The mRNA-Binding Protein KSRP Limits the Inflammatory Response of Macrophages.

KH-type splicing regulatory protein (KSRP) is a single-stranded nucleic acid-binding protein with multiple functions. It is known to bind AU-rich motifs within the 3'-untranslated region of mRNA species, which in many cases encode dynamically regulated proteins like cytokines. In the present study, we investigated the role of KSRP for the immunophenotype of macrophages using bone marrow-derived macrophages (BMDM) from wild-type (WT) and KSRP-/- mice. RNA sequencing revealed that KSRP-/- BMDM displayed significantly higher mRNA expression levels of genes involved in inflammatory and immune responses, particularly type I interferon responses, following LPS stimulation. In line, time kinetics studies revealed increased levels of interferon-γ (IFN-γ), interleukin (IL)-1ß and IL-6 mRNA in KSRP-/- macrophages after 6 h subsequent to LPS stimulation as compared to WT cultures. At the protein level, KSRP-/- BMDM displayed higher levels of these cytokines after overnight stimulation. Matching results were observed for primary peritoneal macrophages of KSRP-/- mice. These showed higher IL-6, tumor necrosis factor-α (TNF-α), C-X-C motif chemokine 1 (CXCL1) and CC-chemokine ligand 5 (CCL5) protein levels in response to LPS stimulation than the WT controls. As macrophages play a key role in sepsis, the in vivo relevance of KSRP deficiency for cytokine/chemokine production was analyzed in an acute inflammation model. In agreement with our in vitro findings, KSRP-deficient animals showed higher cytokine production upon LPS administration in comparison to WT mice. Taken together, these findings demonstrate that KSRP constitutes an important negative regulator of cytokine expression in macrophages.

Effect of the antirheumatic medication methotrexate (MTX) on biomechanical compressed human periodontal ligament fibroblasts (hPDLFs).

BACKGROUND: The aim of this study was to investigate the in vitro effect of the antirheumatic drug methotrexate (MTX) on biomechanically compressed human periodontal ligament fibroblasts (hPDLFs), focusing on the expression of interleukin 6 (IL-6), as its upregulation is relevant to orthodontic tooth movement. METHODS: Human PDLFs were subjected to pressure and simultaneously treated with MTX. Cell proliferation, viability and morphology were studied, as was the gene and protein expression of IL-6. RESULTS: Compared with that in untreated fibroblasts, IL-6 mRNA expression in mechanically compressed ligament fibroblasts was increased (two to sixfold; ****p < 0.0001). Under compression, hPDLFs exhibited a significantly more expanded shape with an increase of cell extensions. MTX with and without pressure did not affect IL-6 mRNA expression or the morphology of hPDLFs. CONCLUSION: MTX has no effect on IL-6 expression in compressed ligament fibroblasts.

Sex and Gender Differences in the Pharmacology of the Overactive Urinary Bladder.

Dysfunction of the lower urinary tract in general and the overactive bladder syndrome (OAB) in particular are prevalent and have major impact on the quality of life of the afflicted patients and their partners. We concisely review sex and gender differences in patients and animal models in physiological bladder function, its alterations in disease (mostly OAB), and its responses to treatment. Women appear to have a smaller functional bladder capacity and, therefore, must void more often than men. On the other hand, men have a greater bladder outlet resistance, which is partly attributed to a longer urethra and partly to the presence of the prostate. Sex and gender differences in bladder contractility appear small and were not found consistently. The ability of bladder smooth muscle to relax may be somewhat smaller in females. However, females are heavily underrepresented in experimental studies on bladder function. Stress urinary incontinence is found predominantly in women (particularly those after childbirth). OAB is similarly prevalent in men and women. Females seek treatment much more often and are overrepresented in clinical trials. Treatment responses in OAB patients are similar in both genders for oral medications, but improvements upon injections of onabotulinum toxin type A appear smaller in men. We conclude that there is no evidence for major sex and gender differences in bladder dysfunction as related to OAB and its treatment responses, but female animals are heavily underrepresented in experimental studies.

A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types.

