calcineurin levels were assayed by immunoprecipitation. RESULTS: Phenylephrine (PE, an alpha(1)-adrenoceptor agonist) increased VSMC proliferation and cell number. Prazosin (an alpha(1)-adrenoceptor antagonist), cyclosporin A (CsA, an inhibitor of calcineurin) and chelerythrine (an inhibitor of PKC) decreased PE-induced proliferation and cell number. Additional treatment of VSMC with CsA or chelerythrine further inhibited proliferation and cell number in the chelerythrine-pretreatment group and the CsA-pretreatment group. CsA and chelerythrine alone had no effect on either absorbance or cell number. CsA decreased PE-induced calcineurin levels and activity. NFATc1 was translocated from the cytoplasm to the nucleus upon treatment with PE. This translocation was reversed by CsA. CsA decreased the PE-induced NFATc1 level in the nucleus. PE increased NFAT's DNA binding activity and NFAT-dependent reporter gene expression. CsA blocked these effects. CONCLUSION: CsA partially suppresses PE-induced VSMC proliferation by inhibiting calcineurin activity and NFATc1 nuclear translocation. The calcineurin-NFATc1 pathway is involved in the hyperplastic growth of VSMC induced by phenylephrine.
Project description:Platelet-derived growth factor BB induced cyclin D1 expression in a time- and nuclear factor of activated T cells (NFAT)-dependent manner in human aortic smooth muscle cells (HASMCs), and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G(1) to S phase. Selective inhibition of NFATc1 by its small interfering RNA also blocked HASMC proliferation and migration. Characterization of the cyclin D1 promoter revealed the presence of several NFAT binding sites, and the site at nucleotide -1333 was found to be sufficient in mediating platelet-derived growth factor BB-induced cyclin D1 promoter-luciferase reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in an NFATc1-dependent manner. Balloon injury-induced cyclin D1-CDK4 activity requires NFAT activation, and adenovirus-mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury-induced vascular wall remodeling events, including smooth muscle cell migration from the medial to luminal region, their proliferation in the intimal region, and neointima formation. Together, these results provide more mechanistic evidence for the role of NFATs, particularly NFATc1, in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the renarrowing of artery after angioplasty.
Project description:Phenotypic switch of vascular smooth muscle cells (VSMCs) is characterized by increased expressions of VSMC synthetic markers and decreased levels of VSMC contractile markers, which is an important step for VSMC proliferation and migration during the development and progression of cardiovascular diseases including atherosclerosis. Chicoric acid (CA) is identified to exert powerful cardiovascular protective effects. However, little is known about the effects of CA on VSMC biology. Herein, in cultured VSMCs, we showed that pretreatment with CA dose-dependently suppressed platelet-derived growth factor type BB (PDGF-BB)-induced VSMC phenotypic alteration, proliferation and migration. Mechanistically, PDGF-BB-treated VSMCs exhibited higher mammalian target of rapamycin (mTOR) and P70S6K phosphorylation, which was attenuated by CA pretreatment, diphenyleneiodonium chloride (DPI), reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) and nuclear factor-?B (NF?B) inhibitor Bay117082. PDGF-BB-triggered ROS production and p65-NF?B activation were inhibited by CA. In addition, both NAC and DPI abolished PDGF-BB-evoked p65-NF?B nuclear translocation, phosphorylation and degradation of Inhibitor ?B? (I?B?). Of note, blockade of ROS/NF?B/mTOR/P70S6K signaling cascade prevented PDGF-BB-evoked VSMC phenotypic transformation, proliferation and migration. CA treatment prevented intimal hyperplasia and vascular remodeling in rat models of carotid artery ligation in vivo. These results suggest that CA impedes PDGF-BB-induced VSMC phenotypic switching, proliferation, migration and neointima formation via inhibition of ROS/NF?B/mTOR/P70S6K signaling cascade.
