In endotoxin (LPS)-mediated sepsis, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production alter multiple functions, including cardiac contractility, vasomotor tone, intestinal epithelial permeability, and leukocyte recruitment. While the molecular pathways which upregulate iNOS in endotoxemia have been extensively characterized, little is known of the corresponding pathways which downregulate iNOS. Utilizing both in vivo murine and in vitro murine macrophage and rat hepatocyte models of LPS stimulation, we have demonstrated that NO feedback inhibits its own synthesis by increasing gene transcription and promoter activation of osteopontin (OPN), a potent trans-repressor of iNOS expression. This negative feedback pathway of NO-dependent OPN gene transcription and protein synthesis has not been previously described. We hypothesize that transcription of OPN, a potent trans-repressor of iNOS expression, is NO-dependent in LPS-stimulated murine macrophages. In the immortalized ANA-1 murine macrophage cell line, we propose to functionally map the OPN promoter in the context of LPS stimulated NO production.
Specific Aim 1. To define NO-dependent cis-acting transcriptional control regions, we will utilize OPN promoter-reporter constructs with deletion analysis and site-directed mutagenesis.
Specific Aim 2. To define NO-dependent trans-acting regulation, we will isolate the NO-dependent transcription factor and its cDNA clone by using the biotin-strepavidin affinity method and screening a cDNA expression library, respectively, using the DNA recognition site as a probe.
Specific Aim 3. To determine the S-nitrosylation status of our transcription factor and its effect upon DNA binding, we will use CuCl-cysteine coupled chemiluminescence.
Specific Aim 4. To confirm relevancy in the LPS-stimulated macrophage, we will inhibit translation of the transcription factor mRNA with antisense techniques and alternatively, over- express the transcription factor by transient transfection.
Specific Aim 5. To confirm in vivo relevancy, we will utilize a murine OPN-knockout model of endotoxemia to demonstrate lack of iNOS inhibition in the absence of OPN. Our studies will define OPN production as a unique and as yet, poorly characterized, NO- dependent transcriptional pathway which inhibits iNOS expression in the setting of endotoxemia.
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