Sepsis is a disease process representing the systemic response to severe infection. In a subset of sepsis patients, toxins such as lipopolysaccharide (LPS) are released by infecting organisms (gram-negative bacteria) and activate immune cells of the host, including macrophages, to produce pro-inflammatory cytokines. In septic shock, exaggerated amounts of these pro-inflammatory cytokines are released, resulting in refractory hypotension, tissue hypoperfusion, and organ dysfunction. The inducible form of nitric oxide synthase (NOS2) plays an important role in endotoxemia/sepsis through overproduction of nitric oxide (NO). NO has been implicated in a number of pathophysiologic mechanisms of endotoxemia/sepsis, including vasoplegia, induction and maintenance of an inflammatory response, and immune modulation. Previous work in our laboratory has revealed that transcriptional upregulation of murine NOS2 by interleukin (IL)-1beta is facilitated by the interaction (promoter region -85 to -54) of nuclear factor (NF)-kappaB and high mobility group (HMG)-I/Y, an architectural transcription factor known to bind DNA at AT-rich sites. We have shown previously that transforming growth factor (TGF)-betal can inhibit the induction of NOS2 and improve outcome during endotoxemia. Moreover, TGF-beta1 downregulates the expression of HMG-I/Y, potentially contributing to this beneficial TGF-beta1 response during endotoxemia. Beyond the downstream portion (bp -85 to -54) of the NOS2 promoter, AT-rich sequences are also present in an upstream enhancer region (bp -985 to -910) implicated in the LPS and interferon (IFN)gamma synergistic induction of NOS2. The same enhancer region contains consensus binding sites for IRF-1 (via an ISRE site), Stat-1 alpha (via a GAS site), and NF-kappaB, all of which have been studied in the context of NOS2 induction. Taking into account the work of our laboratory and others, we believe that HMG-I/Y modulates transcription factor binding and enhanceosome formation responsible for LPS/inflammatory cytokine-induced NOS2 transactivation and NO production. We hypothesize that disruption of HMG-I/Y binding, and a subsequent decrease (but not elimination) in NOS2 gene expression, will have a beneficial effect during endotoxemia and polymicrobial sepsis (cecal ligation and puncture model) in mice. Thus, the goals of this proposal are 1) to determine whether disruption of HMG-I/Y binding to DNA in vitro and in vivo alters murine and human NOS2 gene expression and animal survival under conditions of endotoxemia, 2) to elucidate the role of HMG-I/Y and its effect on NOS2 expression and animal survival during endotoxemia and polymicrobial sepsis using a transgenic mouse expressing a dominant-negative form of HMG-I/Y in the vasculature, and 3) to further elucidate the role of TGF-beta1 in down regulating NOS2 transcriptional activity by inducing the expression of Elk-3, a member of the Ets family of proteins.
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