Structure-Activity Relationship of Staurosporine Analogs in Regulating Expression of Endothelial Nitric-Oxide Synthase Gene
Abstract
In human umbilical vein endothelial cells and in human umbilical vein endothelial cell-derived EA.hy 926 cells, staurosporine (Stsp) and its glycosidic indolocarbazole analogs 7-hydroxystaurosporine (UCN-01) and 4′-N-benzoyl staurosporine (CGP 41251) enhanced nitric-oxide synthase (NOS) III mRNA expression (analyzed by RNase protection assay), protein expression (determined by Western blot), and activity [measured by rat fetal lung fibroblast (RFL-6) reporter cell assay] in a concentration- and time-dependent manner. In contrast, the bisindolylmaleimide analogs GF 109203X, Ro 31–8220 and Gö 6983 had no effect on NOS III expression, and Gö 6976, a methyl- and cyanoalkyl-substituted nonglycosidic indolocarbazole derivative of Stsp, even reduced NOS III expression in a concentration-dependent fashion. The up-regulation of NOS III expression by Stsp and analogs appears to be a transcriptional event because Stsp, 7-hydroxystaurosporine, and CGP 41251 enhanced the activity of a 1.6-kb human NOS III promoter fragment transiently transfected into EA.hy 926 endothelial cells. Stsp and analogs did not affect the stability of the NOS III mRNA. Stsp is known as a potent protein kinase (PK) inhibitor. Data obtained with other kinase inhibitors (and stimulators) indicated, however, that the effect of Stsp and analogs on NOS III expression was unrelated to the activities of PKC, PKA, PKG, or tyrosine kinase(s). Stsp analogs such as CGP 41251 also counteracted the NOS III mRNA-decreasing effect of tumor necrosis factor-α. These findings demonstrate that Stsp analogs represent a new class of compounds positively interacting with the transcription of the endothelial NOS III gene. Such compounds may prove useful in the prophylaxis and therapy of vascular disease.
Footnotes
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Send reprint requests to: Dr. Ulrich Förstermann, Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany. E-mail:Ulrich.Forstermann{at}Uni-Mainz.de
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This work was supported by the Collaborative Research Center SFB 553 (Project A1 to U.F.) from the Deutsche Forschungsgemeinschaft, Bonn, Germany.
- Abbreviations:
- NO
- nitric oxide
- NOS
- nitric-oxide synthase
- TNF-α
- tumor necrosis factor-α
- PKC
- protein kinase C
- Stsp
- staurosporine
- UCN-01
- 7-hydroxystaurosporine
- CGP 41251
- 4′-N-benzoyl staurosporine
- GF 109203X
- {3-[1-[(3-dimethylaminopropyl)-1H-indol-3-yl]-4-(1H-indol-3-yl)]maleimide}
- Ro 31-8220
- {3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-4-(1-methyl-1H-indol-3-yl)]maleimide}
- Gö 6983
- {3-[1-[(3- dimethylaminopropyl)-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)]maleimide}
- Gö 6976
- {12-(2-cyanoethyl)-13-methyl-5-oxo-7H-indolo[2,3-b]pyrrolo[3,4-c]carbazole}
- PMA
- phorbol-12-myristate-13-acetate
- SOD
- superoxide dismutase
- IBMX
- 3-isobutyl-1-methylxanthine
- HUVEC
- human umbilical vein endothelial cell
- DTT
- dithiothreitol
- PMSF
- phenylmethylsulfonyl fluoride
- PAGE
- polyacrylamide gel electrophoresis
- TBS
- Tris-buffered saline
- SV-40
- simian virus 40
- PET
- β-phenyl-1,N2-ethenoguanosine
- RFL-6
- rat fetal lung fibroblast cells
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- Received June 9, 1999.
- Accepted November 10, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
