Abstract
The induction of human inducible nitric-oxide synthase (iNOS) expression depends (among other factors) on activation of the signal transducer and activator of transcription 1 (STAT1) pathway. Therefore, the STAT1 pathway may be an appropriate target for the development of inhibitors of iNOS expression. HeLa S3 cells transiently transfected with a γ-activated site (GAS)/interferon-stimulated response element-driven reporter gene construct were used as the primary screening system. Using this system, three novel inhibitors of interferon-γ-dependent gene expression, namely, sporogen, S14-95, andS-curvularin, were isolated from differentPenicillium species. These three compounds also inhibited cytokine-induced, GAS-dependent reporter gene expression in stably transfected human A549/8-pGASLuc cells, confirming the data obtained with the above-mentioned screening system. Furthermore, in A549/8 cells, sporogen, S14-95, and S-curvularin inhibited cytokine-induced activity of the human iNOS promoter [a 16-kilobase (kb) fragment in stably transfected A549/8-pNOS2(16)Luc cells], cytokine-induced iNOS mRNA expression, and cytokine-induced nitric oxide (NO) production in a concentration-dependent manner. The proliferation of A549/8 cells, and the activity of the human eNOS promoter (a 3.5-kb fragment in stably transfected ECV-pNOS III-Hu-3500-Luc cells), were only influenced marginally by the three compounds. Sporogen, S14-95, and S-curvularin also inhibited cytokine-induced activation of STAT1α in A549/8 cells. In conclusion, sporogen, S14-95, and S-curvularin represent new transcriptionally based inhibitors of iNOS-dependent NO production, acting on the Janus tyrosine kinase-STAT pathway. These compounds may represent lead structures for the development of drugs inhibiting iNOS-dependent overproduction of NO in pathophysiological situations.
Footnotes
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This work was supported by Grant 8312-38 62 61/322a,b from the Innovation Foundation of the State of Rhineland-Palatinate, Grant Kl 1020/4-1 from the Deutsche Forschungsgemeinschaft (to H.K.), and by the Collaborative Research Center SFB 553 (Project A7 to H.K.). This article contains data from the thesis work of Y.Y.
- Abbreviations:
- NO
- nitric oxide
- NOS
- nitric-oxide synthase
- eNOS
- endothelial nitric-oxide synthase
- iNOS
- inducible nitric-oxide synthase
- NF-κB
- nuclear factor-κB
- JAK
- janus kinase
- STAT
- signal transducer and activator of transcription
- IFN
- interferon
- kb
- kilobase(s)
- GAS
- γ-activated site
- BSA
- bovine serum albumin
- IL
- interleukin
- TNF
- tumor necrosis factor
- FCS
- fetal calf serum
- DMEM
- Dulbecco's modified Eagle's medium
- ISRE
- interferon-γ–stimulated responsive element
- CM
- cytokine mixture
- nt
- nucleotide
- SEAP
- secreted alkaline phosphatase
- OD
- optical density
- MTS
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
- TBS
- Tris-buffered saline
- Received August 12, 2002.
- Accepted November 4, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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