肝癌癌蛋白p28~（GANK）正反馈调节β-catenin信号通路的作用和机制研究
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摘要
第一部分肝癌癌蛋白p28~(GANK)正反馈调节β-catenin信号通路的作用和机制研究
研究背景
肝细胞性肝癌(简称肝癌,Hepatocellular Carcinoma,HCC)是我国最常见的高发肿瘤之一,其发生发展是由体内多基因参与、多步骤协同的复杂过程。目前,尽管已经鉴定了一些肝癌相关的抑癌基因,如p53和p16~(INK4a),并明确了它们在肝癌中的特异性失活,但是在肝癌发生中特异性激活的癌基因却少见报道。2000年日本科学家Fujita等人应用消减杂交法从人肝细胞癌组织高表达的基因中筛选出一条编码重复ankyrin(ANK)序列的新基因,命名为gankyrin。我们检索基因库后发现gankyrin基因与1998年Hori等人发现的p28(Nas6p)基因序列完全一致,遂将其命名为p28~(GANK)。癌基因p28~(GANK)编码的蛋白是人26S蛋白酶体(26S proteasome)调节亚单位19S/PA700复合物的一种非ATP酶亚基,其核酸序列中含有5个串联排列的ANK重复单位,其保守的ankyrin序列介导蛋白与蛋白间的相互作用。26S蛋白酶体中的20S蛋白小体是降解体内某些错误折叠蛋白或细胞周期蛋白的场所,目前尚不清楚癌蛋白p28~(GANK)在26S蛋白酶体降解蛋白过程中的具体分子作用机制。前期研究发现癌蛋白p28~(GANK)参与CDK4/CyclinD1/p16~(INK4a)/Rb1/E2F-1信号传导通路的调节,此通路的失调控在肝癌的发生发展过程中具有相当重要的作用;外源性转入癌基因p28~(GANK)使其高表达后的NIH/3T3细胞株可以使裸鼠荷瘤。Nagao等人通过酵母双杂交发现黑色素瘤抗原MAGE-A4能与癌蛋白p28~(GANK)特异性的结合并抑制p28~(GANK)成瘤活性。
β-连环蛋白(β-catenin)是一种重要的肿瘤相关基因,除了在细胞粘附中有重要作用外,还是Wnt信号通路的重要功能分子。通过在不同时间、空间上的差异表达调节一系列下游基因c-myc和cyclin-d1等的表达水平,参与细胞增殖、凋亡以及器官发育。近来研究发现,β-catenin在肝癌发生早期呈现异常聚集,并能够促进肝癌的发生以及早期肝癌细胞的快速生长。因此探讨β-catenin在肝癌发生发展中的作用具有重要价值。已有研究表明提高野生型p53水平可以明显下调β-catenin蛋白水平。结合新近研究发现p28~(GANK)能与泛素蛋白连接酶MDM2相互作用,介导野生型p53蛋白的泛素化及降解,提示在肝细胞癌的发生发展中可能存在着癌蛋白p28~(GANK)与β-catenin的相互联系。因此我们探讨了Wnt/β-catenin信号通路激活对p28~(GANK)表达的影响以及p28~(GANK)对β-catenin信号通路的调节作用。
实验方法:
1.采用Genomatix在线分析软件分析p28~(GANK)启动子区并构建p28~(GANK)报告基因质粒;
2.运用双荧光素酶报告基因系统检测生长因子刺激对p28~(GANK)转录活性的影响;采用瞬时转染方法观察Ras对p28~(GANK)转录活性的影响;应用报告基因系统结合ERK与PI3K信号通路抑制剂确定生长因子与Ras对p28~(GANK)表达的调节通路;采用瞬时转染β-catenin和c-Myc表达质粒确定它们对p28~(GANK)表达的影响;
3.通过Luciferase报告基因质粒pGL-OT(含有β-catenin/TCF4复合物特异结合区)与不同剂量p28~(GANK)质粒细胞共转染实验,确定p28~(GANK)对β-catenin/TCF4转录活性的影响;
4.运用western blot及核浆分离技术,检测HEK293细胞瞬转p28~(GANK)后,β-catenin在胞核胞浆中的分布变化;
5.运用western blot和转染技术研究生长因子、Ras、AKT、β-catenin、c-Myc对p28~(GANK)蛋白表达调节作用;应用RT-PCR技术研究生长因子、Ras、AKT、β-catenin、c-Myc等对p28~(GANK)表达的调节作用;
6.运用染色质免疫共沉淀(ChIP)技术研究β-catenin和c-Myc与28~(GANK)启动子区相互作用关系;
7.应用腺病毒介导的RNA干扰技术下调表达肝癌细胞系中p28~(GANK)的水平,探讨其对β-catenin转录活性的作用,并进一步研究了p28~(GANK)对自身转录水平的影响。
8.应用临床肝癌组织标本应用western blot和免疫组织化学方法检测β-catenin、c-Myc、cyclinD1表达水平与p28~(GANK)蛋白表达水平之间的关系。
结果:
1.肝细胞生长因子和表皮生长因子刺激明显上调饥饿处理后的HEK293和HepG2细胞中p28~(GANK)表达水平;瞬时转染Ras表达质粒显著上调p28~(GANK)mRNA和蛋白水平;
2.PI3K抑制剂处理显著抑制细胞本底水平p28~(GANK)的表达,也能够阻断生长因子或Ras激活对p28~(GANK)表达的上调作用;瞬时转染PI3Kp85质粒和野生或持续激活的AKT表达质粒均能够上调p28~(GANK)的表达水平;
3.报告基因、RT-PCR或western blot实验证明瞬时转染表达β-catenin和c-Myc都能够在转录和蛋白水平上调p28~(GANK)表达;染色质免疫沉淀实验发现β-catenin可以与p28~(GANK)启动子区相互结合;
4.HEK293细胞瞬时转染p28~(GANK)质粒可显著增强β-catenin/TCF4转录活性,而在HepG2、3B中抑制p28~(GANK)表达则可显著降低其转录活性;
5.HEK293细胞中瞬时转染p28~(GANK)表达质粒可提高β-catenin入核蛋白水平,并在一定程度上稳定细胞中β-catenin总蛋白水平;腺病毒介导的干扰p28~(GANK)表达明显降低β-catenin蛋白水平,从而抑制了其下游的c-Myc和cyclinD1的表达水平;
6.Western blot和免疫组化实验分析表明p28~(GANK)的蛋白水平在肝癌组织样品中与β-catenin、c-Myc和cyclinD1呈同向性表达;
7.在HEK293和Hep3B中p28~(GANK)正向调节自身表达。
结论:
生长因子刺激或Ras激活可以上调肝癌癌蛋白p28~(GANK)的表达,这种激活作用可能是通过激活PI3K-AKT通路完成的。β-catenin和c-Myc的上调表达都能够促进p28~(GANK)的表达,并且染色质免疫沉淀实验证实β-catenin/TCF4可以与p28~(GANK)启动子直接结合。进一步实验表明上调的p28~(GANK)可以反过来进一步促进β-catenin蛋白的稳定从而上调β-catenin/TCF4转录活性。在肝癌组织样品中p28~(GANK)的蛋白水平与β-catenin、c-Myc和cyclinD1呈同向性表达。上述结果支持我们提出的p28~(GANK)正反馈调节β-catenin信号通路的结论。
第二部分信号调节蛋白α负性调节Ⅰ型干扰素产生的作用和机制研究
研究背景
信号调节蛋白α(signal regulatory proteinα,SIRPα)是一种广泛存在的抑制性受体,属于免疫球蛋白受体超家族。其胞外区具有三个Ig样结构域和多个糖基化位点,胞内区带有免疫受体酪氨酸抑制基元(ITIMs),其中含四个酪氨酸残基,被磷酸化后能够募集含SH2结构域的蛋白磷酸酶SHP-1和SHP-2。