Project description:Excessive migration of vascular smooth muscle cells (VSMCs) after vascular injury contributes to the development of occlusive vascular disease. Inhibition of VSMC migration is a validated therapeutic modality for occlusive vascular diseases, such as atherosclerosis and restenosis. We investigated the inhibitory effect of chebulinic acid (CBA) on cell migration and matrix metalloproteinase (MMP)-2 activation in platelet-derived growth factor (PDGF)-BB-induced mouse and human VSMCs. CBA significantly inhibited PDGF-BB-induced migration in mouse and human VSMCs, without inducing cell death. Additionally, CBA significantly blocked PDGF-BB-induced phosphorylation of the PDGF receptor (PDGF-R), Akt, and extracellular signal-regulated kinase (ERK)1/2 by inhibiting the activation of the PDGF-BB signalling pathway. In both mouse and human VSMCs, CBA inhibited PDGF-induced MMP-2 mRNA and protein expression as well as the proteolytic activity of MMP-2. Moreover, CBA suppressed sprout outgrowth formation of VSMCs from endothelium-removed aortic rings as well as neointima formation following rat carotid balloon injury. Taken together, our findings indicated that CBA inhibits VSMC migration by decreasing MMP-2 expression through PDGF-R and the ERK1/2 and Akt pathways. Our data may improve the understanding of the antiatherogenic effects of CBA in VSMCs.
Project description:The proliferation and migration of vascular smooth muscle cells (VSMCs) are essential in the pathogenesis of various vascular diseases, such as atherosclerosis and restenosis. Among the mediators of VSMC during atherosclerosis development, platelet-derived growth factor (PDGF)-BB is a potent mitogen for VSMCs and greatly contributes to the intimal accumulation of VSMCs. <i>Glossogyne tenuifolia</i> (GT, Xiang-Ru) is a traditional antipyretic and hepatoprotective herb from Penghu Island, Taiwan. This study evaluated the inhibitory effect of GT ethanol extract (GTE) and GT water extract (GTW) on proliferative and migratory activities in PDGF-BB-induced VSMCs. The experimental results demonstrated that GTE significantly inhibited the PDGF-BB-stimulated VSMC proliferation and migration, as shown by MTT, wound healing, and Boyden chamber assays. GTE was found to have a much more potent inhibitory activity than GTW. Based on the Western blot analysis, GTE significantly blocked the PDGF-BB-induced phosphorylation of NF-?B and mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-regulated kinase (ERK), p38, and JNK, in VSMCs. In addition, GTE retarded the PDGF-BB-mediated migration through the suppression of matrix metalloproteinase (MMP)-2 and MMP-9 expression in VSMCs. Three main ingredients of GT-chlorogenic acid, luteolin-7-glucoside, and luteolin-all showed significant anti-proliferative effects on PDGF-BB-induced VSMCs. As a whole, our findings indicated that GTE has the potential to be a therapeutic agent to prevent or treat restenosis or atherosclerosis.
Project description:BACKGROUND AND PURPOSE:The increased proliferation and migration of vascular smooth muscle cells (VSMCs) after arterial injury contributes greatly to the pathogenesis of neointimal hyperplasia. As a major component of epigenetics, histone methylation plays an important role in several cardiovascular diseases. However, its role in restenosis is still unclear. EXPERIMENTAL APPROACH:Human aortic VSMCs were challenged with PDGF-BB, and total histones were extracted and analysed by HPLC/MS. For the in vivo study, rats were subjected to wire-guided common carotid injury. KEY RESULTS:PDGF-BB markedly increased the H3K27me3 level, as demonstrated by use of HPLC/MS and confirmed by western blot analysis. Enhancer of zeste homologue 2 (EZH2), the histone H3K27 methyltransferase component of polycomb repressive complex 2, was also up-regulated by PDGF-BB in VSMCs, and in the neointimal hyperplasia induced by wire injury of the rat carotid artery. Furthermore, inhibiting H3K27me3 by treatment with 3-?M UNC1999, an EZH2/1 inhibitor, significantly suppressed PDGF-BB-induced VSMC proliferation compared with the PDGF-BB-treated group. Consistently, neointimal formation was significantly attenuated by oral or perivascular administration of UNC1999 compared with the sham group. Mechanistically, the increase in H3K27me3 inhibited the transcription of the cyclin-dependent kinase inhibitor p16INK4A and thus promoted VSMC proliferation. CONCLUSIONS AND IMPLICATIONS:Vascular injury elevated the expression of EZH2 and the downstream target H3K27me3, which suppressed p16INK4A expression in VSMCs and promoted VSMC proliferation and neointimal hyperplasia. EZH2 inhibition might be a potential therapeutic target for restenosis.
