Serum after Partial Hepatectomy Stimulates iNOS Gene Transcription via Downstream NF-κB Site

Abstract

It has been known that the expression of inducible nitric oxide synthase (iNOS) is up-regulated during hepatic regeneration. The present study characterized the molecular mechanisms involved in the transcriptional activation of iNOS gene by using the serum after partial hepatectomy (post-PH serum) in vitro. The post-PH serum rapidly induced iNOS mRNA expression, which was blocked by anti-tumor necrosis factor-α (TNF-α) antibody in BNL CL.2 cells, murine embryonic liver cell line. In addition, EMSAs using a NF-κB-specific oligomer showed that the up-regulated iNOS mRNA expression in cells treated with post-PH serum correlated with transient activation of NF-κB complex (p50/p65 heterodimer). Transient transfection of BNL CL.2 cells with iNOS promoter linked to a CAT reporter gene showed the transcriptional activation of iNOS promoter by post-PH serum. Furthermore, site-directed mutational analysis of the two NF-κB sites individually or in combination revealed that iNOS expression by post-PH serum is regulated by the downstream NF-κB site, but not by upstream NF-κB site. Taken together, these results suggest that the downstream NF-κB site acts as an essential component for the iNOS expression by post-PH serum during hepatic regeneration.