摘要
目的：apelin是近年发现的生物活性肽，通过与它的特异性受体APJ相互作用而发挥重要的生理和病理功能。apelin和APJ均在心血管系统表达,包括血管内皮细胞(endothelial cells, EC)和血管平滑肌细胞(vascularsmooth muscle cells, VSMC)。apelin可调节血管张力，引起血压下降或阻力血管舒张。体外实验证实，apelin引起血管舒张主要是通过作用于内皮细胞上的APJ受体及促进一氧化氮合酶合成NO来实现的。另有研究表明，apelin也可以直接作用于平滑肌细胞而引起血管收缩，apelin还可通过正性肌力作用提高心肌的收缩力。此外，在EC和VSMC中，apelin是一个强效的血管生成因子和细胞分裂原，apelin及其受体APJ在心血管正常发育中起到非常重要的作用。apelin-APJ系统异常与高血压的发病及其并发症心力衰竭密切相关。在EC、VSMC和心肌细胞中，缺氧和血管生成素I可以诱导apelin表达。然而，在心血管系统中，调节apelin表达的分子机制目前仍知之甚少。
Krüppel样因子5（Krüppel-like factor5，KLF5）是一种含有锌指结构的转录因子，是血管紧张素Ⅱ(angiotensin II, Ang II)的靶基因及心血管重塑的重要调节者，KLF5在调节VSMC表型方面也扮演重要的角色。重要的是，KLF5可以与许多其他的转录因子，如c-Jun、维甲酸受体α(retinoicacid receptor α, RARα)、CREB(cAMP-response element binding protein,CREB)结合蛋白(CBP)和PPAR-δ(peroxisome proliferators-activatedreceptor-δ, PPAR-δ)相互作用，共同调节许多基因的表达。另外，血管损伤后KLF5在血管平滑肌细胞中表达上调，在心血管重构过程中激活许多基因，如血小板源性生长因子（platelet-derived growth factorA/B，PDGFA/B）、早期生长反应基因-1(early growth response gene-1, Egr-1)、纤溶酶原激活物抑制剂1(plasminogen activator inhibitor-1, PAI-1)和血管内皮生长因子(vascular endothelial growth factor-1, VEGF)受体等。另外，我们也发现，KLF5可与RARα和HDAC2(histone deacetylase2, HDAC2)在基础条件下形成复合体，结合在p21的启动子上并抑制其表达，而Am80诱导可使KLF5脱乙酰基并且从p21启动子上解离，失去其抑制作用并导致p21启动子的激活。
Am80是一种人工合成的维甲酸类似物，是RARα的激动剂，已经在临床上用于治疗急性早幼粒细胞性白血病。RARα属于核受体超家族成员，当与配体，如Am80或全反式维甲酸（all-trans retinoic acid，ATRA）结合后，可促进VSMC分化和抑制VSMC增殖。因为血管平滑肌细胞的生物学行为是由转录因子网络和一系列的心血管活性物质调节控制，比如apelin和ATRA等，因此我们预测，促增殖转录因子，如KLF5和Sp1（transcriptionfactor stimulating protein-1, Sp1）和促分化转录因子如RARα，在调控apelin基因表达中存在着相互作用。
本研究旨在阐明Am80调节apelin基因表达的分子机制。
方法：细胞培养，腺病毒及质粒载体的构建，定点突变，小干扰RNA转染，RNA提取及定量RT-PCR分析，Western印迹分析，荧光素酶报告基因分析，染色质免疫共沉淀分析，寡核苷酸pull-down分析，GSTpull-down分析，免疫共沉淀分析，BrdU掺入分析，细胞迁移分析，鬼笔环肽细胞骨架染色，免疫组化染色等。
结果：1. KLF5和Sp1介导Am80诱导的apelin基因表达
KLF5和Sp1在多种细胞的增殖和分化中，都发挥着重要的调控作用。Am80作为RARα的激动剂，可以抑制VSMC的表型转化和增殖，但Am80抑制VSMC表达转化和增殖与KLF5和Sp1之间的关系尚不清楚。在本部分研究中，我们观察KLF5和Sp1在Am80诱导apelin表达中的作用。结果如下：1.1Am80在VSMC中上调apelin基因mRNA和蛋白表达水平
基因芯片分析结果表明，在Am80处理的VSMC组中，apelin mRNA表达水平比对照组升高7.48倍。为了验证微阵列分析的结果，我们应用RT-PCR、实时定量PCR及Western印迹分析方法进一步检测apelin在转录和翻译水平的表达情况。结果显示，Am80可显著诱导apelin基因转录和翻译，且呈时间（0、2、6、12、24h）和剂量（0、2、4、8、16μM）依赖性。1.2KLF5通过与apelin启动子上的TCE位点1结合来调节Am80诱导的apelin转录
