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。

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.

中文摘要..........................................................................1
ABSTRACT.....................................................................2
ABBERATIONS..............................................................4
INTRODUCTION............................................................6
EXPERIMENTAL PROCEDURES..............................10
RESULTS......................................................................14
DISCUSSION................................................................20
FIGURE LEGENDS......................................................23
FUGURES......................................................................26
REFERENCES...............................................................38

Argon Y, Simen BB (1999): GRP94, an ER chaperone with protein and peptide binding properties. Semin Cell Dev Biol 10:495-505.
Berridge MJ, Bootman MD, Roderick HL (2003): Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517-29.
Boehning D, Patterson RL, Snyder SH (2004): Apoptosis and calcium: new roles for cytochrome c and inositol 1,4,5-trisphosphate. Cell Cycle 3:252-4.
Brewer JW, Cleveland JL, Hendershot LM (1997): A pathway distinct from the mammalian unfolded protein response regulates expression of endoplasmic reticulum chaperones in non-stressed cells. Embo J 16:7207-16.
Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS (2004): Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol 287:C817-33.
Buggy JJ (1998): Binding of alpha-melanocyte-stimulating hormone to its G-protein-coupled receptor on B-lymphocytes activates the Jak/STAT pathway. Biochem J 331 ( Pt 1):211-6.
Carballo M, Conde M, El Bekay R, Martin-Nieto J, Camacho MJ, Monteseirin J, Conde J, Bedoya FJ, Sobrino F (1999): Oxidative stress triggers STAT3 tyrosine phosphorylation and nuclear translocation in human lymphocytes. J Biol Chem 274:17580-6.
Chang WM, Chen KD, Chen LY, Lai MT, Lai YK (2003): Mitochondrial calcium-mediated reactive oxygen species are essential for the rapid induction of the grp78 gene in 9L rat brain tumour cells. Cell Signal 15:57-64.
Chang YS, Lee LC, Sun FC, Chao CC, Fu HW, Lai YK (2006a): Involvement of calcium in the differential induction of heat shock protein 70 by heat shock protein 90 inhibitors, geldanamycin and radicicol, in human non-small cell lung cancer H460 cells. J Cell Biochem 97:156-65.
Chang YS, Lo CW, Sun FC, Chang MD, Lai YK (2006b): Differential expression of Hsp90 isoforms in geldanamycin-treated 9L cells. Biochem Biophys Res Commun 344:37-44.
Cheriyath V, Desgranges ZP, Roy AL (2002): c-Src-dependent transcriptional activation of TFII-I. J Biol Chem 277:22798-805.
Drummond IA, Lee AS, Resendez E, Jr., Steinhardt RA (1987): Depletion of intracellular calcium stores by calcium ionophore A23187 induces the genes for glucose-regulated proteins in hamster fibroblasts. J Biol Chem 262:12801-5.
Gadina M, Hilton D, Johnston JA, Morinobu A, Lighvani A, Zhou YJ, Visconti R, O'Shea JJ (2001): Signaling by type I and II cytokine receptors: ten years after. Curr Opin Immunol 13:363-73.
Goetz MP, Toft DO, Ames MM, Erlichman C (2003): The Hsp90 chaperone complex as a novel target for cancer therapy. Ann Oncol 14:1169-76.
Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D (2000): Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6:1099-108.
Hong M, Lin MY, Huang JM, Baumeister P, Hakre S, Roy AL, Lee AS (2005): Transcriptional regulation of the Grp78 promoter by endoplasmic reticulum stress: role of TFII-I and its tyrosine phosphorylation. J Biol Chem 280:16821-8.
Ju H, Venema VJ, Liang H, Harris MB, Zou R, Venema RC (2000): Bradykinin activates the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway in vascular endothelial cells: localization of JAK/STAT signalling proteins in plasmalemmal caveolae. Biochem J 351:257-64.
