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.