BACKGROUND: NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA isoform is detected. However, the expression of NOS2 in differentiating human pluripotent stems (hPSCs) has not been analyzed yet. METHODS: Public available RNA-seq databases were screened for data of hPSCs during differentiation to different target cells. An isoform specific algorithm was used to analyze NOS2 mRNA isoform expression. In addition, we differentiated four different human iPSC cell lines toward cortical neurons and analyzed NOS2 mRNA expression by qRT-PCR and 5'-RACE. The functionality of the NOS2-2 protein was analyzed by transient transfection of expression clones in human DLD1 cells and nitrate measurement in the supernatant of these cells. RESULTS: In RNA-seq databases we detected a transient expression of the NOS2 mRNA during the differentiation of hPSCs to cardiomyocytes, chondrocytes, mesenchymal stromal cells, neurons, syncytiotrophoblast cells, and trophoblasts. NOS2 mRNA isoform specific analyses showed, that the transiently expressed NOS2 mRNA in differentiating hPSC (NOS2-2; "diff-iNOS") differ remarkably from the already described NOS2 transcript found in colon or induced islets (NOS2-1; "immuno-iNOS"). Also, analysis of the NOS2 mRNA- and protein expression during the differentiation of four different hiPSC lines towards cortical neurons showed a transient expression of the NOS2 mRNA and NOS2 protein on day 18 of the differentiation course. 5'-RACE experiments and isoform specific qRT-PCR analyses revealed that only the NOS2-2 mRNA isoform was expressed in these experiments. To analyze the functionality of the NOS2-2 protein, we transfected human DLD-1 cells with tetracycline inducible expression clones encoding the NOS2-1- or -2 coding sequence. After induction of the NOS2-1 or -2 mRNA expression by tetracycline a similar nitrate production was measured proofing the functionality of the NOS2-2 protein isoform. CONCLUSIONS: Our data show that a differentiation specific NOS2 isoform (NOS2-2) is transiently expressed during differentiation of hPSC. Video Abstract.

The KH-type splicing regulatory protein (KSRP) regulates type III interferon expression post-transcriptionally.

Type III interferons (IFNs) are the latest members of the IFN family. They play an important role in immune defense mechanisms, especially in antiviral responses at mucosal sites. Moreover, they control inflammatory reactions by modulating neutrophil and dendritic cell functions. Therefore, it is important to identify cellular mechanisms involved in the control of type III IFN expression. All IFN family members contain AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) of their mRNAs that determine mRNA half-life and consequently the expressional level of these cytokines. mRNA stability is controlled by different proteins binding to these AREs leading to either stabilization or destabilization of the respective target mRNA. The KH-type splicing regulatory protein KSRP (also named KHSRP) is an important negative regulator of ARE-containing mRNAs. Here, we identify the interferon lambda 3 (IFNL3) mRNA as a new KSRP target by pull-down and immunoprecipitation experiments, as well as luciferase reporter gene assays. We characterize the KSRP-binding site in the IFNL3 3'-UTR and demonstrate that KSRP regulates the mRNA half-life of the IFNL3 transcript. In addition, we detect enhanced expression of IFNL3 mRNA in KSRP-/- mice, establishing a negative regulatory function of KSRP in type III IFN expression also in vivo Besides KSRP the RNA-binding protein AUF1 (AU-rich element RNA-binding protein 1) also seems to be involved in the regulation of type III IFN mRNA expression.

Regulation of human inducible nitric oxide synthase expression by an upstream open reading frame.

The human inducible nitric oxide synthase (iNOS) gene contains an upstream open reading frame (uORF) in its 5'-untranslated region (5'-UTR) implying a translational regulation of iNOS expression. Transfection experiments in human DLD-1 cells revealed that the uORF although translatable seems not to inhibit the translation start at the bona fide ATG. Our data clearly show that human iNOS translation is cap-dependent and that the 5'-UTR of the iNOS mRNA contains no internal ribosome entry site. Translation of the bona fide coding sequence is most likely mediated by a leaky scanning mechanism. The 5'-UTR is encoded by exon 1 and exon 2 of the iNOS gene with the uORF stop codon located in front of the first intron indicating an involvement of the nonsense mediated RNA decay (NMD) in iNOS regulation. SiRNA-mediated down-regulation of Upf1 resulted in enhanced endogenous cytokine iNOS expression in human DLD-1 cells. Transfection of constructs containing iNOS exon 1, intron 1 and exon 2 in front of a luciferase gene showed a clear effect of the mutation of the uORF-ATG on luciferase reportergene expression. Our data indicate that the uORF in the 5'-UTR sequence of human iNOS gene reduces its expression via the NMD mechanism.