Kroemer G, Zamzami N, Susin SA (1997): Mitochondrial control of apoptosis. Immunol Today 18:44-51.
Lai MT, Huang KL, Chang WM, Lai YK (2003): Geldanamycin induction of grp78 requires activation of reactive oxygen species via ER stress responsive elements in 9L rat brain tumour cells. Cell Signal 15:585-95.
Li WW, Alexandre S, Cao X, Lee AS (1993): Transactivation of the grp78 promoter by Ca2+ depletion. A comparative analysis with A23187 and the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. J Biol Chem 268:12003-9.
Lievremont JP, Rizzuto R, Hendershot L, Meldolesi J (1997): BiP, a major chaperone protein of the endoplasmic reticulum lumen, plays a direct and important role in the storage of the rapidly exchanging pool of Ca2+. J Biol Chem 272:30873-9.
Livak KJ, Schmittgen TD (2001): Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-8.
Luo S, Baumeister P, Yang S, Abcouwer SF, Lee AS (2003): Induction of Grp78/BiP by translational block: activation of the Grp78 promoter by ATF4 through and upstream ATF/CRE site independent of the endoplasmic reticulum stress elements. J Biol Chem 278:37375-85.
Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, Roifman CM (1996): Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 379:645-8.
Nguyen A, Chen P, Cai H (2004): Role of CaMKII in hydrogen peroxide activation of ERK1/2, p38 MAPK, HSP27 and actin reorganization in endothelial cells. FEBS Lett 572:307-13.
Paris S, Denis H, Delaive E, Dieu M, Dumont V, Ninane N, Raes M, Michiels C (2005): Up-regulation of 94-kDa glucose-regulated protein by hypoxia-inducible factor-1 in human endothelial cells in response to hypoxia. FEBS Lett 579:105-14.
Park OK, Schaefer TS, Nathans D (1996): In vitro activation of Stat3 by epidermal growth factor receptor kinase. Proc Natl Acad Sci U S A 93:13704-8.
Pearl LH, Prodromou C (2000): Structure and in vivo function of Hsp90. Curr Opin Struct Biol 10:46-51.
Rao RV, Ellerby HM, Bredesen DE (2004): Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ 11:372-80.
Schroder M, Kaufman RJ (2005): ER stress and the unfolded protein response. Mutat Res 569:29-63.
Shiu RP, Pouyssegur J, Pastan I (1977): Glucose depletion accounts for the induction of two transformation-sensitive membrane proteinsin Rous sarcoma virus-transformed chick embryo fibroblasts. Proc Natl Acad Sci U S A 74:3840-4.
Shu CW, Cheng NL, Chang WM, Tseng TL, Lai YK (2005): Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C. J Cell Biochem 94:1199-209.
Simon AR, Rai U, Fanburg BL, Cochran BH (1998): Activation of the JAK-STAT pathway by reactive oxygen species. Am J Physiol 275:C1640-52.
Song MS, Park YK, Lee JH, Park K (2001): Induction of glucose-regulated protein 78 by chronic hypoxia in human gastric tumor cells through a protein kinase C-epsilon/ERK/AP-1 signaling cascade. Cancer Res 61:8322-30.
Stahl N, Boulton TG, Farruggella T, Ip NY, Davis S, Witthuhn BA, Quelle FW, Silvennoinen O, Barbieri G, Pellegrini S, et al. (1994): Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science 263:92-5.
Vercesi AE, Kowaltowski AJ, Grijalba MT, Meinicke AR, Castilho RF (1997): The role of reactive oxygen species in mitochondrial permeability transition. Biosci Rep 17:43-52.
Wallace TA, Xia SL, Sayeski PP (2005): Jak2 tyrosine kinase prevents angiotensin II-mediated inositol 1,4,5 trisphosphate receptor degradation. Vascul Pharmacol 43:336-45.
Waris G, Tardif KD, Siddiqui A (2002): Endoplasmic reticulum (ER) stress: hepatitis C virus induces an ER-nucleus signal transduction pathway and activates NF-kappaB and STAT-3. Biochem Pharmacol 64:1425-30.
Way KJ, Chou E, King GL (2000): Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol Sci 21:181-7.
Zhou Y, Lee AS (1998): Mechanism for the suppression of the mammalian stress response by genistein, an anticancer phytoestrogen from soy. J Natl Cancer Inst 90:381-8.