干扰素(interferon IFN)是一类广泛表达具有潜在抗病毒感染的细胞因子。但是过度失控的诱导产生IFN还将易导致自身免疫性疾病的发生。所以,对于IFN表达系统的调控对于维持机体内稳态相当重要。IFN的诱导产生依赖于机体模式识别(pattern recognition receptors PRRs)受体的激活。天然免疫系统识别病毒双链RNA可以通过两条途径:TOLL样受体(toll like receptors,TLR)3和胞浆途径(RIG-I/MDA5)。TLR3作为一种重要的模式识别受体,主要表达于巨噬细胞和树突状细胞内涵体内,能够识别细胞吞噬进入的dsRNA。RIG-I/MDA5能够识别病毒复制过程中产生的dsRNA。TLR3和RIG-I/MDA5都能够与dsRNA的类似物poly(I:C)(polyriboinosinic:polyribocytidylic acid)结合,从而介导Ⅰ型IFN产生。本课题主要探讨了SIRPα对poly(I:C)活化巨噬细胞的调节作用和机制。
SIRPα在免疫细胞只选择性地表达在巨噬细胞和树突状细胞等非淋巴细胞。我们研究发现poly(I:C)刺激巨噬细胞SIRPα蛋白表达水平下降,提示该蛋白对巨噬细胞激活及Ⅰ型IFN产生可能存在抑制作用。为进一步了解该蛋白在天然免疫功能中的作用和机制,本课题通过转染并筛选克隆建立稳定高低表达该蛋白的巨噬细胞系及瞬时干扰小鼠体内分离的巨噬细胞中的SIRPα表达,探讨了SIRPα对poly(I:C)诱导的小鼠巨噬细胞活化的影响,全面探讨了SIRPα对TLR3和胞浆途径介导的Ⅰ型IFN产生的调节作用;并探讨SIRPα对巨噬细胞MAPKs信号分子,分泌细胞因子的影响,并进一步探讨了SIRPα发挥上述作用的可能机制,确定了SIRPα在Ⅰ型干扰素诱导产生中的重要调节作用,并为尚未阐明的抑制性受体家族胞内信号转导机制提供新的线索。
实验方法
1.建立稳定转染空载体,高表达Myc-SIRPα及稳定干扰SIRPα的巨噬细胞系:RAW-VT,RAW-OV,RAW-KD,设计,合成并鉴定具有干扰效果的stealthRNA,分别干扰SIRPα,和SHP-2;
2.采用磷酸化特异性抗体检测不同SIRPα表达对poly(I:C)刺激后p38,JNK,ERK,IRF3及AKT磷酸化水平的变化的影响来反映SIRPα对通路活化的影响;
3.运用荧光素酶报告基因系统检测SIRPα对ISRE、PRDⅡ、PRDⅢ-Ⅰ、RANTES和NF-κB转录活性的作用;
4.采用Real-time PCR方法检测sirpa基因转录水平变化和RT-PCR检测ifn-β及其相关诱导基因变化;
5.通过ELISA检测细胞因子变化;
6.采用免疫沉淀的方法确定与SIRPα相互作用的蛋白。
7.采用瞬时干扰SHP-2后检测其对巨噬细胞释放细胞因子和对信号通路磷酸化的影响确定这个蛋白在巨噬细胞激活中的作用。
8.采用免疫共沉淀的方法检测SIRPα不同表达是否影响SHP-2和下游相互作用蛋白的结合。
结果
1.Poly(I:C)刺激后对SIRPα表达的影响:不同的小鼠巨噬细胞系和小鼠体内分离的巨噬细胞给予不同剂量的poly(I:C)和不同时程的poly(I:C)刺激后均可见SIRPα表达明显下调,Real-time PCR结果表明poly(I:C)引起的SIRPα表达下调主要是转录水平的下调。
2.SIRPα对poly(I:C)刺激后IRF3磷酸化及IFN相关转录因子活性的影响:研究发现SIRPα高表达可显著抑制poly(I:C)诱导的巨噬细胞细胞IRF3磷酸化水平,并抑制IFN相关转录因子结合位点PRDⅡ、PRDⅢ-Ⅰ及ISRE活性。SIRPα低表达则可显著提高巨噬细胞IRF3磷酸化水平和IFN相关转录因子活性。
3.SIRPα对TLR3激活的IFN-β及诱导基因表达的影响:报告基因,PCR分析和细胞因子测定结果表明,SIRPα对TLR3激活的Trif通路诱导IFN-β及其诱导基因的表达和其他促炎因子都有明显的抑制作用。
4.SIRPα对poly(I:C)刺激后的MAPK、NF-κB和JAK-STAT信号途径的作用:SIRPα低表达可显著提高poly(I:C)引起的MAPK信号通路的激活,ERK、JNK和p38磷酸化水平都显著增强。SIRPα低表达组IκBα磷酸化增强,NF-kB活性明显高于对照组。SIRPα表达抑制组巨噬细胞STAT1磷酸化水平显著高于对照组,iNOS活性及诱导产生的NO水平明显高于对照组。
5.SIRPα对poly(I:C)激活的胞浆信号途径影响:HEK293细胞中瞬时表达SIRPα可显著抑制瞬转poly(I:C)引起的IRF3的磷酸化水平;SIRPα高表达显著抑制瞬转poly(I:C)引起的IFN-β的表达;HEK293中SIRPα表达显著抑制poly(I:C)引起STAT1磷酸化水平;RT-PCR分析表明SIRPα高表达显著抑制瞬转poly(I:C)引起的IFN-β及其诱导基因的转录。
6.SIRPα的抑制作用依赖于ITIM基序的磷酸化及与SHP2的结合:报告基因分析表明不同剂量SIRPα表达不能抑制Trif或TBK1激活的IFN-β表达。SIRPαITIM突变体不能抑制poly(I:C)引起的IFN-β的表达,证明这种抑制作用依赖于ITIM基序的磷酸化并可能有SHP2的参与。
7.SIRPα对TLR3和胞浆途径IFN-β表达的影响机制:P13K抑制剂可显著抑制TLR3和胞浆途径介导的ISRE活性及IFN-β表达水平。说明PI3K信号途径可能在IFN表达中有正性调节作用。SIRPα的表达可抑制TLR3和胞浆途径介导的poly(I:C)引起AKT的磷酸化水平。免疫共沉淀实验发现SIRPα可以与PI3Kp85亚基形成复合体,并且这种结合是依赖于poly(I:C)刺激的。所以SIRPα发挥抑制作用可能正是通过磷酸化后募集PI3K,引起AKT不完全活化,这直接导致了IRF3的磷酸化水平降低,从而减少了IFN-β的表达。
结论
本研究从分子水平全面深入研究了信号调节蛋白SIRPα对TLR3和胞浆途径介导的Ⅰ型干扰素表达的调节作用,发现SIRPα对poly(I:C)诱导的Ⅰ型干扰素及其它细胞因子分泌等生物学行为具有负向调控能力,详细探讨了其影响相关信号转导通路的作用机制,证明其可能部分通过抑制PI3K-AKT信号通路调节作用而发挥负向调控作用。
PartⅠThe oncoprotein p28~(GANK) establishes a positive feedback loop inβ-catenin signalling
Hepatocelluar carcinoma(HCC) is one of the most prevalent malignant cancers in our country.The development and progression of HCC is a complicated process involving multiple genes and multiple steps in human bodies.Up to now,though some suppressors in HCC were identified such as p53 and p16~(INK4a),and confirmed their specific inactivation,few specific activated oncogenes in HCC was found and reported.In 2000,a novel gene named gankyrin with repeated sequences coding "ankyrin" motif was cloned in human HCC by Japanese scientists Fujita and his colleagues using subtractive hybridization method.We found this gankyrin gene was identical to the p28(Nas6p) gene reported by Hori in 1998 by searching the GenBank data,thus named it as p28~(GANK) in our work.