我们利用计算机程序发现，在apelin启动子的-793/-1区域包含3个TCE（transforming growth factor-β control element，TCE）位点。为了检测这3个位点是否对KLF5和Am80应答，我们构建了5’-端连续缺失的apelin启动子报告基因进行荧光素酶报告基因的分析。结果表明，过表达KLF5显著提高apelin的启动子活性，而Am80的诱导可以进一步提高apelin启动子的活性。
为了证实这3个TCE位点是否为Am80和KLF5诱导apelin表达所必需，我们突变这3个不同的TCE位点后再进行报告基因分析。荧光素酶活性测定结果表明,突变TCE位点2(-173到-523)和TCE位点3(-523到-793)不影响KLF5对apelin启动子的激活，但突变TCE位点1(-173-1)完全阻断了KLF5对apelin启动子的激活作用。1.3KLF5和Sp1共同调节apelin的表达
为了进一步验证KLF5在apelin表达中的重要性，我们分别用腺病毒表达载体pAd-KLF5感染VSMC和应用小干扰RNA（si-KLF5）转染VSMC，以此来过表达和敲低KLF5在VSMC中的表达。结果显示，过表达KLF5显著增加apelin的表达，Am80处理后apelin的转录和翻译进一步增加；相反，敲低KLF5抑制Am80对apelin表达的诱导作用。以上结果说明，KLF5介导Am80诱导的apelin表达。
此外，由于Sp/KLF家族成员能够识别相同的CACCC(TCE)或者GC盒元件，因此，我们试图探索Sp1是否也能调节apelin的表达。为了验证这一假说，我们分别应用腺病毒表达载体pAd-Sp1感染VSMC和应用小干扰RNA（si-Sp1）转染VSMC，以此来过表达和敲低Sp1在VSMC中的表达。我们发现，过表达或敲低Sp1对apelin表达的影响与过表达和敲低KLF5的结果是一致的。1.4KLF5和Sp1通过与apelin启动子上的TCE位点1结合介导Am80诱导的apelin表达
为了验证Sp1是否也像KLF5那样能够结合在TCE位点1上，我们进行染色质免疫共沉淀分析。结果显示，Am80促进Sp1和KLF5在TCE位点1上的结合；与染色质免疫共沉淀实验结果相一致，寡核苷酸pull-down实验分析也证实KLF5和Sp1结合在TCE位点1上，TCE位点1突变，阻断它们的结合。2. Am80通过促进RARα与KLF5和Sp1相互作用而诱导apelin表达
因为Am80通过其特异性受体RARα介导它的生理效应，因此我们试图探索Am80是否通过促进RARα与KLF5和Sp1相互作用来调节apelin表达。在本部分研究中，我们发现KLF5和Sp1通过将RARα募集到apelin启动子的TCE位点1上而协调激活apelin的转录。结果如下：2.1RARα与KLF5和Sp1协同激活apelin启动子的活性
为了探明RARα对apelin启动子活性的影响，我们用apelin启动子报告基因及KLF5、Sp1和RARα表达质粒共转染VSMC后进行荧光素酶活性分析。结果显示，单纯过表达KLF5或Sp1或同时过表达两种或三种转录因子，均可增强apelin启动子的转录活性，当同时表达3种转录因子并给予Am80刺激后，apelin启动子的转录活性进一步增强。以上结果表明，RARα和KLF5及Sp1协同激活apelin基因转录。
用apelin启动子报告基因及不同转录因子的表达质粒共转染VSMC后进行荧光素酶活性分析。结果显示，共转染等量的Sp1及不同量的KLF5表达质粒后，随着KLF5表达量的增加，Sp1对apelin启动子的转录激活作用逐渐增强。同样，共转染等量的KLF5及不同量的Sp1表达质粒，随着Sp1表达量的增加，apelin启动子的活性亦增强。这些结果表明，KLF5和Sp1协同激活apelin的转录。2.2Am80促进RARα与KLF5-Sp1复合物结合
因为RARα与KLF5和Sp1协同激活apelin启动子的活性，所以我们通过免疫共沉淀和GST pull-down实验检测它们三者之间是否存在相互作用。结果显示，在未给予Am80刺激的VSMC中, RARα与KLF5和Sp1可以在一定程度上结合，给予Am80刺激后，RARα与KLF5-Sp1复合物结合活性增强。2.3Am80促进RARα-KLF5-Sp1复合物结合在apelin启动子的TCE位点1上
