ZBP-89, Sp1, and nuclear factor-kappa B regulate epithelial neutrophil-activating peptide-78 gene expression in Caco-2 human colonic epithelial cells.
We reported previously that human colonic epithelial cells produce the C-X-C chemokine epithelial neutrophil-activating peptide-78 (ENA-78) and that its expression is up-regulated in ulcerative colitis. The aim of this study was to investigate the transcriptional regulation of ENA-78 gene expression in Caco-2 intestinal epithelial cells. Reporter gene transfection and electrophoretic mobility shift assay studies demonstrated that cooperation between two regions of the ENA-78 promoter were required for maximal gene expression in interleukin-1beta-stimulated Caco-2 cells. Binding of activated p50/p65 nuclear factor-kappaB to nucleotides -82 to -91 was essential for interleukin-1beta-dependent gene transcription, whereas binding of constitutively expressed zinc-requiring nuclear factors to nucleotides -125 to -134 (site A) was required for basal gene expression. Scanning mutagenesis of site A demonstrated overlapping binding elements at this locus. One site (CTCCCCC) bound Sp1 and Sp3, and overexpression of Sp1 (but not Sp3) up-regulated basal ENA-78 transcription. Another site (CCCCTCCCCC) was found to bind the zinc finger nuclear factor ZBP-89, and overexpression of this protein significantly repressed ENA-78 reporter gene activity. This study demonstrates that ENA-78 gene expression in Caco-2 intestinal epithelial cells is subject to complex regulation involving the coordinate binding of ZBP-89, Sp1, and nuclear factor-kappaB to the ENA-78 promoter.
Keates
A C
AC
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA. akeates@caregroup.harvard.edu
Keates
S
S
Kwon
J H
JH
Arseneau
K O
KO
Law
D J
DJ
Bai
L
L
Merchant
J L
JL
Wang
T C
TC
Kelly
C P
CP
D000818:Animals; D001665:Binding Sites; D018938:Caco-2 Cells; D054365:Chemokine CXCL5; D019743:Chemokines, CXC; D003106:Colon / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004330:Drosophila; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D016209:Interleukin-8 / Q000031:analogs & derivatives* / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*; D015032:Zinc / Q000378:metabolism
C486263:CXCL5 protein, human; D054365:Chemokine CXCL5; D019743:Chemokines, CXC; D004268:DNA-Binding Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C110951:ZNF148 protein, human; D008156:Luciferases; D015032:Zinc