PKR及MAPK在雙股RNA誘發基因表現及細胞凋亡的角色

摘要前言︰雙股RNA為RNA病毒感染時的中間產物，已知會活化PKR及MAPK，調控基因表現及細胞凋亡，但MAPK的活化和PKR的關係尚未完全明瞭，此外雙股RNA如何引發細胞凋亡的詳細機制尚不清楚，有可能和PKR活化導致蛋白質合成受抑制有關，此外粒線體路徑在雙股RNA引發細胞凋亡的貢獻，以及相關促細胞凋亡分子 (如: Bax) 如何活化，其分子機制尚未完全清楚。研究目的︰利用poly-IC分析雙股RNA誘發基因表現以及細胞凋亡的可能機制。探討JNK在雙股RNA誘發細胞凋亡的角色，及JNK與PKR之間的關係，粒線體路徑在雙股RNA誘發細胞凋亡的貢獻，以及相關促細胞凋亡分子如何活化。實驗方法︰利用MAPK及PKR的抑制劑以及RT-PCR分析抗病毒反應相關基因表現的調控機制。利用HO33342及propidium iodide (PI) 染劑以正立螢光顯微鏡觀察細胞存活與死亡。利用染色體萃取技術及洋菜膠水平電泳分析DNA片段化的情形。利用蛋白質電泳分析MAPK磷酸化及caspase 3切割。利用細胞質與粒線體分離技術及蛋白質電泳分析cytochrome c 及Bax的分佈情形。結果：poly-IC所誘發IP-10、PKR、OAS1等基因表現可被p38及NF-kB、PKR抑制劑SB203580、MG132、2-AP所抑制。poly-IC可誘發JNK及Bax活化、cytochrome c的釋出、pro-caspase 3的切割、DNA片段化情形，分別可被PKR及JNK抑制劑 (SP600125)、粒線體保護劑 (TAT-BH4) 所抑制。蛋白質合成抑制劑 cycloheximide能增強poly-IC所誘發JNK磷酸化及細胞凋亡，PKR抑制劑則能降低poly-IC所誘發JNK磷酸化。結論：poly-IC所誘發的基因表現主要是透過PKR、NF-kB、p38之協同所調控。poly-IC引起的細胞死亡方式包含細胞凋亡及細胞壞死，與實際病毒感染可能有所不同。poly-IC誘發caspase 3活化是由PKR-JNK及粒線體的細胞凋亡路徑所調控。JNK的活化可能和poly-IC造成PKR活化，引發蛋白質合成被抑制有關。由於JNK及Bax活化皆較早，JNK可能透過調控Bax的轉移達到調控poly-IC所誘發的細胞凋亡，因此Bax在啟動粒腺體細胞凋亡路徑中扮演重要角色。此外poly-IC所誘發抗病毒基因及細胞凋亡是兩種互不相容的反應，由此可見p38和JNK相對的活化情形可作為雙股RNA引發抗病毒基因表現或是細胞凋亡的指標。

關鍵字

雙股RNA ；細胞凋亡；基因表現；訊息傳遞

並列摘要

Abstract Background: It is known that double-stranded RNA (dsRNA), a replication intermediate of several viruses, causes activation of PKR and MAPK which are involved in the induction of gene expression and apoptosis. The relationship of PKR and MAPK is not completely clear. Whether activation of apoptotic pathway is caused by the inhibition of protein synthesis, the contribution of mitochondrial pathways, and regulation of pro-apoptotic molecules are still unclear. Aims of study: (1) Using polyinosinic: polycytidylic acid (poly-IC) to study the possible mechanisms of dsRNA-induced gene expression and apoptosis. (2) To study the roles of JNK, PKR and their relationship in dsRNA-induced apoptosis. (3) To study the contribution of mitochondrial pathway and regulation of related molecules in poly-IC-induced apoptosis. Methods: Poly-IC-induced gene expression was analyzed by RT-PCR using inhibitors for MAPK and PKR. Cell viability was observed with fluorescent microcopy using HO33342 and propidium iodide staining. DNA fragmentation was analyzed by extraction of chromosomal DNA and detected on agarose electrophoresis. Western blot was used to analyze phosphorylation of MAPK and cleavage of pro-caspase 3. The distribution of cytochrome c and Bax was analyzed by Western blot using cytosolic and mitochondrial fractions of protein extracts. Results: Poly-IC-induced expression of IP-10, PKR and OAS1 was suppressed by the inhibitors of p38 (SB203580), NF-kB (MG132) and PKR (2-AP). Poly-IC caused activation of JNK, translocation of Bax and release of cytochrome c from mitochondria, cleavage of pro-caspase 3, which were suppressed by the inhibitors of PKR (2-AP), JNK (SP600125), and protected by TAT-BH4. The inhibitor of protein synthesis cycloheximide enhanced poly-IC-induced JNK phosphorylation and apoptosis. On the other hand, poly-IC-induced JNK phosphorylation was suppressed by the inhibitor of PKR. Conclusions: Poly-IC-induced gene expression is mainly regulated by PKR, NF-kB and p38. Poly-IC caused apoptosis and necrosis, which may be different from that induced by viral infection. Poly-IC-induced activation of caspase 3 is mainly regulated by PKR, JNK and mitochondrial apoptotic pathway. Poly-IC-induced JNK phosphorylation is correlated with the inhibition of protein synthesis during PKR activation. Poly-IC-induced apoptosis is mediated by JNK probably through activation of Bax. Thus Bax may be a key molecule in the activation of mitochondrial apoptotic pathway. Since induction of gene expression and apoptosis are mutually exclusive responses, which are respectively regulated by p38 and JNK. Thus, during poly:IC-induced apoptosis activated JNK may be inhibitory to p38.

並列關鍵字

double-stranded RNA ； apoptosis ； gene expression ； signal transduction

參考文獻

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PKR及MAPK在雙股RNA誘發基因表現及細胞凋亡的角色

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