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.

Endothelial dysfunction in tristetraprolin-deficient mice is not caused by enhanced tumor necrosis factor-α expression.

Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP(-/-) mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP(-/-) mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP(-/-) mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP(-/-) mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP(-/-) animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP(-/-) animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.

The fungal lactone oxacyclododecindione is a potential new therapeutic substance in the treatment of lupus-associated kidney disease.

Recently oxacyclododecindione (Oxa), a macrocyclic lactone isolated from the imperfect fungus Exserohilum rostratum, has been described as a potent transcription inhibitor of inducible proinflammatory and profibrotic genes in cell culture models. As kidney disease in systemic lupus erythematosus is characterized by aberrant expression of inflammatory mediators and infiltration of immune cells, we investigated the effect of Oxa in MRL-Fas(lpr) mice, a model of systemic lupus erythematosus. These mice develop a spontaneous T-cell and macrophage-dependent autoimmune disease including severe glomerulonephritis that shares features with human lupus. Comparable to the results of in vitro models, we found a reduced expression of the cytokines IFN-γ, IL-6, and TNF-α and the chemokines CCL2, RANTES, and CSF-1 on mRNA and protein level in the kidney of Oxa-treated MRL-Fas(lpr) mice. Accordingly, Oxa treatment reduced the infiltration of immune cells and the frequency of activated proinflammatory T cells in the kidney. Moreover, kidney disease, measured by histopathology, IgG and collagen deposition, and proteinuria, was ameliorated in Oxa-treated MRL-Fas(lpr) mice compared with the control group. Thus, Oxa is a new effective anti-inflammatory compound, which may serve as base structure for the development of new therapeutics for the treatment of chronic inflammatory and/or fibrotic diseases.

Artichoke, cynarin and cyanidin downregulate the expression of inducible nitric oxide synthase in human coronary smooth muscle cells.

Artichoke (Cynara scolymus L.) is one of the world's oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS) in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects of artichoke on iNOS expression in human coronary artery smooth muscle cells (HCASMC). Incubation of HCASMC with a cytokine mixture led to an induction of iNOS mRNA expression. This iNOS induction was concentration- and time-dependently inhibited by an artichoke leaf extract (1-100 µg/mL, 6 h or 24 h). Consistently, the artichoke leaf extract also reduced cytokine-induced iNOS promoter activation and iNOS protein expression. In addition, treatment of HCASMC with four well-known artichoke compounds (cynarin > cyanidin > luteolin ≈ cynaroside) led to a downregulation iNOS mRNA and protein expression, with cynarin being the most potent one. In conclusion, artichoke contains both eNOS-upregulating and iNOS-downregulating compounds. Such compounds may contribute to the beneficial effects of artichoke and may per se have therapeutic potentials.

Chronic inflammatory cardiomyopathy of interferon γ-overexpressing transgenic mice is mediated by tumor necrosis factor-α.

