Structure-activity relationship of staurosporine analogs in regulating expression of endothelial nitric-oxide synthase gene

In human umbilical vein endothelial cells and in human umbilical vein endot helial cell-derived EA.hy 926 cells, staurosporine (Stsp) and its glycosidi c indolocarbazole analogs 7-hydroxystaurosporine (UCN-01) and 4'-N-benzoyl staurosporine (CGP 41251) enhanced nitric-oxide synthase (NOS) III mRNA exp ression (analyzed by RNase protection assay), protein expression (determine d by Western blot), and activity [measured by rat fetal lung fibroblast (RF L-6) reporter cell assay] in a concentration- and time-dependent manner. In contrast, the bisindolylmaleimide analogs GF 109203X, Ro 31-8220 and Go 69 83 had no effect on NOS III expression, and Go 6976, a methyl- and cyanoalk yl-substituted nonglycosidic indolocarbazole derivative of Stsp, even reduc ed NOS III expression in a concentration- dependent fashion. The up-regulat ion of NOS III expression by Stsp and analogs appears to be a transcription al event because Stsp, 7-hydroxystaurosporine, and CGP 41251 enhanced the a ctivity 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 stab ility of the NOS III mRNA. Stsp is known as a potent protein kinase (PK) in hibitor. Data obtained with other kinase inhibitors (and stimulators) indic ated, however, that the effect of Stsp and analogs on NOS III expression wa s 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 ef fect of tumor necrosis factor-alpha. These findings demonstrate that Stsp a nalogs represent a new class of compounds positively interacting with the t ranscription of the endothelial NOS III gene. Such compounds may prove usef ul in the prophylaxis and therapy of vascular disease.