Evidence for common mechanisms in the transcriptional control of type II nitric oxide synthase in isolated hepatocytes - Requirement of NF-kappa B activation after stimulation with bacterial cell wall products and phorbol esters

Incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS), S-[2,3-bis(palmitoyloxy)(2-R,S)-propyl] -N-palmitoyl-(R)-Cys-Ser-Lys(4) (TPP), a synthetic lipopeptide present in bacterial cell wall lipoproteins, or with phorbol 12,13-dibutyrate (PDBu) induced an increase in nitric oxide synthesis through the expression of type II nitric oxide synthase (iNOS), Transfection of hepatocytes with a HindII fragment corresponding to the promoter region of the murine iNOS gene (from nucleotide -1588 to +165) resulted in the expression of the reporter gene when cells were stimulated with these factors, The transcription factors activated by these stimuli involved an increase in the nuclear content of proteins that bind to kappa B, AP-1, GAS, and SIE sequences, Inhibition of NF-kappa B activation with pyrrolidine dithiocarbamate eliminated the expression of iNOS in hepatocytes stimulated with LPS, TPP, or PDBu. In addition to this, transfection of hepatocytes with promoter mutants in which a sequential 2-base pair change within the kappa B sites was introduced (position -971 to -961 and -85 to -75, respectively), resulted in approximately 17 and 35%, respectively, of the activity of the naive promoter, Simultaneous mutation of both kappa B sites abolished the promoter activity. Analysis of the proteins involved in kappa B binding showed the presence of p50/p65 dimers in the nuclei of activated cells at the time that an important decrease of I kappa B-alpha was observed soon after cell stimulation with LPS, TPP, or PDBu. However, only LPS was able to decrease the amount of I kappa B-beta. These results suggest that LPS, TPP, and PDBu, although activating different signal transduction pathways, use a common mechanism mediating iNOS expression in cultured hepatocytes.