Hartmut Kleinert

Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (> 90% for isoforms I and III, > 80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO synthase can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not regulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzymes and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is constitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endothelial cells keeps blood vessels dilated, prevents the adhesion of platelets and white cells, and probably inhibits vascular smooth muscle proliferation.

Expressional control of the ‘constitutive’ isoforms of nitric oxide synthase (NOS I and NOS III)

Nitric oxide synthase (NOS) exists in three established isoforms. NOS I (NOS1, ncNOS) was originally discovered in neurons. This enzyme and splice variants thereof have since been found in many other cells and tissues. NOS II (NOS2, iNOS) was first identified in murine macrophages, but can also be induced in many other cell types. NOS III (NOS3, ecNOS) is expressed mainly in endothelial cells. Whereas NOS II is a transcriptionally regulated enzyme, NOS I and NOS III are considered constitutively expressed proteins. However, evidence generated in recent years indicates that these two isoforms are also subject to expressional regulation. In view of the important biological functions of these isoforms, changes in their expression may have physiological and pathophysiological consequences. This review recapitulates compounds and conditions that modulate the expression of NOS I and NOS III, summarizes transcriptional and posttranscriptional effects that underlie these changes, and—where known—describes the molecular mechanisms leading to changes in transcription, RNA stability, or translation of these enzymes.—Förstermann, U., Boissel, J.‐P., Kleinert, H. Expressional control of the ‘constitutive’ isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J. 12, 773–790 (1998)

Isoforms of nitric oxide synthase. Properties, cellular distribution and expressional control.

NOt, the smallest known bioactive product of mammalian cells, can be produced by most cell types. Despite its almost ubiquitous occurrence, this simple molecule can act in a fairly specific manner controlling vital functions such as neurotransmission or vascular tone (via activation of soluble guanylyl cyclase) [1,2], gene transcription [3] and mRNA translation (via iron-responsive elements) [4]. NO can produce post-translational modifications of proteins (via ADP-ribosylation) [5] and is capable of destroying parasites and tumor cells by inhibiting iron-containing enzymes [6] or directly interacting with the DNA of these cells. [7, 8]. In view of this multitude of molecular targets, effectors and functions of NO, it is clearly important to understand the mechanisms by which cells accomplish and regulate their NO production. In 1991, the first commentary on Isoforms of Nitric Oxide Synthase was published [9]. At that time only protein chemistry data were available to classify the NOS isozymes. While the basic characterization withstood the test of time, some aspects (such as the subclassification of NOS I and a postulated inducible NOS IV [9]) had to be abandoned as knowledge progressed. During the last 4 years, information on NOS has increased to such an extent that this new commentary had to be restricted to some important biochemical aspects of the NOS enzymes, namely their protein and cDNA structure, their cellular distribution, and the mechanism controlling their expression. Functional aspects of the enzymes have been reviewed elsewhere [2].

Nitric oxide synthase: expression and expressional control of the three isoforms.

Three isozymes of nitric oxide synthase (NOS) have been identified. Their cDNA- and protein structures as well as their genomic DNA structures have been described. NOS I (ncNOS, originally discovered in neurons) and NOS III (ecNOS, originally discovered in endothelial cells) are low output, Ca2+-activated enzymes whose physiological function is signal transduction. NOS II (iNOS, originally discovered in cytokine-induced macrophages) is a high output enzyme which produces toxic amounts of NO that represent an important component of the antimicrobial, antiparasitic and antineoplasic activity of these cells. Depending on the species, NOS II activity is largely (human) or completely (mouse and rat) Ca2+-independent. In the human species, the NOS isoforms I, II and III are encoded by three different genes located on chromosomes 12, 17 and 7, respectively. The amino acid sequences of the three human isozymes (deduced from the cloned cDNAs) show less than 59% identity. Across species, amino acid sequences are more than 90% conserved for NOS I and III, and greater 80% identical for NOS II. All NOS produce NO by oxidizing a guanidino nitrogen of L-arginine utilizing molecular oxygen and NADPH as co-substrates. All isoforms contain FAD, FMN and heme iron as prosthetic groups and require the cofactor BH4. NOS I and III are constitutively expressed in various cells. Nevertheless, expression of these isoforms is subject to regulation. Expression is enhanced by e.g. estrogens (for NOS I and III), shear stress, TGF-β1, and (in certain endothelial cells) high glucose (for NOS III). TNF-α reduces the expression of NOS III by a post-trancriptional mechanism destabilizing the mRNA. The regulation of the NOS I expression seems to be very complex as reflected by at least 8 different promoters transcribing 8 different exon 1 sequences which are expressed differently in different cell types. Expression of NOS II is mainly regulated at the transcriptional level and can be induced in many cell types with suitable agents such as LPS, cytokines, and other compounds. Whether some cells can express NOS II constitutively is still under debate. Pathways resulting in the induction of the NOS II promoter may vary in different cells. Activation of transcription factor NF-κB seems to be an essential step for NOS II induction in most cells. The induction of NOS II can be inhibited by a wide variety of immunomodulatory compounds acting at the transcriptional levels and/or post-transcriptionally.

