Julia Art

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).

Transcriptional and post-transcriptional regulation of iNOS expression in human chondrocytes.

Chondrocytes are important for the development and maintenance of articular cartilage. However, both in osteoarthritis (OA) and rheumatoid arthritis (RA) chondrocytes are involved in the process of cartilage degradation and synthesize important immunomodulatory mediators, including nitric oxide (NO) generated by the inducible NO synthase (iNOS). To uncover the role of iNOS in the pathomechanisms of OA and RA, we analyzed the regulation of iNOS expression using immortalized human chondrocytes as a reproducible model. In C-28/I2 chondrocytes, iNOS expression was associated with the expression of the chondrocyte phenotype. Peak induction by a cytokine cocktail occurred between 6 and 8h and declined by 24h. Inhibition of p38MAPK, NF-kappaB and the JAK2-STAT-1alpha pathways resulted in a reduction of iNOS expression. In contrast to other cell types, the cytokine-mediated induction of the human iNOS promoter paralleled the induction rate of the iNOS mRNA expression in C-28/I2 chondrocytes. However, in addition post-transcriptional regulation of iNOS expression by the RNA binding protein KSRP seems to operate in these cells. As seen in other chondrocyte models, glucocorticoids were not able to inhibit cytokine-induced iNOS expression in C-28/I2 cells, due to the lack of the glucocorticoid receptor mRNA expression. In this model of glucocorticoid-resistance, the new fungal anti-inflammatory compound S-curvularin was able to inhibit cytokine-induced iNOS expression and iNOS-dependent NO-production. In summary, we demonstrate for the first time that differentiated human immortalized C-28/I2 chondrocytes are a representative cell culture model to investigate iNOS gene expression in human joint diseases.

Effects of nitroglycerin or pentaerithrityl tetranitrate treatment on the gene expression in rat hearts: evidence for cardiotoxic and cardioprotective effects

Nitroglycerin (NTG) and pentaerithrityl tetranitrate (PETN) are organic nitrates used in the treatment of angina pectoris, myocardial infarction, and congestive heart failure. Recent data show marked differences in the effects of NTG and PETN on the generation of reactive oxygen species. These differences are attributed to different effects of NTG and PETN on the expression of antioxidative proteins like the heme oxygenase-I. To analyze the expressional effects of NTG and PETN in a more comprehensive manner we performed whole genome expression profiling experiments using cardiac total RNA from NTG- or PETN-treated rats and DNA microarrays containing oligonucleotides representing 27,044 rat gene transcripts. The data obtained show that NTG and PETN together significantly modify the expression of >1,600 genes (NTG 532, PETN 1212). However, the expression of only a small group of these genes (68) was modified by both treatments, indicating marked differences in the expressional effects of NTG and PETN. NTG treatment resulted in the enhanced expression of genes that are believed to be markers for cardiotoxic processes. In addition, NTG treatment reduced the expression of genes described to code for cardioprotective proteins. In sharp contrast, PETN treatment enhanced the expression of cardioprotective genes and reduced the expression of genes believed to perform cardiotoxic effects. In conclusion, our data suggest that NTG treatment results in the induction of cardiotoxic gene expression networks leading to an activation of mechanisms that result in pathological changes in cardiomyocytes. In contrast, PETN treatment seems to activate gene expression networks that result in cardioprotective effects.

Similar Regulation of Human Inducible Nitric-oxide Synthase Expression by Different Isoforms of the RNA-binding Protein AUF1

