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Originally published In Press as doi:10.1074/jbc.M212704200 on January 6, 2003

J. Biol. Chem., Vol. 278, Issue 11, 9298-9308, March 14, 2003
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Interferon-gamma Induces p11 Gene and Protein Expression in Human Epithelial Cells through Interferon-gamma -activated Sequences in the p11 Promoter*

Xiu-li HuangDagger , Rafal PawliczakDagger §, Xiang-lan YaoDagger , Mark J. CowanDagger , Mark T. GladwinDagger , M. J. Walter, M. J. Holtzman, Patricia MadaraDagger , Carolea LogunDagger , and James H. ShelhamerDagger ||

From the Dagger  Critical Care Medicine Department of the Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, the § Department of Clinical Immunology and Allergy, Medical University of Lodz, Lodz, 92213 Poland, and the  Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

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 ~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 ionophor-stimulated arachidonic acid release suggesting that at least in part IFN-gamma -stimulated p11 expression may serve a counterregulatory role.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Warren G. Magnuson Clinical Center, Critical Care Medicine Dept., Bldg. 10, Rm. 7D43, 10 Center Dr., MSC1662, Bethesda, MD 20892-1662. Tel.: 301-496-9320; Fax: 301-480-3389.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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