NF-IL6beta(hC/EBPdelta)為一轉錄因子並屬於C/EBP家族成員，目前已知 NF-IL6beta具有調控細胞分化、細胞凋亡以及發炎的功能。本實驗室的研究成果顯示在人類肝癌細胞 (HepG2)中大量表現NF-IL6beta蛋白可以促進人類 PPAR-gamma2基因啟動區的活化和 PPAR-gamma2 mRNA及蛋白質表現的增加，然而 PPAR-gamma2基因的轉錄與NF-IL6brta之彼此間詳細調控機轉仍然未明
，因此我們想探討NF-IL6beta調控PPAR-gamma2基因的分子機制為何。此外在IL-6處理下，HepG2 cells中其NF-IL6beta與PPAR-gamma2有先後活化的關係，因此IL-6可否藉由活化 NF-IL6beta進而調控 PPAR-gamma2的表現也是本篇論文要探討的課題。
　　經由電腦軟體分析 PPAR-gamma2基因啟動區後預測出三個可能的C/EBP蛋白結合位置，而在凝膠電泳位移實驗也顯示NF-IL6beta的確可以結合在PPAR-gamma2基因啟動區之C/EBP結合位置，經由DAPA實驗更進一步確定IL-6可增加NF-IL6beta於PPAR-gamma2基因啟動區之結合量，並可能藉由此結合能力來調控 PPAR-gamma2基因啟動區的活化。經由PPAR-gamma2基因啟動區5’端段切之報導基因分析也發現，不論是NF-IL6beta大量表現或是IL-6處理下PPAR-gamma2基因啟動區活化的能力皆會隨著其C/EBP結合位置之喪失而降低，也顯示我們這些C/EBP結合位置對NF-IL6beta活化PPAR-gamma2轉錄活性之重要性。此外先前本實驗室的研究發現SUMO-1可以對NF-IL6beta進行修飾，此修飾對經由NF-IL6beta活化的COX-2基因扮演一個負向調控的角色，因此我們也想知道 sumoylated NF-IL6beta對人類PPAR-gamma2基因的影響，實驗結果顯示sumoylated NF-IL6beta會減弱PPAR-gamma2基因的活化能力，且在細胞增生實驗中我們也發現sumoylated NF-IL6beta會減少抑制細胞生長的能力，推測可能與PPAR-gamma可調控細胞凋亡之功能有關。因此在本論文中我們確立了NF-IL6beta對PPAR-gamma2基因調控扮演重要的角色。

　NF-IL6beta(hC/EBPdelta)belongs to a family of transcription regulators C/EBP that has been implicated in mediating the acute phase response to inflammatory stimuli and in controlling of adipogenesis or apoptosis. We have found that overexpression of NF-IL6beta in human hepatoma cells (HepG2) could increase the expression of PPAR-gamma2 mRNA and protein synthesis through the activation of PPAR-gamma2 promoter. However little is known about the molecular mechanism of PPAR-gamma2 regulated by NF-IL6beta. In this study, we characterized the molecular mechanism of NF-IL6beta involving in the PPAR-gamma2 gene regulation. Furthermore, we found that IL-6 activate NF-IL6beta and PPAR-gamma2 in a time-dependent manner in HepG2 cells. We also studied that if IL-6 activated PPAR-gamma2 through the activation of NF-IL6beta.
By analyzing the PPAR-gamma2 promoter region by computer program, we found that there are three possible C/EBP binding sites. The result of gel shift assay showed that NF-IL6beta directly interacted with these C/EBP binding sites in the promoter region. Furthermore, IL-6 increased the binding of NF-IL6beta to PPAR-gamma2 promoter region in DAPA, and might activate the promoter activity through binding to these C/EBP binding sites. Reporter assays for 5’-deletion of PPAR-gamma2 promoter revealed that the activation of PPAR-gamma2 promoter activity was lost with the reduction of C/EBP sites during NF-IL6beta overexpression or IL-6 treatment. Moreover, the sumoylated NF-IL6beta attenuated PPAR-gamma2 promoter activity, and also reduced the growth arrest ability. Taken together, we proposed that C/EBP sites play important roles in PPAR-gamma2 promoter regulated by NF-IL6beta.

Ahdjoudj, S., Kaabeche, K., Holy, X., Fromigue, O., Modrowski, D., Zerath, E. and Marie, PJ. Transforming growth factor-beta inhibits CCAAT/enhancer-binding protein expression and PPAR-gamma activity in unloaded bone marrow stromal cells. Exp Cell Res. 303: 138-147, 2005.

Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., and Kishimoto, T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9: 1897-906, 1990.

Akira, S. and Kishimoto, T. IL-6 and NF-IL6 in acute phase response and viral infection. Immunol Rev. 127: 25–50, 1992.

Alam, T., An, M. R., and Papaconstantinou, J. Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response. J. Biol. Chem. 267: 5021-5024, 1992.

Banerjee, S. S., Feinberg, M. W., Watanabe, M., Gray, S., Haspel, R. L., Denkinger, D. J., Kawahara, R., Hauner, H. and Jain, M. K. The Kruppel-like factor KLF2 inhibits peroxisome proliferator-activated receptor-gamma expression and adipogenesis. J Biol Chem. 278: 2581-2584, 2003.

Bannach, F. G., Gutierrez-Fernandez, A., Parmer R. J. And Miles, L. A. Interleukin-6-induced plasminogen gene expression in murine hepatocytes is mediated by transcription factor CCAAT/enhancer- binding proteinbeta (C/EBPbeta). J Thromb Haemost. 2: 2205-12, 2004.

Berger, J., Tanen, M., Elbrecht, A., Hermanowski-Vosatka, A., Moller, D.E. and Wright, S.D. Peroxisome proliferator-activated receptor-γ ligands inhibit adipocyte 11beta-hydroxysteroid dehydrogenase type 1 expression and activity, J Biol Chem, 276: 12629-12635, 2001.

Billiard, J., Grewal, S. S., Lukaesko, L., Stork, P. J., and Rotwein, P. Hormonal Control of Insulin-like Growth Factor I Gene Transcription in Human Osteoblasts. Dual Actions of cAMP-dependent Protein Kinase on CCAAT/enhancer-binding protein delta. J. Biol. Chem. 276: 3123831246, 2001.

Breed, D. R., Margraf, L. R., Alcorn, J. L., and Mendelson, C. R. Transcription factor C/EBPdeltain fetal lung: developmental regulation and effects of cyclic adenosine 3',5'-monophosphate and glucocorticoids. Endocrinology 138, 5527-5534, 1997.

Bull, A.W. The role of peroxisome proliferator-activated receptor γ in colon cancer and inflammatory bowel disease, Arch Pathol Lab Med, 127: 121-1123, 2003.

Cantwell, C. A., Sterneck, E. and Johnson, P. F. Interleukin-6-specific activation of the C/EBPdelta gene in hepatocytes is mediated by Stat3 and Sp1. Mol Cell Biol. 18: 2108-2117, 1997.

Cao, Z., Umek, R. M., and McKnight, S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 5: 1538-1552, 1991.

Cassel, T. N., Nordlund-Moller, L., Andersson, O., Gustafsson, J. A., and Nord, M. C/EBPalphaand C/EBPdeltaactivate the clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites. Am. J. Respir. Cell Mol. Biol. 22, 469-480, 2000.

Clarke, S. L., Robinson, C. E. and Gimble, J. M. CCAAT/enhancer-binding proteins directly modulate transcription from the peroxisome proliferator activated receptor-gamma2 promoter. Biochem. Biophys. Res. Commun. 240: 99-103, 1997.

Custafson, B., Jack, M. M., Cushman, S. W. and Smith, U. Adiponectin gene activation by thiazolidinediones requires PPAR-gamma2, but not C/EBPalpha-evidence for differential regulation of the aP2 and adiponectin genes Biochem. Biophys. Res. Commun. 308: 933-939, 2003.

Demetri, G. D., Fletcher, C. D., Mueller, E., Sarraf, P., Naujoks, R. and Campbell, N. Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-gamma ligand troglitazone in patients with liposarcoma, Proc Natl Acad Sci USA, 96: 3951-3956, 1999.

Dong, J., Fujii, S., Li, H., Nakabayashi, H., Sakai, M., Nishi, S., Goto, D., Furumoto, T., Imagawa, S., Zaman, T. A. and Kitabatake, A. Interleukin-6 and mevastatin regulate plasminogen activator inhibitor-1 through CCAAT/enhancer-binding protein delta. Arterioscler Thromb Vasc Biol. 25:1078-84, 2005.

Elberg, G., Gimble, J. M. and Tsai, S. Y. Modulation of the murine peroxisome proliferator-activated receptor-gamma2 promoter activity by CCAAT/ enhancer-binding proteins. J Biol Chem, 275: 27815-27822, 2000.

Eibl, G., Wente, M. N., Reber, H. A., and Hines, O. J. Peroxisome proliferator-activated receptor-gamma induces pancreatic cancer cell apoptosis Biochem. Biophys. Res. Commun. 287: 522-529, 2001.