We recently described a model of inflammatory cardiomyopathy in interferon (IFN)-γ overexpressing transgenic mice stably circulating IFN-γ in the serum referred to as SAP--IFN-γ mice. SAP-IFN-γ transgenic mice show cardiac infiltration by mononuclear leukocytes, culminating in dilated cardiomyopathy characterized by an increase of left ventricular end diastolic diameter and reduction of fractional shortening. We hypothesized that the pathological mechanism underlying SAP-IFN-γ cardiomyopathy might be mediated by (auto)immune processes or tumor necrosis factor (TNF)-α synthesis from IFN-γ-activated macrophages. To verify these hypotheses, we crossed SAP-IFN-γ transgenic mice with immunodeficient Rag1(-/-) or TNF-α(-/-) knockout mice and analyzed the cardiac phenotype of the resulting double-mutant offspring. Immunodeficient Rag1(-/-) SAP-IFN-γ mice had a decreased impaired life span and intensive cardiac inflammatory reactions, showing that the cardiotoxic IFN-γ effect operative in SAP-IFN-γ mice was not mediated by an adaptive immune mechanism. SAP-IFN-γ TNF-α(-/-) hearts showed virtually no histopathological alterations, a significant reduction of cardiac infiltration by CD11c(+) dendritic cells and F4/80(+) macrophages, almost complete normalization of cardiac troponin T levels in serum and of left ventricular end diastolic diameter and fractional shortening, and a dramatic increase of life span, compared with SAP-IFN-γ transgenic controls. Thus, myocarditis and cardiomyopathy developing in IFN-γ-overexpressing transgenic mice is, to a significant degree, mediated by TNF-α. TNF-α-mediated cardiotoxicity in SAP-IFN-γ transgenic mice is independent of changes of apoptosis.

Human inducible nitric oxide synthase (iNOS) expression depends on chromosome region maintenance 1 (CRM1)- and eukaryotic translation initiation factor 4E (elF4E)-mediated nucleocytoplasmic mRNA transport.

Human inducible nitric oxide synthase (iNOS) is regulated on the expressional level mostly by post-transcriptional mechanisms modulating the mRNA stability. Another important step in the control of eukaryotic gene expression is the nucleocytoplasmic mRNA transport. Most cellular mRNAs are exported via the TAP/Nxt complex of proteins. However, some mRNAs are transported by a different mechanism involving the nuclear export receptor CRM1. Treatment of DLD-1 cells with the CRM1 inhibitor leptomycin B (LMB) or anti-CRM1 siRNAs reduced cytokine-induced iNOS expression. We could demonstrate that the iNOS mRNA is exported from the nucleus in a CRM1-dependent manner. Since CRM1 itself does not possess any RNA binding affinity, an adapter protein is needed to mediate CRM1-dependent mRNA export. Western blot experiments showed that the eukaryotic translation initiation factor eIF4E is retained in the nucleus after LMB treatment. Blockade of eIF4E by ribavirin or overexpression of the promyelocytic leukemia protein (PML) decreased iNOS expression due to reduced iNOS mRNA export from the nucleus. Transfection experiments provide evidence that the 3'-untranslated region of the iNOS mRNA is involved in eIF4E-mediated iNOS mRNA transport. In summary, CRM1 and eIF4E seem to play an important role in the nucleocytoplasmic export of human iNOS mRNA.

Post-transcriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(A)-binding protein (PABP).

Affinity purification using the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide synthase (iNOS) mRNA identified the cytosolic poly(A)-binding protein (PABP) as a protein interacting with the human iNOS 3'-UTR. Downregulation of PABP expression by RNA interference resulted in a marked reduction of cytokine-induced iNOS mRNA expression without changes in the expression of mRNAs coding for the major subunit of the RNA polymerase II (Pol 2A) or ß2-microglobuline (ß2M). Along with the mRNA also iNOS protein expression was reduced by siPABP-treatment, whereas in the same cells protein expression of STAT-1α, NF-κB p65, or GAPDH was not altered. Reporter gene analyses showed no change of the inducibility of the human 16kb iNOS promoter in siPABP cells. In contrast, the siPABP-mediated decline of iNOS expression correlated with a reduction in the stability of the iNOS mRNA. As the stability of the Pol 2A and ß2M mRNA was not changed, siPABP-treatment seems to have a specific effect on iNOS mRNA decay. UV-crosslinking experiments revealed that PABP interacts with one binding site in the 5'-UTR and two different binding sites in the 3'-UTR of the human iNOS mRNA. Mutation or deletion of the binding site in the 5'-UTR but not in the 3'-UTR reduced luciferase expression in DLD-1 cells transfected with iNOS-5'-UTR or iNOS-3'-UTR luciferase reporter constructs. In summary, our data demonstrate that PABP by binding to specific sequence elements in the 5'-UTR post-transcriptionally enhances human iNOS mRNA stability and thereby iNOS expression.

