Expression of inducible nitric oxide synthase (iNOS) is central to many of the systemic effects associated with sepsis. iNOS expression and nitric oxide (NO) production alter multiple functions, including cardiac contractility, vasomotor tone, intestinal epithelial permeability, and leukocyte recruitment. Utilizing both in vivo and in vitro murine models of endotoxemia (LPS), we have previously demonstrated that NO feedback inhibits its own synthesis by increasing transcription of osteopontin (OPN), a potent trans-repressor of iNOS expression. In this competitive renewal, we propose to characterize the pathway by which OPN feeds back to downregulate iNOS transcription. In in vivo, ex vivo, and in vitro murine models of LPS- and cecal ligation and puncture (CLP) mediated sepsis, our studies show that: 1) OPN acts with STAT-interacting LIM (SLIM) protein to ubiquitinate (Ub) an essential iNOS transcription factor, STAT1, for 26s proteasome mediated degradation and inhibit STAT1 dependent iNOS expression, 2) STAT1 is not ubiquitinated in the absence of SLIM, and 3) survival of OPN null or SLIM null mice is significantly decreased indicating the functional relevance of this pathway in the pathophysiology of sepsis. We hypothesize that OPN acts through SLIM as an E3 ubiquitin ligase to degrade STAT1 protein and inhibit iNOS transcription in sepsis. We will focus on the following specific aims which are critical to defining the mechanisms underlying OPN mediated STAT1 degradation in murine models of LPS and CLP mediated sepsis. 1) We will identify the OPN-regulated E3 ligase which transfers ubiquitin to STAT1, focusing initially on SLIM protein. 2) We will define the role of OPN in regulating expression and/or activation of SLIM. 3) We will confirm in vivo relevance of the OPN-STAT1-Ub pathway in murine models of LPS stimulation and/or CLP that incorporate OPN null and SLIM null animals. The role of OPN in the regulation of STAT1 dependent protein expression in sepsis has not been previously explored. Our proposed studies will utilize iNOS as a specific example of a STAT1 dependent protein to define OPN as a unique and as yet, poorly characterized, trans-activator of STAT1 degradation. Characterization of this regulatory pathway may identify potential regulatory targets for therapy in septic shock.

Public Health Relevance

Death as the result of severe infection and injury remains a critical problem in the U.S. This research examines the regulation of a major player in the breakdown of normal bodily functions following major infection and injury. If successful, our studies may result in prevention of this breakdown.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065113-09
Application #
8308608
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Dunsmore, Sarah
Project Start
2002-07-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
9
Fiscal Year
2012
Total Cost
$286,559
Indirect Cost
$94,881
Name
Loyola University Chicago
Department
Surgery
Type
Schools of Medicine
DUNS #
791277940
City
Maywood
State
IL
Country
United States
Zip Code
60153
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Talbot, Lindsay Jones; Mi, Zhiyong; Bhattacharya, Syamal Dave et al. (2011) Pharmacokinetic characterization of an RNA aptamer against osteopontin and demonstration of in vivo efficacy in reversing growth of human breast cancer cells. Surgery 150:224-30
Bhattacharya, Syamal D; Garrison, Juline; Guo, Hongtao et al. (2010) Micro-RNA-181a regulates osteopontin-dependent metastatic function in hepatocellular cancer cell lines. Surgery :
Guo, Hongtao; Mi, Zhiyong; Bowles, Dawn E et al. (2010) Osteopontin and protein kinase C regulate PDLIM2 activation and STAT1 ubiquitination in LPS-treated murine macrophages. J Biol Chem 285:37787-96
Mi, Zhiyong; Guo, Hongtao; Russell, M Benjamin et al. (2009) RNA aptamer blockade of osteopontin inhibits growth and metastasis of MDA-MB231 breast cancer cells. Mol Ther 17:153-61
Diesen, Diana L; Kuo, Paul C (2009) Nitric Oxide and Redox Regulation in the Liver: Part I. General Considerations and Redox Biology in Hepatitis. J Surg Res :
Guo, Hongtao; Wai, Philip Y; Mi, Zhiyong et al. (2008) Osteopontin mediates Stat1 degradation to inhibit iNOS transcription in a cecal ligation and puncture model of sepsis. Surgery 144:182-8

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