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J. Biol. Chem., Vol. 281, Issue 33, 23958-23968, August 18, 2006

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Phosphatidylinositol 3-Kinase-dependent Suppression of the Human Inducible Nitric-oxide Synthase Promoter Is Mediated by FKHRL1^*

Arnold S. Kristof¹, Jill Fielhaber, Alexandra Triantafillopoulos, Shino Nemoto^¶, and Joel Moss

From the Pulmonary-Critical Care Medicine Branch and ^¶Cardiovascular Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the Critical Care and Respiratory Divisions, Royal Victoria Hospital and Meakins-Christie Laboratories, McGill University, Montreal, Quebec H3A 1A1, Canada

The synthesis of nitric oxide by inducible nitric-oxide synthase (iNOS) plays an important role in the innate immune response by promoting microbial killing and cell damage. In response to inflammatory cytokines and bacterial products, the human iNOS (hiNOS) gene undergoes rapid transcriptional activation via binding of stimulatory transcription factors (e.g. AP-1 and NF-B) to its 5 '-flanking region. However, maximal hiNOS promoter induction was suppressed via an unknown phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. We hypothesized that inhibition of the transcription factor FKHRL1 by the PI3K/protein kinase B pathway attenuates hiNOS promoter induction by bacterial lipopolysaccharide and interferon-gamma (LPS/IFN-). Human lung epithelial adenocarcinoma (A549) cells were transiently transfected with an 8.3-kb hiNOS promoter luciferase reporter construct. Co-expression of dominant-negative protein kinase B potentiated LPS/IFN--stimulated hiNOS promoter activity. In response to LPS/IFN-, FKHRL1 was phosphorylated in a PI3K- and time-dependent fashion. Co-expression of constitutively active FKHRL1 increased hiNOS promoter activity and mRNA levels. Dominant-negative siRNA expression showed that FKHRL1 was necessary for the inhibitory effects of PI3K on hiNOS induction. The same effect was observed upon mutation of a consensus FKHRL1-binding site in the hiNOS promoter. By gel-shift analysis, the corresponding oligonucleotide probe bound endogenous FKHRL1 in an LPS/IFN-- and PI3K-sensitive fashion. Regulation of the hiNOS promoter by FKHRL1 represents a potentially important molecular mechanism by which the PI3K pathway might suppress pro-inflammatory and proapoptotic responses to cytokines and bacterial products.

Received for publication, December 30, 2005 , and in revised form, April 25, 2006.

^* This work was supported by the Division of Intramural Research, NHLBI, National Institutes of Health (to J. M., A. S. K., and S. N.) and a Canadian Institutes for Health Research Operating Grant (to A. S. K., J. F., and A. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: McGill University, Royal Victoria Hospital, 687 Pine Ave. W., Rm. L3.05, Montreal, Quebec H3A 1A1, Canada. Tel.: 514-843-1664; Fax: 514-843-1686; E-mail: arnold.kristof{at}muhc.mcgill.ca.

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