Regulation of the expression of inducible nitric oxide synthase

Kleinert, Hartmut; Pautz, Andrea; Linker, Katrin; Schwarz, Petra

doi:10.1016/j.ejphar.2004.07.030

Cited by 571 publications

(420 citation statements)

References 116 publications

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“…This sequence is also relatively UC-rich but does not contain the "classical" UCUU-motif. In summary, besides the ARE sequences already proven to be essential for iNOS mRNA stability (22)(23)(24)(25), these data reveal a second important sequence motif necessary for the post-transcriptional regulation of human iNOS expression. To prove the importance of PTB binding to the iNOS 3Ј-UTR in intact cells, we analyzed the effect of PTB on the expression of a luciferase reporter mRNA with (indicated as Luc-UTR) or without (indicated as Luc) the iNOS 3Ј-UTR under control of a tetracycline-inducible promoter.…”

Section: Discussionmentioning

confidence: 99%

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The Polypyrimidine Tract-binding Protein (PTB) Is Involved in the Post-transcriptional Regulation of Human Inducible Nitric Oxide Synthase Expression

Pautz¹,

Linker²,

Hubrich

et al. 2006

Journal of Biological Chemistry

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Human inducible nitric oxide synthase (iNOS) expression is regulated by transcriptional and post-transcriptional mechanisms. We have recently shown that the multifunctional RNAbinding proteins KH-type splicing regulatory protein and tristetraprolin are critically involved in the post-transcriptional regulation of human iNOS expression. Several reports have shown that KH-type splicing regulatory protein colocalizes with the polypyrimidine tract-binding protein (PTB), and both RNAbinding proteins seem to interact with the same mRNAs. Therefore we analyzed the involvement of PTB in human iNOS expression. In human DLD-1 cells, cytokine incubation necessary to induce iNOS expression did not change PTB localization or expression. However, intracellular binding of PTB to the human iNOS mRNA increased after cytokine stimulation. Overexpression of PTB resulted in enhanced cytokine-induced iNOS expression. Accordingly, small interfering RNA-mediated knock down of PTB reduced cytokine-dependent iNOS expression. Recombinant PTB displayed binding to an UC-rich sequence in the 3-untranslated region of the human iNOS mRNA. Transfection experiments showed that PTB mediates its effect on iNOS expression via binding to this region. The underlying mechanism is based on a modulation of iNOS mRNA stability. In summary, human iNOS is the first example of a human pro-inflammatory gene regulated by PTB on the level of mRNA stability.Post-transcriptional mechanisms represent an important part of the regulation of gene expression. In particular, genes whose expression has to be controlled precisely (e.g. oncogenes, pro-inflammatory genes) are regulated at the post-transcriptional level, mostly by modulation of mRNA stability (1, 2). Inherently unstable mRNAs that code for cytokines, transcription factors, proto-oncogenes, and pro-inflammatory mediators often contain AU-rich elements (AREs) 3 in their 3Ј-untranslated regions (3Ј-UTRs). These AREs are targets for transacting proteins regulating mRNA stability and translation (1, 3). The family of ARE-binding proteins includes the embryonic lethal abnormal vision protein family members (most importantly HuR), the ARE/poly(U)-binding/degradation factor 1 (AUF-1, also named hnRNP D), the KH-type splicing regulatory protein (KSRP), tristetraprolin, the T cell-restricted intracellular antigen (TIA-1), and the T cell-restricted intracellular antigen-related protein (1, 3). In mammalian cells, the AREsequences mediate mRNA decay mainly by recruitment of the exosome (a multisubunit particle with 3Ј to 5Ј nuclease activity) to the mRNAs, thereby promoting their rapid degradation. However, the mammalian exosome does not seem to recognize the ARE-containing RNAs on its own but requires certain AREbinding proteins (like KSRP or tristetraprolin) for this interaction (4, 5). The polypyrimidine tract-binding protein (PTB), also known as hnRNP I, is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing (6), internal ribosome entry site-driven translatio...

