進階搜尋


 
系統識別號 U0026-0812200913462743
論文名稱(中文) 探討ARNT在人類子宮頸上皮癌A431中與c-Jun/Sp1複合體共同調控EGF誘導12(S)-lipoxygenase基因表現
論文名稱(英文) ARNT cooperates with c-Jun/Sp1 to regulate EGF-induced 12(S)-lipoxygenase gene expression in A431 cells
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 95
學期 2
出版年 96
研究生(中文) 陳淑婷
研究生(英文) Shu-Ting Chen
學號 s2694402
學位類別 碩士
語文別 中文
論文頁數 56頁
口試委員 口試委員-呂增宏
指導教授-陳炳焜
指導教授-張文昌
中文關鍵字 上皮增生因子  基因表現 
英文關鍵字 EGF  gene expression 
學科別分類
中文摘要 目前已知EGF可經由c-Jun/Sp1此複合體調控與癌症密切相關的基因如12(S)-lipoxygenase 和p21WAF1/CIP1的表現,在此c-Jun透過跟Sp1交互作用而結合到沒有AP1的結合序列的啟動區上,進而去調控該基因的表現。然而在EGF訊息傳遞路徑下,是否尚有其他未知的因子可以經由這樣的機制來調控基因的表現呢?於是我們利用蛋白質體學的技術,分析在EGF處理下與Sp1結合情況有明顯改變的蛋白質,發現ARNT此一屬於hypoxia inducible factor-1的轉錄因子在EGF處理下與Sp1結合有明顯的增加。這可能暗示著ARNT可能在EGF的訊息傳遞路徑下扮演了某些角色,基於此,我們便試著去探討ARNT是否有可能會參與在EGF所調控的基因表現過程中。首先從免疫沉澱的實驗中得知EGF可促進 ARNT與c-Jun、Sp1 的交互作用,而在DNA親和免疫沈澱分析中得知ARNT/c-Jun/Sp1複合體可以結合到12(S)-lipoxygenase啟動區上。將細胞中的Sp1 knock down後發現ARNT與12(S)-lipoxygenase啟動區結合有抑制的情形,且 overexpression ARNT並無法活化Sp1 binding site突變的12(S)-lipoxygenase啟動區,顯示ARNT調控12(S)-lipoxygenase的基因表現需透過Sp1。使用ARNT siRNA將ARNT knock down後發現EGF所induce的12(S)-lipoxygenase啟動區的活性及mRNA都有抑制的現象,而大量表現ARNT則會活化該啟動區活性,證實了ARNT可透過transcriptional level來調控該基因的表現。而在ARNT/c-Jun/Sp1調控12(S)-lipoxygenase的基因表現中,c-Jun為主要的轉錄活化因子負責轉錄活化基因的表現,ARNT對基因的轉錄活化作用在此並不是那麼重要。在免疫沉澱的實驗中,我們發現c-Jun與Sp1的交互作用會因ARNT knock down而減少,因此ARNT很有可能經由影響c-Jun與Sp1的交互作用而參與在12(S)-lipoxygenase的基因調控中。除了12(S)-lipoxygenase 外,其他由c-Jun/Sp1所調控的基因,如p21WAF1/CIP1也有可能受到相同的調控機制,此結果顯示ARNT可以在normoxia的情形下參與c-Jun/Sp1所調控的基因表現,此研究也為hypoxia inducible factor促進腫瘤生長的機制提供了一個新的解釋。
英文摘要 It is well documented that EGF regulates the gene expression including 12(S)-lipoxygenase and p21WAF1/CIP1 through the transcription factor c-Jun and Sp1. However, the signaling molecules involved in EGF-responsive gene expression are not totally calified. Here we used the immunoprecipitation and followed by MALDI-TOF to analyze Sp1-associated proteins. We found that ARNT, a hypoxia related transcription factor, co-immunoprecipitated with Sp1. This result suggested that ARNT may play a role in EGF signaling event under physicial condiction. We found that EGF stimulated the formation of c-Jun/ARNT/Sp1 complexs as well as the binding of the transcription factor complex to the gene promoter in a time-dependent manner. ARNT small interfering RNA (siRNA) inhibited the promoter activity and the mRNA level of 12(S)-lipoxygenase in cells treated with EGF. Furthermore, overexpression of ARNT increased the 12(S)-lipoxygenase promoter activity. We also identified that Sp1 binding site of 12(S)-lipoxygenase promoter was responsible for overexpressing ARNT activation. In addition, ARNT apparently acted via c-Jun because expression of c-Jun mutant nullified the action of ARNT. Contrary to the transcriptional activity of c-Jun, ARNT mediated the interaction between c-Jun and Sp1 in EGF-treated cells. Furthermore, ARNT siRNA also inhibited EGF-induced expression of p21WAF1/CIP1. These results revealed that ARNT mediated c-Jun/Sp1-regulated gene expression under normoxic condition and provided an additional mechanism in considering the HIF factor as regulator of tumor growth.
