The Role And Mechanism Of Am80in Regulation Of Apelin Gene Expression

Objective: Apelin is a bioactive peptide with very important pathologicaland physiological functions by binding and activating its specific receptor APJ(angiotensin II receptor-like1). Apelin and APJ are expressed in thecardiovascular system, including vascular endothelial cells (ECs) and vascularsmooth muscle cells (VSMCs). Apelin regulates vascular tone in vivo, causinga decrease in blood pressure or vasodilation of resistance vessles. In vitro,apelin causes vasodilation of human vessels largely via acting on apelinreceptors on the endothelium in a nitric oxide-dependent manner. Apelin alsoleads to vasoconstriction of human vessels in vitro by a direct action onVSMCs. Apelin induces an increased myocardial contractility by exerting apositive inotropic effect in rats and mice in vivo. In vitro studies indicated thatapelin is a potent positive inotropic agent by a direct action on cardiac tissuein rat and human. In addition, apelin is a potent angiogenic factor and mitogenof ECs and VSMCs. Apelin and its receptor are required for normalcardiovascular development. Abnormalities in apelin-APJ signaling may beinvolved in the pathogenesis of hypertension and its complications such asheart failure. Apelin expression is induced by hypoxia and angiopoietin-I inECs, VSMCs and hearts. However, the molecular mechanisms influencingapelin expression are still largely unknown in the cardiovascular system.Krüppel-like factor5(KLF5), a zinc finger-containing transcription factor,is a target for angiotensin II signaling and an essential regulator ofcardiovascular remodeling. KLF5plays an important role in the control ofVSMC phenotype after vascular injury. Importantly, KLF5can interact withmany other transcription factors, such as c-Jun, RARα, CREB binding protein(CBP) and peroxisome proliferators-activated receptor-δ(PPAR-δ), andregulates the expression of many genes involved in cell proliferation, differentiation, angiogenesis and carcinogenesis. In the vasculature, KLF5expression is up-regulated in VSMCs after vascular injury and can activatemany genes inducible during cardiovascular remodeling, such asplatelet-derived growth factorA/B (PDGF-A/B), early growth responsegene-1(Egr-1), plasminogen activator inhibitor-1(PAI-1), and vascularendothelial growth factor (VEGF) receptors. We found that KLF5forms acomplex with HDAC2and RARα at the p21promoter to inhibit its expressionunder basal conditions. Am80treatment induces KLF5deacetylation anddissociation from the p21promoter, losing its inhibitory effect on p21promoter and leading to the activation of p21expression.Am80, a synthetic retinoid, is a retinoic acid receptor alpha(RARα)-specific agonist that has been safely used to treat acute promyelocyticleukemia. RARα is a member of the nuclear receptor superfamily, and whenbound by its ligands, such as Am80or all-trans retinoic acid (ATRA),promotes VSMC differentiation and inhibits VSMC proliferation. Since thebiology and pathobiology of VSMCs are governed by the activity of atranscription factor network and cardiovascular activity-regulating substances,including apelin and ATRA, we predict that functional interactions betweenthe pro-proliferative transcription factors, such as KLF5and transcriptionfactor stimulating protein-1(Sp1), and pro-differentiation factors, such asRARα, could occur in the regulation of apelin gene expression by Am80.In this study, we aimed to elucidate whether and how Am80modulates theinteraction of RARα with KLF5and Sp1as well as regulates apelin expressionin VSMCs.Methods:1. Cell culture and treatment;2. RNA preparation andquantitative reverse transcription polymerase chain reaction (qRT-PCR);3.Western blotting;4. Adenovirus expression vector and plasmid constructs;5.Site-directed mutagenesis;6. Small interfering RNA (siRNA) transfection;7.Luciferase assay;8. Oligonucleotide pull-down assay;9. Chromatinimmunoprecipitation (ChIP) assay;10. Co-immunoprecipitation (Co-IP) assay;11. Glutathione transferase (GST) pull-down assay;12. Cell proliferation assay;13. Cell migration assay;14. Phalloidin staining for actin stress fibers;15. Balloon injury and siRNA transfection of rat carotid artery;16.Immunohistochemistry and image analysisResult:1. KLF5and Sp1mediates Am80-induced apelin expressionKLF5and Sp1plays important roles in a variety of cellular processesincluding proliferation and differentiation, however, synthetic retinoid Am80,a RARα-specific agonist, inhibits phenotypic modulation and proliferation ofVSMCs. In this study, We show that both KLF5and Sp1regulatesAm80-induced apelin expression in VSMCs. Results are as follows:1.1Am80upregulates apelin mRNA and protein levels in VSMCsGene chip map illustrated that apelin mRNA levels were7.48-foldupregulated in Am80-treated VSMCs. To validate the microarray analysisresults, RT-PCR, qRT-PCR, and western blotting were used to further examineapelin expression in terms of transcription and translation levels in response toAm80signaling. When VSMCs were treated with4μΜ Am80for varioustimes, apelin mRNA and protein levels obviously increased in