Nitric oxide production by nitric oxide synthase 2 (NOS2) has been implicated in epithelial cell injury from oxidative and immunologic stress. The NOS2 gene is transcriptionally activated by lipopolysaccharide (LPS) and cytokines in medullary thick ascending limb of Henle's loop (MTAL) cells and other cell types. The 5'-flanking region of the NOS2 gene contains a consensus element for CCAAT/enhancer binding proteins (C/EBP) at 150 to 142 that we hypothesized contributes to NOS2 trans-activation in the mouse MTAL cell line ST-1. Gel shift assays demonstrated LPS + interferon- (IFN-) induction of C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBP, but not C/EBP, C/EBP, or C/EBP. NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box exhibited lower activities in response to LPS + IFN- compared with wild-type NOS2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN- activation of the NOS2 promoter. In trans-activation assays, overexpression of C/EBP stimulated basal NOS2 promoter activity. Thus C/EBP appears to be an important trans-activator of the NOS2 gene in the MTAL.

gene transcription; kidney; cell signaling; promoter; transcription factors

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