Heat shock enhances transcriptional activation of the murine-inducible nitric oxide synthase gene

There is considerable interest in determining the conditions leading to enhanced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) biosynthesis. Using in vivo footprinting, we demonstrate that heat shock of murine macrophages concurrent with lipopolysaccharide (LPS) treatment stimulated changes in guanine methylation sensitivity at ?898/9, at a putative partial heat shock element (HSE) and at -893/4, a site bordering an E-box, within the iNOS gene enhancer, suggesting inducible occupation by transcription factors at these regions. LPS treatment accompanied by heat shock provoked increased iNOS gene transcription, increased levels of iNOS protein, and increased production of NO compared with LPS treatment alone. Electrophoretic mobility shift analysis revealed low constitutive levels of specific binding to an E-box and a partial HSE within the iNOS enhancer. Binding to the E-box was increased by LPS treatment or by heat shock, achieving a greater increase by a combination of both treatments. The proteins occupying this site were identified as belonging to the USF family of transcription factors. Heat shock or LPS increased binding to the HSE, and the factor responsible for this interaction was identified as heeat shock factor-1 (HSF-1). Mutations at the HSE revealed the importance of HSF-1 in the induction of iNOS by LPS. Thus, our data reveal two novel regulatory sites in the murine iNOS gene, one of which is implicated in enhancing iNOS expression via LPS stimulation, and provide the first evidence that heat shock enhances transcription of the iNOS gene. These results could have implications in the host response mechanism to fever-associated gram-negative infection