核受体PPARγ和FXR的抗炎机制研究
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摘要
炎症是机体为了抵抗外界病原物质的浸入或者细胞损伤物质而产生的保护性应答反应，由此导致一系列复杂的炎症调节作用，并最终达到组织结构或功能的恢复。而核受体与炎症因子基因启动子区中DNA特异位点的直接结合，于转录水平上实现对炎症因子信号激活引起的一系列反应应答调控作用。核受体是一个结构特异的配体依赖性转录因子超家族，与目的基因的启动子DNA序列特异性结合，对维持细胞稳态及各种反应的蛋白分子表达从转录水平上起着调控作用。而本论文则主要以核受体PPARγ与FXR为研究对象，以窥核受体在炎症反应中抗炎作用的分子机制。PPARγ方面主要通过以LPS诱导的急性肺损伤小鼠模型（野生型小鼠及A2AR基因敲除鼠）、LPS诱导的小鼠单核巨噬炎症模型细胞Raw264.7为研究对象，对炎症模型中PPARγ对腺受体A2AR表达调控分子机制研究为主探讨PPARγ抗炎的分子机制。而FXR方面，则在实验室以前研究的基础上，以在炎症反应各个过程中均占有重要地位的血管内皮细胞，以细胞系EA.hy926为模型，探讨了FXR对血栓调节蛋白TM表达调控分子机制，以此丰富FXR在抗炎活动中的机制。
本论文以LPS刺激建立ALI小鼠模型，通过HE染色、免疫组化分析中性粒细胞浸润程度等手段检测ALI小鼠肺组织损伤程度及炎症的严重程度，并采用小鼠单核巨噬细胞系细胞Raw264.7判断激活PPARγ或A2AR后，对炎症的抑制作用。Real-time、Western Blot等常规手段也被用于检测炎症因子mRNA表达水平、PPARγ或A2AR表达水平变化。在此基础上，还采用基因表达调控研究中常见的载体构建技术、置换点突变技术、EMSA、ChIP实验等探索PPARγ-A2AR正反馈环路中两分子上调的分子机制。通过以上实验技术从体内/体外水平上证实了PPARγ-A2AR正反馈环路存在，并证实了此环路具有抑炎作用，进而从基因表达调控的角度阐述了此环路存在所依赖的分子机制，揭示了PPARγ通过与A2AR基因启动子区DR10PPRE(-218~-197)位点直接结合上调A2AR基因的转录水平从而上调A2AR蛋白的表达，而A2AR的激活继而引发了A2AR-PKA通路的激活，导致在此通路中磷酸化的CREB与PPARγ启动子区+4~+11CRE-like位点的直接结合作用，从而上调PPARγ转录活性并上调PPARγ蛋白表达上调依次完成PPARγ-A2AR正反馈环路且进一步完成在炎症反应中的抑制作用。因而以PPARγ与A2AR的正反馈环路为例探讨了在生命活动调控作用中均占据重要地位的核受体超家族与G蛋白偶联受体蛋白家族之间的联系，并为以急性肺损伤为例的病症中抗炎治疗提供了一个新的策略。
本论文的第二部分则集中于FXR对血栓调节蛋白TM的上调分子机制研究。本室以前的研究已明确在血管内皮细胞中，激活FXR可明显上调TM分子mRNA及蛋白表达水平，同时也可明显增强TM分子活化，本论文则同样以上述通过基因表达调控实验技术：荧光素酶检测实验、置换点突变实验、EMSA实验以及ChIP实验在以前研究的基础上，明确了激活的FXR可通过直接结合于TM分子启动子区IR8位点增强TM基因启动子活性，并上调TM转录水平，从而达到上调TM表达的作用。这为FXR与TM在抑炎作用、抗纤维化以及心血管疾病的治疗中，提供了新的思路。
Inflammation is a protective host response to external challenge or cellular injury thatleads to the activation of a complex array of inflammatory mediators, finalizing therestoration of tissue structure and function[1]. An important general mechanism responsible forthis activity is referred to as transrepression, in which nuclear receptors interfere withsignal-dependent activation of inflammatory response genes through protein-proteininteractions with coregulatory proteins and promoter-bound transcription factors, rather thandirect, sequence-specific interactions with DNA. Nuclear receptors comprise a superfamily ofstructurally conserved, ligand-dependent transcription factors that regulate diverse aspects ofdevelopment and homeostasis by both positively and negatively regulating gene expression.Our studies foucused on two classical NRs, PPARγ and FXR, employing the LPS-inducedAcute lung injury mice （wild-type and A2AR KO mice）, LPS-induced murine macrophagesRaw264.7cells and HUVECs, discussed the underlying mechaniasms of regulation of thepotential inhibitor A2AR and TM respectively in anti-inflammation effection.
