Characterization of the human intestinal CD98 promoter and its regulation by interferon-

Yan Y, Dalmasso G, Sitaraman S, Merlin D. Characterization of the human intestinal CD98 promoter and its regulation by interferon. Am J Physiol Gastrointest Liver Physiol 292: G535–G545, 2007. First published October 5, 2006; doi:10.1152/ajpgi.00385.2006.—Growing evidence that epithelial CD98 plays an important role in intestinal inflammation focused our interest to investigate the transcriptional regulation of CD98. Our mouse-based in vivo and in vitro experiments revealed that epithelial colonic CD98 mRNA expression was transcriptionally increased in intestinal inflammation. We then isolated and characterized a 5 -flanking fragment containing the promoter region required for CD98 gene transcription. Primer extension and rapid amplification of 5 -cDNA ends were used to map a transcriptional initiation site 129 bp upstream from the translational start codon (ATG). Direct sequencing of the 5 -flanking region revealed the presence of four GC-rich stimulating protein (Sp)1 binding domains, one NFB binding domain, and no TATA box. Binding of Sp1 [Sp1( 874), SP1( 386), Sp1( 187), and Sp1( 177)] and NFB [NFB( 213)] to the promoter was confirmed by EMSA and supershift assays. Furthermore, chromatin immunoprecipitation experiments showed the in vivo DNA-Sp1 and DNA-NFB interactions under basal and IFN-stimulated conditions. Reporter genes driven by serially truncated and site-mutated CD98 promoters were used to examine basal and IFN-responsive transcription in transiently transfected Caco2-BBE cells. Our results revealed that Sp1( 187), Sp1( 177), and the NFB binding site were essential for basal and IFN-stimulated CD98 promoter activities, whereas Sp1( 874) and Sp1( 386) were not. The results from additional site-mutated CD98 promoters suggested that Sp1( 187), Sp1( 177), and the NFB site may cooperate in mediating basal and IFN-stimulated CD98 promoter activities. Finally, we demonstrated that a reduction of Sp1 or NFB expression reduced CD98 protein expression in unstimulated and IFN-stimulated Caco2-BBE cells. Collectively, these findings indicate that the Sp1 and NFB transcription factors are likely to play a significant role in IFN-mediated transcriptional regulation of CD98 in the intestinal epithelium, providing new insights into the regulation of CD98 expression in intestinal inflammation.