CCAAT/enhancer binding protein-beta trans-activates murine nitric oxide synthase 2 gene in an MTAL cell line

Nitric oxide production by nitric oxide synthase 2 (NOS2) has been implicat ed in epithelial cell injury from oxidative and immunologic stress. The NOS 2 gene is transcriptionally activated by lipopolysaccharide (LPS) and cytok ines in medullary thick ascending limb of Henle's loop (MTAL) cells and oth er 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 w e hypothesized contributes to NOS2 trans-activation in the mouse MTAL cell line ST-1. Gel shift assays demonstrated LPS + interferon-gamma (IFN-gamma) induction of C/EBP family protein-DNA complexes in nuclei harvested from t he cells. Supershift assays revealed that the complexes were comprised of C /EBP beta, but not C/EBP alpha, C/EBP delta, or C/EBP epsilon. NOS2 promote r-luciferase genes harboring deletion or mutation of the C/EBP box exhibite d lower activities in response to LPS+IFN-gamma compared with wild-type NOS 2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN-gamma activation of the NOS2 promoter. In trans-a ctivation assays, overexpression of C/EBP beta stimulated basal NOS2 promot er activity. Thus C/EBP beta appears to be an important trans-activator of the NOS2 gene in the MTAL.