論文目次 中文摘要................................................II

英文摘要................................................III

附圖目錄................................................VI

縮寫指引...............................................VII

第一章 緒論............................................1

第二章 實驗材料及方法
來源....................................................8
實驗材料及方法..........................................12

第三章 實驗結果
第一節 EGF誘導ARNT與c-Jun/Sp1交互作用的形成
(一) 確認ARNT跟c-Jun/Sp1的交互關係......................26
(二) 確認ARNT跟12(S)-lipoxygenase啟動區的結合...........26
(三) Sp1在ARNT調控12(S)-lipoxygenase基因表現的重要性....27
第二節 ARNT調控12(S)-lipoxygenase的基因表現
(一) ARNT siRNA抑制 EGF所誘導的12(S)-lipoxygenase mRNA表現......................................................28
(二) ARNT對12(S)-lipoxygenase啟動區活性的影響...........28第三節 探討ARNT與c-Jun調控12(S)-lipoxygenase的基因表現之間的關係
(一) 探討ARNT在 overexpression c-Jun所誘導12(S)-lipoxygenase
基因表現的重要性........................................29
(二) 探討c-Jun在 overexpression ARNT所誘導12(S)-lipoxygenase基因表現的重要性............................29
(三) 探討c-Jun的transactivation domain 在ARNT調控12(S)-lipoxygenase基因表現的重要性啟動區結合的影響............30
(四) 探討c-Jun的transactivation domain 對於ARNT 與12(S)-lipoxygenase啟動區結合的影響............................30
第四節 探討ARNT在c-Jun/Sp1複合體調控12(S)-lipoxygenase的基因表現中所提供的功能
(一) 探討ARNT對於 c-Jun/Sp1複合體穩定的重要性...........31
(二) 探討ARNT在其他由c-Jun/Sp1複合體調控的基因表現之重要性......................................................31
(三) ARNT亦參與在EGF調控p21的基因表現過中..............32

第四章 討論.............................................33

第五章 參考文獻.........................................38

附圖....................................................45
參考文獻 Alfranca, A., Gutierrez, M. D., Vara, A., Aragones, J.,Vidal, F., Landazuri, M. O. c-Jun and hypoxia-inducible factor 1 functionally cooperate in hypoxia-induced gene transcription, Mol. Cell. Biol. 22:12-22, 2002.
Arora, J. K., Lysz, T. W., Zelenka, P. S. A role for 12(S)-HETE in the response of human lens epithelial cells to epidermal growth factor and insulin, Invest Ophthalmol Vis. Sci. 37: 1411-8, 1996.
Beischlag. T. V., Taylor. R.T., Rose. D.W., Yoon. D., Chen. Y., Lee. W. H, Rosenfeld. M. G., Hankinson. O. Recruitment of thyroid hormone receptor/retinoblastoma -interacting protein 230 by the aryl hydrocarbon receptor nuclear translocator is required for the transcriptional response to both dioxin and hypoxia, J.Biol.Chem. 279 : 54620-8, 2004.
