Serum after partial hepatectomy stimulates iNOS gene transcription via downstream NF-kappa B site

It has been known that the expression of inducible nitric oxide synthase (i NOS) is up-regulated during hepatic regeneration. The present study charact erized the molecular mechanisms involved in the transcriptional activation of iNOS gene by using the serum after partial hepatectomy (post-PH serum) i n vitro. The post-PH serum rapidly induced iNOS mRNA expression, which was blocked by anti-tumor necrosis factor-alpha (TNF-alpha) antibody in BNL CL. 2 cells, murine embryonic liver cell line. In addition, EMSAs using a NF-ka ppaB-specific oligomer showed that the up-regulated iNOS mRNA expression in cells treated with post-PH serum correlated with transient activation of N F-kappaB complex (p50/p65 heterodimer). Transient transfection of BNL CL.2 cells with iNOS promoter linked to a CAT reporter gene showed the transcrip tional activation of iNOS promoter by post-PH serum. Furthermore, site-dire cted mutational analysis of the two MF-kappaB sites individually or in comb ination revealed that iNOS expression by post-PH serum is regulated by the downstream NF-kappaB site, but not by upstream NF-kappaB site. Taken togeth er, these results suggest that the downstream NF-kappaB site acts as an ess ential component for the iNOS expression by post-PH serum during hepatic re generation. (C) 2001 Academic Press.