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In primary human umbilical vein endothelial cells (HUVECs), incubation with phorbol-12-myristate-13-acetate (PMA) enhanced basal and bradykinin-stimulated nitric oxide production. In the HUVEC-derived cell line EA.hy 926, PMA and phorbol-12,13-dibutyrate stimulated endothelial nitric oxide synthase (NOS III) mRNA expression in a concentration- and time-dependent manner. Maximal mRNA expression (3.3-fold increase) was observed after 18 hr. NOS III protein and activity were increased to a similar extent. The specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (1 microM), Gö 6976 [12-(2 cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo-[3, 4-c]carbazole] (1 microM), Ro-31-8220 [3-[1-[3(amidinothio)propyl-1H-inoyl-3-yl]3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate] (1 microM), and chelerythrine (3 microM) did not change NOS III expression when applied alone, but they all prevented the up-regulation of NOS III mRNA produced by PMA. Of the PKC isoforms expressed in EA.hy 926 cells (alpha, beta I, delta, epsilon, eta, zeta, lambda, and mu), only PKC alpha and PKC epsilon showed changes in protein expression after PMA treatment. Incubation of EA.hy 926 cells with PMA for 2-6 hr resulted in a translocation of PKC alpha and PKC epsilon from the cytosol to the cell membrane, indicating activation of these isoforms. After 24 hr of PMA incubation, both isoforms were down-regulated. The time course of activation and down-regulation of these two PKC isoforms correlated well with the PMA-stimulated increase in NOS III expression. When human endothelial cells (ECV 304 or EA.hy 926) were transiently or stably transfected with a 3.5-kb fragment of the human NOS III promoter driving a luciferase reporter gene, PMA stimulated promoter activity up to 2.5-fold. On the other hand, PMA did not change the stability of the NOS III mRNA. These data indicate that stimulation of PKC alpha, PKC epsilon, or both by active phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.

Abstract-Estrogens have been found to reduce the incidence of cardiovascular disease that has been ascribed in part to an increased expression and/or activity of the vasoprotective endothelial NO synthase (NOS III). Some reports have shown that the level of expression of this constitutive enzyme can be upregulated by estrogens. The current study investigates the molecular mechanism of the NOS III upregulation in human endothelial EA.hy 926 cells. Incubation of EA.hy 926 cells with 17␤-estradiol or the more stable 17␣-ethinyl estradiol enhanced NOS III mRNA and protein expression up to 1.8-fold, without changing the stability of the NOS III mRNA. There was no enhancement of NOS III mRNA after incubation of EA.hy 926 cells with testosterone, progesterone, or dihydrocortisol or when 17␣-ethinyl estradiol was added together with the estrogen antagonist RU58668, indicating a specific estrogenic response. Nuclear run-on assays indicated that the increase in NOS III mRNA is the result of an estrogen-induced enhancement of NOS III gene transcription. In transient transfection experiments using a 1.6 kb human NOS III promoter fragment (which contains no bona fide estrogen-responsive element, ERE), basal promoter activity was enhanced 1.7-fold by 17␣-ethinyl estradiol. In electrophoretic mobility shift assays, nuclear extracts from estrogen-incubated EA.hy 926 cells showed no enhanced binding activity either for the ERE-like motif in the human NOS III promoter or for transcription factor GATA. However, binding of transcription factor Sp1 (which is essential for the activity of the human NOS III promoter) was significantly enhanced by estrogens. These data suggest that the estrogen stimulation of the NOS III promoter could be mediated in part by an increased activity of transcription factor Sp1. (Hypertension. 1998;31:582-588.)Key Words: 17␣-ethinyl estradiol Ⅲ 17␤-estradiol Ⅲ nitric oxide synthase Ⅲ transcription factor Sp1 S ex differences in the incidence of coronary heart disease are well established. The incidence of coronary heart disease is relatively low among premenopausal women and increases sharply with the occurrence of menopause.

