Induction by staurosporine of nitric oxide synthase expression in vascular smooth muscle cells: role of NF-kappa B, CREB and C/EBP beta.
1. The effect of different protein kinase inhibitors on the expression of the inducible isoform of nitric oxide (NO) synthase (iNOS) was investigated in cultured vascular smooth muscle cells (VSMC) isolated from the rat aorta. 2. The non-selective protein kinase C (PKC) inhibitor, staurosporine, but not the more selective PKC inhibitors, calphostin C and Ro 31-8820, or the tyrosine kinase inhibitors, genistein and erbstatin analogue (erbstatin A), elicited a distinct (up to six fold) up-regulation of iNOS gene expression in these cells, as demonstrated by a parallel increase in iNOS mRNA and protein abundance as well as an accumulation of nitrite (NO2-) in the conditioned medium. Actinomycin D and cycloheximide inhibited the effect of staurosporine, suggesting an involvement of both DNA transcription and de nova protein synthesis. 3. Staurosporine also synergistically potentiated the stimulating effect of interleukin-1 beta (IL-1 beta), but not that of the adenylyl cyclase activator, forskolin, on NO2- production and iNOS protein abundance. Staurosporine, on the other hand, had no effect on the IL-1 beta-mediated increase in iNOS mRNA abundance. The effect of staurosporine on both basal and IL-1 beta-stimulated NO2- production was concentration-dependent with an apparent maximum at 3 nM. Among the other protein kinase inhibitors tested, only calphostin C also enhanced the stimulant effect of IL-1 beta approximately two fold, while genistein, erbstatin A and Ro 31-8220 inhibited rather than potentiated it. 4. Staurosporine did not influence basal activity of the transcription factors CREB and nuclear factor kappa B (NF-kappa B), but increased that of C/EBP. Moreover, staurosporine significantly augmented the activation of C/EBP by IL-1 beta and forskolin. 5. These findings suggest that in cultured VSMC a staurosporine-sensitive protein kinase exists, which is unlikely to be related to PKC, that prevents iNOS gene expression presumably by suppressing basal C/EBP activity. They also indicate that NF-kappa B and a member of the C/EBP family of transcription factors, presumably C/EBP beta, act synergistically under basal conditions and possibly also following exposure to IL-1 beta in the up-regulation of iNOS gene expression in these cells. Targeting of the activation of C/EBP beta may thus represent an interesting approach to interfere selectively with the cytokine-induced over-production of NO in acute and chronic inflammatory conditions.
Hecker
M
M
Centre of Physiology, J.W. Goethe University Clinic, Frankfurt, Germany.
Preiss
C
C
Schini-Kerth
V B
VB
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000201:enzymology; D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007375:Interleukin-1 / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology; D016328:NF-kappa B / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D009687:Nuclear Proteins / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011921:Rats, Inbred WKY; D019311:Staurosporine / Q000494:pharmacology*; D014157:Transcription Factors / Q000502:physiology*
D022762:CCAAT-Enhancer-Binding Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D005576:Colforsin; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D011493:Protein Kinase C; D019311:Staurosporine