Bogatcheva, N. V., Sergeeva, M. G., Dudek, S. M., Verin, A. D. Arachidonic acid cascade in endothelial pathobiology, Microvas. Res. 69: 107 – 127, 2005.
Chang, W. C., Ning, C. C., Lin, M. T., Huang, J. D. Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells, J. Biol. Chem. 267: 3657-66, 1992.
Chang, W. C., Liu, Y. W., Ning, C. C., Suzuki, H., Yoshimoto, T., Yamamoto, S. Induction of arachidonate 12-lipoxygenase mRNA by epidermal growth factor in A431 cells, J. Biol. Chem. 268: 18734-9, 1993.
Chang, W.C. Cell signaling and gene regulation of human 12(S)-lipoxygenase expression, Prostaglandins Other Lipid Mediat. 71: 277-85, 2003.
Chang, W. C., Chen, B. K. Transcription factor Sp1 functions as an anchor protein in gene transcription of human 12(S)-lipoxygenase, Biochem. Biophys. Res. Commun. 338: 117-21, 2005.
Chen, B.K., Chang, W.C. Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase, Proc. Natl. Acad. Sci. U S A. 97: 10406-11, 2000.
Chen, B.K., Kung, H.C., Tsai, T.Y., Chang, W.C. Essential role of mitogen-activated protein kinase pathway and c-Jun induction in epidermal growth factor-induced gene expression of human 12-lipoxygenase, Mol. Pharmacol. 57: 153-61,2000.
Cheng. J., Perkins, N. D., Yeh, E. T. Differential regulation of c-Jun-dependent transcription by SUMO-specific proteases, J. Biol. Chem. 280 : 14492-8, 2005.
Citri, A., Yarden, Y. EGF-ERBB signalling: towards the systems level,Nat. Rev. Mol. Cell. Biol. 7: 505-16, 2006
Czech, M. P. ARNT misbehavin' in diabetic beta cells, Nat. Med. 12: 39-40, 2006.
Eguchi. H., Ikuta. T., Tachibana. T., Yoneda. Y., Kawajiri. K. A nuclear localization signal of human aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor 1beta is a novel bipartite type recognized by the two components of nuclear pore-targeting complex, J. Biol. Chem. 272 : 17640-7, 1997.
Ema, M., Morita, M., Ikawa, S., Tanaka, M., Matsuda, Y., Gotoh, O., Saijoh, Y., Fujii, H., Hamada, H., Kikuchi, Y., Fujii-Kuriyama, Y. Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis, Mol. Cell. Biol. 16 : 5865-75, 1996.
Gao, X., Grignon, D. J., Chbihi, T., Zacharek, A., Chen, Y. Q., Sakr, W., Porter, A. T., Crissman, J. D., Pontes, J. E., Powell, I. J., et al. Elevated 12-lipoxygenase mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer, Urology 46 : 227-37, 1995.
Geng, S., Mezentsev, A., Kalachikov, S., Raith, K., Roop, D. R., Panteleyev, A. A. Targeted ablation of Arnt in mouse epidermis results in profound defects in desquamation and epidermal barrier function. J. Cell. Sci. 119 : 4901-12, 2006.
Gunton, J. E., Kulkarni, R. N., Yim, S., Okada, T., Hawthorne, W.J., Tseng, Y. H., Roberson, R. S., Ricordi, C., O'Connell, P.J., Gonzalez, F. J., Kahn, C.R. Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes, Cell 122: 337-49, 2005.
Hagmann, W., Gao, X., Timar, J., Chen, Y. Q., Strohmaier, A. R., Fahrenkopf, C., Kagawa, D., Lee, M., Zacharek, A., Honn, K. V. 12-Lipoxygenase in A431 cells: genetic identity, modulation of expression, and intracellular localization, Exp. Cell. Res. 228: 197-205,1996.
