| Summary: | 碩士 === 國立成功大學 === 藥理學研究所 === 92 === CCAAT/enhancer-binding protein d (C/EBPd) belongs to a family of transcription regulators C/EBP that has been implicated in mediating the acute phase response to inflammatory stimuli and in controling of adipogenesis. It was reported that LPS activated the transcription of C/EBPd in mouse macrophage cell line RAW264.7. However, little is known about the molecular mechanism of C/EBPd induced by LPS. In this study, we used trichostatin A (TSA), a histone deacetylase inhibitor, and cyclosporin A (CsA), a phosphatase 2B inhibitor, to assess whether any post-translational modification of acetylation or phosphorylation are involved in the regulation of LPS-induced C/EBPd gene. In this report, we found that TSA inhibited LPS-induced protein and mRNA expression of C/EBPd, and CsA inhibited LPS-induced protein expression of C/EBPd. Moreover, we also studied the LPS responsive element in the C/EBPd promoter. Analysis of the 5'-deletion mutants revealed that the 5'-flanking region of C/EBPd promoter sequences between -345 to +29 bp were sufficient for basal and LPS-inducibility. Furthermore, analysis of point mutation or double mutation of C/EBPd promoter revealed that Sp1 (-120/-115 bp) and CRE (-45/-38 bp) sites were essential for LPS responsiveness. Gel shift assay was used to identify potential regulators of these LPS responsive elements. The results showed that Sp1 and Sp3 bound to the promoter region of Sp1 site (-120/-115 bp); CREB and c-Jun bound to the promoter region of CRE site. Additionally, overexpression of c-Jun and Sp1, but not of CREB increased the promoter activity of C/EBPd gene. These results suggested that the Sp1 (-120/-115 bp) and the CRE (-45/-38 bp) sites are essential for LPS-induced transcriptional activity of C/EBPd gene. Both of Sp1 and c-Jun were important factors which were able to enhance the transcriptional activity of C/EBPd gene�|
|
|---|
