當未折疊或折疊錯誤的蛋白質累積在內質網時，細胞會產生內質網壓力（ER stress）。此時內質網可以引發未折疊蛋白質反應（ Unfolded Protein Response, UPR）以幫助內質網恢復正常功能。JAB1在早先研究中被發現為AP-1轉錄因子的輔助因子。隨後亦發現JAB1能調控細胞週期激酶抑制因子p27kip1的出核與分解。先前的研究中指出內質網壓力可能對JAB1的調控產生影響，但機制未明。本研究，我們希望探討在內質網壓力的影響下JAB1如何受到調控。本次實驗中，我們選用tunicamycin，一種胺型蛋白質醣基化抑制劑，當作內質網壓力引發因子。西方墨點法和反轉錄聚合酶與聚合酶鏈反應顯示受到tunicamycin處理48小時的Hep3B細胞，其JAB1蛋白質與訊息核醣核酸的表現量受到抑制。連續性減短的JAB1基因啟動子活性測試實驗指出-405 bp到-223 bp這段區域很可能對tunicamycin引發的內質網壓力具反應性。電腦程式預測有許多候選因子可能會參與該區域對於JAB1的基因調控。點突變JAB1基因啟動子活性測試指出tunicamycin引發的內質網壓力對於JAB1基因啟動子的影響區域位於-342/-338和-331/-327。染色體免疫沉澱法試驗指出tunicamycin引發的內質網壓力抑制JAB1基因啟動子活性是藉由增加SP1和DNMT3b結合到JAB1基因啟動子-342/-338和-331/-327區域的SP1結合位。甲基化特異性聚合酶鏈反應指出tunicamycin引發的內質網壓力造成JAB1基因啟動子-342/-338和-331/-327區域的SP1結合位甲基化。綜合以上實驗，我們的研究指出tunicamycin引發的內質網壓力在Hep3B細胞內對於JAB1的抑制性基因調控是藉由增加SP1和DNMT3b結合到JAB1基因啟動子以及促使啟動子甲基化以抑制JAB1的表現。

Endoplasmic reticulum (ER) stress is the condition that unfolded or misfolded proteins accumulated in the ER which leads to the solubility stress. ER can activate the unfolded protein response (UPR) to restore the ER homeostasis. JAB1 was originally identified as the coactivator of AP-1 transcription factor. JAB1 was then discovered to mediate the cyclin-dependent-kinase inhibitor p27kip1 nuclear exportation and degradation. Previous studies demonstrate that ER stress may affect the regulation of JAB1, but the mechanism is still unknown. In this study, we want to investigate how JAB1 is regulated in ER stress. We applied tunicamycin, a protein N-glycosylation inhibitor, as the ER stress inducer. Western blot and reverse transcription PCR revealed that treatment with tunicamycin for 48 hours in Hep3B induced ER stress and repressed JAB1 protein and mRNA expression. Serial deletion of the JAB1 promoter activity assay revealed that the region from -405 bp to -223 bp may be responsive in the tunicamycin-induced ER stress. Computational prediction suggested that there are several candidate factors may join the regulation of JAB1 in this region. Site-directed mutation of JAB1 promoter assay revealed that the tunicamycin-induced ER stress repressed JAB1 promoter activity through the sites at -342/-338 and -331/-327 in JAB1 promoter. Chromatin immunoprecipitation assay suggested that tunicamycin-induced ER stress repressed the JAB1 promoter activity through increasing the SP1 and DNMT3b binding to the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter. Methylation specific PCR showed that the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter were methylated in tunicamycin-induced ER stress. Taken together, we demonstrated that tunicamycin-induced ER stress repressed the JAB1 gene expression in Hep3B through increasing the binding of SP1 and DNMT3b to the SP1 binding sites and inducing promoter methylation to repress JAB1 expression.

