Journal Article

Nitric oxide induces expression of cyclooxygenase-2 in mouse skin through activation of NF-κB

Kyung-Soo Chun, Hyun-Ho Cha, Jun-Wan Shin, Hye-Kyung Na, Kwang-Kyun Park, Won-Yoon Chung and Young-Joon Surh

in Carcinogenesis

Volume 25, issue 3, pages 445-454
Published in print March 2004 | ISSN: 0143-3334
Published online March 2004 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgh021
Nitric oxide induces expression of cyclooxygenase-2 in mouse skin through activation of NF-κB

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Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are frequently overexpressed in tumor tissues or transformed cells. In the present work, we assessed the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of iNOS and COX-2 in mouse skin. Topical application to the dorsal skin of female ICR mice of 10 nmol TPA led to maximal induction of iNOS and COX-2 protein expression at ∼2 and 4 h, respectively. When applied topically onto shaven backs of mice 30 min prior to TPA, the NOS inhibitor aminoguanidine (AG) inhibited the expression of COX-2 protein at the pharmacologically effective dose. Pretreatment with a more specific iNOS inhibitor, NG-nitro-l-arginine-methyl ester, also suppressed TPA-induced COX-2 expression. Immunohistochemical analysis of TPA-treated mouse skin using an anti-nitrotyrosine antibody reveals enhanced levels of nitrotyrosine protein localized in epidermal and dermal layers. Topical application of NO donors, such as sodium nitroprusside (SNP) and S-nitroso-N-acetyl-d,l-penicillamine, induced expression of COX-2 in mouse skin, which was attenuated by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide. SNP treatment stimulated NF-κB activation in mouse skin, which was associated with the degradation of IκBα. Topical application of inhibitors of NF-κB, such as pyrrolidine dithiocarbamate or N-α-p-tosyl-l-lysine chloromethylketone, inhibited the SNP-induced COX-2 expression. SNP induced a weak but concentration-related increase in COX-2 expression in cultured mouse keratinocytes, which was abolished by treatment with SN50, a specific inhibitor of nuclear translocation of NF-κB. Mouse keratinocytes treated with SNP exhibited an elevated NF-κB-driven COX-2 promoter activity. Topical application of AG (10 µmol) prior to each TPA treatment after initiation reduced the multiplicity of papillomas by 44% at 22 weeks. In conclusion, up-regulation of COX-2 by NO may be mediated by activation of NF-κB in mouse skin, which provides a molecular mechanism by which COX-2 is induced during tumor promotion.

Keywords: AG, aminoguanidine; carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide; COX, cyclooxygenase; DMBA, 7,12-dimethylbenz[a]anthracene; EMSA, electrophoretic mobility shift assay; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NAME, NG-nitro-l-arginine-methyl ester; NOS, nitric oxide synthase; NP-40, Nonidet P-40; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline containing 0.1% Tween-20; PDTC, pyrrolidine dithiocarbamate; PGE2, prostaglandin E2; PMSF, phenylmethylsulfonyl fluoride; SNAP, S-nitroso-N-acetyl-d,l-penicillamine; SNP, sodium nitroprusside; TPA, 12-O-tetradecanoylphorbol-13-acetate; TPCK, N-α-p-tosyl-l-lysine chloromethylketone

Journal Article.  7043 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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