Haque, M. S., Arora, J. K., Dikdan, G. Lysz, T. W., Zelenka, P. S. The rabbit lens epithelial cell line N/N1003A requires 12-lipoxygenase activity for DNA synthesis in response to EGF, Mol. Vis. 15: 8, 1999.
Henson, E. S., Gibson, S. B. Surviving cell death through epidermal growth factor (EGF) signal transduction pathways: implications for cancer therapy. Cell Signal. 18: 2089-97, 2006.
Hung, J. J., Wu, C. Y., Liao, P. C., Chang, W. C. Hsp90alpha recruited by Sp1 is important for transcription of 12(S)-lipoxygenase in A431 cells, J. Biol. Chem. 280: 36283-92, 2005.
Hung, J. J., Wang, Y. T., Chang, W. C. Sp1 deacetylation induced by phorbol ester recruits p300 to activate 12(S)-lipoxygenase gene transcription, Mol. Cell. Biol. 26: 1770-85, 2006.
Isaacs, J. S., Jung, Y. J., Neckers, L. Aryl hydrocarbon nuclear translocator (ARNT) promotes oxygen-independent stabilization of hypoxia-inducible factor-1alpha by modulating an Hsp90-dependent regulatory pathway, J. Biol. Chem. 279: 16128-35, 2004.
Iversen, L., Kragballe, K., Arachidonic acid metabolism in skin health and disease, Prostaglandins Other Lipid Mediat. 63: 25–42, 2000.
Kandouz , M., Honn, K. V. Eicosanoids regulation of transcription factors in PC-3 prostate cancer cells, Adv. Exp. Med. Biol. 507: 563-8, 2002
Kandouz , M., Nie, D., Pidgeon, G. P., Krishnamoorthy, S., Maddipati, K. R., Honn, K. V. Platelet-type 12-lipoxygenase activates NF-kappaB in prostate cancer cells, Prostaglandins Other Lipid Mediat. 71: 189-204, 2003.
Kewley, R. J., Whitelaw, M. L., Chapman-Smith, A. The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int. J. Biochem. Cell Biol. 36:189-204, 2004.
Kobayashi, A., Sogawa, K., Fujii-Kuriyama, Y. Cooperative interaction between AhR.Arnt and Sp1 for the drug-inducible expression of CYP1A1 gene, J. Biol. Chem. 271: 12310-6, 1996.
Kobayashi. A., Numayama-Tsuruta. K., Sogawa. K., Fujii-Kuriyama. Y.
CBP/p300 functions as a possible transcriptional coactivator of Ah receptor nuclear translocator (Arnt), J. Biochem. 122 : 703-10, 1997.
Lee, K. H., Park, J. W., Chun, Y. S. Non-hypoxic transcriptional activation of the aryl hydrocarbon receptor nuclear translocator in concert with a novel hypoxia-inducible factor-1alpha isoform. Nucleic Acids Res. 32: 5499-511, 2004.
Levine. S. L., Perdew. G. H. Aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) activity is unaltered by phosphorylation of a periodicity/ARNT/single-minded (PAS)-region serine residue,
Mol. Pharmacol. 59 : 557-66, 2001.
Levine. S. L., Perdew. G. H. Okadaic acid increases ARNT homodimer transactivation potential, Cell. Biol. Toxicol. 18 : 109-20, 2002.
Levisetti, M. G., Polonsky, K. S. Diabetic pancreatic beta cells ARNT all they should be, Cell Metab. 2: 78-80, 2005.
Li, Y. M., Zhou, B. P., Deng, J., Pan, Y., Hay, N., Hung, M. C. A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells. Cancer Res. 65: 3257-63, 2005.
Liu, Y. W., Asaoka, Y., Suzuki, H., Yoshimoto, T., Yamamoto, S., Chang, W. C. Induction of 12-lipoxygenase expression by epidermal growth factor is mediated by protein kinase C in A431 cells, J. Pharmacol. Exp. Ther. 271: 567-73, 1994.
Liu(a), Y. W., Arakawa, T., Yamamoto, S., Chang, W. C., Transcriptional activation of human 12-lipoxygenase gene promoter is mediated through Sp1 consensus sites in A431 cells, Biochem. J. 324: 133-40, 1997.