目錄

英文摘要 i

中文摘要 ii

Introduction 1

Specific aim 8

Materials and methods 9

Results 22

Discussion 28

Figure 33

Reference 46

Reference

1. Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nature Reviews Drug Discovery 2008;7:1013-30.
2. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nature Reviews Molecular Cell Biology 2007;8:519-29.
3. Kadowaki H, Nishitoh H, Ichijo H. Survival and apoptosis signals in ER stress: the role of protein kinases. Journal of Chemical Neuroanatomy 2004;28:93-100.
4. Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe? Nature Reviews Cancer 2004;4:966-77.
5. Chamovitz DA. Revisiting the COP9 signalosome as a transcriptional regulator. EMBO Reports 2009;10:352-8.
6. Richardson KS, Zundel W. The emerging role of the COP9 signalosome in cancer. Molecular Cancer Research 2005;3:645-53.
7. Claret FX, Hibi M, Dhut S, Toda T, Karin M. A new group of conserved coactivators that increase the specificity of AP-1 transcription factors. Nature 1996;383:453-7.
8. Tomoda K, Kubota Y, Kato J. Degradation of the cyclin-dependent-kinase inhibitor p27kip1 is instigated by Jab1. Nature 1999;398:160-5.
9. Kouvaraki MA, Korapati AL, Rassidakis GZ, Tian L, Zhang Q, Chiao P, Ho L, Evans DB, Claret FX. Potential role of Jun activation domain-binding protein 1 as a negative regulator of p27kip1 in pancreatic adenocarcinoma. Cancer Research 2006;66:8581-9.
10. Fukumoto A, Tomoda K, Kato NY, Nakajima Y, Kato J, et al. Depletion of Jab1 inhibits proliferation of pancreatic cancer lines. FEBS Letters 2006;580:5836-44.
11. Berg JP, Zhou Q, Breuhahn K, Schirmacher P, Patil MA, Chen X, Schafer N, Holler TT, Fisher HP, Buttner ,Gutgenmann I, et al. Inverse expression of Jun activation domain binding protein1 and cell cycle inhibitor p27kip1: influence on proliferation in hepatocellular carcinoma. Human Pathology 2007;38:1621-27.
12. Sui L, Dong Y, Ohno M, Watanabe Y,Sugimoto K, Tai Y, Tokuda M. Jab1 expression is associated with inverse expression of p27kip1 and poor prognosis in epithelial ovarian tumors. Clinical Cancer Research 2001;7:4130-35.
13. Patil MA, Gutgenmann I, Zhang J, Ho C, Cheung ST, Ginzinger D, Li R, Dykema KJ, So S, Fan ST, Kakar S, Furge KA, Buttner R, Chen X. Array-based comparative genomic hybridization reveals recurrent chromosomal aberrations and Jab1 as a potential target for 8q gain in hepatocellular carcinoma. Carcinogenesis 2005;26:2050-7.
14. Tomoda K, Kubota Y, Arata Y, Mori S, Maeda M, Tanaka T, Yoshida M, Kato NY, Kato JY. The cytoplasmic shuttling and subsequent degradation of p27kip1 mediated by Jab1/CSN5 and the COP9 signalosome complex. Journal of Biological Chemistry 2002;277:2302-10.
15. Hoozemans JJM, Stieler J, Haastert ESV, Veerhuis R, Rozemuller AJM, Bass F, Eikelenboom P,Arendt T, Scheper W. The unfolded protein response affects neuronal cell cycle protein expression: Implications for Alzheimer’s disease pathogenesis. Experimental Gerontology 2006;41:380-6.
16. Chiang PC, Hsu JL, Yeh TC, Pan SL, Guh JH. Elucidation of susceptible factors to endoplasmic reticulum stress-mediated anticancer activity in human hepatocellular carcinoma. Naunyn Schmiedebergs Arch Pharmacol 2008;377:167-77.
17. Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 2001;107:881-91.
18. Brewer JW, Hendershot LM, Sherr CJ, Diehl JA. Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression. Proceedings of National Academy of Sciences of the United States of America 1999;96:8505-10.
19. Donati G, Imbriano C, Mantovani R. Dynamic recruitment of transcription factors and epigenetic changes on the ER stress response gene promoters. Nucleic Acids Research 2006;34:3116-27.
20. Abdelrahim M, Liu S, Safe S. Induction of endoplasmic reticulum-induced stress genes in Panc-1 pancreatic cancer cells is dependent on Sp proteins. Journal of Biological Chemistry 2005;280:16508-13.
21. Hsu MC, Chang HC, Hung WC. HER-2/neu represses the metastasis suppressor RECK via ERK and Sp transcription factors to promote cell invation. Journal of Biological Chemistry 2006;281:4718-25.
22. Bartoszewski R, Rab A, Twitty G, Stevenson L, Fortnberry J, Piotrowski A, Dumanski J, Bebok Z. The mechanism of cystic fibrosis transmembrane conductance regulator transcriptional repression during the unfolded protein response. Journal of Biological Chemistry 2008;283:12154-65.
23. Oono K, Yoneda T, Manabe T, Yamagishi S, Matsuda S, Hitomi J, Miyata S, Mizuno T, Imaizumi K, Katayama T, Tohyama M. JAB1 participates in unfolded protein responses by association and dissociation with IRE1. Neurochemistry International 2004;45:765-72.
24. Hsieh YH, Su IJ, Wang HC, Tsai JH, Huang YJ, Chang WW, Lai MD, Lei, HY, Huang W. Hepatitis B virus pre-S2 mutant surface antigen induces degradation of cyclin-dependent kinase inhibitor p27kip1 through c-Jun activation domain-binding protein 1. Molecular Cancer Research 2007;5:1063-72.
25. Hsu MC, Chang HC, Hung WC. HER-2/neu transcriptionally activates Jab1 expression via the AKT/β-catenin pathway in breast cancer cells. Endocrine Related Cancer 2007;14:655-67.
26. Hsu MC, Huang CC, Chang HC, Hu TH, Hung WC. Overexpression of Jab1 in hepatocellular carcinoma and its inhibition by peroxisome proliferators-activated receptorγ ligands In vitro and In vivo. Clinical Cancer Research 2008;14:4045-52.