In order to investigate the interaction between PPARγ and A2AR, we carried out ourstudies as followed:
1. PPARγ activation attenuate lung damages of mice with ALI in an A2AR-dependentmanner.
2. A2AR deficiency blocks the inhibitory effect of PPARγ activation on inflammatorycytokine expression.
3. PPARγ agonist upregulate A2AR expression in lung tissues of mice with ALI andLPS-stimulated murine macrophages.
4. A2AR activation positively regulates PPARγ mRNA and protein expressions in aPKA-dependent manner.
In above parties, comparisons between histopathological evaluation, immunofluorescence analysis and test of lung water content of lung tissues of wild-type mice and A2AR geneknockout mice, which were treated with or without agonist and antagonist of A2AR orPPARγ showed that PPARγ activation attenuate lung damages of mice with ALI in anA2AR-dependent manner, while activation of A2AR could obviously prevent the inhibitionof LPS effect on the expression of PPARγ. Real-time, Western blot assays were employedto demonstrate the positive expression loop between PPARγ and A2AR in vivo and in vitro.
5. PPARγ activation enhances the transcriptional activation of A2AR promoter.
6. PPARγ directly targets A2AR in the A2AR promoter region.
7. CREB, the downstream of PKA, directly mediates the A2AR-induced upregulationof PPARγ.
In above parties, Luciferase reporter assays, point mutation technology, EMSA andChIP assays were showed that PPARγ activation enhance the transcriptional activation ofA2AR via a DR10PPRE in-218to-197sites in A2AR promoter region in murinemacrophages. And CREB, phosphorylated in A2AR-PKA-CREB pathway as a downstreamtranscription factor, directly mediated the A2AR-induced upregulation of PPARγ by bindingto a CRE-like site locating in+4to+11sites in PPARγ promoter region.
8. Combined treatment of PPARγ agonist ROSI and A2AR agonist CGS21680exertsbetter protective effect than the single administration of them.
In the above part, we used immunofluorescence to detecte the infliltration ofneutrophil in the lung tissues of LPS-induced ALI mice. And the combined treatement ofROSI and CGS21680achieved a better protective effect than the single administration ofthem.
Taken together, our findings suggest PPARγ and A2AR form a positive feedback loopto inhibit inflammation and attenuate lung damages in ALI. This transcriptional regulationof PPARγ and A2AR appears to be critical in controlling their endogenous ligand’s (such as15d-PGJ2to PPARγ while adenosine to A2AR) response under pathophysiologicalconditions. In addition, it provides novel evidence for understanding the molecular basis ofPPARγ and A2AR expressions and their functions in ALI, as well as the action of the tworeceptors-related drugs for ALI.
For another, we focused our studies on the regulation of FXR effect onThrombomdulin (TM), which is a significant mediator molecules expressed on vascular endothelial cells. Endothelial cells (ECs) dysfunction induced a variety of diseases such asinflammation, vascular diseases, fibrosis, thrombosis and even spontaneous metastasis[21-22].
In the studies of Li. et al., we have already demonstrated that activation of FXR by itsnatural ligand CDCA or synthetic ligand GW4064lead to both a significant up-regulationof TM expression and an increase of TM activity. Furthermore, FXR induces TMexpression maybe though directly binding to the IR8FXRE sites in the promoter region ofTM. Then we performed directly sites mutant, electrophoretic mobility shift assay (EMSA)and ChIP assays to inquire into the underlying mechanisms of the regulation and clearifiedthis transcriptional regulation of TM appears to be achieved via a novel IR8FXRE in thepromoter region of TM. Additionally, it provides novel angel to investigate FXR and TMfunctions in the inflammation, fibrosis, and other vascular diseases.
