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* Department of Surgery, Duke University Medical Center, Durham, NC 27710; and
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115
In systemic inflammation induced by endotoxin (LPS), the macrophage produces the majority of the circulating NO metabolites. However, while the molecular pathways which up-regulate iNOS expression have been extensively studied in the macrophage, little is known of the parallel counterregulatory pathways which repress or inhibit macrophage iNOS expression. Using both in vivo and in vitro murine models of endotoxin (LPS) stimulation, we have previously demonstrated that NO feedback inhibits its own synthesis by increasing transcription of osteopontin (OPN), a potent transrepressor of inducible NO synthase expression. In this current study, using a system of LPS-treated RAW264.7 macrophages, we go on to demonstrate that OPN increases STAT1 ubiquitination and subsequent 26s proteasome-mediated degradation to inhibit STAT1 dependent iNOS promoter activity, transcription, and protein expression. In addition, we identify STAT-interacting LIM protein as the critical STAT ubiquitin E3 ligase critical for STAT1 degradation in this setting. OPN has not been linked previously to STAT1 degradation. This regulation of STAT1 degradation underlies OPN's effect as an inhibitor of iNOS gene transcription. These are novel findings and define OPN as a unique and as yet, poorly characterized, transactivator of STAT1 degradation by the ubiquitin-proteasome system.
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.
1 This work was supported by National Institutes of Health Grants R01-GM65113 and R01-AI44629 (to P.C.K.) and R01-AI506296 (to M.J.G.).
2 Address correspondence and reprint requests to Dr. Paul C. Kuo, 110 Bell Building, Duke University Medical Center, Box 3522, Durham, NC 27710. E-mail address: kuo00004{at}mc.duke.edu
3 Abbreviations used in this paper: iNOS, inducible NO synthase; BMM, bone marrow macrophage; ChIP, chromatin immunoprecipitation; DN, dominant negative; E1, Ub-activating enzyme; E2, Ub-conjugating enzyme; E3, Ub ligase; GAS, IFN-
-regulated STAT1
binding site; HA, hemagglutinin; hnRNP, heterogeneous nuclear ribonucleoprotein; MEF, murine embryonic fibroblast; OPN, osteopontin; P-STAT1, phosphorylated Tyr701 STAT1; PIAS, protein inhibitor of STAT; PVDF, polyvinylidene difluoride; Q-PCR, quantitative real-time PCR; siRNA, small interfering RNA; siRNA MM, mismatch hairpin siRNA; SLIM, STAT-interacting LIM; SOCS, suppressor of cytokine signaling; Tap1, transporter associated with Ag processing 1; Ub, ubiquitin.
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