p28~(GANK)(also known as PSMD10,p28,Nas6p,and Gankyrin),a non-ATPase subunit of the 19S/PA700 complex from the 26S proteasome,contains five ankyrin repeats mediating proteins interaction.20S proteasome is a protease degrading some incorrectly folding proteins or cell cycle regulators.Little is known about the mechanism how oncoprotein p28~(GANK) relates with proteins degradation by 26S proteasome,p28~(GANK) regulates the CDK4/CyclinD1/p16~(INK4a)/Rb1/E2F-1 pathway which connects with tumor development and progression,and p28~(GANK)-transfected cells are tumorigenic in nude mice.Recently,Nagao et al has identified MAGE-A4 binds to gankyrin and suppresses its oncogenic activity through yeast two-hybrid screen,p28~(GANK) also directly binds to MDM2,which increases p53-MDM2 association,inducing the ubiquitylation and degradation of p53.
Theβ-catenin signalling pathway has a critical role in cell-fate determination,tissue homeostasis and tumorigenesis.In unstimulated cells,the cytoplasmic concentration ofβ-catenin is kept very low owing to phosphorylation by CK1 and GSK3βkinases. Activation of the Wnt pathway blocksβ-catenin degradation so thatβ-catenin accumulates and translocates to the nucleus,where it interacts with T-cell factor (TCF)/lymphoid enhancer factor(LEF) transcription factors.About 50%-70%of all HCC examined showed an abnormalβ-catenin protein accumulation in the cytoplasm and nucleus.However,in HCC theβ-catenin mutation rate is 13%-26%,the Axin mutation rate is 5%-10%and rare mutation of APC has been reported so far.This led us to speculate that other factors might be involved in the regulatingβ-catenin in HCC.
To investigate the factors which might influence p28~(GANK) expression,we cloned a 1226 bp fragment of the 5'-flanking region of human p28~(GANK) gene into pGL3 luciferase reporter vector,which analyzed using Gene2Promoter(Genomatix) online software.To elucidate whether p28~(GANK) upregulation was correlated with cell cycle progression in normal or HCC cell lines,we examined the potential cell-cycle-dependence of p28~(GANK) expression in HepG2 and HEK293 cells after mitogenic stimulation.We found that EGF or HGF stimulation significantly increased p28~(GANK) report gene activity after the stimulation with different doses of either EGF or HGF.We also demonstrated that Ras activation could upregulate p28~(GANK) expression.Use of the PI3K inhibitor LY294002 abolishing the ability of growth factors and Ras activation induced PI3K-Akt cascade irritation and use of PD98059 or U0126 to inhibit ERK kinase cascade activation,we then approached p28~(GANK) expression by detection its promoter activation in HEK293 cells.The results show that PI3K inhibitor LY294002 significantly suppressed EGF or HGF induced p28~(GANK) reporter gene activities,while the ERK inhibitors PD98059 and U0126 show some enhancement instead of any inhibition.Reporter gene assays were performed on HEK293 ceils with introduced exogenousβ-catenin and c-Myc expression on p28~(GANK) expression.To our surprise,they both can significantly increased p28~(GANK) promoter activity.Furthermore,we found that theβ-cantenin/TCF could directly bind to the p28~(GANK) promoter in vivo.Then we asked whether p28~(GANK) expression could affectβ-catenin/TCF-dependent transcription.We found that introduced expression of p28~(GANK) induced activation ofβ-catenin/TCF-mediated transactivation in a concentration-dependent manner.Studies have revealed that elevating levels of wild-type p53 will downregulateβ-catenin in a variety of cell types.It was reported that p28~(GANK) could bind to Mdm2,facilitating p53-Mdm2 binding,and increased ubiquitylation and degradation of p53.To elucidate whether the effect dependent on p53,we detected theβ-catenin-OT activity in p53-negative Hep3B cells.The reporter activities were decreased with declined expression of p28~(GANK) and the RT-PCR assay also indicated that p28~(GANK) reduction resulted in a substantial decrease inβ-catenin-OT reporter activity which was accompanied by the downregulation of c-Myc,as well as cyclinD1.We assessed the correlation betweenβ-catenin signaling and the expression of p28~(GANK) in human primary hepatic turnouts.We found that there was a remarkable correlation between the activationβ-catenin signalling and the expression of p28~(GANK),c-Myc and cyclinD1,which indicated thatβ-catenin signaling-induced p28~(GANK) expression might be an important mechanism for promoting cell cycles in human liver cancers.In order to confirm the positive feedback regulating effect,we examined the effect of p28~(GANK) expression on its own promoter activity in HEK293 and Hep3B cells.To our prediction,p28~(GANK) significantly increased its own expression in HEK293 cells.After endogenous p28~(GANK) knockdown in Hep3B cells,p28~(GANK) promoter activities decreased.
In conclusion,we show that growth factors stimulation or Ras activation could upregulate p28~(GANK) expression through activationβ-catenin signalling.Inaddtion,β-cantenin was transcriptional activator of p28~(GANK) and the induced p28~(GANK) subsequently regulated theβ-canenin/TCF transcription activation.Thus the oncoprotein p28~(GANK) established a positive feedback loop inβ-catenin signaling and involved in tumorigenesis and progression in HCC.
PartⅡSIRPαNegatively Regulates Both TLR3 and Cytoplasmic Pathways in TypeⅠIFN Induction
Signal regulatory proteinα(SIRPα) was originally identified in rat cells by its association with cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase SHP-2 and was later shown to be highly conserved in other mammals,including human,mouse,and cattle.The cytoplasmic region of SIRPαcontains two immunoreceptor tyrosine-based inhibitory motifs(ITIMs) with four tyrosine residues that are phosphorylated in response to a variety of growth factors and ligand binding.This phosphorylation enables recruitment and activation of SHP-1 and SHP-2 that in turn dephosphorylates specific protein substrates involved in mediating various physiological effects.SIRPαis especially abundant in innate immune cells including macrophages and dendritic cells although it is also expressed in other cell types such as neurons and fibroblasts.
Host antiviral responses are initiated through the detection of viral components by host pattern recognition receptors(PRRs).Upon recognition,PRRs activation results in expression of typeⅠinterferon(IFN-α/β),IFN-stimulated genes,and inflammatory cytokines that suppress viral replication and facilitate the adaptive immune responses. Double-stranded RNA(dsRNA),which is produced during replication of many viruses,is one of the viral components recognized by several PRRs,including Toll-like receptor(TLR)3 and the RNA helicases,namely retinoic acid-inducible geneⅠ(RIG-Ⅰ) and melanoma-differentiation-associated gene 5(MDA5).It is accepted that TLR3 detects extra-cellular viral dsRNA internalized into the endosomes,whereas RIG-Ⅰ/MDA5 detects intracellular viral dsRNA.Engagement of these receptors triggers the rapid production of typeⅠIFN and thus activates the innate immune response against infectious virus.