由于KLF5、Sp1和RARα三者之间形成复合物，我们探索Am80是否促进该复合物在apelin启动子上的募集。连续ChIP实验分析显示，用KLF5抗体先沉淀VSMC的染色质片段可以扩增得到含有TCE位点1的DNA片段，Am80处理使KLF5与DNA的结合显著增加。然后，用Sp1或RARα抗体再次沉淀KLF5抗体沉淀得到的染色质片段，二次沉淀物中仍能扩增得到含apelin启动子TCE位点1的DNA片段。以上结果表明，RARα、KLF5和Sp1在apelin启动子的TCE位点1上形成复合物，Am80刺激促进三者在apelin启动子上的募集装配。按相同的方法，我们首先用Sp1抗体沉淀VSMC染色质片段,然后再用KLF5或RARα抗体沉淀Sp1抗体沉淀得到的染色质片段，二次沉淀物中也能扩增得到含有apelin启动子的TCE-位点1的DNA片段，所得到的结果与以上结果相一致。
为了进一步研究RARα在apelin启动子上的募集是否依赖于KLF5-Sp1复合物在apelin启动子TCE位点1的结合，我们应用小干扰RNA技术分别敲低内源性KLF5和Sp1后，应用ChIP分析检测RARα在apelin启动子上的结合情况。结果显示，应用si-RNA技术敲低内源性KLF5和Sp1可显著抑制RARα在apelin启动子上的募集。
另外，我们应用si-RNA技术敲低内源性RARα后进行ChIP分析的结果也表明，Am80刺激明显促进KLF5和Sp1在apelin启动子TCE位点1上的结合；应用si-RARα下调内源性RARα表达可减少Am80诱导的KLF5和Sp1在apelin启动子上的募集。以上结果表明，RARα结合在KLF5-Sp1复合物上，以RARE非依赖的方式调节apelin基因的转录。3. apelin促进VSMC表型转化和增殖
已经证明VSMC的增殖和迁移受到许多因素的调控，但apelin对VSMC表型的调节仍然知道的很少。因此，在本部分研究中，我们探索apelin和Am80在调节VSMC表型中的关系。3.1apelin-13促进VSMC的增殖和迁移
BrdU掺入分析实验结果显示，apelin-13以浓度（0、0.02、0.05、0.1、1μM）和时间（0、2、6、12、24h）依赖的方式促进VSMC的增殖。通过伤口愈合实验分析，我们观察apelin-13对VSMC平面迁移能力的影响。结果显示，apelin-13可显著增强VSMC的迁移能力；western印迹结果显示，与迁移密切相关的基质金属蛋白酶-2（matrix metalloproteinase-2,MMP-2）表达也明显升高。
同时，我们通过免疫印迹实验发现，apelin-13可以以浓度（0、0.02、0.05、0.1、1μM）和时间（0、2、6、12、24h）依赖的方式促进增殖标志基因PCNA和cyclinD1表达；而分化标志基因SM α-actin的表达明显减少。鬼笔环肽染色显示，apelin-13处理的VSMC中肌丝明显减少；Am80处理明显使VSMC形态伸长，形成许多粗大的、沿细胞长轴平行排列的应力纤维。这些结果提示，apelin-13促进VSMC向增殖表型转化。3.2apelin拮抗Am80诱导的VSMC分化
为进一步研究apelin对VSMC表型的影响，我们应用si-RNA技术敲低内源性apelin后再用Am80处理细胞。免疫印迹实验结果显示，敲低内源性apelin后再用Am80处理的VSMC与未敲低apelin直接用Am80处理的细胞相比，SM22α的表达升高而PCNA的表达水平降低。同时，鬼笔环肽染色显示，敲低内源性apelin后再用Am80处理的VSMC伸长更加明显，并形成更多的沿细胞长轴平行排列的应力纤维。结果提示，apelin可以在一定程度上拮抗Am80诱导的VSMC分化。3.3敲低apelin抑制球囊损伤诱导的血管新生内膜的增生
利用大鼠颈总动脉球囊损伤模型，我们观察敲低apelin后对血管新生内膜形成的影响。HE染色结果显示，损伤14天后，与模型组相比，apelin敲低动物的血管内膜/中膜（I/M）比值明显降低。免疫组织化学染色结果显示，apelin敲低动物的血管新生内膜和中膜中PCNA的表达明显减少。结果表明，apelin可促进血管新生内膜的形成。
结论：
1. Am80上调apelin基因mRNA和蛋白表达水平。
2. KLF5和Sp1与apelin启动子上的TCE位点1结合来调节Am80诱导的
apelin转录。
3. Am80通过促进RARα与KLF5和Sp1相互作用诱导apelin表达。
4. apelin促进VSMC表型转化和增殖。
5. apelin促进血管新生内膜的形成。
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 analysis
Result:1. KLF5and Sp1mediates Am80-induced apelin expression