Fajas, L., Auboeuf, D., Raspe, E., Schoonjans, K., Lefebvre, A. M., Saladin, R., Najib, J., Laville, M., Fruchart, J. C., Deeb, S., Vidal-Puig, A., Flier, J., Briggs, MR., Staels, B., Vidal, H. and Auwerx J. The organization, promoter analysis, and expression of the human PPAR-gamma Gene. J Biol Chem, 272: 18779-18789, 1997.

Fu, M., Zhang, J., Lin, Y., Zhu, X., Ehrengruber, M. U. and Chen, Y. E. Early growth response factor-1 is a critical transcriptional mediator of peroxisome proliferator-activated receptor-1 gene expression in human aortic smooth muscle cells. J Biol Chem. 277: 26808-26814, 2002.

Gearing, K. L., Gottlicher, M., Teboul, M., Widmark, E. and Gustafsson, J. A. Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. Proc Natl Acad Sci USA, 90: 1440-1444, 1993.

Gery, S., Tanosaki, S., Hofmann, W. K., Koppel, A. and Koeffler, HP. C/EBP expression in a BCR-ABL-positive cell line induces growth arrest and myeloid differentiation. Oncogene, 24: 1589-1597, 2005.

Heaney, A.P. Novel pituitary ligands: peroxisome proliferators activating receptor-γ. Cedars-Sinai Research Institute, Division of Endocrinology. Pituitary, 6: 153-159, 2003.

Hogan, J. C. and Stephens, J. M. The identification and characterization of a STAT 1 binding site in the PPAR-2 promoter. Biochem Biophys Res Commun. 287: 484-92, 2001.

Huang, A. M., Montagna, C., Sharan, S., Ni, Y., Ried, T., and Sterneck, E. Loss of CCAAT/enhancer binding protein deltapromotes chromosomal instability. Oncogene 23: 1549-1557, 2004.

Imagawa, S., Yamamoto, M. and Miura, Y. Negative regulation of the erythropoietin gene expression by the GATA transcription factors. Blood. 89: 1430-1439, 1997.

Jiang, J. G. and Zarnegar, R. A novel transcriptional regulatory region within the core promoter of the hepatocyte growth factor gene is responsible for its inducibility by cytokines via the C/EBP family of transcription factors. Mol Cell Biol. 17: 5758-5770, 1997.

Johnson, P. F., and McKnight, S. L. Eukaryotic transcriptional regulatory proteins. Annu. Rev. Biochem. 58: 799-839, 1989.

Juge-Aubry, C., Pernin, A., Favez, T., Burger, A.G., Wahli, W. and Meier, C.A. DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. Importance of the 5′-flanking region, J Biol Chem, 272: 25252-25259, 1997.

Kinoshita, S., Akira, S. and Kishimoto, T. A member of the C/EBP family, NF-IL6beta, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc. Natl. Acad. Sci. U.S.A. 89: 1473–1476, 1992.

Kintscher, U., Wakino, S., Bruemmer, D., Goetze, S., Graf, K., Hsueh, WA. and Law, R. E. TGF-1 induces peroxisome proliferator-activated receptor-gamma1 and gamma2 expression in human THP-1 monocytes. Biochem Biophys Res Commun. 297: 794-799, 2002.

Koeffler, H. P. Peroxisome proliferator-activated receptor γ and cancers, Clin Cancer Res, 9: 1-9, 2003.

Kota, B. P., Huang, T. H-W. and Roufogalis, B. D. An overview on biological mechanisms of PPARs. Pharmacological Research. 51: 85-94, 2005.

Landschulz, W. H., Johnson, P. F., Adashi, E. Y., Graves, B. J. and McKnight, S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 2: 786–800, 1988.

Laudet, V. and Hanni, C. Evolution of the nuclear receptor gene super family. EMBO J,11: 1003-1013, 1992.

Lekstrom-Himes, J. and Xanthopoulos, K. G. Biological role of the CCAAT/ enhancer-binding protein family of transcription factors. J. Biol. Chem. 273: 28545–28548, 1998.

Leibowitz, S. B. and Kantoff, P. W. Differentiating agents and the treatment of prostate cancer: vitamin D3 and peroxisome proliferator-activated receptor-γ ligands, Semin Oncol, 30: 698-708, 2003.

Li, M. Y., Deng, H., Zhao, J. M., Dai, D. and Tan, X. Y. PPAR-gammapathway activation results in apoptosis and COX-2 inhibition in HepG2 cells. World J. Gastroenterol. 9: 1220-1226, 2003.