SIRPαhas been shown to negatively or positively regulate MAPKs signaling initiated either by tyrosine kinase-coupled receptors for growth factors or by cell adhesion to extracellular matrix.Moreover,the expression of dominant negative form of SIRPαstimulates NF-κB activity and makes the cells resistant to TNF specific apoptosis.In our previousely study,we demonstrated the role of SIRPαin regulating TLR4-mediated signaling during innate immune responses.However,it is not known whether SIRPαis involved in regulating typeⅠIFN induction signaling.In the present study,we found that SIRPαexpression was lost after stimulation with polyriboinosinic:polyribocytidylic acid(poly(I:C)),a synthetic analogue of dsRNA. We also provide evidence that SIRPαfunctions as a specific inhibitor of dsRNA-activated signaling,transcription factor activation and gene induction in both the TLR3 and the cytoplasmic pathways in cells.Furthermore,the phosphorylation on the tyrosine residues of ITIM motifs in SIRPαand its binding to the p85 subunit of PI3K in a ligand-dependent fashion are required for its inhibitory function.Thus,we demonstrate an important function for SIRPαas an inhibitory protein in both TLR3 and cytoplasmic pathways in typeⅠIFN induction.
To explore the involvement of SIRPαin regulating the induction of typeⅠIFN,we examined SIRPαprotein and mRNA levels after poly(I:C) treatment in mouse macrophage cell line(RAW 264.7) as well as in mouse peritoneal washout macrophages(PWCs).The results show that poly(I:C) treatment triggered a dosage-dependent decline in expression of SIRPαin protein extracts from RAW 264.7 and PWCs.Moreover,poly(I:C) treatment at 5μg/ml resulted in a decline in expression of SIRPαafter 6 hr(2-fold by densitometry),and SIRPαremained lower than control levels after 24 hr.quantitative real-time PCR analysis showed that there was nearly a 1.5-fold reduction of SIRPαmRNA expression after 30 min of exposure to poly(I:C) and treatment.Stimulation of TLR3 with poly(I:C) leads to IRF3 phosphorylation on multiple phosphorylation acceptor(phospho-acceptor) sites.We therefore investigated the possible involvement of SIRPαin regulating the activity of IRF3.We first synthesized small interfering RNAs(siRNA) targeting mouse SIRPαin order to suppress the endogenous SIRPαexpression in PWCs.We also constructed a shRNA vector that specifically downregulated SIRPαand then stably transfected it into RAW264.7 macrophage cells.The transfections were referred to here as SIRPI-KD(knockdown),OV(overexpression) and VT(vector) macrophages respectively.In response to poly(I:C),SIRPα-siRNA macrophages exhibited an enhanced phosphorylation of IRF3 whereas cells transfected with negative-control oligonucleotides had a much lower IRF3 activation in PWCs.There was an enhanced phosphorylation of IRF3 in SIRPα-KD compared to VT treated with poly(I:C). Therefore,we examined the effects of knockdown of SIRPαexpression on the activation of reporter genes of the ISRE,PRDⅢ-Ⅰand PRDⅡ.We found that all reporter genes were strongly activated in SIRPα-KD macrophages as compared with SIRPα-VT cells.To examine whether IFN-βand IFN-βinducible gene expression was indeed suppressed by SIRPα,we stimulated with poly(I:C) and analyzed the expression of IFN-βand IFN-βinducible genes,including ifit-1(encoding IFIT-1), ccl-5(encoding RANTES) and mip-1α(encoding MIP-1α).As expected, poly(I:C)-induced expression of these genes were higher in SIRPα-KD macrophages, whereas overexpression of SIRPαresulted in a reduction of the expression of these genes.We also analyzed the production of IFN-β,TNF-αand IL-6 after poly(I:C) treatment.All the three cytokines were significantly enhanced in SIRPα-siRNA PWCs compared to negative-control PWCs,as measured by ELISA.To further investigate the direct involvement of SIRPαin virus-induced typeⅠIFN production, we examined the effects of SIRPαknockdown on MAPK,NF-κB and STAT1 pathways which are all required for cytokine production during TLR3 signaling. SIRPα-siRNA macrophages displayed enhanced phosphorylation of p38,JNK and ERK,whereas negative-control macrophages displayed impaired activation.Because the phosphorylation of IκBαwas essential for the activation of NF-κB,we then examined the phosphorylation status of IκBαand NF-κB activity in macrophages with SIRPαknockdown.There was enhanced phosphorylation of IκBαand NF-κB activity in SIRPα-KD macrophages after poly(I:C) stimulation,which was consistent with the higher NF-κB activity,responsible PRDⅡactivity in SIRPα-KD macrophages.Furthermore,tyrosine phosphorylation of STAT1 was also displayed enhanced phosphorylation in PWCs transfected with SIRPα-siRNA.
TypeⅠIFN and inflammatory cytokines are induced when poly(I:C) is administrated into the cytoplasm by lipofection.Notably,this induction is independent of TLR3 and dependent on RIG-Ⅰ/MDA5.To determine the effects of SIRPα,we used HEK293 cells lacking TLR3 expression.HEK293 cells were transfected with a SIRPαexpression plasmid or an empty plasmid and 24 hr later cells were re-transfected with poly(I:C) at different time points.Cells transfected with SIRPαexpression plasmids were found to exhibit decreased phosphorylation of IRF3 compared with cells transfected with empty plasmids.We next examined the effects of SIRPαon RIG-Ⅰ/MDA5-dependent gene induction at the level of promoter activation.We co-transfected cells with different doses of SIRPαexpression plasmids together with ISRE,PRDⅢ-Ⅰ,IFN-βor RANTES reporter genes for 24 hr and then re-transfected with poly(I:C) for another 6 hr.Consistent with the effect of SIRPαon RIG-Ⅰ/MDA5-dependent IRF3 activation,ISRE,PRDⅢ-Ⅰ,IFN-βand RANTES gene reporter activities were severely impaired in SIRPα-transfected cells in a dose-dependent manner.Impaired tyrosine-phosphorylated STAT1 was also observed in HEK293 cells transfected with SIRPαexpression plasmid.
To further investigate whether the inhibition of typeⅠIFN induction by SIRPαwas dependent on the tyrosine phosphorylation in its ITIM motif,we adopted a SIRPα-4Y expression plasmid.HEK293 cells were co-transfected with different doses of SIRPα-WT or SIRPα-4Y expression plasmids together with ISRE,PRDⅢ-Ⅰ, RANTES or IFN-βreporter genes and then transfected with poly(I:C).Only SIRPα-WT inhibited poly(I:C) transfection-induced activation of ISRE,PRDⅢ-Ⅰ, RANTES and IFN-βreporter genes,while the SIRPα-4Y exhibited no inhibitory effects.Activation of PI3K plays a key role in the recruitment and activation of a wide variety of lipid and protein kinase-signaling cascades.The activity of PI3K has also been reported to be essential for IRF3-induced gene induction by dsRNA.Hence we investigated whether SIRPαmediated its negative regulatory effects via coupling PI3K.The requirement of phosphoinositide 3-kinase(PI3K) activity for the induction of IFN-βand IFN-β-inducible genes by dsRNA is supported by the observation that a PI3K inhibitor failed to activate IFN-βand IFN-β-inducible gene expression.Moreover,the activity of PI3K downstream kinase AKT was downregulated in the SIRPα-overexpression RAW264.7 and HEK293 cells.Given the observations that SIRPαwas tyrosine-phosphorylated in response to dsRNA stimulation and PI3K was required for gene induction in the production of typeⅠIFN-β,we investigate whether PI3K could interact with SIRPα.Our results demonstrated that PI3K was recruited to SIRPαafter poly(I:C) stimulation, suggesting that the recruitment was dependent on the phosphorylation of the tyrosine residue in the cytoplasmic domain of SIRPα.
In conclusion,we have shown here that SIRPαplays a critically negative role in typeⅠIFN induction.SIRPαmay accomplish its inhibitory function in typeⅠIFN induction,in part,through its association and sequestration of the signal transducer PI3K.Therefore SIRPαmay be an extremely beneficial and potentially exploitable molecule to inhibit immune responses such as autoimmunity or to enhance the immune response during viral infections.