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Section: Discussionmentioning

confidence: 99%

“…The 3Ј-UTR of the human iNOS mRNA contains five ARE sequences and destabilizes the mRNA of a heterologous reporter gene in human A549 or DLD-1 cells (22). In recent studies, we showed that KSRP, HuR, and tristetraprolin are essentially involved in the post-transcriptional regulation of human iNOS expression in a complex manner (22)(23)(24)(25).…”

mentioning

confidence: 99%

The Polypyrimidine Tract-binding Protein (PTB) Is Involved in the Post-transcriptional Regulation of Human Inducible Nitric Oxide Synthase Expression

Pautz¹,

Linker²,

Hubrich

et al. 2006

Journal of Biological Chemistry

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“…Traditionally, eNOS has been considered to be a low output enzyme that synthesizes low levels of NO, believed to serve the physiological role of a secondary messenger. Conversely, it is believed that iNOS is a high output NOS whose expression is inducible at the transcriptional level and that upon expression, iNOS is always active and synthesizes high levels of NO (4,5). Catalytic activity of iNOS is almost 10 times higher than eNOS (39,40).…”

Section: Discussionmentioning

confidence: 99%

“…Upon stimulation of several different cell types (such as endothelial cells and macrophages) with various cytokines or bacterial lipopolysaccharide, iNOS transcription is induced, and, within several hours, iNOS protein is expressed. It is believed that once induced to express, iNOS is always active and produces NO until the protein is degraded (4,5). Interestingly, constitutive expression of low levels of iNOS has been reported in various tissues, such as kidneys (6), paranasal sinuses (7), and blood platelets (8,9).…”

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confidence: 99%

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Marjanovic¹,

Stojanović²,

Brovkovych

et al. 2008

Journal of Biological Chemistry

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Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

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“…All three are comprised of an Nterminal heme-containing oxygenase domain, an intervening calmodulin (CaM) binding sequence, and a C-terminal reductase domain that contains FMN and FAD (3). The three NOS enzymes have different gene expression patterns, protein interactions, posttranslational modifications, and catalytic behaviors that enable specific roles in biology (4)(5)(6)(7)(8). Of note, the NO synthesis activity of eNOS is one-10th that of iNOS and one-sixth that of nNOS.…”

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confidence: 99%

A connecting hinge represses the activity of endothelial nitric oxide synthase

Haque

Panda

Tejero

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K mO2 for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.electron flux ͉ heme reduction ͉ kox N itric oxide (NO) is a widespread signal molecule in biology (1, 2). Three nitric oxide synthases (NOSs) generate NO in mammals [inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS)]. All three are comprised of an Nterminal heme-containing oxygenase domain, an intervening calmodulin (CaM) binding sequence, and a C-terminal reductase domain that contains FMN and FAD (3). The three NOS enzymes have different gene expression patterns, protein interactions, posttranslational modifications, and catalytic behaviors that enable specific roles in biology (4-8). Of note, the NO synthesis activity of eNOS is one-10th that of iNOS and one-sixth that of nNOS. This activity is associated with a slower heme reduction in eNOS (9, 10). Which protein features enable these differences, and why they evolved, are still unclear.Studies of eNOS and nNOS chimeras established that their NO synthesis activities and heme reduction rates are primarily a function of the reductase domain (9, 11). For example, a chimera comprised of an eNOS oxygenase domain fused to an nNOS reductase domain had an NO synthesis activity and heme reduction rate that were similar to those of wild-type nNOS. This result implies that protein structural elements within the reductase domain are largely responsible for the different catalytic behaviors of eNOS and nNOS. While addressing this issue, we became interested in two hinge elements that connect the FMN subdomain of NOS to the rest of the enzyme (Fig. 1A). During catalysis, these hinge elements position the FMN subdomain to receive electrons from the NADPH-FAD module, and then may guide its interactions with the NOS oxygenase domain for electron transfer to the heme (12-17). In this way, the hinge elements could determine the rate of heme reduction and NO synthesis activity.The hinge that connects the FMN subdomain to the rest of the nNOS reductase domain is visible in the reductase crystal structure...

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Regulation of the expression of inducible nitric oxide synthase

Cited by 571 publications

References 116 publications

The Polypyrimidine Tract-binding Protein (PTB) Is Involved in the Post-transcriptional Regulation of Human Inducible Nitric Oxide Synthase Expression

The Polypyrimidine Tract-binding Protein (PTB) Is Involved in the Post-transcriptional Regulation of Human Inducible Nitric Oxide Synthase Expression

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

A connecting hinge represses the activity of endothelial nitric oxide synthase

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