KLF5and 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 VSMCs
Gene 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 promoter
Using 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 expression
To 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 promoter
To 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 promoter
Because 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 promoteractivity
To 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 Sp1
Although 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 promoter
Because 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 modulation
It 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 migration
Apelin-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 phenotype
To 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 hyperplasia
After 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.
引文
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1O'Dowd BF, Heiber M, Chan A, et al. A human gene that shows identitywith the gene encoding the angiotensin receptor is located onchromosome11. Gene,1993,136(1-2):355-360
2Tatemoto K, Hosoya M, Habata Y, et al. Isolation and characterizationof a novel endogenous peptide ligand for the human APJ receptor.Biochem Biophys Res Commun,1998,251(2):471-476
3De Mota N, Reaux-Le Goazigo A, El Messari S, et al. Apelin, a potentdiuretic neuropeptide counteracting vasopressin actions throughinhibition of vasopressin neuron activity and vasopressin release. ProcNatl Acad Sci U S A,2004,101(28):10464-10469
4Kawamata Y, Habata Y, Fukusumi S, et al. Molecular properties ofapelin: tissue distribution and receptor binding. Biochim Biophys Acta,2001,1538(2-3):162-171
5Lee DK, Ferguson SS, George SR, et al. The fate of the internalizedapelin receptor is determined by different isoforms of apelin mediatingdifferential interaction with beta-arrestin. Biochem Biophys ResCommun,2010,395(2):185-189
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