NFκB-p65 dependent transcriptional regulation of glycosyltransferases in human colon adenocarcinoma HT-29 by stimulation with tumor necrosis factor α

Regulation of fucosyltransferases (FUTs) and sialyltransferases (STs) in a human colon adenocarcinoma cell line HT-29 and nuclear factor kappa B (NF kappa B)-p65 knockdown HT-29 cells was investigated after stimulation with tumor necrosis factor alpha (TNF alpha) using real time PCR. TNF alpha stimulation induced the biphasic increases in expression of NF kappa B-p65, ST3Gal 1, 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 NF kappa B-p65 knockdown HT-29 cells, the biphasic and transient increases in all of these mRNA expression induced with TNFa were diminished. On the other hand, NF kappa B-p65 siRNA enhanced the constitutive expression levels of ST3GalNAc IV mRNA which was suppressed by TNFa. Transcription activities of ST3Gal I reporter gene from nt - 1050 5'-flanking region to translation initiation site which has consensus NF kappa B binding sites were up-regulated by stimulation with TNFa in HT-29 cells. The promoter activities for deletion constructs of each NF kappa B 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 TNF alpha enhanced ST3Gal I gene expression through NF kappa B binding sites in HT-29 cells. Combination of stimulation with TNF alpha and NF kappa B knockdown with siRNA is useful for determination of NF kappa B dependent transcriptional regulation.