Liu, Y. W., Tseng, H. P., Chen, L. C., Chen, B. K. and Chang, W. C. Functional cooperation of simian virus 40 promoter factor 1 and CCAAT/enhancer-binding protein beta and delta in lipopolysaccharide -induced gene activation of IL-10 in mouse macrophages. J Immunol. 171: 821-828, 2003.
.
Liu, J., Lu, H., Huang, R., Lin, D., Wu, X., Lin, Q., Wu, X., Zheng, J., Pan, X., Peng, J., Song, Y., Zhang, M., Hou, M. and Chen, F. Peroxisome proliferators-activated receptor-gammaligands induced cell growth inhibition and its influence on matrix metalloproteinase activity in human myeloid leukemia cells. Cancer Chemother Pharmacol. 2005.

McKnight, S. L. CCAAT/enhancer binding protein. In: McKnight S. L., Yamamoto K. R., editors. Transcriptional regulation. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. 771-795, 1992.

Marx, N., Sukhova, G., Murphy, C., Libby, P. and Plutzky, J. Macrophages in human atheroma contain PPAR: differentiation-dependent peroxisome proliferator-activated receptor-gamma(PPAR-gamma) expression and reduction of MMP-9 activity through PPAR-gammaactivation in mononuclear phagocytes in vitro, Am J Pathol, 153: 17-23, 1998.

Nagamine, M., Okumura, T., Tanno, S., Sawamukai, M., Motomura, W., Takahashi, N., and Kohgo, Y. PPAR-gammaligand-induced apoptosis through a p53-dependent mechanism in human gastric cancer cells. Cancer Sci. 94: 338-343, 2003.

Niu, X., Renshaw-Gegg, L., Miller, L. and Guiltinan, M. J. Bipartite determinants of DNA-binding specificity of plant basic leucine zipper proteins, Plant Molecular Biology 41: 1–13, 1999.

Ohshima, T., Koga, H. and Shimotohno, K. Transcriptional activity of peroxisome proliferator-activated receptor-gammais modulated by SUMO-1 modification. J Biol Chem. 279: 29551-29557, 2004.

O'Rourke, J. P., Hutt, J. A. and DeWille, J. Transcriptional regulation of C/EBPdelta in G(0) growth-arrested mouse mammary epithelial cells. Biochem. Biophys. Res. Commun. 262: 696–701, 1999.

Pasceri, V., Wu, H. D., Willerson, J. T. and Yeh, E. T. Modulation of vascular inflammation in vitro and in vivo by peroxisome proliferator-activated receptor-gamma activators, Circulation, 101: 235-238, 2000.

Poli, V. The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J. Biol. Chem. 273: 29279–29282, 1998.

Ramji, D. P., and Foka, P. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem. J. 365: 561575, 2002.

Ramji, D. P., Vitelli, A., Tronche, F., Cortese, R. and Ciliberto, G. The two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBPdelta/NF-IL6beta, are induced by IL-6 to promote acute phase gene transcription via different mechanisms. Nucleic Acids Res. 21: 289–294, 1993.

Rangwala, S. M., Rhoades, B., Shapiro, J. S., Rich, A. S., Kim, J. K. and Shulman, G. I. Genetic modulation of PPAR-γ phosphorylation regulates insulin sensitivity, Dev Cell, 5: 657-663, 2003.

Shao, D. and Lazer, M. A. Peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein α, and cell cycle status regulate the adipocyte differentiation, J Biol Chem, 272: 21473-21478, 1997.

Shi, X. M., Blair, H. C., Yang, X., McDonald, J. M. and Cao, X. Tandem repeat of C/EBP binding sites mediates PPAR-gamma2 gene transcription in glucocorticoid-induced adipocyte differentiation. J Cell Biochem. 76: 518-527.

Shi, X., Shi, W., Li, Q., Song, B., Wan, M., Bai, S. and Cao, X. A glucocorticoid-induced leucine-zipper protein, GILZ, inhibits adipogenesis of mesenchymal cells. EMBO Rep. 4: 374-380, 2003.

Shibasaki, M., Takahashi, K., Itou, T., Bujo, H. and Saito, Y. A PPAR agonist improves TNF-α-induced insulin resistance of adipose tissue in mice, Biochem Biophys Res Commun, 309: 419-424, 2003.

Shimaya, A., Noshiro, O., Hirayama, R., Yoneta, T., Niigata, K. and Shikama, H. Insulin sensitizer YM268 ameliorates insulin resistance by normalizing the decreased content of GLUT4 in adipose tissue of obese Zucker rats, Eur J Endocrinol, 137: 693-700, 1997.

