STAT3 抑制第一型干擾素反應機轉的研究

The suppressive effect of STAT3 on type I interferon-mediated response

鄭子珮(Tzu-Pei Cheng)

指導教授 ： 李建國

國立臺灣大學/醫學院/免疫學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00518

摘要

第一型干擾素 (type I IFN) 對於抗病毒扮演非常重要的角色，它可以活化訊號傳導與轉錄子 (STAT) 1, STAT2 和 STAT3。第一型干擾素會藉由 STAT1 及 STAT2 來正向調控抗病毒蛋白產生。而 STAT3 在第一型干擾素訊號傳遞中所扮演的角色還不是很清楚，直到最近發現缺乏 STAT3 的細胞在第一型干擾素刺激下會增加促進干擾素誘導基因 (ISG) 的表現以及增強抗病毒的能力，這顯示 STAT3 會負調控第一型干擾素所引起的抗病毒能力。然而其詳細的機制還需進一步研究。我們利用放回 STAT1, STAT3, 以及 STAT1 和 STAT3 同時放回的 STAT1/STAT3 雙基因剔除鼠胚胎纖維母細胞株 (MEFs) 來研究 STAT3 負調控的機制。我們發現放回的 STAT1 及 STAT3 蛋白的表現量與正常小鼠胚胎纖維母細胞株相似，此外利用反轉錄及時聚合酶鏈式反應 (RT-QPCR) 和基因表現微陣列分析 (microarray) 發現同時放回 STAT1 和 STAT3 與只放回 STAT1的小鼠胚胎纖維母細胞株相比，STAT3 確實可以抑制第一型干擾素誘導STAT1下游基因的產生及抗病毒反應。我們更進一步利用染色質免疫沉澱法 (ChIP assay) 證明在給予第一型干擾素下，STAT3 會阻礙干擾素促進因子3複合物 (IFN-stimulated gene factor 3 complex, ISGF3) 被引導到位於 MDA5 及 IFIT1的干擾素反應片段 (ISRE) 上。由於 STAT3 的 N 端 (胺基酸 1 到 134) 足以造成抑制作用，因此我們進一步釐清 STAT3 中兩個可能被乙醯化位於 49 和 87 的離胺酸 (lysine) 是否參與抑制作用。將 STAT3 上這兩個位置單獨或同時由離胺酸突變為精胺酸 (Arginine) 會阻礙 STAT3 的促進轉錄的能力。然而只將位於 49 的胺基酸由離胺酸換成精胺酸或兩個都換成精胺酸則會抑制 STAT3 負調控第一型干擾素的能力。有趣的是突變為精胺酸的 STAT3 無法被第一型干擾素誘導乙醯化。綜合以上結果顯示 STAT3 會藉由阻斷促進干擾素因子 3 複合物被引導到促進干擾素因子的啟動子 (promoter) 而影響第一型干擾素的反應。而其詳細之機制則可能與 STAT3 N- 端第 49 及 87 兩個精胺酸的乙醯化有關係。

關鍵字

訊號傳導與轉錄子3 ； 干擾素 ； 微陣列分析

並列摘要

Type I IFNs, activating STAT1, STAT2, and STAT3, are crucial for antiviral response. Stimulation of type I IFN induces the production of antiviral proteins in a STAT1- and STAT2-dependent manner. However, the function of STAT3 in type I IFN response was not completely understood. Recently, STAT3 was reported to negatively regulate type I IFN-mediated antiviral response. Cells lacking STAT3 displayed enhanced ISGs expression and antiviral activity upon IFN stimulation. However, the detailed mechanism remains elusive. To investigate the underlying mechanism, we used STAT1 and STAT3 double knockout (DKO) MEFs that had been restored with STAT1, STAT3, or both molecules. STAT1 or STAT3 was expressed in the DKO MEFs at a level comparable to WT MEFs. Using expression microarray and RT-QPCR, we also found that in STAT1/STAT3-restored MEFs, STAT3 could negatively regulate STAT1-dependent type I IFN-mediated gene induction and antiviral response as compared with STAT1-restored MEFs. Furthermore, using ChIP assay we showed that STAT3 impeded the recruitment of ISGF3 complex to ISRE of MDA5 and IFIT1, two IFN-stimulated genes (ISGs), following type I IFN stimulation. Since NH2-terminal domain (amino acids 1 to 134) of STAT3 was able to confer suppressive effect, we further dissect the involvement of two potential acetylation sites 49K and 87K in suppressive activating ofSTAT3. Single or double K to R mutations of these two sites in STAT3 blocked its transactivation ability. However, only K49R or RR mutants inhibited the suppressive effect of STAT3 on type I IFN response. Interestingly, IFNα-dependent acetylation of STAT3 was abolished in RR mutant STAT3. Together, these results indicate that STAT3 may affect type I IFN response by blocking the recruitment of ISGF3 complex to the promoter of ISGs. Acetylation of K49 and K87 in NTD of STAT3 may be involved in the negative regulator of the IFN response.

並列關鍵字

STAT3 ； IFN ； microarray

參考文獻

Akira, S., Nishio, Y., Inoue, M., Wang, X.J., Wei, S., Matsusaka, T., Yoshida, K., Sudo, T., Naruto, M., and Kishimoto, T. (1994). Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell 77, 63-71.

Antunes, F., Marg, A., and Vinkemeier, U. (2011). STAT1 signaling is not regulated by a phosphorylation-acetylation switch. Mol. Cell. Biol. 31, 3029-3037.

Bach, E.A., Aguet, M., and Schreiber, R.D. (1997). The IFN gamma receptor: a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. 15, 563-591.

Baechler, E.C., Batliwalla, F.M., Karypis, G., Gaffney, P.M., Ortmann, W.A., Espe, K.J., Shark, K.B., Grande, W.J., Hughes, K.M., Kapur, V., et al. (2003). Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc. Natl. Acad. Sci. U. S. A. 100, 2610-2615.

Biron, C.A. (2001). Interferons alpha and beta as immune regulators--a new look. Immunity 14, 661-664.