研究背景
肝细胞性肝癌(简称肝癌,Hepatocellular Carcinoma,HCC)是我国最常见的高发肿瘤之一,其发生发展是由体内多基因参与、多步骤协同的复杂过程。目前,尽管已经鉴定了一些肝癌相关的抑癌基因,如p53和p16~(INK4a),并明确了它们在肝癌中的特异性失活,但是在肝癌发生中特异性激活的癌基因却少见报道。2000年日本科学家Fujita等人应用消减杂交法从人肝细胞癌组织高表达的基因中筛选出一条编码重复ankyrin(ANK)序列的新基因,命名为gankyrin。我们检索基因库后发现gankyrin基因与1998年Hori等人发现的p28(Nas6p)基因序列完全一致,遂将其命名为p28~(GANK)。癌基因p28~(GANK)编码的蛋白是人26S蛋白酶体(26S proteasome)调节亚单位19S/PA700复合物的一种非ATP酶亚基,其核酸序列中含有5个串联排列的ANK重复单位,其保守的ankyrin序列介导蛋白与蛋白间的相互作用。26S蛋白酶体中的20S蛋白小体是降解体内某些错误折叠蛋白或细胞周期蛋白的场所,目前尚不清楚癌蛋白p28~(GANK)在26S蛋白酶体降解蛋白过程中的具体分子作用机制。前期研究发现癌蛋白p28~(GANK)参与CDK4/CyclinD1/p16~(INK4a)/Rb1/E2F-1信号传导通路的调节,此通路的失调控在肝癌的发生发展过程中具有相当重要的作用;外源性转入癌基因p28~(GANK)使其高表达后的NIH/3T3细胞株可以使裸鼠荷瘤。Nagao等人通过酵母双杂交发现黑色素瘤抗原MAGE-A4能与癌蛋白p28~(GANK)特异性的结合并抑制p28~(GANK)成瘤活性。
β-连环蛋白(β-catenin)是一种重要的肿瘤相关基因,除了在细胞粘附中有重要作用外,还是Wnt信号通路的重要功能分子。通过在不同时间、空间上的差异表达调节一系列下游基因c-myc和cyclin-d1等的表达水平,参与细胞增殖、凋亡以及器官发育。近来研究发现,β-catenin在肝癌发生早期呈现异常聚集,并能够促进肝癌的发生以及早期肝癌细胞的快速生长。因此探讨β-catenin在肝癌发生发展中的作用具有重要价值。已有研究表明提高野生型p53水平可以明显下调β-catenin蛋白水平。结合新近研究发现p28~(GANK)能与泛素蛋白连接酶MDM2相互作用,介导野生型p53蛋白的泛素化及降解,提示在肝细胞癌的发生发展中可能存在着癌蛋白p28~(GANK)与β-catenin的相互联系。因此我们探讨了Wnt/β-catenin信号通路激活对p28~(GANK)表达的影响以及p28~(GANK)对β-catenin信号通路的调节作用。
实验方法:
1.采用Genomatix在线分析软件分析p28~(GANK)启动子区并构建p28~(GANK)报告基因质粒;
2.运用双荧光素酶报告基因系统检测生长因子刺激对p28~(GANK)转录活性的影响;采用瞬时转染方法观察Ras对p28~(GANK)转录活性的影响;应用报告基因系统结合ERK与PI3K信号通路抑制剂确定生长因子与Ras对p28~(GANK)表达的调节通路;采用瞬时转染β-catenin和c-Myc表达质粒确定它们对p28~(GANK)表达的影响;
3.通过Luciferase报告基因质粒pGL-OT(含有β-catenin/TCF4复合物特异结合区)与不同剂量p28~(GANK)质粒细胞共转染实验,确定p28~(GANK)对β-catenin/TCF4转录活性的影响;
4.运用western blot及核浆分离技术,检测HEK293细胞瞬转p28~(GANK)后,β-catenin在胞核胞浆中的分布变化;
5.运用western blot和转染技术研究生长因子、Ras、AKT、β-catenin、c-Myc对p28~(GANK)蛋白表达调节作用;应用RT-PCR技术研究生长因子、Ras、AKT、β-catenin、c-Myc等对p28~(GANK)表达的调节作用;
6.运用染色质免疫共沉淀(ChIP)技术研究β-catenin和c-Myc与28~(GANK)启动子区相互作用关系;
7.应用腺病毒介导的RNA干扰技术下调表达肝癌细胞系中p28~(GANK)的水平,探讨其对β-catenin转录活性的作用,并进一步研究了p28~(GANK)对自身转录水平的影响。
8.应用临床肝癌组织标本应用western blot和免疫组织化学方法检测β-catenin、c-Myc、cyclinD1表达水平与p28~(GANK)蛋白表达水平之间的关系。
结果:
1.肝细胞生长因子和表皮生长因子刺激明显上调饥饿处理后的HEK293和HepG2细胞中p28~(GANK)表达水平;瞬时转染Ras表达质粒显著上调p28~(GANK)mRNA和蛋白水平;
2.PI3K抑制剂处理显著抑制细胞本底水平p28~(GANK)的表达,也能够阻断生长因子或Ras激活对p28~(GANK)表达的上调作用;瞬时转染PI3Kp85质粒和野生或持续激活的AKT表达质粒均能够上调p28~(GANK)的表达水平;
3.报告基因、RT-PCR或western blot实验证明瞬时转染表达β-catenin和c-Myc都能够在转录和蛋白水平上调p28~(GANK)表达;染色质免疫沉淀实验发现β-catenin可以与p28~(GANK)启动子区相互结合;
4.HEK293细胞瞬时转染p28~(GANK)质粒可显著增强β-catenin/TCF4转录活性,而在HepG2、3B中抑制p28~(GANK)表达则可显著降低其转录活性;
5.HEK293细胞中瞬时转染p28~(GANK)表达质粒可提高β-catenin入核蛋白水平,并在一定程度上稳定细胞中β-catenin总蛋白水平;腺病毒介导的干扰p28~(GANK)表达明显降低β-catenin蛋白水平,从而抑制了其下游的c-Myc和cyclinD1的表达水平;
6.Western blot和免疫组化实验分析表明p28~(GANK)的蛋白水平在肝癌组织样品中与β-catenin、c-Myc和cyclinD1呈同向性表达;
7.在HEK293和Hep3B中p28~(GANK)正向调节自身表达。
结论:
生长因子刺激或Ras激活可以上调肝癌癌蛋白p28~(GANK)的表达,这种激活作用可能是通过激活PI3K-AKT通路完成的。β-catenin和c-Myc的上调表达都能够促进p28~(GANK)的表达,并且染色质免疫沉淀实验证实β-catenin/TCF4可以与p28~(GANK)启动子直接结合。进一步实验表明上调的p28~(GANK)可以反过来进一步促进β-catenin蛋白的稳定从而上调β-catenin/TCF4转录活性。在肝癌组织样品中p28~(GANK)的蛋白水平与β-catenin、c-Myc和cyclinD1呈同向性表达。上述结果支持我们提出的p28~(GANK)正反馈调节β-catenin信号通路的结论。
第二部分信号调节蛋白α负性调节Ⅰ型干扰素产生的作用和机制研究
研究背景
信号调节蛋白α(signal regulatory proteinα,SIRPα)是一种广泛存在的抑制性受体,属于免疫球蛋白受体超家族。其胞外区具有三个Ig样结构域和多个糖基化位点,胞内区带有免疫受体酪氨酸抑制基元(ITIMs),其中含四个酪氨酸残基,被磷酸化后能够募集含SH2结构域的蛋白磷酸酶SHP-1和SHP-2。