本论文以LPS刺激建立ALI小鼠模型，通过HE染色、免疫组化分析中性粒细胞浸润程度等手段检测ALI小鼠肺组织损伤程度及炎症的严重程度，并采用小鼠单核巨噬细胞系细胞Raw264.7判断激活PPARγ或A2AR后，对炎症的抑制作用。Real-time、Western Blot等常规手段也被用于检测炎症因子mRNA表达水平、PPARγ或A2AR表达水平变化。在此基础上，还采用基因表达调控研究中常见的载体构建技术、置换点突变技术、EMSA、ChIP实验等探索PPARγ-A2AR正反馈环路中两分子上调的分子机制。通过以上实验技术从体内/体外水平上证实了PPARγ-A2AR正反馈环路存在，并证实了此环路具有抑炎作用，进而从基因表达调控的角度阐述了此环路存在所依赖的分子机制，揭示了PPARγ通过与A2AR基因启动子区DR10PPRE(-218~-197)位点直接结合上调A2AR基因的转录水平从而上调A2AR蛋白的表达，而A2AR的激活继而引发了A2AR-PKA通路的激活，导致在此通路中磷酸化的CREB与PPARγ启动子区+4~+11CRE-like位点的直接结合作用，从而上调PPARγ转录活性并上调PPARγ蛋白表达上调依次完成PPARγ-A2AR正反馈环路且进一步完成在炎症反应中的抑制作用。因而以PPARγ与A2AR的正反馈环路为例探讨了在生命活动调控作用中均占据重要地位的核受体超家族与G蛋白偶联受体蛋白家族之间的联系，并为以急性肺损伤为例的病症中抗炎治疗提供了一个新的策略。
本论文的第二部分则集中于FXR对血栓调节蛋白TM的上调分子机制研究。本室以前的研究已明确在血管内皮细胞中，激活FXR可明显上调TM分子mRNA及蛋白表达水平，同时也可明显增强TM分子活化，本论文则同样以上述通过基因表达调控实验技术：荧光素酶检测实验、置换点突变实验、EMSA实验以及ChIP实验在以前研究的基础上，明确了激活的FXR可通过直接结合于TM分子启动子区IR8位点增强TM基因启动子活性，并上调TM转录水平，从而达到上调TM表达的作用。这为FXR与TM在抑炎作用、抗纤维化以及心血管疾病的治疗中，提供了新的思路。
Inflammation is a protective host response to external challenge or cellular injury thatleads to the activation of a complex array of inflammatory mediators, finalizing therestoration of tissue structure and function[1]. An important general mechanism responsible forthis activity is referred to as transrepression, in which nuclear receptors interfere withsignal-dependent activation of inflammatory response genes through protein-proteininteractions with coregulatory proteins and promoter-bound transcription factors, rather thandirect, sequence-specific interactions with DNA. Nuclear receptors comprise a superfamily ofstructurally conserved, ligand-dependent transcription factors that regulate diverse aspects ofdevelopment and homeostasis by both positively and negatively regulating gene expression.Our studies foucused on two classical NRs, PPARγ and FXR, employing the LPS-inducedAcute lung injury mice （wild-type and A2AR KO mice）, LPS-induced murine macrophagesRaw264.7cells and HUVECs, discussed the underlying mechaniasms of regulation of thepotential inhibitor A2AR and TM respectively in anti-inflammation effection.
In order to investigate the interaction between PPARγ and A2AR, we carried out ourstudies as followed:
1. PPARγ activation attenuate lung damages of mice with ALI in an A2AR-dependentmanner.
2. A2AR deficiency blocks the inhibitory effect of PPARγ activation on inflammatorycytokine expression.
3. PPARγ agonist upregulate A2AR expression in lung tissues of mice with ALI andLPS-stimulated murine macrophages.
4. A2AR activation positively regulates PPARγ mRNA and protein expressions in aPKA-dependent manner.
In above parties, comparisons between histopathological evaluation, immunofluorescence analysis and test of lung water content of lung tissues of wild-type mice and A2AR geneknockout mice, which were treated with or without agonist and antagonist of A2AR orPPARγ showed that PPARγ activation attenuate lung damages of mice with ALI in anA2AR-dependent manner, while activation of A2AR could obviously prevent the inhibitionof LPS effect on the expression of PPARγ. Real-time, Western blot assays were employedto demonstrate the positive expression loop between PPARγ and A2AR in vivo and in vitro.
5. PPARγ activation enhances the transcriptional activation of A2AR promoter.
6. PPARγ directly targets A2AR in the A2AR promoter region.
7. CREB, the downstream of PKA, directly mediates the A2AR-induced upregulationof PPARγ.
In above parties, Luciferase reporter assays, point mutation technology, EMSA andChIP assays were showed that PPARγ activation enhance the transcriptional activation ofA2AR via a DR10PPRE in-218to-197sites in A2AR promoter region in murinemacrophages. And CREB, phosphorylated in A2AR-PKA-CREB pathway as a downstreamtranscription factor, directly mediated the A2AR-induced upregulation of PPARγ by bindingto a CRE-like site locating in+4to+11sites in PPARγ promoter region.
8. Combined treatment of PPARγ agonist ROSI and A2AR agonist CGS21680exertsbetter protective effect than the single administration of them.
In the above part, we used immunofluorescence to detecte the infliltration ofneutrophil in the lung tissues of LPS-induced ALI mice. And the combined treatement ofROSI and CGS21680achieved a better protective effect than the single administration ofthem.