Liu(b), Y. W., Chen, B. K., Chen, C. J., Arakawa, T., Yoshimoto, T., Yamamoto, S., Chang, W. C. Epidermal growth factor enhances transcription of human arachidonate 12-lipoxygenase in A431 cells. Biochim. Biophys. Acta. 1344: 38-46, 1997.
Long. W. P., Chen. X., Perdew. G. H. Protein kinase C modulates aryl hydrocarbon receptor nuclear translocator protein-mediated transactivation potential in a dimer context, J. Biol. Chem. 274 : 12391-400, 1999.
Lopez-Perez. M., Salazar. E. P., A role for the cytoskeleton in STAT5 activation in MCF7 human breast cancer cells stimulated with EGF. Int.J. Biochem. Cell. Biol. 38 : 1716-28, 2006.
Maltepe, E., Schmidt, J. V., Baunoch, D., Bradfield, C.A., Simon, M.C. Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature 386: 403-7, 1997.
McCabe, N. P., Selman, S. H., Jankun, J. Vascular endothelial growth factor production in human prostate cancer cells is stimulated by overexpression of platelet 12-lipoxygenase, Prostate. 66: 779-87, 2006.
Moffett, P., Pelletier, J. Different transcriptional properties of mSim-1 and mSim-2. FEBS Lett. 466:80-6, 2000.
Nie , D., Hillman, G. G., Geddes, T., Tang, K., Pierson, C., Grignon, D. J., Honn, K. V. Platelet-type 12-lipoxygenase regulates angiogenesis in human prostate carcinoma, Adv. Exp. Med. Biol. 469: 623-30, 1999.
Nie , D., Krishnamoorthy, S., Jin, R., Tang, K., Chen, Y., Qiao, Y., Zacharek, A., Guo, Y., Milanini, J., Pages, G., Honn, K. V. Mechanisms regulating tumor angiogenesis by 12-lipoxygenase in prostate cancer cells, J. Biol. Chem. 281: 18601-9, 2006.
Ottino, P., Taheri, F., Bazan, H. E. Growth factor-induced proliferation in corneal epithelial cells is mediated by 12(S)-HETE. Exp. Eye. Res. 5: 613-22, 2003.
Peng. X. H., Karna, P., Cao, Z., Jiang, B. H., Zhou, M., Yang, L. Cross-talk
between epidermal growth factor receptor and hypoxia-inducible factor-1alpha signal pathways increases resistance to apoptosis by up-regulating survivin gene expression, J. Biol.Chem. 281: 25903-14, 2006.
Pidgeon, G. P., Kandouz, M., Meram, A., Honn, K. V. Mechanisms Controlling Cell Cycle Arrest and Induction of Apoptosis after 12-Lipoxygenase Inhibition in Prostate Cancer Cells, Cancer Research 62: 2721–2727, 2002.
Pidgeon, G. P., Tang, K., Cai, Y. L., Piasentin, E., Honn, K. V. Overexpression of platelet-type 12-lipoxygenase promotes tumor cell survival by enhancing alpha(v)beta(3) and alpha(v)beta(5) integrin expression. Cancer Res. 63: 4258-67, 2003.
Pore, N., Jiang, Z., Gupta, A., Cerniglia, G., Kao, G. D., Maity, A. EGFR tyrosine kinase inhibitors decrease VEGF expression by both hypoxia-inducible factor (HIF)-1-independent and HIF-1-dependent mechanisms, Cancer Res. 66: 3197-204, 2006.
Preston, I. R., Hill, N. S., Warburton, R. R., Fanburg, B. L., Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation, Am. J. Physiol. Lung. Cell. Mol. Physiol. 290: 367–374, 2006.
Rankin, E. B., Higgins, D. F., Walisser, J. A., Johnson, R. S., Bradfield, C. A, Haase, V. H. Inactivation of the arylhydrocarbon receptor nuclear translocator (Arnt) suppresses von Hippel-Lindau disease-associated vascular tumors in mice, Mol. Cell. Biol. 25: 3163-72, 2005.
