title: Characterization of the human intestinal CD98 promoter and its regulation by interferon-gamma.
abstract: 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 NF-kappaB binding domain, and no TATA box. Binding of Sp1 [Sp1(-874), SP1(-386), Sp1(-187), and Sp1(-177)] and NF-kappaB [NF-kappaB(-213)] to the promoter was confirmed by EMSA and supershift assays. Furthermore, chromatin immunoprecipitation experiments showed the in vivo DNA-Sp1 and DNA-NF-kappaB interactions under basal and IFN-gamma-stimulated conditions. Reporter genes driven by serially truncated and site-mutated CD98 promoters were used to examine basal and IFN-gamma-responsive transcription in transiently transfected Caco2-BBE cells. Our results revealed that Sp1(-187), Sp1(-177), and the NF-kappaB binding site were essential for basal and IFN-gamma-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 NF-kappaB site may cooperate in mediating basal and IFN-gamma-stimulated CD98 promoter activities. Finally, we demonstrated that a reduction of Sp1 or NF-kappaB expression reduced CD98 protein expression in unstimulated and IFN-gamma-stimulated Caco2-BBE cells. Collectively, these findings indicate that the Sp1 and NF-kappaB transcription factors are likely to play a significant role in IFN-gamma-mediated transcriptional regulation of CD98 in the intestinal epithelium, providing new insights into the regulation of CD98 expression in intestinal inflammation.
authors.affiliation: Div of Digestive Diseases, Dept of Medicine, Emory Univ, Atlanta, GA 30322, USA.
authors.forename: Yutao
authors.initials: Y
authors.lastname: Yan
authors.forename: Guillaume
authors.initials: G
authors.lastname: Dalmasso
authors.forename: Shanthi
authors.initials: S
authors.lastname: Sitaraman
authors.forename: Didier
authors.initials: D
authors.lastname: Merlin
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018938:Caco-2 Cells; D047369:Chromatin Immunoprecipitation; D003092:Colitis / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D027261:Fusion Regulatory Protein-1 / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection
chemical_list: D027261:Fusion Regulatory Protein-1; D016328:NF-kappa B; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D007371:Interferon-gamma; D008156:Luciferases