The ARE/poly-(U) binding factor 1 (AUF1), a protein family consisting of four isoforms, is believed to mediate mRNA degradation by binding to AU-rich elements (ARE). However, evidence exists that individual AUF1 isoforms may stabilize ARE-containing mRNAs. The 3′-untranslated region of the human inducible nitric-oxide synthase (iNOS) contains five AREs, which promote RNA degradation. We have recently shown that the RNA-binding protein KSRP is critically involved in the decay of the iNOS mRNA. In this study we examined the effects of the individual AUF1 isoforms on iNOS expression. Overexpression of each AUF1 isoform reduces iNOS expression on mRNA and protein levels to the same extent by modulation of mRNA stability. Accordingly, knockdown of all or individual AUF1 isoforms by an RNA interference approach enhances iNOS expression. The AUF1 effect on iNOS expression is dependent on the iNOS 3′-untranslated region sequence, as demonstrated in transfection experiments with a reporter mRNA. Binding studies showed that all AUF1 isoforms interact with the same AU-rich region in the iNOS-3′-untranslated region. Cytokine stimulation altered intracellular AUF1 binding activities. These data demonstrate that AUF1 is an important factor that promotes iNOS mRNA degradation. Furthermore, all individual AUF1 isoforms act in a similar manner.

Regulation of Human Mitochondrial Aldehyde Dehydrogenase (ALDH-2) Activity by Electrophiles in Vitro

Recently, mitochondrial aldehyde dehydrogenase (ALDH-2) was reported to reduce ischemic damage in an experimental myocardial infarction model. ALDH-2 activity is redox-sensitive. Therefore, we here compared effects of various electrophiles (organic nitrates, reactive fatty acid metabolites, or oxidants) on the activity of ALDH-2 with special emphasis on organic nitrate-induced inactivation of the enzyme, the biochemical correlate of nitrate tolerance. Recombinant human ALDH-2 was overexpressed in Escherichia coli; activity was determined with an HPLC-based assay, and reactive oxygen and nitrogen species formation was determined by chemiluminescence, fluorescence, protein tyrosine nitration, and diaminonaphthalene nitrosation. The organic nitrate glyceryl trinitrate caused a severe concentration-dependent decrease in enzyme activity, whereas incubation with pentaerythritol tetranitrate had only minor effects. 4-Hydroxynonenal, an oxidized prostaglandin J2, and 9- or 10-nitrooleate caused a significant inhibition of ALDH-2 activity, which was improved in the presence of Mg2+ and Ca2+. Hydrogen peroxide and NO generation caused only minor inhibition of ALDH-2 activity, whereas peroxynitrite generation or bolus additions lead to severe impairment of the enzymatic activity, which was prevented by the thioredoxin/thioredoxin reductase (Trx/TrxR) system. In the presence of glyceryl trinitrate and to a lesser extent pentaerythritol tetranitrate, ALDH-2 may be switched to a peroxynitrite synthase. Electrophiles of different nature potently regulate the enzymatic activity of ALDH-2 and thereby may influence the resistance to ischemic damage in response to myocardial infarction. The Trx/TrxR system may play an important role in this process because it not only prevents inhibition of ALDH-2 but is also inhibited by the ALDH-2 substrate 4-hydroxynonenal.

Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol.

The Anti-Inflammatory Fungal Compound (S)-Curvularin Reduces Proinflammatory Gene Expression in an In Vivo Model of Rheumatoid Arthritis

In previous studies, we identified the fungal macrocyclic lactone (S)-curvularin (SC) as an anti-inflammatory agent using a screening system detecting inhibitors of the Janus kinase/signal transducer and activator of transcription pathway. The objective of the present study was to investigate whether SC is able to decrease proinflammatory gene expression in an in vivo model of a chronic inflammatory disease. Therefore, the effects of SC and dexamethasone were compared in the model of collagen-induced arthritis (CIA) in mice. Total genomic microarray analyses were performed to identify SC target genes. In addition, in human C28/I2 chondrocytes and MonoMac6 monocytes, the effect of SC on proinflammatory gene expression was tested at the mRNA and protein level. In the CIA model, SC markedly reduced the expression of a number of proinflammatory cytokines and chemokines involved in the pathogenesis of CIA as well as human rheumatoid arthritis (RA). In almost all cases, the effects of SC were comparable with those of dexamethasone. In microarray analyses, we identified additional new therapeutic targets of SC. Some of them, such as S100A8, myeloperoxidase, or cathelicidin, an antimicrobial peptide, are known to be implicated in pathophysiological processes in RA. Similar anti-inflammatory effects of SC were also observed in human C28/I2 chondrocyte cells, which are resistant to glucocorticoid treatment. These data indicate that SC and glucocorticoid effects are mediated via independent signal transduction pathways. In summary, we demonstrate that SC is a new effective anti-inflammatory compound that may serve as a lead compound for the development of new drugs for the therapy of chronic inflammatory diseases.