Taken together, our findings suggest PPARγ and A2AR form a positive feedback loopto inhibit inflammation and attenuate lung damages in ALI. This transcriptional regulationof PPARγ and A2AR appears to be critical in controlling their endogenous ligand’s (such as15d-PGJ2to PPARγ while adenosine to A2AR) response under pathophysiologicalconditions. In addition, it provides novel evidence for understanding the molecular basis ofPPARγ and A2AR expressions and their functions in ALI, as well as the action of the tworeceptors-related drugs for ALI.
For another, we focused our studies on the regulation of FXR effect onThrombomdulin (TM), which is a significant mediator molecules expressed on vascular endothelial cells. Endothelial cells (ECs) dysfunction induced a variety of diseases such asinflammation, vascular diseases, fibrosis, thrombosis and even spontaneous metastasis[21-22].
In the studies of Li. et al., we have already demonstrated that activation of FXR by itsnatural ligand CDCA or synthetic ligand GW4064lead to both a significant up-regulationof TM expression and an increase of TM activity. Furthermore, FXR induces TMexpression maybe though directly binding to the IR8FXRE sites in the promoter region ofTM. Then we performed directly sites mutant, electrophoretic mobility shift assay (EMSA)and ChIP assays to inquire into the underlying mechanisms of the regulation and clearifiedthis transcriptional regulation of TM appears to be achieved via a novel IR8FXRE in thepromoter region of TM. Additionally, it provides novel angel to investigate FXR and TMfunctions in the inflammation, fibrosis, and other vascular diseases.
引文
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1. Lingling Chen and Gordon G.Carmichael.2010. Long noncoding RNAs in mammaliancells:What, Where and Why? Wires RNA.1,2-21.
2. Carninci P, Yasuda J, Hayashizaki Y.2008. Multifaceted mammalian transcriptome.Curr Opin Cell Biol.20,274–280.
3. Bartel DP.2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell.116,281–297.
4. Carthew RW, Sontheimer EJ.2009. Origins and Mechanisms of miRNAs and siRNAs.Cell.136,642–655.
5. Aravin AA, Hannon GJ, Brennecke J.2007. The PiwipiRNA pathway provides anadaptive defense in the transposon arms race. Science.318,761–764.
6. Okazaki Y, Furuno M, Kasukawa T, Adachi J, Bono H, et al. Analysis of the mousetranscriptome based on functional annotation of60,770full-length cDNAs. Nature2002,420:563–573.
7. Bertone P, Stolc V, Royce TE, Rozowsky JS, Urban AE, et al. Global identification ofhuman transcribed sequences with genome tiling arrays. Science.2004,306:2242–2246.
8. Birney E, Stamatoyannopoulos JA,Dutta A, Guigo R, Gingeras TR, et al. Identificationand analysis of functional elements in1%of the human genome by the ENCODE pilotproject. Nature2007,447:799–816.
9. Kapranov P, Cheng J, Dike S,Nix DA, Duttagupta R, et al. RNA maps reveal new RNAclasses and a possible function for pervasive transcription. Science2007,316:1484–1488.
10. Dechao Bu, Kuntao Yu, Silong Sun, Chaoyong Xie, Geir Skogerb, Ruoyu Miao, HuiXiao, Qi Liao, Haitao Luo, Guoguang Zhao, Haitao Zhao, Zhiyong Liu, Changning Liu,Runsheng Chen, and Yi Zhao.2011.NONCODE v3.0: integrative annotation of longnoncoding RNAs. Nucleic Acids Research.40, D210-215.
11. Jia,H., Osak,M., Bogu,G.K., Stanton,L.W., Johnson,R. and Lipovich,L.2010. Genome-wide computational identification and manual annotation of human long noncodingRNA genes. RNA.16,1478–1487.
12. Prensner,J.R., Iyer,M.K., Balbin,O.A., Dhanasekaran,S.M.,Cao,Q., Brenner,J.C., Laxman,B.,Asangani,I.A., Grasso,C.S. and Kominsky,H.D.2011.Transcriptome sequencing across aprostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in diseaseprogression. Nat. Biotechnol.,29,742–749.
13. Cabili,M.N., Trapnell,C., Goff,L., Koziol,M., Tazon-Vega,B., Regev,A. and Rinn,J.L.2011. Integrative annotation of human large intergenic noncoding RNAs reveals globalproperties and specific subclasses. Genes Dev.,25,1915–1927.
