CCAAT/enhancer-binding protein δ( C/EBPδ)為轉錄因子C/EBP 家族的其中一個成員，在受到發炎物質刺激時，調控與發炎反應有關之基因表現。由本實驗室先前的研究已知，在老鼠巨噬細胞(RAW264.7)中，LPS可誘導 C/EBPδ基因表現。而LPS 誘導C/EBPδ基因表現之機制已被發現。在LPS 的刺激下同時可透過ERK/JNK/p38 signal pathway 及NF-κB signal pathway 來分別增加c-Jun 及c-Rel 作用在C/EBPδ基因啟動區，進而調控C/EBPδ基因表現。然而根據本實驗室先前的研究已初步知道在histone deacetylase inhibitor-trichostatin A(TSA)作用下，可抑制LPS 所誘導 C/EBPδ基因表現，但其詳細作用機制如何仍未釐清。因此本實驗主要探討：在老鼠巨噬細胞RAW264.7 中，TSA 抑制LPS 所誘導C/EBPδ基因表現的作用機制?實驗證明，於TSA 作用下，可呈現劑量相關性抑制LPS 所誘導 C/EBPδ蛋白質及RNA表現。經由實驗證明我們得知，TSA的作用並不是經由NF-κB 及ERK signal pathway 調控LPS 所誘導C/EBPδ基因表現。且於TSA 作用下並不會影響LPS 誘導c-Jun 和c-Rel蛋白質表現量，及c-Jun 和c-Rel 轉錄因子各別在CRE binding site(為c-Jun binding site) 及 APRE binding site (為c-Rel binding site )之DNA 結合能力。但根據本實驗室先前研究，調控12-LOX 基因表現機制當中，c-Jun會透過Sp1 而結合到基因啟動區進而啟動12-LOX 基因表現。我們經由DNA Affinity Precipitation Assay 證明，在調控C/EBPδ基因表現機制當中，於TSA 作用下，會抑制由LPS 所誘導c-Jun 於-129/-96 基因啟動區之結合力(-129/-96:為含有Sp1 binding site 但不含c-Jun binding site 之基因序列)。因此我們初步推測於TSA 作用下會藉由抑制c-Jun經由Sp1 結合到基因啟動區的結合力，進而降低C/EBPδ基因表現量。而Reporter assay data 亦顯示-129/-96 區段對於TSA 調控LPS 誘導C/EBPδ基因表現的確扮演一個重要的角色。此外，TSA 為Histone deacetylase inhibitor，又根據本實驗室先前研究已知TSA 可藉由調控 Sp1 乙醯化，而進一步抑制標的蛋白表現。因此我們著手於Sp1 Acetylation assay，發現的確在LPS作用下可以將Sp1 去乙醯化，且增加Sp1 與p300 蛋白質之間的結合，而在合併給予TSA 作用下會將Sp1 回復為乙醯化狀態，且同時降低Sp1 與p300 蛋白質之間的結合。根據以上已知的相關證據，推測TSA 抑制LPS 的機制中，LPS 作用下，會將Sp1 去乙醯化而增加p300 及c-Jun 轉錄因子間接結合至-129/-96 基因啟動區，進而啟動C/EBPδ基因表現；而在TSA作用下會抑制掉Sp1 去乙醯化機制，進而抑制C/EBPδ基因表現。

C/EBPδ is an important transcription regulatory factor that has been implicated in inflammatory acute phase response. It is also reported that LPS could induce C/EBPδ gene expression in mouse macrophage cell line RAW264.7. Our previous study reveals that LPS-induced C/EBPδ gene activation is through the activation of the pathway of ERK/JNK/p38 and NF-κB, followed by the recruitment of c-Rel and c-Jun to the promoter of C/EBPδ. However, we found that TSA, a histone deacetylase inhibitor, could inhibit LPS-induced C/EBPδ protein in a dose-dependent manner. But mechanism of the inhibitory effect of TSA on LPS-induced C/EBPδ gene expression is still unclear. Herein we demonstrated that the effect of TSA in C/EBPδ are not due to the repression of ERK or NF-κB pathway. We also found that TSA treatment did not affect the levels of c-Jun and c-Rel in LPS-induced cells. However, TSA could inhibit the recruitment of c-Jun to promoter(-129/-96) of C/EBP δ . TSA also partially inhibited c-Rel-DNA binding activity with –129/-96 fragment. Furthermore, we also found that the acetylation of Sp1 was inhibited under LPS treatment, and increases the recruitment of p300 to the promoter. However,this effect of LPS in recruitment of p300 was reversed by TSA treatment. Therefore, to increase the acetylation of Sp1 by TSA treatment inhibited the transcriptional activity of C/EBPδ through the inhibition of recruitment of c-Jun and p300 to gene promoter.

