酪胺酸激脢抑制劑AG490於9L大鼠腦瘤細胞中引起葡萄糖調控蛋白78及94之誘發表現-透過引發細胞內鈣離子擾動及過氧化逆境壓力的機制

TYROSINE KINASE INHIBITOR AG490 INDUCES GRP78 AND GRP94 IN 9L RBT CELLS: INVOLVEMENT OF INTRACELLULAR CALCIUM DISTURBANCES AND OXIDATIVE STRESS

高睿君(Jui-Chun Kao)

指導教授 ： 黎耀基

國立清華大學/生命科學院/生物科技研究所/碩士(2006年)

摘要

過去我們實驗室發現以未達致死藥量的膠達納黴素(geldanamycin, GA)處理9L大鼠腦瘤細胞(rat brain tumour, RBT)會引發內質網逆境反應(ER stress), 並且伴隨活性氧物質(reactive oxygen species, ROS)的產生。然而GA對錯誤折疊蛋白質反應途徑(unfolded protein response, UPR)的影響卻仍未知。最近的研究結果顯示細胞內產生活性氧物質的過程中或是受外在物質過氧化氫(H2O2)的刺激下, JAK/STAT訊息傳導路徑容易被活化。在此篇研究中, 我們利用酪胺酸激脢Janus kinase-2 (JAK2)的專一抑制劑AG490來觀察其對GA引發內質網保護子蛋白GRP78的表現與內質網逆境下的訊號傳遞的機制有無影響。實驗方法是將處理GA或是AG490的9L大鼠腦瘤細胞以即時定量聚合酶連鎖反應(real-time quantitative RT-PCR)、西方墨點分析與同位素標定新合成蛋白質實驗來對葡萄糖調控蛋白78和94的信號核糖核酸(mRNA)及蛋白質做定量分析。在實驗中我們發現9L大鼠腦瘤細胞在受到AG490單處理下即特別地誘發內質網保護子葡萄糖調控蛋白78和94基因; 除此之外, 鈣離子螯和劑BAPTA-AM、粒腺體uniporter的抑制劑RR、抗氧化劑NAC和粒腺體通透性通道(PT pore)抑制劑CyA會降低AG490誘發葡萄糖調控蛋白78和94基因活化的現象; 因此, 我們推測AG490引起內質網逆境的機制牽涉了細胞內鈣離子擾動與氧化壓力的產生。並且絲氨酸/酥胺酸激脢(serine/threonine kinase)抑制劑H7、蛋白質激脢A (PKA)的抑制劑KT5720和對異構蛋白質激脢C (PKC)有專一抑制效果的Gö6983和Gö6976同樣可以抑制AG490誘發葡萄糖調控蛋白78和94基因的活化。然而Gö6983和Gö6976並未在GA誘發葡萄糖調控蛋白78和94的信號核糖核酸機制中有抑制效果; 因此我們推測AG490和GA在內質網逆境下所引發的訊號傳遞可能不同。簡言之, 在9L大鼠腦瘤細胞中, 內質網逆境誘導劑AG490會引起細胞內鈣離子的擾動與活氧性物質的產生, 並且透過典型蛋白質激脢C (cPKC)和PKA的參與以誘發葡萄糖調控蛋白78和94基因的活化。

關鍵字

9L大鼠腦瘤細胞 ； 葡萄糖調控蛋白78 ； 葡萄糖調控蛋白94

並列摘要

Previously, our group found that geldanamycin (GA) with sublethal dose provokes the ER stress in rat brain tumour 9L (9L RBT) cells, which followed by the generation of reactive oxygen species (ROS). However, the effect of GA on the unfolded protein response (UPR) signaling pathway remains unclear. Recently, activation of JAK/STAT pathway has been observed in response to generation of intracellular ROS and exogenous hydrogen peroxide (H2O2).
Here, we investigated the effect of AG490, the specific inhibitor of Janus kinase-2 (JAK2), on the expression of grp78 coding for ER stress protein and the mechanistic relationship of GA signaling to ER stress. The mRNA and protein level of grp78 and grp94 were examined by real-time quantitative RT-PCR, Western blotting analysis and metabolic labeling experiment in 9L RBT cells treated with GA or AG490. In this study, we firstly discovered that AG490 only could specifically transactivate the ER-resident molecular chaperones GRP78 and GRP94 in 9L RBT cells. In addition, calcium chelator BAPTA-AM, mitochondrial uniporter inhibitor ruthenium red (RR), antioxidant N-acetylcysteine (NAC), and the inhibitor of mitochondrial PT pore, cyclosporin A (CyA), abolished the grp78 and grp94 induction by AG490. Therefore, it suggests that intracellular calcium disturbances and oxidative stress are involved in AG490-induced ER stress.
Furthermore, serine/threonine kinase inhibitor H7, PKA inhibitor KT5720, and additional PKC isozyme-selective inhibitors including Gö6983 and Gö6976 could diminished the AG490-induced upregulation of grp78 and grp94 genes. However, the similar suppression effect of Gö6983 and Gö6976 was not observed on the GA-mediated induction of grp78 and grp94 mRNA. Thus, we suggest that the signaling pathway of AG490-induced ER stress response might different from GA. In conclusion, AG490, as an ER stress inducer, might evoke intracellular calcium disturbances and generation of ROS, which lead to activation of cPKC and PKA for upregulation of grp78 and grp94 genes in 9L RBT cells.

參考文獻

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