Osteopontin regulates ubiquitin-proteasome degradation of STAT1

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Macrophage and parenchymal cell 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 LPS stimulation, 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 acts to downregulate iNOS transcription. Using in vitro and in vivo murine models of LPS stimulation and/or cecal ligation and puncture (CLP) mediated sepsis, our preliminary data indicate that OPN increases STAT1 ubiquitination and subsequent 26s proteasome mediated STAT1 degradation to inhibit STAT1 dependent iNOS promoter activity, transcription, and protein expression. STAT1 is an essential activator of LPS and/or pro-inflammatory cytokine-mediated iNOS transcription. Our studies indicate OPN to be a novel regulator of activated STAT1 degradation in the context of LPS stimulation. The role of OPN in the regulation of STAT1 dependent protein expression has not been previously explored. We hypothesize that OPN accelerates ubiquitin (Ub)-dependent STAT1 protein degradation to inhibit iNOS transcription. 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 STAT-interacting LIM (SLIM) protein. 2) We will define the role of OPN in regulating expression and/or activation of SLIM (or the appropriate E3 ligase). 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. 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, transactivator of STAT1 degradation. Characterization of this regulatory pathway may identify potential regulatory targets for therapy in septic shock.