Evidence for Common Mechanisms in the Transcriptional Control of Type II Nitric Oxide Synthase in Isolated Hepatocytes

doi:10.1074/jbc.271.47.30114

Evidence for Common Mechanisms in the Transcriptional Control of Type II Nitric Oxide Synthase in Isolated Hepatocytes

REQUIREMENT OF NF-κB ACTIVATION AFTER STIMULATION WITH BACTERIAL CELL WALL PRODUCTS AND PHORBOL ESTERS*

From the Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain

^‡ To whom correspondence should be addressed. E-mail: boscal{at}eucmax.sim.ucm.es.

Abstract

Incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS), S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys₄ (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 κB, AP-1, GAS, and SIE sequences. Inhibition of NF-κB 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 κB 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 κB sites abolished the promoter activity. Analysis of the proteins involved in κB binding showed the presence of p50/p65 dimers in the nuclei of activated cells at the time that an important decrease of IκB-α was observed soon after cell stimulation with LPS, TPP, or PDBu. However, only LPS was able to decrease the amount of IκB-β. These results suggest that LPS, TPP, and PDBu, although activating different signal transduction pathways, use a common mechanism mediating iNOS expression in cultured hepatocytes.

Footnotes

↵* This work was supported by Grant PM95-007 from the Comisión Interministerial de Ciencia y Tecnología, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

iNOS

type II nitric oxide synthase

LPS

lipopolysaccharide

TPP

S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys₄

PDBu

phorbol 12,13-dibutyrate

IFN-γ

interferon-γ

EMSA

electrophoretic mobility shift assay

CAT

chloramphenicol acetyltransferase

GAS

γ-activated site

PBS

phosphate-buffered saline.
- Received January 24, 1996.
- Revision received August 6, 1996.