Shojima, N., Sakoda, H., Ogihara, T., Fujishiro, M., Katagiri, H. and Anai, M. Humoral regulation of resistin expression in 3T3-L1 and mouse adipose cells, Diabetes, 51: 1737-1744, 2002.

Sterneck, E., Paylor, R., Jackson-Lewis, V., Libbey, M., Przedborski, S., Tessarollo, L., Crawley, J. N. and Johnson, P. F. Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer-binding protein delta. Proc. Natl. Acad. Sci. U.S.A. 95: 10908–10913, 1998.

Sugahara, K., Sadohara, T., Sugita, M., Iyama, K. and Takiguchi, M. Differential expression of CCAAT/enhancer-binding protein family in rat alveolar epithelial cell proliferation and in acute lung injury. Cell Tissue Res. 297, 261270, 1999.

Takiguchi, M. The C/EBP family of transcription factors in the liver and other organs. Int. J. Exp. Pathol. 79: 369391, 1998.

Tanaka, T., Yoshida, N., Kishimoto, T. and Akira, S. Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene. EMBO J. 16: 7432–7443, 1997.

Tong, Q., Tsai, J., Tan, G., Dalgin, G. and Hotamisligil, G. S. Interaction between GATA and the C/EBP family of transcription factors is critical in GATA-mediated suppression of adipocyte differentiation. Mol Cell Biol. 25: 706-715, 2005.

Tontonz, P., Hu, E. and Speiegelman, B. M. Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor-γ, Curr Opin Genet Dev. 5: 571-576, 1995.

Tsubouchi, Y., Sano, H., Kawahito, Y., Mukai, S., Yamada, R., Kohno, M., Inoue, K., Hla, T. and Kondo, M. Inhibition of human lung cancer cell growth by the peroxisome proliferator-activated receptor-gamma agonists through induction of apoptosis. Biochem. Biophys. Res. Commun. 270: 400-405, 2000.

Tsujie, M., Nakamori, S. and Okami, J. Thiazolidinediones inhibit growth of gastrointestinal, biliary, and pancreatic adenocarcinoma cells through activation of the peroxisome proliferator-activated receptor γ/retinoid X receptor α pathway, Exp Cell Res, 289: 143-151, 2003.

Umayahara, Y., Ji, C., Centrella, M., Rotwein, P. and McCarthy, T. L. CCAAT/enhancer-binding Protein delta activates Insulin-like growth factor-I gene transcription in osteoblasts. identification of a novel cyclic AMP signaling pathway in bone. J. Biol. Chem. 272: 31793-31800, 1997.

Wahli, W., Braissant, O. and Desvergne, B. Peroxisome proliferator activated receptors: transcriptional regulators of adipogenesis, lipid metabolism and more. Chem Biol. 2: 261-266,1995.

Wang, J.M., Ko, C.Y., Chen, L.C. and Chang, W.C. (2005) Functional role of NF-IL6β and its sumoylation and acetylation modifications in promoter activation of cyclooxygenase-2 in A431 cells. Submitted to Nucleic Acids Res.

Wang, J.M., Tseng, J.T. and Chang, W.C. Induction of human NF-IL6 by epidermal growth factor is mediated through the p38 signaling pathway and CREB activation in A431 cells. Mol Biol Cell. 16: 3365-3376, 2005.

Williams, S. C., Baer, M., Dillner, A. J. and Johnson, P. F. CRP2 (C/EBPbeta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J. 14: 3170–3183, 1995.

Wu, Z., Rosen, E. D., Brun, R., Hauser, S., Adelmant, G. and Troy, A. E. Cross-regulation of C/EBPalphaand PPAR-gammacontrols the transcriptional pathway of adipogenesis and insulin sensitivity. Mol Cell, 3: 151-158, 1999.

Yamada, T., Tsuchiya, T., Osada, S., Nishihara, T. and Imagawa, M. CCAAT/enhancer-binding protein delta gene expression is mediated through downstream binding sites. Biochem. Biophys. Res. Commun. 242: 88–92, 1998.

Yang, T. T. C. and Chow, C. W. Transcription cooperation by NFAT- C/EBP composite enhancer complex. J. Biol. Chem. 278: 15874-15885, 2003.

Zhu, Y., Sanders, M. A., Yeh, H., Deng, W. G. and Wu, K. K. Dynamic regulation of cyclooxygenase-2 promoter activity by isoform of CCAAT/enhancer-binding proteins. J. Biol. Chem. 277: 6923-6928, 2002.