NFkappaB-p65 dependent transcriptional regulation of glycosyltransferases in human colon adenocarcinoma HT-29 by stimulation with tumor necrosis factor alpha.
Regulation of fucosyltransferases (FUTs) and sialyltransferases (STs) in a human colon adenocarcinoma cell line HT-29 and nuclear factor kappaB (NFkappaB)-p65 knockdown HT-29 cells was investigated after stimulation with tumor necrosis factor alpha (TNFalpha) using real time PCR. TNFalpha stimulation induced the biphasic increases in expression of NFkappaB-p65, ST3Gal I, FUT IV, ST3Gal IV and ST6GalNAc III mRNAs and the transient increase in expression of ST6Gal I mRNA and the decrease in ST3GalNAc IV mRNA. In NFkappaB-p65 knockdown HT-29 cells, the biphasic and transient increases in all of these mRNA expression induced with TNFalpha were diminished. On the other hand, NFkappaB-p65 siRNA enhanced the constitutive expression levels of ST3GalNAc IV mRNA which was suppressed by TNFalpha. Transcription activities of ST3Gal I reporter gene from nt -1050 5'-flanking region to translation initiation site which has consensus NFkappaB binding sites were up-regulated by stimulation with TNFalpha in HT-29 cells. The promoter activities for deletion constructs of each NFkappaB binding sites were determined using dual luciferase assay. The results indicated that constitutive promoter activities were detected at nt -120 5'-flanking translation initiation site and TNFalpha enhanced ST3Gal I gene expression through NFkappaB binding sites in HT-29 cells. Combination of stimulation with TNFalpha and NFkappaB knockdown with siRNA is useful for determination of NFkappaB dependent transcriptional regulation.
Higai
Koji
K
Department of Clinical Chemistry, School of Pharmaceutical Sciences, Toho University, Chiba, Japan. koji@phar.toho-u.ac.jp
Ishihara
Saki
S
Matsumoto
Kojiro
K
D000230:Adenocarcinoma / Q000201:enzymology* / Q000473:pathology; D001483:Base Sequence; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology; D017931:DNA Primers; D016695:Glycosyltransferases / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation
D017931:DNA Primers; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016695:Glycosyltransferases