Hypomorphic function of STAT2 in ENU mutaginized mice and the crosstalk between STAT2 and TLR signaling

第一型干擾素在宿主先天性免疫系統中對抗病毒或細菌感染是重要的細胞激素。動物遺傳實驗證據顯示，第一型干擾素的訊息傳遞主要是透過STAT1和STAT2蛋白來傳遞。透過篩選中研院基因突變鼠動物模式核心實驗室所提供的突變鼠，我們找到了一隻對於第一型干擾素反應出現問題的突變鼠。這隻對於第一型干擾素之所以會有低反應的老鼠的主要原因，因為有一個T到A的點突變發生在STAT2基因上的4～5內含子（Intron）的剪接供體（Splicing donor），進而造成STAT2蛋白表現嚴重地減少。利用低功能STAT2蛋白的突變老鼠和細胞，我們研究STAT2在干擾素與類鐸受體（TLR）交互訊息傳遞中扮演的角色。首先，利用轉錄與轉譯抑制物（actinomycin D and cycloheximide）去加到STAT2突變的細胞上，我們證明STAT2蛋白過低表現不是因為訊息RNA或蛋白質穩定度的降低所造成。此外，STAT2突變鼠與細胞對於腦心肌炎病毒（EMCV）感染也具有高感受性，此現象與STAT2剔除老鼠相似。再者，STAT2突變老鼠與細胞在加入類鐸受體配合體（ligand）刺激之後，呈現出的發炎反應也是降低的，此證據暗示STAT2在與類鐸受體交互訊息傳遞中扮演了正向調控的角色。為了證明STAT2突變老鼠的表現型的研究是因為受到STAT2基因的點突變所造成，而非其他基因的突變所造成，我們在STAT2突變的纖維細胞株中放回老鼠與人類的STAT2。結果顯示兩種STAT2都可以使突變細胞回復第一型干擾素反應。總言，我們找到了另一種STAT2突變的老鼠，這突變鼠的結果，同樣支持STAT2在抗病毒反應上的重要性。我們也同時發現STAT2可以正向調控類鐸受體的訊息傳遞，但機制有待更進一步研究。Type I interferons (IFNs) are critical cytokines for innate immunity to protect host from viral and bacterial infections. Genetic evidence suggests that the action of type I IFN is mainly mediated by its downstream signal mediators STAT1 and STAT2. From screening the ENU-mutagenized mice generated in the Mouse Mutagenesis Program Core Facility at Academia Sinica, we have identified a mutant mouse displaying impaired type I IFN responses. The hyporesponsiveness of IFNα/β in the mutant mouse is due to a T to A point mutation in the splicing donor of intron 4~5 of STAT2 gene, leading to dramatically reduced production of full length STAT2 protein. Using STAT2 mutant mice and cells, we investigate the hypomorphic functions of STAT2 and its role in the crosstalk between IFNs and TLR signaling. First, by treating cells with actinomycin D or cycloheximide, we confirm that the impaired production of STAT2 protein is not due to reduced stability of its mRNA or protein. In addition, the STAT2 mutant mice and cells were highly susceptible to EMCV infection, which is similar to what has been reported in STAT2KO mice. Furthermore, STAT2 mutant mice and cells display diminished inflammatory response after TLR ligands, suggesting that STAT2 may crosstalk with stimulation of TLR signaling and play a positive regulator. To confirm that the hypomorphic phenotypes of mutant mice are due to the point mutation in STAT2 gene but not mutations else where, we restore a functional mouse and human STAT2 gene in STAT2 mutant embryonic fibroblasts. Both STAT2 genes are able to rescue type I IFN responses. Taken together, we have found another mutant allele of STAT2 to support the importance of STAT2 in antiviral responses. We also demonstrate that STAT2 can positively regulate TLR signaling by which mechanisms need to be further clarified.Chapter I Introduction................................................................................................ 1art 1 background............................................................................................................ 1.1 Interferons and IFN receptors................................................................................ 1.2 JAK-STAT family................................................................................................... 1.3 Type I and type II IFN signaling............................................................................ 2.4 Role of STAT2 in type I IFN signaling.................................................................. 3.5 Antiviral effects of IFN.......................................................................................... 4.6 TLR signaling......................................................................................................... 4art 2 Rationales and objectives...................................................................................... 5hapter II Materials and Methods.............................................................................. 8.1 Mice and cells........................................................................................................ 8.2 Isolation of mRNA, preparation of cDNA and QPCR......................................... 10.3 Construction of MigR1-muST2 and MigR1-huST2............................................ 12.4 Transfection and retroviral delivery..................................................................... 13.5 Nonsense-mediated mRNA decay assay.............................................................. 14.6 Electroporation...................................................................................................... 14.7 Western blotting................................................................................................... 15.8 FACS analysis...................................................................................................... 15.9 Antiviral state assay.............................................................................................. 16.10 D-galactosamine plus R848 survival assay.......................................................... 16.11 Encephalomyocarditis virus (EMCV) survival assay........................................... 16.12 Northern blotting.................................................................................................. 17hapter III Results.................................................................................................... 18.1 STAT2 mutant mice are hypomorphic but not null mutant.................................. 18.2 Reduced expression of STAT2 protein in the mutant mic is not due to decreased mRNA stability.............................................................................................................. 19.3 Reduced expression of STAT2 protein in mutant mice is not due to decreased stability of STAT2 protein............................................................................................. 20.4 STAT2 mutant mice and cells are extremely susceptible to viral infection in vivo and in vitro..................................................................................................................... 21.5 Crosstalk of TLR and JAK-STAT signaling......................................................... 22.6 Activation of STAT1 is normal in the mutant splenocytes in response to IFNα... 23.7 Restoration of STAT2 rescues IFNα response in STAT2 mutant cells................. 24hapter IV Discussion.............................................................................................. 27igures........................................................................................................................... 30igure 1. Reduced IFNα responses in STAT2 mutant splenocytes................................ 31igure 2. Hypothetic stop codons formation in STAT2 mutant cells............................. 32igure 3. Comparable half-life of STAT2 mRNA in WT and STAT2 mutnat splenocytes..................................................................................................................... 33igure 4. Comparable level of STAT2 mRNA in WT and STAT2 mutant splenocytes..................................................................................................................... 34igure 5. Stability of STAT2 protein is comparable between WT and STAT2 mutant MEF............................................................................................................................... 35igure 6. Impaired antiviral responses to EMCV infection in STAT2 mutant MEF................................................................................................................................ 36igure 7. STAT2 mutant mice are highly susceptible to EMCV infection.................... 37igure 8. The expression profiles of different proinflammatory cytokines in STAT2 mutnat and WT splenocytes after TLR-ligand treatment............................................... 38igure 9. STAT2 mutant mice are more resistant to TLR ligand-induced toxic shock than WT and heterozygous mice................................................................................... 39igure 10. Comparable p-STAT1 expression in WT and STAT2 mutant cells after IFNα or IFNγ stimulation....................................................................................................... 40igure 11. Ectopic expression of mouse STAT2 in STAT2 mutant splenocytes........... 41igure 12. Transfection of MigR1, MigR1-huST2, and MigR1-muST2 in 293T cells................................................................................................................................ 42igure 13. Retroviral transduction of MigR1, MigR1-huST2, and MigR1-muST2 in STAT2 mutant MEFs..................................................................................................... 43igure 14. Induction of antiviral genes in restored STAT2 mutant MEFs by IFNα stimulation..................................................................................................................... 44eferences..................................................................................................................... 4