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.

Berger, S. L. Histone modifications in transcriptional regulation. Curr. Opin.Genet. Dev. 12: 142-148, 2002.

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.

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.

Cardinaux, J. R., Allaman I., and Magistretti P. J. Pro-inflammatory cytokines induce the transcription factors C/EBPbeta and C/EBPdelta in astrocytes. Glia. 29: 91-97, 2000.

Chen, B. K., and 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: 10406-10411, 2000.

Dauphinee, S. M., and Aly Karsan. Lipopolysaccharide signaling in endothelial. Cells. Laboratory Investigation 86: 9–22, 2006.

Davies, G. E., Sabatakos, G., Cryer, A., and Ramji, D. P. The ovine CCAAT-enhancer binding protein δ gene: cloning, characterization, and species-specific autoregulation. Biochem. Biophys. Res. Commun. 271: 346–352, 2000.

Guha, M., and Mackman N. LPS induction of gene expression in human monocytes. Cell Signal. 13: 85-94. 2001.

Han, K. K., and Martinage, A. Post-translational chemical modification(s) of proteins. Int. J. Biochem. 24: 19-28, 1992.

Hebbes, T. R., Thorne, A. W., and Crane Robinson, C. A direct link between core histone acetylation and transcriptionally active chromatin. EMBO J. 7: 1395-1402. 1988.

Hill, C. S., and Treisman, R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80: 199-211, 1995.

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

Hunter, T., and Karin, M. The regulation of transcription by phosphorylation. Cell 70: 375 387, 1992.

Hutt, J. A., O'Rourke, J. P. , and DeWille, J. Signal transducer and activator of transcription 3 activates CCAAT enhancer-binding protein delta gene transcription in G0 growth-arrested mouse mammary epithelial cells and in involuting mouse mammary gland. J. Biol. Chem. 275: 29123-31, 2000.

Hung, J. J., Wang, Y. T., and Chang, W. C. Sp1 Deacetylation Induced by Phorbol Ester Recruits p300 To Activate 12(S)-Lipoxygenase Gene Transcription. Mol. Cell. Biol. 26: 1770-1785, 2006.

Kinoshita, S., Akira, S., and Kishimoto, T. A member of the C/EBP family, NF-IL6β, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc. Natl. Acad. Sci. U.S.A. 89: 1473–1476, 1992.

Kuo, M. H., and Allis, C. D. Roles of histone acetyltransferase and deacetylases in gene regulation. Bioessays 20: 615-626, 1998.

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.

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.

Liu, Y. W., Chen, C. C., Tseng, H. P., and Chang,W.C. Lipopolysaccharide induced transcriptional activation of interleukin-10 is mediated by MAPK and NF-kappaB-induced CCAAT/enhancer-binding protein delta in mouse macrophages. Cell Signal. 18: 1492-500, 2006.

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.

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

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

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

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/EBPβ and/or C/EBP δ gene. EMBO J. 16: 7432–7443, 1997.

Umayahara, Y., Ji, C., Centrella, M., Rotwein, P., and McCarthy, T. L. CCAAT/Enhancer-binding Protein 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.

Velazquez, O. C., Jabbar, A., DeMatteo, R. P., and Rombeau, J. L. Butyrate inhibits seeding and growth of colorectal metastases to the liver in mice. Surgery 120: 440-447, 1996.