© 1998 American Heart Association, Inc.
Original Contributions |
NO Inhibits Cytokine-Induced iNOS Expression and NF-
B Activation by Interfering With Phosphorylation and Degradation of IB-
From the Endocrine-Hypertension Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
Correspondence to Yukio Hirata, MD, Endocrine-Hypertension Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Abstract—Nitric oxide (NO) is
known to have antiatherogenic and anti-inflammatory properties, but its
effects on the cytokine-induced nuclear factor-kappa B
(NF-B) activation pathway in relation to the regulation of inducible
nitric oxide synthase (iNOS) gene in vascular smooth muscle cells
(VSMCs) remain elusive. To elucidate the roles of NO in the regulation
of cytokine-induced NF-B activation and consequent iNOS
gene expression, we studied the effects of NO donors
[(±)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide
(NOR3) and sodium nitroprusside] on interleukin (IL)-1ß–induced
NF-B activation and IB- degradation and subsequent iNOS
expression in rat VSMCs. Northern blot and Western blot
analyses demonstrated that NO donors decreased IL-1ß–induced
iNOS mRNA and protein expression. Electrophoretic mobility shift assay
using synthetic oligonucleotide corresponding to the
downstream NF-B site of rat iNOS promoter as a probe showed that
NOR3 inhibited IL-1ß–induced NF-B activation and its nuclear
translocation, as demonstrated with immunocytochemical study. These
effects were independent of guanylate cyclase activation;
an inhibitor of soluble guanylate cyclase
(1H-oxadiazolo-1,2,4-[4,3-]quinoxaline-1-one) had
no effect on NOR3-induced inhibition of NF-B activation or iNOS mRNA
expression by IL-1ß, and a cGMP derivative (8-bromo-cGMP) failed to
mimic the effects of NO donors. Western blot analysis using
anti–IB- and anti–phospho-IB- antibodies revealed that
IL-1ß induced a transient degradation of IB- preceded by a
rapid appearance of phosphorylated IB-, both of
which were completely blocked by NOR3. A proteasome
inhibitor (MG115) blocked IL-1ß–induced transient
degradation of IB- and stabilized the appearance of
phosphorylated IB- stimulated by IL-1ß. NOR3
inhibited the appearance of IL-1ß–induced
phosphorylated IB- even in the presence of MG115.
Our results indicate that an inhibitory action by NO on
cytokine-induced NF-B activation and iNOS gene
expression is due to its direct blockade on
phosphorylation and subsequent degradation of IB-
via the cGMP-independent pathway in rat VSMCs.
Key Words: NF-B • IL-1ß • inducible nitric oxide synthase • IB-
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