Promoter analysis of Interleukin 19

• Main Authors: Feng-Wei Chen, 陳逢維
• Other Authors: Ming-Shi Chang
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/y6v75h

碩士 === 國立成功大學 === 生物化學研究所 === 90 === 　　Cytokines are important molecules of hormones. Most of the cytokines are small to medium sized proteins which mediate potent biological effects on most cell types. Originally they are identified as being important in inflammatory processes, the development and maintenance of immune respones and haematopoiesis. 　　A family of interleukin-10 (IL-10)-related cytokines has been emerged which include IL-19, IL-20, IL-22 [IL-10-related T-cell-derived inducible factor (IL-TIF)], IL-24 [melanoma differentiation-associated antigen 7 (MDA-7)] and IL-26 (AK155). Whereas IL-10 is a well-studied pleiotropic immunosuppressive and immun-stimulatory cytokine. It inhibits the production of IL-1, IL-6 and TNF-α. IL-22/IL-TIF can mediate acute-phase response signals in hepatocytes and IL-20 induces the hyperproliferation of keratinocytes. IL-24 is related to the suppression of tumor cell growth. But the function of IL-19 and IL-26 are still unclear. The genes for IL-10, IL-19, IL-20 and IL-24 are found within a 200kb region of chromosome 1 and the two other family members IL-22 and IL-26 are found on chromosome 12. 　　IL-19 was first discoveried in year 2000. It shares 21% amino acid identity with the amino acid sequence of IL-10. In the previous study, it was reported that IL-10 functions as an inhibitor in immune system especial on monocyte、T cell and B cell and the production of cytokines ,such as IFN-γ. The expression of IL-19 mRNA could be induced by LPS and GM-CSF treatment in monocytes. The exon/intron structure of IL-19 is similar to that of the human IL-10 gene, comprising five exons and four introns within the coding region of the IL-19 cDNA. There are at least two distinct IL-19 mRNA species that differ in their 5'-sequences. By 5’RACE experiment, we identified the transcription start site and constructed 5 potential promoter fragments of different length upstream of exon1. We termed these five constructs to PSA、PSB、PSC、PSD、PSE and transfected these plasmid into MDCK and 293 cell lines. The results of luciferase activity showed that the PSE region expressed highest promoter activity in both MDCK and 293 cells. To find the binding protein and binding region of IL-19 promoter, we used EMSA experiment to define the binding sequence between promoter region and transcription factor. The EMSA data proved that a specific region of PSE can bind to some transcription factors contained in the nuclear extract and control the luciferase activity. In the region of nt.-141~-146 and nt.-42~-51, the HNF-5 and TFIID-MBP may bind to the DNA sequence. Furthermore, we mutated the six nucleotide of -141~-146 region in reporter vector. We found the luciferase activity of this mutant decreased 70％ activity compared to the wild type control. When we treated cells with several reagents:LPS､TGF-β､IFN-γ､IL-6､GM-CSF. We found that GM-CSF could enhance the IL-19 promoter activity in 293 cell line.