Interferon-gamma induces p11 gene and protein expression in human epithelial cells through interferon-gamma-activated sequences in the p11 promoter.
The effect of interferon (IFN)-gamma on p11 expression was studied in two human epithelial cell lines (BEAS-2B and HeLa). Treatment with IFN-gamma resulted in increased steady-state levels of p11 mRNA and protein expression, with a time-dependent and dose-dependent effect. Transient transfection experiments of a reporter gene construct containing 1498 bp of the 5'-flanking region of the p11 promoter demonstrated that IFN-gamma induced p11 gene expression at the transcriptional level. These effects were inhibited at the promoter and protein levels by a specific JAK-2 kinase inhibitor, AG-490. Functional analysis of the p11 promoter indicates that two gamma-activated sequence elements (GAS) located at positions 1219 and 1090 are important for the induction of the p11 promoter by IFN-gamma. Transfection of mutated reporter constructs demonstrated that the mutation at the GAS-2 site (1090) inhibited the p11 promoter activity, with a reduction of about approximately 73% and mutation at the GAS-3 site (1219) eliminated about 26% of the p11 promoter activity. A STAT1 dominant negative mutant vector at Tyr-701 (JAK kinase phosphorylation site) blocked the effect of IFN-gamma on the p11 promoter activity. IFN-gamma induced a rapid tyrosine phosphorylation and nuclear translocation of STAT1 protein, which is involved in the binding to the GAS-2 site in the p11 promoter by EMSA analysis. These data suggest that IFN-gamma-induced p11 expression is mediated through the binding of STAT1 to GAS sites in the p11 promoter. Inhibition of p11 expression by inhibitory antisense RNAs (iRNA) treatment resulted in enhanced IFN-gamma and calcium ionophore-stimulated arachidonic acid release suggesting that at least in part IFN-gamma-stimulated p11 expression may serve a counterregulatory role.
Huang
Xiu-li
XL
Critical Care Medicine Department of the Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
Pawliczak
Rafal
R
Yao
Xiang-lan
XL
Cowan
Mark J
MJ
Gladwin
Mark T
MT
Walter
M J
MJ
Holtzman
M J
MJ
Madara
Patricia
P
Logun
Carolea
C
Shelhamer
James H
JH
D017306:Annexin A2; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D017353:Gene Deletion; D005799:Genes, Dominant; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007371:Interferon-gamma / Q000378:metabolism*; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D009418:S100 Proteins; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D000099248:S100 Calcium Binding Protein A10
D017306:Annexin A2; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D007371:Interferon-gamma; D016376:Oligonucleotides, Antisense; D012313:RNA; D012333:RNA, Messenger; D012260:Ribonucleases; D009418:S100 Proteins; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D000099248:S100 Calcium Binding Protein A10; C494079:STAT1 protein, human