干扰素(interferon IFN)是一类广泛表达具有潜在抗病毒感染的细胞因子。但是过度失控的诱导产生IFN还将易导致自身免疫性疾病的发生。所以,对于IFN表达系统的调控对于维持机体内稳态相当重要。IFN的诱导产生依赖于机体模式识别(pattern recognition receptors PRRs)受体的激活。天然免疫系统识别病毒双链RNA可以通过两条途径:TOLL样受体(toll like receptors,TLR)3和胞浆途径(RIG-I/MDA5)。TLR3作为一种重要的模式识别受体,主要表达于巨噬细胞和树突状细胞内涵体内,能够识别细胞吞噬进入的dsRNA。RIG-I/MDA5能够识别病毒复制过程中产生的dsRNA。TLR3和RIG-I/MDA5都能够与dsRNA的类似物poly(I:C)(polyriboinosinic:polyribocytidylic acid)结合,从而介导Ⅰ型IFN产生。本课题主要探讨了SIRPα对poly(I:C)活化巨噬细胞的调节作用和机制。
SIRPα在免疫细胞只选择性地表达在巨噬细胞和树突状细胞等非淋巴细胞。我们研究发现poly(I:C)刺激巨噬细胞SIRPα蛋白表达水平下降,提示该蛋白对巨噬细胞激活及Ⅰ型IFN产生可能存在抑制作用。为进一步了解该蛋白在天然免疫功能中的作用和机制,本课题通过转染并筛选克隆建立稳定高低表达该蛋白的巨噬细胞系及瞬时干扰小鼠体内分离的巨噬细胞中的SIRPα表达,探讨了SIRPα对poly(I:C)诱导的小鼠巨噬细胞活化的影响,全面探讨了SIRPα对TLR3和胞浆途径介导的Ⅰ型IFN产生的调节作用;并探讨SIRPα对巨噬细胞MAPKs信号分子,分泌细胞因子的影响,并进一步探讨了SIRPα发挥上述作用的可能机制,确定了SIRPα在Ⅰ型干扰素诱导产生中的重要调节作用,并为尚未阐明的抑制性受体家族胞内信号转导机制提供新的线索。
实验方法
1.建立稳定转染空载体,高表达Myc-SIRPα及稳定干扰SIRPα的巨噬细胞系:RAW-VT,RAW-OV,RAW-KD,设计,合成并鉴定具有干扰效果的stealthRNA,分别干扰SIRPα,和SHP-2;
2.采用磷酸化特异性抗体检测不同SIRPα表达对poly(I:C)刺激后p38,JNK,ERK,IRF3及AKT磷酸化水平的变化的影响来反映SIRPα对通路活化的影响;
3.运用荧光素酶报告基因系统检测SIRPα对ISRE、PRDⅡ、PRDⅢ-Ⅰ、RANTES和NF-κB转录活性的作用;
4.采用Real-time PCR方法检测sirpa基因转录水平变化和RT-PCR检测ifn-β及其相关诱导基因变化;
5.通过ELISA检测细胞因子变化;
6.采用免疫沉淀的方法确定与SIRPα相互作用的蛋白。
7.采用瞬时干扰SHP-2后检测其对巨噬细胞释放细胞因子和对信号通路磷酸化的影响确定这个蛋白在巨噬细胞激活中的作用。
8.采用免疫共沉淀的方法检测SIRPα不同表达是否影响SHP-2和下游相互作用蛋白的结合。
结果
1.Poly(I:C)刺激后对SIRPα表达的影响:不同的小鼠巨噬细胞系和小鼠体内分离的巨噬细胞给予不同剂量的poly(I:C)和不同时程的poly(I:C)刺激后均可见SIRPα表达明显下调,Real-time PCR结果表明poly(I:C)引起的SIRPα表达下调主要是转录水平的下调。
2.SIRPα对poly(I:C)刺激后IRF3磷酸化及IFN相关转录因子活性的影响:研究发现SIRPα高表达可显著抑制poly(I:C)诱导的巨噬细胞细胞IRF3磷酸化水平,并抑制IFN相关转录因子结合位点PRDⅡ、PRDⅢ-Ⅰ及ISRE活性。SIRPα低表达则可显著提高巨噬细胞IRF3磷酸化水平和IFN相关转录因子活性。
3.SIRPα对TLR3激活的IFN-β及诱导基因表达的影响:报告基因,PCR分析和细胞因子测定结果表明,SIRPα对TLR3激活的Trif通路诱导IFN-β及其诱导基因的表达和其他促炎因子都有明显的抑制作用。
4.SIRPα对poly(I:C)刺激后的MAPK、NF-κB和JAK-STAT信号途径的作用:SIRPα低表达可显著提高poly(I:C)引起的MAPK信号通路的激活,ERK、JNK和p38磷酸化水平都显著增强。SIRPα低表达组IκBα磷酸化增强,NF-kB活性明显高于对照组。SIRPα表达抑制组巨噬细胞STAT1磷酸化水平显著高于对照组,iNOS活性及诱导产生的NO水平明显高于对照组。
5.SIRPα对poly(I:C)激活的胞浆信号途径影响:HEK293细胞中瞬时表达SIRPα可显著抑制瞬转poly(I:C)引起的IRF3的磷酸化水平;SIRPα高表达显著抑制瞬转poly(I:C)引起的IFN-β的表达;HEK293中SIRPα表达显著抑制poly(I:C)引起STAT1磷酸化水平;RT-PCR分析表明SIRPα高表达显著抑制瞬转poly(I:C)引起的IFN-β及其诱导基因的转录。
6.SIRPα的抑制作用依赖于ITIM基序的磷酸化及与SHP2的结合:报告基因分析表明不同剂量SIRPα表达不能抑制Trif或TBK1激活的IFN-β表达。SIRPαITIM突变体不能抑制poly(I:C)引起的IFN-β的表达,证明这种抑制作用依赖于ITIM基序的磷酸化并可能有SHP2的参与。
7.SIRPα对TLR3和胞浆途径IFN-β表达的影响机制:P13K抑制剂可显著抑制TLR3和胞浆途径介导的ISRE活性及IFN-β表达水平。说明PI3K信号途径可能在IFN表达中有正性调节作用。SIRPα的表达可抑制TLR3和胞浆途径介导的poly(I:C)引起AKT的磷酸化水平。免疫共沉淀实验发现SIRPα可以与PI3Kp85亚基形成复合体,并且这种结合是依赖于poly(I:C)刺激的。所以SIRPα发挥抑制作用可能正是通过磷酸化后募集PI3K,引起AKT不完全活化,这直接导致了IRF3的磷酸化水平降低,从而减少了IFN-β的表达。
结论
本研究从分子水平全面深入研究了信号调节蛋白SIRPα对TLR3和胞浆途径介导的Ⅰ型干扰素表达的调节作用,发现SIRPα对poly(I:C)诱导的Ⅰ型干扰素及其它细胞因子分泌等生物学行为具有负向调控能力,详细探讨了其影响相关信号转导通路的作用机制,证明其可能部分通过抑制PI3K-AKT信号通路调节作用而发挥负向调控作用。
PartⅠThe oncoprotein p28~(GANK) establishes a positive feedback loop inβ-catenin signalling
Hepatocelluar carcinoma(HCC) is one of the most prevalent malignant cancers in our country.The development and progression of HCC is a complicated process involving multiple genes and multiple steps in human bodies.Up to now,though some suppressors in HCC were identified such as p53 and p16~(INK4a),and confirmed their specific inactivation,few specific activated oncogenes in HCC was found and reported.In 2000,a novel gene named gankyrin with repeated sequences coding "ankyrin" motif was cloned in human HCC by Japanese scientists Fujita and his colleagues using subtractive hybridization method.We found this gankyrin gene was identical to the p28(Nas6p) gene reported by Hori in 1998 by searching the GenBank data,thus named it as p28~(GANK) in our work.