1 In human epithelial-like DLD-1 cells, nitric oxide synthase (NOS) II expression was induced by interferon-g (100 u ml 71 ) alone and, to a larger extent, by a cytokine mixture (CM) consisting of interferon-g, interleukin-1b (50 u ml 71 ) and tumor necrosis factor-a (10 ng ml 71 ). 2 CM-induced NOS II expression was inhibited by tyrphostin B42 (mRNA down to 1%; nitrite production down to 0.5% at 300 mM) and tyrphostin A25 (mRNA down to 24%, nitrite production down to 1% at 200 mM), suggesting the involvement of janus kinase 2 (JAK-2). Tyrphostin B42 also blocked the CM-induced JAK-2 phosphorylation (kinase assay) and reduced the CM-stimulated STAT1a binding activity (gel shift analysis). 3 CM reduced the nuclear binding activity of transcription factor AP-1. A heterogenous group of compounds, that stimulated the expression of c-fos/c-jun, enhanced the nuclear binding activity of AP-1. This group includes the protein phosphatase inhibitors calyculin A, okadaic acid, and phenylarsine oxide, as well as the inhibitor of translation anisomycin. All of these compounds reduced CM-induced NOS II mRNA expression (to 9% at 50 nM calyculin A; to 28% at 500 nM okadaic acid; to 18% at 10 mM phenylarsine oxide; and to 19% at 100 ng ml 71 anisomycin) without changing NOS II mRNA stability. In cotransfection experiments, overexpression of c-Jun and c-Fos reduced promoter activity of a 7 kb DNA fragment of the 5'-¯anking sequence of the human NOS II gene to 63%. 4 Nuclear extracts from resting DLD-1 cells showed signi®cant binding activity for transcription factor NF-kB, which was only slightly enhanced by CM. The NF-kB inhibitors dexamethasone (1 mM), 3,4-dichloroisocoumarin (50 mM), panepoxydone (5 mg ml 71 ) and pyrrolidine dithiocarbamate (100 mM) produced no inhibition of CM-induced NOS II induction. 5 We conclude that in human DLD-1 cells, the interferon-g ± JAK-2-STAT1a pathway is important for NOS II induction. AP-1 (that is downregulated by CM) seems to be a negative regulator of NOS II expression. NF-kB, which is probably important for basal activity of the human NOS II promoter, is unlikely to function as a major e ector of CM in DLD-1 cells.

Transcription factor NF-B is essential for the induction of nitric oxide synthase (NOS) II (iNOS) by bacterial lipopolysaccharide in murine macrophages (Xie, Q. W., Kashiwabara, Y., and Nathan, C. (1994) J. Biol. Chem. 269, 4705-4708). In 3T3 fibroblasts, agents other than cytokines are efficacious inducers of NOS II expression. In addition to cytokines such as interferon-␥ or tumor necrosis factor-␣, protein kinase C-stimulating agents such as tetradecanoylphorbol-13-acetate, or cyclic AMPelevating agents such as forskolin and 8-bromo-cAMP markedly increased NOS II mRNA (measured by S1 nuclease and RNase protection analyses), NOS II protein (determined by Western blotting), and NOS activity (measured by chemiluminescence detection of NO 2 ؊ ). Transforming growth factor-␤1 (which is an inhibitor of NOS II induction in other cell types) potentiated NOS II mRNA expression produced by all inducing agents listed, whereas dexamethasone, pyrrolidine dithiocarbamate and 3,4-dichloroisocoumarin (inhibitors of NF-B activation) suppressed NOS II mRNA induction in response to all stimulants. In electrophoretic mobility shift assays, nuclear protein extracts from 3T3 cells stimulated with any of the inducing agents significantly slowed the migration of an NF-B-binding oligonucleotide, whereas nuclear extracts from untreated control cells did not. These experiments indicate that NF-B is the key control element for the induction of NOS II in response to at least three different second messenger pathways in 3T3 cells. Nitric oxide (NO)1 is a short-lived bioactive molecule participating in the physiology and/or pathophysiology of many organ systems (1). The expression of the inducible isoform of nitric oxide synthase (NOS II or iNOS) is regulated mainly at the transcriptional level (2). Inflammatory stimuli such as bacterial lipopolysaccharide (LPS) and cytokines induce the expression of this enzyme in many cell types. Interestingly, in some cells, agents other than cytokines are efficacious inducers of NOS II expression. For example, in rat mesangial cells, cAMPelevating agents stimulate NOS II expression (3). Phorbol ester induction of NOS II has been reported for rat peritoneal macrophages (4). In murine BALB 3T3 fibroblasts, NOS II is expressed in response to forskolin, dibutyryl cAMP, or tetradecanoylphorbol-13-acetate (TPA) (5). Analyses of the cloned murine NOS II promoter (6 -8) have revealed the presence of numerous consensus sequences for the binding of transcription factors. Of these potentially relevant transcription factors, nuclear factor-B (NF-B) (6, 9) and interferon regulatory factor (10, 11) have been shown to be functionally important for NOS II induction. The molecular mechanisms utilized by other second messenger pathways are still unclear. In rat mesangial cells, the inhibitor of NF-B activation, pyrrolidine dithiocarbamate (PDTC), blocked NOS II expression induced by interleukin-1␤ (IL-1␤), but not the expression stimulated by 8-bromo-cAMP, suggesting two different induction pathways (12).In the current ...