atime-dependent manner. Moreover, Am80also dose-dependently increasedapelin mRNA and protein levels.1.2KLF5mediates Am80-induced apelin transcription via direct binding toTCE site-1in the apelin promoterUsing computer programs, we found that the-793/-1bp region containsthree TCE sites within the apelin promoter. To test whether this region isresponsive to KLF5and Am80, we constructed progressive5’-deletionconstructs of the apelin promoter containing the TCE site fused to thePGL3-basic reporter construct. Using transfection of these constructs, weexamined the ability of Am80and KLF5to activate the expression of eachconstruct in VSMC. The results showed that KLF5overexpressionsignificantly elevated the activities of the apelin promoter of the three differentconstructs. When Am80was added, the relative activity of the differentconstructs further increased. To assess whether the TCE sites in the apelin promoter region wererequired for Am80-induced apelin expression, we co-transfected the promoterconstructs in which the different TCE sites were mutated, respectively, withKLF5expression plasmid GFP-KLF5. The luciferase activity assay resultsshowed that mutation of site-2(-173to-523) or site-3(-523to-793) did notaffect apelin promoter activity and that only the mutation of site-1(-173to-1)blocked the activation of the apelin promoter by KLF5.1.3KLF5and Sp1mediates apelin expressionTo further verify the importance of KLF5in apelin expression, we infectedVSMCs with pAd-KLF5or transfected VSMCs with siRNA targeting KLF5(si-KLF5) to overexpress or knock down KLF5expression. Overexpression ofKLF5markedly increased the basal and Am80-induced apelin expression interms of transcription and translation levels. Conversely, knockdown of KLF5blocked Am80-induced apelin expression and also reduced its basal expression.These results suggest that KLF5played a key role in the regulation of apelinexpression by Am80.In addition, because members of the Sp/KLF family recognize the sameDNA-binding sequences containing CACCC-box (TCE) or GC-rich elements,we sought to determine whether Sp1can also regulate apelin expression. Totest this hypothesis, we infected VSMCs with pAd-Sp1or transfected VSMCswith siRNA targeting Sp1(si-Sp1) to overexpress or knock down Sp1expression. We find that the effect of Sp1overexpression or Sp1knowdownon apelin expression was consistent with KLF5overexpression or knockdown.1.4KLF5and Sp1mediates Am80-induced apelin expression via TCE site-1in the apelin promoterTo validate whether both KLF5and Sp1directly bind to TCE site-1, aChIP assay was carried out. The results showed that Am80treatment increasedthe binding of KLF5and Sp1to site-1. Consistent with the results of the ChIPassay, the oligonucleotide pull-down assay showed that the binding of KLF5and Sp1to site-1was increased by Am80stimulation, while mutation in site-1interrupted binding. 2. Am80activates apelin expression by promoting interaction of RARαwith KLF5and Sp1prebound to the TCE of the apelin promoterBecause the effects of Am80are mediated via its specific receptor RARα,we sought to further define the relationship between RARα, KLF5, and Sp1inthe regulation of apelin expression. In this study we showed RARα wasrecruited to the apelin promoter via its interaction with KLF5and Sp1, whichare associated with TCE site-1, to cooperatively activate apelin transcription.Results are as follows:2.1RARα cooperates with KLF5and Sp1and activated apelin promoteractivityTo evaluate the additive effects of the interaction of RARα with KLF5andSp1on apelin promoter activity, VSMCs were co-transfected with the apelinpromoter reporter, along with various combinations of expression plasmids forRARα, KLF5, and Sp1, and then treated with or without Am80, followed by aluciferase assay. Transient expression of KLF5or Sp1alone, as well as two orthree combinations of these expression plasmids, increased apelin promoteractivity to a certain extent. The strongest activation was observed when allthree expression plasmids for RARα, KLF5, and Sp1were co-transfected inthe presence of Am80. Altogether, these results demonstrated that RARα,KLF5, and Sp1cooperatively activated the apelin promoter.To further investigate the effect of KLF5cooperated with Sp1on apelinpromoter activity, VSMCs were also co-transfected with a constant amount ofpEGFP-Sp1plasmid and increasing amounts of pEGFP-KLF5plasmid, alongwith the apelin promoter-reporter construct pGL3-apelin-luc. The stimulatoryeffect of Sp1on the apelin promoter gradually increased with increasingamounts of pEGFP-KLF5. Likewise, when VSMCs were co-transfected with aconstant amount of pEGFP-KLF5and increasing amounts of pEGFP-Sp1, Sp1enhanced the stimulatory effect of KLF5on the apelin promoter in aconcentration-dependent manner.2.2Am80induces the interaction of RARα with KLF5and Sp1Although Am80stimulation did not affect KLF5expression in VSMCs grown in DMEM containing2%FBS, Sp1and RARα protein levelstime-dependently increased after Am80treatment. Because RARα, KLF5, andSp1cooperatively activated the apelin promoter, we sought to determine ifinteractions exist between RARα, KLF5, and