p28~(GANK)(also known as PSMD10,p28,Nas6p,and Gankyrin),a non-ATPase subunit of the 19S/PA700 complex from the 26S proteasome,contains five ankyrin repeats mediating proteins interaction.20S proteasome is a protease degrading some incorrectly folding proteins or cell cycle regulators.Little is known about the mechanism how oncoprotein p28~(GANK) relates with proteins degradation by 26S proteasome,p28~(GANK) regulates the CDK4/CyclinD1/p16~(INK4a)/Rb1/E2F-1 pathway which connects with tumor development and progression,and p28~(GANK)-transfected cells are tumorigenic in nude mice.Recently,Nagao et al has identified MAGE-A4 binds to gankyrin and suppresses its oncogenic activity through yeast two-hybrid screen,p28~(GANK) also directly binds to MDM2,which increases p53-MDM2 association,inducing the ubiquitylation and degradation of p53.
Theβ-catenin signalling pathway has a critical role in cell-fate determination,tissue homeostasis and tumorigenesis.In unstimulated cells,the cytoplasmic concentration ofβ-catenin is kept very low owing to phosphorylation by CK1 and GSK3βkinases. Activation of the Wnt pathway blocksβ-catenin degradation so thatβ-catenin accumulates and translocates to the nucleus,where it interacts with T-cell factor (TCF)/lymphoid enhancer factor(LEF) transcription factors.About 50%-70%of all HCC examined showed an abnormalβ-catenin protein accumulation in the cytoplasm and nucleus.However,in HCC theβ-catenin mutation rate is 13%-26%,the Axin mutation rate is 5%-10%and rare mutation of APC has been reported so far.This led us to speculate that other factors might be involved in the regulatingβ-catenin in HCC.
To investigate the factors which might influence p28~(GANK) expression,we cloned a 1226 bp fragment of the 5'-flanking region of human p28~(GANK) gene into pGL3 luciferase reporter vector,which analyzed using Gene2Promoter(Genomatix) online software.To elucidate whether p28~(GANK) upregulation was correlated with cell cycle progression in normal or HCC cell lines,we examined the potential cell-cycle-dependence of p28~(GANK) expression in HepG2 and HEK293 cells after mitogenic stimulation.We found that EGF or HGF stimulation significantly increased p28~(GANK) report gene activity after the stimulation with different doses of either EGF or HGF.We also demonstrated that Ras activation could upregulate p28~(GANK) expression.Use of the PI3K inhibitor LY294002 abolishing the ability of growth factors and Ras activation induced PI3K-Akt cascade irritation and use of PD98059 or U0126 to inhibit ERK kinase cascade activation,we then approached p28~(GANK) expression by detection its promoter activation in HEK293 cells.The results show that PI3K inhibitor LY294002 significantly suppressed EGF or HGF induced p28~(GANK) reporter gene activities,while the ERK inhibitors PD98059 and U0126 show some enhancement instead of any inhibition.Reporter gene assays were performed on HEK293 ceils with introduced exogenousβ-catenin and c-Myc expression on p28~(GANK) expression.To our surprise,they both can significantly increased p28~(GANK) promoter activity.Furthermore,we found that theβ-cantenin/TCF could directly bind to the p28~(GANK) promoter in vivo.Then we asked whether p28~(GANK) expression could affectβ-catenin/TCF-dependent transcription.We found that introduced expression of p28~(GANK) induced activation ofβ-catenin/TCF-mediated transactivation in a concentration-dependent manner.Studies have revealed that elevating levels of wild-type p53 will downregulateβ-catenin in a variety of cell types.It was reported that p28~(GANK) could bind to Mdm2,facilitating p53-Mdm2 binding,and increased ubiquitylation and degradation of p53.To elucidate whether the effect dependent on p53,we detected theβ-catenin-OT activity in p53-negative Hep3B cells.The reporter activities were decreased with declined expression of p28~(GANK) and the RT-PCR assay also indicated that p28~(GANK) reduction resulted in a substantial decrease inβ-catenin-OT reporter activity which was accompanied by the downregulation of c-Myc,as well as cyclinD1.We assessed the correlation betweenβ-catenin signaling and the expression of p28~(GANK) in human primary hepatic turnouts.We found that there was a remarkable correlation between the activationβ-catenin signalling and the expression of p28~(GANK),c-Myc and cyclinD1,which indicated thatβ-catenin signaling-induced p28~(GANK) expression might be an important mechanism for promoting cell cycles in human liver cancers.In order to confirm the positive feedback regulating effect,we examined the effect of p28~(GANK) expression on its own promoter activity in HEK293 and Hep3B cells.To our prediction,p28~(GANK) significantly increased its own expression in HEK293 cells.After endogenous p28~(GANK) knockdown in Hep3B cells,p28~(GANK) promoter activities decreased.
In conclusion,we show that growth factors stimulation or Ras activation could upregulate p28~(GANK) expression through activationβ-catenin signalling.Inaddtion,β-cantenin was transcriptional activator of p28~(GANK) and the induced p28~(GANK) subsequently regulated theβ-canenin/TCF transcription activation.Thus the oncoprotein p28~(GANK) established a positive feedback loop inβ-catenin signaling and involved in tumorigenesis and progression in HCC.
PartⅡSIRPαNegatively Regulates Both TLR3 and Cytoplasmic Pathways in TypeⅠIFN Induction
Signal regulatory proteinα(SIRPα) was originally identified in rat cells by its association with cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase SHP-2 and was later shown to be highly conserved in other mammals,including human,mouse,and cattle.The cytoplasmic region of SIRPαcontains two immunoreceptor tyrosine-based inhibitory motifs(ITIMs) with four tyrosine residues that are phosphorylated in response to a variety of growth factors and ligand binding.This phosphorylation enables recruitment and activation of SHP-1 and SHP-2 that in turn dephosphorylates specific protein substrates involved in mediating various physiological effects.SIRPαis especially abundant in innate immune cells including macrophages and dendritic cells although it is also expressed in other cell types such as neurons and fibroblasts.
Host antiviral responses are initiated through the detection of viral components by host pattern recognition receptors(PRRs).Upon recognition,PRRs activation results in expression of typeⅠinterferon(IFN-α/β),IFN-stimulated genes,and inflammatory cytokines that suppress viral replication and facilitate the adaptive immune responses. Double-stranded RNA(dsRNA),which is produced during replication of many viruses,is one of the viral components recognized by several PRRs,including Toll-like receptor(TLR)3 and the RNA helicases,namely retinoic acid-inducible geneⅠ(RIG-Ⅰ) and melanoma-differentiation-associated gene 5(MDA5).It is accepted that TLR3 detects extra-cellular viral dsRNA internalized into the endosomes,whereas RIG-Ⅰ/MDA5 detects intracellular viral dsRNA.Engagement of these receptors triggers the rapid production of typeⅠIFN and thus activates the innate immune response against infectious virus.
