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Notch受体与EB病毒感染及平滑肌细胞的基因调控
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摘要
Notch是一个跨膜受体。Notch受体通过与膜上配体的相互作用而被激活,在哺乳类其配体特指Jagged/Delta。Notch受体至少含有三个位点(S1-S3),在此位点蛋白水解酶使跨膜受体裂解。在成熟期间,300 kDa的蛋白前体在S1位点被Furine样的转化酶所裂解。Notch受体一配体的相互作用诱导进一步分离,首先在S2位点排除Notch受体细胞外部分(Notch extracellular domain,NEC),然后在S3位点释放Notch受体细胞内部分(Notch intracellular domain,Notch-IC)。分离以后,Notch-IC就转移进入核内,与RBP-J(在哺乳动物中又称CBFl)相互作用并调节基因的表达。在哺乳动物中已有4个Notch基因(Notch1,Notch2,Notch3和Notch4)被描述,它们编码只有单一跨膜区的受体。几个哺乳类动物的靶基因通过Notch-IC和RBP-J/CBF1的相互作用而被诱导,像HES or Hes(the hairy enhancer of split)和HRT or Hey(hairy-related transcription factor,Hairy相关转录因子)。
     EB病毒(Epstein-Barr virus,EBV)通过引起B淋巴细胞潜伏感染,在人类建立了终生持续型感染状态,并可以导致淋巴细胞性及上皮性恶性肿瘤。近来研究显示,EBV感染的B淋巴细胞永生化依赖于病毒携带的一个B淋巴细胞转化基本调节因子EBNA2(EB病毒核心抗原2)。尽管在基因序列上没有相关性,Notch受体和EBNA2却共同具有一些生化和功能上的特性,例如都相互作用于Notch信号通路上的核心因子,RBP-J kappa(又称CBF1)。然而,在调节下游信号通路基因表达方面,Notch与EBNA2相互关系还不清楚。
     本研究选择Notch2受体和EBNA2基因为目标,应用HeLa细胞和Cos7细胞与不同的载体瞬间联合转染,实时定量反转录-PCR(QRTPCR),CBF1和HES1启动子荧光素酶分析及Notch2-RNAi干扰技术,探讨Notch2-IC与EBNA2相互作用关系。
     在现有的研究中,我们发现过度表达的Notch2-IC或EBNA2载体可以明显上调CBF1启动子表达,CBF1启动子突变后(3XMT),N2IC则完全丧失对CBF1启动子的上调作用,而EBNA2则部分丧失对CBF1启动子的上调作用,说明由Notch-IC和EBNA2调节的启动子系列有重叠但不等同。同时研究结果显示,N2IC和EBNA2均能有效地激活HES1启动子活性表达,说明Notch与EBNA2可以共同作用于Notch基因家族的下游信号通路。令人感兴趣的是,Notch2-RNAi(干扰Notch2使无其活性)可以削弱EBNA2介导的CBF1和HES1表达。
     我们的结果证实,在EBNA2介导的信号通路中,活化的Notch2起关键性作用,提示Notch信号(尤其内源性Notch)可能参与EBV感染过程中EBNA2调节。
     第二部分Notch1与Notch2受体在控制血管平滑肌细胞生长过程中的相反作用:HES1的调节作用
     Notch受体家族在决定细胞的最终命运及血管形成方面起重要作用。Notch受体与配体混合物存在的多样性表明每个Notch受体可能具有不同的作用。在这里,我们报道了Notch-1与Notch-2在血管平滑肌细胞生长调节过程中新的作用机制。
     实验方法包括:用不同血管平滑肌细胞(Vascular Smooth Muscle Cell,VSMC)与不同的载体瞬间联合转染,实时定量反转录-PCR(QRTPCR),[~3H]脱氧胸腺嘧啶核苷接合实验,蛋白质印迹(Western blotting),人p27~(KIPI)启动子荧光素酶分析,染色质免疫沉淀(ChIP)及HES1-RNAi干扰技术。同时构建了A7r5(胚胎期大鼠主动脉平滑肌细胞)稳定表达Notch1-IC和Notch2-IC细胞株及人p27~(KIPI)启动子荧光素酶-HES1结合位点定点突变载体。
     我们观察到,Notch-1细胞内与激活相关的关键区(N1-IC)高表达可通过提高细胞周期抑制剂p27~(KIPI)的表达,从而抑制血管平滑肌细胞生长。相反,Notch-2细胞内与激活相关的关键区(N2-IC)则表现为刺激血管平滑肌生长及诱导p27~(KIPI)表达水平降低。p27~(KIPI)启动子5′端大约3.5kb的启动子区域基因删除图谱分析结果表明:存在于-774与-549bp之间区域的cis-物质对N1-IC与N2-IC参与的p27~(KIPI)转录激活的不同的调节作用起关键作用,并且此区域包含有假定的HES1联结区,实际上N1-IC对HES1的表达起上调作用,N2-IC则无此作用。而且,HES1在N1-IC与N2-IC参与的p27~(KIPI)基因转录的不同反应中起重要的调节作用。此作用已经通过一系列的实验得到了验证,包括直接定位点突变和染色质免疫沉淀及RNAi对HES1的基因干扰作用。更进一步,干扰HES1基因会引起N1-IC诱导的生长抑制作用与N2-IC刺激的平滑肌生长作用得到潜在性的削弱。
     以上结果表明:Notch-1与Notch-2受体分别调控众多与Notch转录关联的HES1的重要下游转录因子中的一个效应基因,因此,在平滑肌生长调节中起完全相反的作用。
Notch is a transmembrane receptor. Notch receptors are activated by interaction with membrane bound ligands, which in vertebrates are designated Jagged / Delta. Notch receptors have at least three sites (S1 to S3), where they are proteolytically cleaved . During maturation, the 300 kDa precursor protein is cleaved by a furine-like convertase at S1; Ligand-receptor interactions induce two further cleavages which removes most of the Notch extracellular domain (NEC) at S2 and releases Notch intracellular domain (Notch-IC) at S3. After cleavage, Notch-IC is translocated to the nucleus, where it interacts with RBP-J(or CBF1) and modulates gene expression. In mammals four Notch-genes (Notch 1-4) have been described, encoding receptors with a single transmembrane domain. Several mammalian target genes have been described, which are induced through the interaction of Notch-IC with RBP-J, like the hairy enhancer of split (HES or Hes ) and hairy-related transcription factor (HRT or Hey)
     Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans by latently infecting B cells, with a potential development of both lymphoid and epithelial tumors. Recent studies have demonstrated that immortalization of B cells infected by Epstein-Barr virus (EBV) depends on the virally encoded Epstein-Barr virus nuclear antigen 2 (EBNA2) protein ,a essential mediator for B cell transformation. Although there is less homologue between Notch receptor and EBNA2 genes, they share several biochemical and functional properties, such as interaction with RBP-J kappa (or CBF1) a nuclear component of the Notch signaling pathway. However, it is not clear whether Notch interacts with EBNA2 in driving the downstream signaling.