14. Taft,R.J., Pang,K.C., Mercer,T.R., Dinger,M. and Mattick,J.S.2010. Non-coding RNAs:regulators of disease. J. Pathol.,220,126–139.
15. Mercer,T.R., Dinger,M.E. and Mattick,J.S.2009. Long non-coding RNAs: insights intofunctions. Nat. Rev. Genet.,10,155–159.
16. Banfai, B., Jia, H., Khatun, J., Wood, E., Risk, B., Gundling, W. E., Jr et al.2012. Longnoncoding RNAs are rarely translated in two human cell lines. Genome Res.22,1646–1657.
17. Sleutels, F., Zwart, R.&Barlow, D. P.2002. The non-coding Air RNA is required forsilencing autosomal imprinted genes. Nature,415,810–813.
18. Pandey, R. R., Mondal, T., Mohammad, F., Enroth, S., Redrup, L., Komorowski, J. et al.2008.
19. Kcnq1ot1antisense noncoding RNA mediates lineage-specific transcriptional silencingthrough chromatin-level regulation. Mol. Cell,32,232–246.
20. Vallot, C., Huret, C., Lesecque, Y., Resch, A.,Oudrhiri, N., Bennaceur-Griscelli, A. et al.2013. XACT, a long noncoding transcript coating the active X chromosome in humanpluripotent cells. Nat. Genet.45,239–241.
21. Irina V. Novikova, Scott P. Hennelly, Chang-Shung Tung and Karissa Y. Sanbonmatsu.2013. Rise of the RNA Machines: Exploring the Structure of Long Non-Coding RNAs.JMB.
22. Xu, B., Yang,W.H.,Gerin, I., Hu,C.D.,Hammer,G.D.&Koenig, R. J.2009. Dax-1andsteroid receptor RNA activator (SRA) function as transcriptional coactivators forsteroidogenic factor1in steroidogenesis. Mol. Cell. Biol.29,1719–1734.
23. Colley, S. M.&Leedman, P. J.2009. SRA and its binding partners: an expanding rolefor RNAbinding coregulators in nuclear receptor-mediated gene regulation. Crit. Rev.Biochem. Mol. Biol.44,25–33.
24. Yao, H., Brick, K., Evrard, Y., Xiao, T., Camerini-Otero, R. D.&Felsenfeld, G.2010.Mediation of CTCF transcriptional insulation by DEAD-box RNAbinding protein p68and steroid receptor RNA activator SRA. Genes Dev.24,2543–2555.
25. Lucks, J. B., Mortimer, S. A., Trapnell, C., Luo, S., Aviran, S., Schroth, G. P. et al.2011.Multiplexed RNA structure characterization with selective2′-hydroxyl acylationanalyzed by primer extension sequencing (SHAPE-Seq). Proc. Natl Acad. Sci. USA,108,11063–11068.
26. Chris P. Ponting, Peter L. Oliver, and Wolf Reik.2009.Evolution and functions of longnoncoding RNAs. Cell.136,629-641.
27. Mercer, T.R., Dinger, M.E., Sunkin, S.M., Mehler, M.F., and Mattick, J.S.2008.Specific expression of long noncoding RNAs in the mouse brain. Proc. Natl. Acad. Sci.USA105,716–721.
28. Babak, T., Blencowe, B.J., and Hughes, T.R.2007. Considerations in the identificationof functional RNA structural elements in genomic alignments.BMC Bioinformatics8,33.
29. Kapranov, P., Cheng, J., Dike, S., Nix, D.A., Duttagupta, R., Willingham, A.T., Stadler, P.F.,Hertel, J., Hackermuller, J., Hofacker, I.L., et al.2007. RNA maps reveal new RNA classesand a possible function for pervasive transcription.Science316,1484–1488.
30. Goodrich, J.A., and Kugel, J.F.2006. Non-coding-RNA regulators of RNA polymeraseII transcription. Nat. Rev. Mol. Cell Biol.7,612–616.
31. Carninci, P., Kasukawa, T., Katayama, S., Gough, J., Frith, M.C., Maeda, N.,Oyama, R.,Ravasi, T., Lenhard, B., Wells, C., et al.2005. The transcriptional landscape of themammalian genome. Science309,1559–1563.
32. Conley, A.B., Miller, W.J., and Jordan, I.K.2008. Human cis natural antisensetranscripts initiated by transposable elements. Trends Genet.24,53–56.
33. Katayama, S.,Tomaru, Y.,Kasukawa, T.,Waki, K.,Nakanishi, M.,Nakamura, M.,Nishida,H., Yap, C.C., Suzuki, M.,Kawai, J., et al.2005. Antisense transcription in themammalian transcriptome. Science309,1564–1566.
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