Q.W. Xie

The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa B/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS-treated mouse macrophages.

Nitric oxide synthases (NOSs)' (L-arginine, NADPH:oxygen oxidoreductases (nitric oxide forming); EC 1.14.13.39) are unique among eukaryotic enzymes in being dimeric, calmodulin-dependent or calmodulin-containing cytochrome P450-like hemoproteins that combine reductase and oxygenase catalytic domains in one monomer, bear both FAD and FMN, and carry out a 5-electron oxidation of a non-aromatic amino acid (L-arginine) with the aid of tetrahydrobiopterin (1).Eukaryotes as phylogenetically ancient and diverse as Limulus polyphemus (2) and Rhodnius prolixus (3) make NO, and species as distantly related as Drosophila' and man (&lo) have yielded NOS cDNAs.Biosynthesis of nitric oxide (NO) appears to be within the repertoire of most mammalian cell types ( l l ) , provided they are appropriately stimulated.NO has been known for the past 6 years to mediate aspects of macrophage cytotoxicity, regulate blood pressure, and participate in neurotransmission (11,12), yet a steady stream of reports continues to enlarge NO'S sphere of action.For example, endogenous NO can contribute to destruction of the host's joints (13, 14) and kidneys ( 14) or to its survival from viral infection (15).Accordingly, regulation of NO biosynthesis has attracted intensive study and is the subject of this review.Other recent surveys deal with the enzymology of NOSs (1, 16) and their physiologic roles (11,12). RecapResearch through 1992 described two constitutive, calmodulindependent NOSs (cNOSs).One is apparently restricted to endothelium.The other is found in central and peripheral neurons, neuroblastomas (7), human but not rat skeletal muscle (6), p cells of pancreatic islets, and epithelial cells of bronchioli, uterus, and stomach (17).The cNOSs lie dormant until and so long as a rise in intracellular Ca2+ sustains the binding of calmodulin, leading to NO release over several minutes (18).An inducible NOS is expressed in many cell types after challenge with immunologic or inflammatory stimuli (11) and thereupon generates large amounts of NO over periods as long as 5 days (19).Tonic activation of this isoform may reflect its ability to bind calmodulin even at the trace levels of Ca2+ found in resting cells (20).This isoform was termed iNOS to signify its independence of elevated Ca2+ and exogenous calmodulin (21).These elements of the picture remain in place.Now, however, the canvas is crowded with new details. How Many NOSs?The human endothelial cNOS gene, residing at 7q35-7q36, comprises 26 exons that span 21 kb (22,23).The 28-exon neural cNOS gene on human chromsome 12q24.2extends over 100 kb (7).3 Hu-

AIMS: The Notch1 signaling pathway is implicated in multiple inflammatory diseases. However, the role of Notch1 signaling in alcoholic steatohepatitis (ASH) has not been fully investigated. We aimed to determine whether Notch1-Nuclear factor-κB (NF-κB) signaling mediates oxidative stress-induced ASH. METHODS: In vitro, three cell lines were used: the HepG2 cells, HepG2 cells transfected with a control vector (Neo cells) and HepG2 cells transfected with a cytochrome P4502E1-expression vector (2E1 cells), which allows the cells to undergo oxidative stress in response to ethanol. All three cell lines were incubated with ethanol with/without Notch1 inhibitor treatment, oxidative stress marker, steatohepatitis marker and Notch1-NF-κB signaling were assessed. To further test Notch1-NF-κB signaling in vivo, rats were fed with ethanol, ethanol plus Notch1 inhibitor or an isocaloric diet for 8 weeks. Hepatitis, oxidative stress and Notch1-NF-κB activity in the liver were assessed to further verify the in vitro results. RESULTS: Ethanol was shown to induce oxidative stress and steatohepatitis with remarkably elevated Notch1-NF-κB expression in 2E1 cells rather than HepG2 and Neo cells. Notch1 inhibitor was non-toxic in the three cell lines and had a protective effect against markers of ASH. Similarly, chronic alcohol administration in vivo induced alcoholic hepatitis, oxidative stress and elevated Notch1-NF-κB expression in rats, while Notch1 inhibitor attenuated alcoholic liver injury. CONCLUSION: These findings provide direct in vitro and in vivo evidence that the oxidative stress-induced ASH is mediated by the Notch1-NF-κB signaling pathway, which can be effectively reversed by Notch1 inhibitor.