Abstract
Human inducible nitric-oxide synthase (iNOS) expression is regulated both at transcriptional and post-transcriptional levels. In the present study, the effect of Jun N-terminal kinase (JNK) on human iNOS expression was investigated. In A549/8 human alveolar epithelial cells, both the inhibition of JNK by a pharmacological inhibitor anthra[1,9-cd]pyrazol-6(2H)-one1,9-pyrazoloanthrone (SP600125) and small interfering RNA (siRNA)-mediated down-regulation of JNK led to a reduction of iNOS mRNA and protein expression. iNOS promoter activity was not affected by these treatments. Hence, JNK seems to regulate iNOS expression through post-transcriptional mechanisms by stabilizing iNOS mRNA. Our laboratory has shown recently that a cytokine-induced RNA binding protein tristetraprolin (TTP) is a major positive regulator of human iNOS expression by stabilizing iNOS mRNA. Therefore, the effect of JNK inhibition by SP600125 or down-regulation by siRNA on TTP expression was investigated. Both SP600125 and siRNA targeted at JNK resulted in a reduction of TTP protein expression without affecting the amount of TTP mRNA. These data suggest a post-transcriptional control of TTP expression by JNK. Moreover, the modulation of JNK signaling by SP600125 or siRNA did not change p38 phosphorylation. In summary, the results suggest that JNK regulates human iNOS expression by stabilizing iNOS mRNA possibly by a TTP-dependent mechanism.
Footnotes
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This work was supported by grants from Academy of Finland, Emil Aaltonen Foundation, Finland, and Tampere Tuberculosis Foundation, Finland (to R.K.), competitive research funding of the Pirkanmaa Hospital District, Finland (to R.K. and E.M.), and grant 8312 to 8338 62 61/322a,b from the Innovation Foundation of the State of Rhineland-Palatinate and by the Collaborative Research Center SFB 553 (project A7, to H.K.).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.033449.
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ABBREVIATIONS: ARE, AU-rich element; CT, cycle threshold; DMEM, Dulbecco's modified Eagle's medium; DRB, 5,6-dichlorobenzimidazole-1-β-d-ribofuranoside; CM, cytokine mixture containing interferon-γ, interleukin-1β, and tumor necrosis factor-α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; IL, interleukin; iNOS, inducible nitric-oxide synthase; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PTB, polypyrimidine tract binding protein; siRNA, small interfering RNA; shRNA, short hairpin RNA; SP600125, anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone; TNF-α, tumor necrosis factor α; TTP, tristetraprolin; UTR, untranslated region; kb, kilobase(s); qRT-PCR, quantitative reverse transcriptase/real-time polymerase chain reaction; DMSO, dimethyl sulfoxide; KSRP, KH-type splicing regulatory protein.
- Received December 14, 2006.
- Accepted February 22, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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