Project description:The phenotypic switch of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of various vascular diseases, such as atherosclerosis and post-angioplasty restenosis. Small non-coding microRNAs (miRNAs) have emerged as critical modulators of VSMC function. In the present study, miR-26a was significantly increased in cultured VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB) and in arteries with neointimal lesion formation. Moreover, we demonstrated that miR-26a regulates the expression of VSMC differentiation marker genes such as ?-smooth muscle actin (?-SMA), calponin and smooth muscle myosin heavy chain (SM-MHC) in PDGF-BB-treated VSMCs. We further confirmed that the regulatory effect of miR-26a during the phenotypic transition occurs through its target gene Smad1, which is a critical mediator of the pro-contractile signal transmitted by bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF-?). This discovery proposed a new channel for communication between PDGF and the BMP/TGF-? family. We concluded that miR-26a is an important regulator in the PDGF-BB-mediated VSMC phenotypic transition by targeting Smad1. Interventions aimed at miR-26a may be promising in treating numerous proliferative vascular disorders.
Project description:Vascular remodeling diseases (VRD) are mainly characterized by inflammation and a vascular smooth muscle cells (VSMCs) proproliferative and anti-apoptotic phenotype. Recently, the activation of the advanced glycation endproducts receptor (RAGE) has been shown to promote VSMC proliferation and resistance to apoptosis in VRD in a signal transducer and activator of transcription (STAT)3-dependant manner. Interestingly, we previously described in both cancer and VRD that the sustainability of this proproliferative and antiapoptotic phenotype requires activation of the transcription factor NFAT (nuclear factor of activated T-cells). In cancer, NFAT activation is dependent of the oncoprotein provirus integration site for Moloney murine leukemia virus (Pim1), which is regulated by STAT3 and activated in VRD. Therefore, we hypothesized that RAGE/STAT3 activation in VSMC activates Pim1, promoting NFAT and thus VSMC proliferation and resistance to apoptosis. Methods/Results- In vitro, freshly isolated human carotid VSMCs exposed to RAGE activator N?-(carboxymethyl)lysine (CML) for 48 hours had (1) activated STAT3 (increased P-STAT3/STAT3 ratio and P-STAT3 nuclear translocation); (2) increased STAT3-dependent Pim1 expression resulting in NFATc1 activation; and (3) increased Pim1/NFAT-dependent VSMC proliferation (PCNA, Ki67) and resistance to mitochondrial-dependent apoptosis (TMRM, Annexin V, TUNEL). Similarly to RAGE inhibition (small interfering RNA [siRNA]), Pim1, STAT3 and NFATc1 inhibition (siRNA) reversed these abnormalities in human carotid VSMC. Moreover, carotid artery VSMCs isolated from Pim1 knockout mice were resistant to CML-induced VSMC proliferation and resistance to apoptosis. In vivo, RAGE inhibition decreases STAT3/Pim1/NFAT activation, reversing vascular remodeling in the rat carotid artery-injured model.RAGE activation accounts for many features of VRD including VSMC proliferation and resistance to apoptosis by the activation of STAT3/Pim1/NFAT axis. Molecules aimed to inhibit RAGE could be of a great therapeutic interest for the treatment of VRD.
Project description:We applied the transcriptome profiling (RNA-seq) for high-throughput profiling of genes changes in VSMC dedifferentiation. Rat primary VSMCs were divided into 3 groups, control, PDGF-BB, PDGF-BB+PJ34,and mRNA sequence were performed. We found that PDGF-BB could upregualted the genes involved in cell proliferation and migration, and downregulated the VSMC contractile genes, all of which could be reversed by PARP inhibitor PJ34. Then we knockdowned the co-factor Myocardin in VSMCs, and found the above effects of PJ34 were nearly abolished.Our study first provided the transcription changes by RNA-seq in VSMC dedifferentiation, and demonstrated the key roles of PARP1 and the PARylation process in VSMC phenotypic switch. Overall design: Eximination of genes changes in VSMC dedifferentiation