Roos,M.D., Su, K., Baker, J. R., Kudlow, J. E. O glycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions. Mol. Cell. Biol. 17: 6472-80, 1997.
Seth, R.K., Haque, M. S., Zelenka, P. S. Regulation of c-fos induction in lens epithelial cells by 12(S)HETE-dependent activation of PKC, Invest Ophthalmol. Vis. Sci. 42: 3239-46,2001.
The. C. H., Lam. K. K., Loh. C. C., Loo. J. M., Yan. T., Lim. T. M. Neuronal PAS domain protein 1 is a transcriptional repressor and requires arylhydrocarbon nuclear translocator for its nuclear localization, J. Biol. Chem. 281: 34617-29, 2006.
Tojo. M., Matsuzaki. K., Minami. T., Honda. Y., Yasuda. H., Chiba. T., Saya. H., Fujii-Kuriyama. Y., Nakao. M. The aryl hydrocarbon receptor nuclear transporter is modulated by the SUMO-1 conjugation system, J. Biol. Chem, 277 : 46576-85, 2002.
Tomita, S., Sinal, C. J., Yim, S. H., Gonzalez, F. J. Conditional disruption of the aryl hydrocarbon receptor nuclear translocator (Arnt) gene leads to loss of target gene induction by the aryl hydrocarbon receptor and hypoxia-inducible factor 1alpha. Mol . Endocrinol. 14: 1674-81, 2000.
Trotman, L.C., Wang, X., Alimonti, A., Chen, Z., Teruya-Feldstein, J., Yang, H., Pavletich, N. P., Carver, B. S., Cordon-Cardo. C., Erdjument-Bromage. H., Tempst, P., Chi, S. G., Kim, H. J., Misteli, T., Jiang, X., Pandolfi, P. P. Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell. 128: 141-56, 2007.
Virmani, J., Johnson, E. N., Klein-Szanto, A. J., Funk, C. D. Role of 'platelet-type' 12-lipoxygenase in skin carcinogenesis, Cancer Lett. 162: 161-5, 2001.
Wang, F., Hoivik, D., Pollenz, R., Safe, S. Functional and physical interactions between the estrogen receptor Sp1 and nuclear aryl hydrocarbon receptor complexes, Nucleic Acids Res. 26: 3044-52, 1998.
Wang, F., Wang, W., Safe, S. Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex, Biochemistry, 38: 11490-500, 1999.
Wood, S.M., Gleadle, J.M., Pugh, C.W., Hankinson, O., Ratcliffe, P. J.
The role of the aryl hydrocarbon receptor nuclear translocator (ARNT) in hypoxic induction of gene expression. Studies in ARNT-deficient cells.
J. Biol. Chem. 271:15117-23, 1996.
Woods, S.L., Whitelaw, M. L. Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop-helix/per-Arnt-Sim homology transcription factors.J. Biol. Chem. 277: 10236-43, 2002.
Yamamoto, S., Mammalian lipoxygenases: molecular structures and functions, Biochim. Biophy. Acta. 1128: 117-131, 1992.
Yim, S. H., Shah, Y., Tomita, S., Morris, H. D., Gavrilova, O., Lambert, G., Ward, J. M., Gonzalez, F. J. Disruption of the Arnt gene in endothelial cells causes hepatic vascular defects and partial embryonic lethality in mice. Hepatology 44: 550-60, 2006.
Yoshimoto, T., Takahashi, Y. Arachidonate 12-lipoxygenases. Prostaglandins Other Lipid Mediat. 68–69: 245–262, 2002.
Zhou, J., Fariss, R. N., Zelenka, P. S. Synergy of epidermal growth factor and 12(S)-hydroxyeicosatetraenoate on protein kinase C activation in lens epithelial cells, J. Biol. Chem. 278: 5388-98, 2003.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2012-07-23起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2012-07-23起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw