2006 Volume 29 Issue 12 Pages 2372-2377
Regulation of fucosyltransferases (FUTs) and sialyltransferases (STs) in a human colon adenocarcinoma cell line HT-29 and nuclear factor κB (NFκB)-p65 knockdown HT-29 cells was investigated after stimulation with tumor necrosis factor α (TNFα) using real time PCR. TNFα stimulation induced the biphasic increases in expression of NFκB-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 NFκB-p65 knockdown HT-29 cells, the biphasic and transient increases in all of these mRNA expression induced with TNFα were diminished. On the other hand, NFκB-p65 siRNA enhanced the constitutive expression levels of ST3GalNAc IV mRNA which was suppressed by TNFα. Transcription activities of ST3Gal I reporter gene from nt −1050 5′-flanking region to translation initiation site which has consensus NFκB binding sites were up-regulated by stimulation with TNFα in HT-29 cells. The promoter activities for deletion constructs of each NFκB 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 TNFα enhanced ST3Gal I gene expression through NFκB binding sites in HT-29 cells. Combination of stimulation with TNFα and NFκB knockdown with siRNA is useful for determination of NFκB dependent transcriptional regulation.