Regulation of the expression of inducible nitric oxide synthase

Nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) is involved in complex immunomodulatory and antitumoral mechanisms and has been described to have multiple beneficial microbicidal, antiviral and antiparasital effects. However, dysfunctional induction of iNOS expression seems to be involved in the pathophysiology of several human diseases. Therefore iNOS has to be regulated very tightly. Modulation of expression, on both the transcriptional and post-transcriptional level, is the major regulation mechanism for iNOS. Pathways resulting in the induction of iNOS expression vary in different cells or species. Activation of the transcription factors NF-kappaB and STAT-1alpha and thereby activation of the iNOS promoter seems to be an essential step for the iNOS induction in most human cells. However, at least in the human system, also post-transcriptional mechanisms involving a complex network of RNA-binding proteins build up by AUF1, HuR, KSRP, PTB and TTP is critically involved in the regulation of iNOS expression. Recent data also implicate regulation of iNOS expression by non-coding RNAs (ncRNAs).

Estrogens increase transcription of the human endothelial NO synthase gene analysis of the transcription factors involved

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 17beta-estradiol or the more stable 17alpha-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 17alpha-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 17alpha-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.

Involvement of KSRP in the post-transcriptional regulation of human iNOS expression–complex interplay of KSRP with TTP and HuR

We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3′-untranslated region (3′-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3′-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3′-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3′-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation.

Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

ABSTRACT Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3′ untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3′ UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.

Cytokine induction of NO synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-κB-signaling pathways

1 In human epithelial‐like DLD‐1 cells, nitric oxide synthase (NOS) II expression was induced by interferon‐γ (100 u ml−1) alone and, to a larger extent, by a cytokine mixture (CM) consisting of interferon‐γ, interleukin‐1β (50 u ml−1) and tumor necrosis factor‐α (10 ng ml−1). 2 CM‐induced NOS II expression was inhibited by tyrphostin B42 (mRNA down to 1%; nitrite production down to 0.5% at 300 μM) and tyrphostin A25 (mRNA down to 24%, nitrite production down to 1% at 200 μM), 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 STAT1α 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 μM phenylarsine oxide; and to 19% at 100 ng ml−1 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′‐flanking sequence of the human NOS II gene to 63%. 4 Nuclear extracts from resting DLD‐1 cells showed significant binding activity for transcription factor NF‐κB, which was only slightly enhanced by CM. The NF‐κB inhibitors dexamethasone (1 μM), 3,4‐dichloroisocoumarin (50 μM), panepoxydone (5 μg ml−1) and pyrrolidine dithiocarbamate (100 μM) produced no inhibition of CM‐induced NOS II induction. 5 We conclude that in human DLD‐1 cells, the interferon‐γ–JAK‐2‐STAT1α 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‐κB, which is probably important for basal activity of the human NOS II promoter, is unlikely to function as a major effector of CM in DLD‐1 cells.

Lovastatin inhibits Rho-regulated expression of E-selectin by TNFalpha and attenuates tumor cell adhesion.

E‐selectin mediated cell‐cell adhesion plays an important role in inflammatory processes and extravasation of tumor cells. Tumor necrosis factor‐α (TNF‐α) induces E‐selectin gene and protein expression in primary human endothelial cells (HUVEC) and in an endothelial cell line (EA.hy‐926). As shown by ELISA and FACS analyses, HMG‐CoA reductase inhibitors (e.g., lovastatin) impair the TNF‐α stimulated increase in E‐selectin protein expression. Similar results were obtained for E‐selectin mRNA expression and promoter activity, indicating that the effect of lovastatin is based on inhibition of gene expression. The effective inhibitory concentration of lovastatin was in a physiologically relevant range (IC50<0.1 µM). Lovastatin‐mediated block of TNF‐α induced E‐selectin expression is due to inhibition of protein geranylgeranylation rather than farnesylation. Down‐regulation of Rho signaling by coexpression of dominant‐negative Rho mutants (i.e RhoA, RhoB and Rac) impaired TNF‐α driven E‐selectin gene expression, indicating Rho signaling to be essential for transcriptional activation of the E‐selectin gene. Inhibition of E‐selectin expression by lovastatin gives rise to a significant reduction in TNF‐α stimulated adhesion of colon carcinoma cells to HUVEC. Furthermore, low concentration of lovastatin (i.e., ≤1 µM) attenuated TNF‐α induced tumor cell invasion in vitro. The data support the view that statins might be clinically useful in protection against E‐selectin mediated metastasis.