The expression of prodynorphin gene is down-regulated by activation with lipopolysaccharide in U-937 macrophage cells.
Dynorphins are endogenous kappa opioid peptides widely distributed in the central nervous system. A number of recent reports have established their roles in modulating immunological functions. In this study, we investigated the expression of prodynorphin (PDYN) gene, which encodes the precursor of dynorphin peptides, in human macrophage U-937 cell line. PDYN mRNA was expressed at a detectable level, measured with a standard RT-PCR method in U-937 cells, but not in Jurkat and Raji cells, which are human T and B lymphocytes, respectively. Further analyses with RT-PCR assays by using primers covering each exon of the PDYN gene showed that U-937 cells expressed the adult brain-type PDYN mRNA. Most interestingly, activation of U-937 cells with lipopolysaccharide (LPS) led to a decrease in PDYN mRNA levels. This decrease was dependent on both the concentration of LPS and the duration of LPS treatment. In order to test the role of transcription in LPS-mediated down-regulation of PDYN gene expression in U937 cells, the proximal PDYN gene promoter with a length of either approximately 300 base pairs or approximately 900 base pairs was cloned and inserted upstream of luciferase reporter gene. Results from transient transfections and dual luciferase assays showed that PDYN gene promoter activity was decreased by LPS treatment. Taken together, our results suggested the U-937 cells expressed adult brain-type PDYN mRNA which was down-regulated by activation of the cells with LPS due to an inhibition of PDYN gene transcription.
Sun
Baoyong
B
Department of Pharmacology and Physiology, P.O. Box 711, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642-8711, USA.
Tipton
Christopher M
CM
Bidlack
Jean M
JM
D015152:Blotting, Northern / Q000379:methods; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004745:Enkephalins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D005786:Gene Expression Regulation; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D020298:U937 Cells
D004745:Enkephalins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D011498:Protein Precursors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C033660:preproenkephalin