Sp1. Thus,co-immunoprecipitation and GST pull-down assays were performed. Theresults showed that RARα could interact to some extent with Sp1under basalconditions and that Am80stimulation increased their interactions in VSMCs.Interestingly, we also found that Am80markedly enhanced the association ofSp1with KLF5. GST pull-down assays showed that although RARα, KLF5,and Sp1formed a complex without Am80treatment, Am80stimulationpromoted RARα interaction with KLF5and Sp1.2.3Am80promotes the assembly of Sp1, KLF5, and RARα on TCE site-1ofthe apelin promoterBecause KLF5, Sp1, and RARα formed a complex, we sought todetermine whether Am80promoted the recruitment of this complex to theapelin promoter. Therefore, we performed sequential ChIP analysis in whichVSMC chromatin was immunoprecipitated first with anti-KLF5and secondwith anti-Sp1or anti-RARα. Upon Am80signaling activation, the TCE site-1containing region was obviously amplified in the immunoprecipitates pulleddown with anti-Sp1or anti-RARα compared with Am80-untreated cells,suggesting that Am80enhanced the recruitment of the KLF5-Sp1-RARαcomplex to TCE site-1of the apelin promoter. In addition, we also repeatedthe sequential ChIP assays using immunoprecipitation first with anti-Sp1andsecond with anti-KLF5or anti-RARα and obtained the same results.To further test whether the binding of RARα to the apelin promoterdepended on the binding of KLF5and Sp1to the TCE site-1, we performed aChIP assay in VSMCs in which KLF5or Sp1was knocked down by RNAinterference (siRNA). Treatment of VSMCs with Am80caused greaterrecruitment of RARα, whereas knockdown of endogenous KLF5and Sp1bysiRNA significantly attenuated the binding of RARα to the apelin promoterinduced by Am80. In addition, we knocked down endogenous RARα by transfecting VSMCswith siRNA specific for RARα (si-RARα). The ChIP assay showed that Am80markedly promoted the binding of KLF5and Sp1to TCE site-1of the apelinpromoter in si-NS-treated VSMCs; knockdown of endogenous RARα bysi-RARα abrogated Am80-induced recruitment of KLF5and Sp1to the apelinpromoter. These results suggest that RARα was indirectly bound to theKLF5/Sp1binding sites of the apelin promoter by forming a complex withKLF5and Sp1to modulate apelin gene transcription.3. The role of apelin on VSMC phenotypic modulationIt has been known that VSMC proliferation and migration can be regulatedby many factors. However, whether apelin-13or Am80-induced apelin affectVSMC phenotypic change is still unknown. Therefore, in this study, we soughtto further find the relationship between apelin and Am80in VSMC phenotypicmodulation.3.1Apelin-13promotes VSMC proliferation and migrationApelin-13could markedly induce VSMC proliferation in a time-anddose-dependent manner as confirmed by BrdU incorporation experiments.Wounding cell migration assay showed that apelin-13could markedly induceVSMC migration, and western blotting analyses showed that expression ofVSMC migration-related gene MMP-2markedly increased in a time-anddose-dependent manner. Western blotting analyses showed that expression ofVSMC proliferation-related genes PCNA and cyclinD1was markedlyupregulated, whereas expression of differentiation-related genes SMα-actinsignificantly decreased in apelin-13-treated VSMCs. Phalloidin stainingshowed that apelin-13treatment could decrease the actin stress fiberscompared with vehicle (Veh)-treated cells, and that Am80promoted theformation of actin stress fibers, suggesting that the changes in VSMC markergene expression induced by apelin-13caused cellular phenotypic switching.3.2Apelin modulates VSMC phenotypeTo further investigate the effect of apelin on VSMC phenotype modulaiton,we knocked down endogenous apelin by transfecting VSMCs with siRNA against apelin (si-apelin) and then treated cells with Am80. The resultsshowed that knockdown of apelin increased SM22α expression induced byAm80and decreased PCNA level in Am80-treated VSMCs. Phalloidinstaining also showed that knockdown of apelin facilitated actin stress fiberformation, consistent with the results from VSMC marker gene expression.These results suggested that Am80-induced apelin expression counteracted tosome extent Am80-induced VSMC differentiation.3.3Knockdown of apelin inhibits neointimal hyperplasiaAfter neointimal formation was induced by balloon injury, an increasedI/M ratio of carotid arteries was observed at14days. Apelin-knocked downanimals reduced I/M ratio by60%compared with injured models.Immunohistochemistry analysis showed that the positive cells of PCNAstaining were decreased in the neointima of balloon-injured arteries inapelin-knocked down animals. These results suggest a potent promotion effectof apelin on VSMC proliferation in vivo.Conclusion:1. Am80upregulates apelin expression in VSMCs. KLF5and Sp1mediateAm80-induced apelin expression via TCE site-1in the apelin promoter.2. Interaction of RARα with Sp1and KLF5cooperatively activates apelinpromoter activity.3. Am80promotes the assembly of Sp1, KLF5, and RARα on TCE site-1ofthe apelin promoter.4. Apelin-13promotes VSMC proliferation and migration5. Apelin promotes balloon injury-induced neointimal hyperplasia.