The RNA binding protein tristetraprolin influences the activation state of murine dendritic cells

Dendritic cells (DCs) serve to maintain peripheral tolerance under steady state conditions. Upon triggering by activation signals they initiate strong immune responses. The activation of DCs is accompanied by a rapid upregulation of proinflammatory cytokines, which were shown in other cell types to be regulated by mechanisms at the transcriptional and posttranscriptional level. Tristetraprolin (TTP), an important RNA binding protein, is involved in the regulation of mRNA stability of such cytokines. In this study we analyzed the significance of TTP for mouse DCs, which were derived from TTP(-/-) and WT bone marrow progenitor cells (BM-DCs). Unstimulated BM-DCs of TTP(-/-) mice expressed lower levels of mRNAs encoding the costimulatory molecules CD40 and CD86 and surprisingly also the canonical TTP targets TNF-alpha and IL-10 as compared with WT DCs. On the protein level, both DC populations expressed comparable amounts of CD80 and CD86 and of either cytokine, but TTP(-/-) DCs expressed less MHCII than WT DCs. On the other hand, TTP(-/-) DCs displayed elevated expression of other TTP target mRNAs like IL-1beta, c-fos and Mkp-1. Stimulation of BM-DCs of either genotype with lipopolysaccharide resulted in a rapid upregulation to a comparable extent of all molecules monitored so far, except for c-fos mRNA. Subsequent mRNA decay analysis revealed gene-specific differences in mRNA stability, which was influenced by the presence of TTP and the activation state of the DCs. Unstimulated TTP(-/-) DCs exerted a markedly lower allogeneic T cell stimulatory potential than WT DCs. Moreover, TTP(-/-) DCs induced an altered cytokine pattern in cocultures of DCs and T cells. However, allogeneic T cells primed by unstimulated DCs of either genotype were equally refractory to restimulation and suppressed the proliferation of naive T cells to the same extent. Thus, the findings of this study lend support to the interpretation that without external stimulation antigen presenting activity in DCs in the presence of TTP is more pronounced than in its absence and that posttranscriptional regulation contributes to the control of gene expression in DCs.

Chapter 7 – Regulation of the Expression of Inducible Nitric Oxide Synthase

Publisher Summary This chapter reveals how nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) exerts multiple beneficial microbicidal, antiviral, antiparasital, complex immunomodulatory, and antitumoral effects. Aberrant iNOS induction in the wrong place or at the wrong time has detrimental consequences and it is involved in the pathophysiology of several human diseases. Therefore, iNOS has to be regulated very tightly. The inducible isoform of NOS is mainly regulated at the level of expression. The mechanisms regulating iNOS expression involve modulation of promoter activity, mRNA stability and translatability, and protein stability. Modulation of iNOS expression is the major regulation mechanism for iNOS. Both transcriptional and post-transcriptional mechanisms have been shown to regulate iNOS expression. In contrast to the murine system, induction of human iNOS expression normally needs a complex mixture of cytokines. Pathways resulting in the induction of iNOS expression vary in different cells or species. Activation of the transcription factors NF-kB and STAT-1α and thereby activation of the iNOS promoter seems to be an essential step for iNOS induction in most human cells. However, at least in the human system, post-transcriptional mechanisms, involving a complex network of RNA binding proteins built up by AUF1, HuR, KSRP, PTB, and TTP, are also critically involved in the regulation of iNOS expression.