SIRPαhas been shown to negatively or positively regulate MAPKs signaling initiated either by tyrosine kinase-coupled receptors for growth factors or by cell adhesion to extracellular matrix.Moreover,the expression of dominant negative form of SIRPαstimulates NF-κB activity and makes the cells resistant to TNF specific apoptosis.In our previousely study,we demonstrated the role of SIRPαin regulating TLR4-mediated signaling during innate immune responses.However,it is not known whether SIRPαis involved in regulating typeⅠIFN induction signaling.In the present study,we found that SIRPαexpression was lost after stimulation with polyriboinosinic:polyribocytidylic acid(poly(I:C)),a synthetic analogue of dsRNA. We also provide evidence that SIRPαfunctions as a specific inhibitor of dsRNA-activated signaling,transcription factor activation and gene induction in both the TLR3 and the cytoplasmic pathways in cells.Furthermore,the phosphorylation on the tyrosine residues of ITIM motifs in SIRPαand its binding to the p85 subunit of PI3K in a ligand-dependent fashion are required for its inhibitory function.Thus,we demonstrate an important function for SIRPαas an inhibitory protein in both TLR3 and cytoplasmic pathways in typeⅠIFN induction.
To explore the involvement of SIRPαin regulating the induction of typeⅠIFN,we examined SIRPαprotein and mRNA levels after poly(I:C) treatment in mouse macrophage cell line(RAW 264.7) as well as in mouse peritoneal washout macrophages(PWCs).The results show that poly(I:C) treatment triggered a dosage-dependent decline in expression of SIRPαin protein extracts from RAW 264.7 and PWCs.Moreover,poly(I:C) treatment at 5μg/ml resulted in a decline in expression of SIRPαafter 6 hr(2-fold by densitometry),and SIRPαremained lower than control levels after 24 hr.quantitative real-time PCR analysis showed that there was nearly a 1.5-fold reduction of SIRPαmRNA expression after 30 min of exposure to poly(I:C) and treatment.Stimulation of TLR3 with poly(I:C) leads to IRF3 phosphorylation on multiple phosphorylation acceptor(phospho-acceptor) sites.We therefore investigated the possible involvement of SIRPαin regulating the activity of IRF3.We first synthesized small interfering RNAs(siRNA) targeting mouse SIRPαin order to suppress the endogenous SIRPαexpression in PWCs.We also constructed a shRNA vector that specifically downregulated SIRPαand then stably transfected it into RAW264.7 macrophage cells.The transfections were referred to here as SIRPI-KD(knockdown),OV(overexpression) and VT(vector) macrophages respectively.In response to poly(I:C),SIRPα-siRNA macrophages exhibited an enhanced phosphorylation of IRF3 whereas cells transfected with negative-control oligonucleotides had a much lower IRF3 activation in PWCs.There was an enhanced phosphorylation of IRF3 in SIRPα-KD compared to VT treated with poly(I:C). Therefore,we examined the effects of knockdown of SIRPαexpression on the activation of reporter genes of the ISRE,PRDⅢ-Ⅰand PRDⅡ.We found that all reporter genes were strongly activated in SIRPα-KD macrophages as compared with SIRPα-VT cells.To examine whether IFN-βand IFN-βinducible gene expression was indeed suppressed by SIRPα,we stimulated with poly(I:C) and analyzed the expression of IFN-βand IFN-βinducible genes,including ifit-1(encoding IFIT-1), ccl-5(encoding RANTES) and mip-1α(encoding MIP-1α).As expected, poly(I:C)-induced expression of these genes were higher in SIRPα-KD macrophages, whereas overexpression of SIRPαresulted in a reduction of the expression of these genes.We also analyzed the production of IFN-β,TNF-αand IL-6 after poly(I:C) treatment.All the three cytokines were significantly enhanced in SIRPα-siRNA PWCs compared to negative-control PWCs,as measured by ELISA.To further investigate the direct involvement of SIRPαin virus-induced typeⅠIFN production, we examined the effects of SIRPαknockdown on MAPK,NF-κB and STAT1 pathways which are all required for cytokine production during TLR3 signaling. SIRPα-siRNA macrophages displayed enhanced phosphorylation of p38,JNK and ERK,whereas negative-control macrophages displayed impaired activation.Because the phosphorylation of IκBαwas essential for the activation of NF-κB,we then examined the phosphorylation status of IκBαand NF-κB activity in macrophages with SIRPαknockdown.There was enhanced phosphorylation of IκBαand NF-κB activity in SIRPα-KD macrophages after poly(I:C) stimulation,which was consistent with the higher NF-κB activity,responsible PRDⅡactivity in SIRPα-KD macrophages.Furthermore,tyrosine phosphorylation of STAT1 was also displayed enhanced phosphorylation in PWCs transfected with SIRPα-siRNA.
TypeⅠIFN and inflammatory cytokines are induced when poly(I:C) is administrated into the cytoplasm by lipofection.Notably,this induction is independent of TLR3 and dependent on RIG-Ⅰ/MDA5.To determine the effects of SIRPα,we used HEK293 cells lacking TLR3 expression.HEK293 cells were transfected with a SIRPαexpression plasmid or an empty plasmid and 24 hr later cells were re-transfected with poly(I:C) at different time points.Cells transfected with SIRPαexpression plasmids were found to exhibit decreased phosphorylation of IRF3 compared with cells transfected with empty plasmids.We next examined the effects of SIRPαon RIG-Ⅰ/MDA5-dependent gene induction at the level of promoter activation.We co-transfected cells with different doses of SIRPαexpression plasmids together with ISRE,PRDⅢ-Ⅰ,IFN-βor RANTES reporter genes for 24 hr and then re-transfected with poly(I:C) for another 6 hr.Consistent with the effect of SIRPαon RIG-Ⅰ/MDA5-dependent IRF3 activation,ISRE,PRDⅢ-Ⅰ,IFN-βand RANTES gene reporter activities were severely impaired in SIRPα-transfected cells in a dose-dependent manner.Impaired tyrosine-phosphorylated STAT1 was also observed in HEK293 cells transfected with SIRPαexpression plasmid.
To further investigate whether the inhibition of typeⅠIFN induction by SIRPαwas dependent on the tyrosine phosphorylation in its ITIM motif,we adopted a SIRPα-4Y expression plasmid.HEK293 cells were co-transfected with different doses of SIRPα-WT or SIRPα-4Y expression plasmids together with ISRE,PRDⅢ-Ⅰ, RANTES or IFN-βreporter genes and then transfected with poly(I:C).Only SIRPα-WT inhibited poly(I:C) transfection-induced activation of ISRE,PRDⅢ-Ⅰ, RANTES and IFN-βreporter genes,while the SIRPα-4Y exhibited no inhibitory effects.Activation of PI3K plays a key role in the recruitment and activation of a wide variety of lipid and protein kinase-signaling cascades.The activity of PI3K has also been reported to be essential for IRF3-induced gene induction by dsRNA.Hence we investigated whether SIRPαmediated its negative regulatory effects via coupling PI3K.The requirement of phosphoinositide 3-kinase(PI3K) activity for the induction of IFN-βand IFN-β-inducible genes by dsRNA is supported by the observation that a PI3K inhibitor failed to activate IFN-βand IFN-β-inducible gene expression.Moreover,the activity of PI3K downstream kinase AKT was downregulated in the SIRPα-overexpression RAW264.7 and HEK293 cells.Given the observations that SIRPαwas tyrosine-phosphorylated in response to dsRNA stimulation and PI3K was required for gene induction in the production of typeⅠIFN-β,we investigate whether PI3K could interact with SIRPα.Our results demonstrated that PI3K was recruited to SIRPαafter poly(I:C) stimulation, suggesting that the recruitment was dependent on the phosphorylation of the tyrosine residue in the cytoplasmic domain of SIRPα.
In conclusion,we have shown here that SIRPαplays a critically negative role in typeⅠIFN induction.SIRPαmay accomplish its inhibitory function in typeⅠIFN induction,in part,through its association and sequestration of the signal transducer PI3K.Therefore SIRPαmay be an extremely beneficial and potentially exploitable molecule to inhibit immune responses such as autoimmunity or to enhance the immune response during viral infections.
引文
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