A systematic review on renal effects of SGLT2 inhibitors in rodent models of diabetic nephropathy.

We have performed a systematic review of studies reporting on the renal effects of SGLT2 inhibitors in rodent models of diabetes. In 105 studies, SGLT2 inhibitors improved not only the glycemic control but also various aspects of renal function in most cases. These nephroprotective effects were similarly reported whether treatment with the SGLT2 inhibitor started concomitant with the onset of diabetes (within 1 week), early after onset (1-4 weeks) or after nephropathy had developed (>4 weeks after onset) with the latter probably having the greatest translational value. They were observed across various animal models of type 1 and type 2 diabetes/obesity (4 and 23 models, respectively), although studies in the type 2 diabetes model of db/db mice more often had negative data than in other models. Among possibly underlying pathophysiological mechanisms of nephroprotection, treatment with SGLT2 inhibitors had beneficial effects on lipid metabolism, blood pressure, glomerulosclerosis as well as renal tubular fibrosis, apoptosis, oxidative stress, and inflammation. These pathomechanisms highly influence atherosclerosis and renal health, which are two major factors that lead to an enhanced mortality in patients with diabetes and/or chronic kidney disease. Interestingly, renal SGLT2 inhibitor effects did not always correlate with those on glucose homeostasis, particularly in a limited number of direct comparative studies with other anti-diabetic treatments, indicating that nephroprotection may at least partly occur by mechanisms other than improving glycemic control. Our analyses did not provide evidence for different nephroprotective efficacy between SGLT2 inhibitors. Importantly, only four of 105 studies reported on female animals, and none provided direct comparative data between sexes. We conclude that more data on female animals and more direct comparative studies with other anti-diabetic compounds and combinations of treatments are needed.

The macrocyclic lactone oxacyclododecindione reduces fibrosis progression.

Background: Renal fibrosis is one of the most important triggers of chronic kidney disease (CKD), and only a very limited number of therapeutic options are available to stop fibrosis progression. As fibrosis is characterized by inflammation, myofibroblast activation, and extracellular matrix (ECM) deposition, a drug that can address all these processes might be an interesting therapeutic option. Methods: We tested in vivo in an ischemia-reperfusion (I/R) model in C57BL/6 mice and in kidney tubular epithelial cells (TEC) (HK2 cell line and primary cells) whether the natural product oxacyclododecindione (Oxa) reduces fibrosis progression in kidney disease. This was evaluated by Western blot, mRNA expression, and mass spectrometry secretome analyses, as well as by immunohistochemistry. Results: Indeed, Oxa blocked the expression of epithelial-mesenchymal transition marker proteins and reduced renal damage, immune cell infiltration, and collagen expression and deposition, both in vivo and in vitro. Remarkably, the beneficial effects of Oxa were also detected when the natural product was administered at a time point of established fibrotic changes, a situation close to the clinical situation. Initial in vitro experiments demonstrated that a synthetic Oxa derivative possesses similar features. Conclusion: Although open questions such as possible side effects need to be investigated, our results indicate that the combination of anti-inflammatory and anti-fibrotic effects of Oxa make the substance a promising candidate for a new therapeutic approach in fibrosis treatment, and thus in the prevention of kidney disease progression.

A comparison of urinary bladder weight in male and female mice across five models of diabetes and obesity.