     To gain insight into the Notch2 receptor- and EBNA2-operated expression of CBF1 and HES1, in the present study, Notch 2 or/and EBNA2 gain- or loss-of-function approaches were employed to analyze the CBF1 and HES1 promoter activity in vitro using HeLa cell and Cos7 cell.
     We observed that overexpression of either active form of Notch2 (N2IC) and EBNA2 up-regulated CBF1 promoter expression. The N2IC-mediated CBF1 expression was blocked by CBF1 promoter mutation (3XMT); However, EBNA2-mediated CBF1 expression was partially blocked by CBF1 mutation (3XMT). Theses results suggest that a set of cis-elements on CBF1 promoter regulated by Notch-IC and EBNA2 is overlapping but not identical. In addition, as in Notch-triggered signaling, EBNA2 might operate its downstream gene expression through CBF-1-mediated replacement of repressor proteins to release their transactivation domains. Meanwhile, we found Notch2 and EBNA2 triggered HES1 expression, suggesting that EBNA2 is able to activate downstream targets of Notch signaling. Interestingly, the EBNA2-mediated CBF1 and HES1 expression was shut down by the Notch2-RNAi.
     Our data demonstrate that activation of Notch2 is critical for EBNA2-operated signaling, suggesting a possible involvement of Notch signaling (especially endogenous Notch signaling) in EBV infection.
     The Notch receptor family plays an important role in cell fate determination and vasculogenesis. The presence of multiple Notch ligand receptor combinations suggests that each of the Notch receptors may play a distinctive role. Herein we report a novel mechanism underlying the countervailing regulation of vascular smooth muscle cell (VSMC) growth by Notch-1 and Notch-2.
     A series of experimental approaches were applied including transient transfection in different VSMC, quantitative real-time reverse transcription-PCR (QRTPCR), [~3H] thymidine incorporation, Western blotting, p27~(KIP1) promoter analysis, HES1 site-directed mutagenesis analysis, and chromatin immunoprecipitation (ChIP). Meanwhile we generated A7r5 stable cell lines for forced expression of the constitutively active Notch1 or Notch2 intracellular domain (N1-IC) or (N2-IC), and HES1 site-directed mutagenesis of p27~(KIP1) promoter.
     We observed that Nl-IC stable cell line inhibited VSMC growth in association with increased expression of the cell-cycle inhibitor p27~(KIP1). In contrast, N2-IC stable cell line stimulated VSMC growth and induced a decrease in p27~(KIP1) levels. A deletion mapping analysis of about 3.5 kb of the 5' promoter region of the p27~(KIP1) promoter indicated that a cis-element located between -774 to -549 bp is critical for the differential regulation of p27~(KIP1) transcriptional activity by Nl-IC and N2-IC and contained putative HES1 binding sites. Indeed, HES1 expression was up-regulated by N1-IC but not by N2-IC stimulation. Moreover the essential mediator role of HES1 in conferring differential response of N1-IC vs N2-IC on p27~(KIP1) gene transcription was confirmed by a series of experiments that included site-directed mutagenesis, chromatin immunoprecipitation and RNAi-mediated knockdown of HES1. Furthermore, knockdown of HES1 led to a potent attenuation of both N1-IC-induced growth arrest as well as N2-IC-stimulated VSMC growth.
     Taken together, these results indicate that Notch-1 and Notch-2 receptors differentially govern one of the critical downstream effectors of the Notch transcriptional cascade HES1 and thereby induce diametric effects on VSMC growth regulation.
引文
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