Introduction: Diabetes often leads to lower urinary tract dysfunction. The most frequently assessed parameter of urinary bladder dysfunction in animal models of diabetes is an enlargement of the bladder, which is consistently observed in type 1 and less consistently in type 2 diabetes. The vast majority of studies on bladder weight in animal models of diabetes and obesity has been performed in males, and no studies have directly compared this outcome parameter between sexes. Methods: Therefore, we have compared bladder weight and bladder/body weight ratio in five mouse models of obesity and diabetes (RIP-LCMV, db/db, ob/ob (two studies), insulin receptor substrate 2 (IRS2) knock-out mice and mice on a high-fat diet; pre-specified secondary analysis of a previously reported study). Results: In a pooled analysis of the control groups of all studies, females exhibited slightly lower glucose levels, lower body weight, and lower bladder weight, but bladder/body weight ratio was similar in both sexes (0.957 vs. 0.986 mg/g, mean difference 0.029 [-0.06; 0.118]). Among the six diabetic/obese groups, bladder/body weight ratio was similar in both sexes in three but smaller in female mice in three other groups. The mRNA expression of a panel of genes implied in the pathophysiology of bladder enlargement and/or fibrosis and inflammation did not differ systematically between sexes. Conclusions: We conclude that sex differences in diabetes/obesity-associated bladder enlargement may be model dependent.

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 J(2), and 9- or 10-nitrooleate caused a significant inhibition of ALDH-2 activity, which was improved in the presence of Mg(2+) and Ca(2+). 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.

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.

Transcriptional regulation of Nox4 by histone deacetylases in human endothelial cells.

Nox4 is a member of the NADPH oxidase family, which represents a major source of reactive oxygen species (ROS) in the vascular wall. Nox4-mediated ROS production mainly depends on the expression levels of the enzyme. The present study was aimed to investigate the mechanisms of Nox4 transcription regulation by histone deacetylases (HDAC). In human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells, treatment with the pan-HDAC inhibitor scriptaid led to a marked decrease in Nox4 mRNA expression. A similar down-regulation of Nox4 mRNA expression was observed by siRNA-mediated knockdown of HDAC3. HDAC inhibition in endothelial cells was associated with enhanced histone acetylation, increased chromatin accessibility in the human Nox4 promoter region, with no significant changes in DNA methylation. In addition, we provided evidence that c-Jun played an important role in controlling Nox4 transcription. Knockdown of c-Jun with siRNA led to a down-regulation of Nox4 mRNA expression. In response to scriptaid treatment, the binding of c-Jun to the Nox4 promoter region was reduced despite the open chromatin structure. In parallel, the binding of RNA polymerase IIa to the Nox4 promoter was significantly inhibited as well, which may explain the reduction in Nox4 transcription. In conclusion, HDAC inhibition decreases Nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the Nox4 promoter, most likely because of a hyperacetylation-mediated steric inhibition.

Induction of tolerogenic lung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitor ameliorated experimental allergic asthma.

Signal transducer and activator of transcription (STAT)-3 inhibitors play an important role in regulating immune responses. Galiellalactone (GL) is a fungal secondary metabolite known to interfere with the binding of phosphorylated signal transducer and activator of transcription (pSTAT)-3 as well of pSTAT-6 dimers to their target DNA in vitro. Intra nasal delivery of 50 µg GL into the lung of naive Balb/c mice induced FoxP3 expression locally and IL-10 production and IL-12p40 in RNA expression in the airways in vivo. In a murine model of allergic asthma, GL significantly suppressed the cardinal features of asthma, such as airway hyperresponsiveness, eosinophilia and mucus production, after sensitization and subsequent challenge with ovalbumin (OVA). These changes resulted in induction of IL-12p70 and IL-10 production by lung CD11c(+) dendritic cells (DCs) accompanied by an increase of IL-3 receptor α chain and indoleamine-2,3-dioxygenase expression in these cells. Furthermore, GL inhibited IL-4 production in T-bet-deficient CD4(+) T cells and down-regulated the suppressor of cytokine signaling-3 (SOCS-3), also in the absence of STAT-3 in T cells, in the lung in a murine model of asthma. In addition, we found reduced amounts of pSTAT-5 in the lung of GL-treated mice that correlated with decreased release of IL-2 by lung OVA-specific CD4(+) T cells after treatment with GL in vitro also in the absence of T-bet. Thus, GL treatment in vivo and in vitro emerges as a novel therapeutic approach for allergic asthma by modulating lung DC phenotype and function resulting in a protective response via CD4(+)FoxP3(+) regulatory T cells locally.