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 stimulatio

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 modulates expression of extracellular matrix genes linked to fibrosis in kidney mesangial cells

Mesangial cells are thought to be important mediators of glomerular inflammation and fibrosis. Studies have established a direct role for nitric oxide (NO) in the regulation of gene expression in mesangial cells. Representational difference analysis was used to investigate changes in gene expression elicited by the treatment of S-nitroso-L-glutathione in rat mesangial cells. Seven upregulated and 11 downregulated genes were identified. Four out of 11 downregulated genes (connective tissue growth factor, thrombospondin-1, collagen type I all and collagen type I alpha 2) are known to be linked to inflammation and fibrosis. Results were verified across species in mesangial cells treated with a series of NO donors using Northern blot analysis, quantitative real-time PCR and protein analysis methods. Induction of endogenous NO production by cytokine stimulation also triggered regulation of the genes. One example gene, connective tissue growth factor, was studied at the promoter level. Promoter-reporter gene studies in mesangial cells demonstrated that NO acts at the transcriptional level to suppress gene expression. Our results reveal a complex role of NO in regulating gene expression in mesangial cells and suggest an antifibrotic potential for NO

The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

Abstract Background Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). High amounts of NO are synthesized following up-regulation of inducible NO synthase (iNOS). The expression of iNOS is tightly regulated by complex molecular mechanisms, involving both transcriptional and post-transcriptional processes. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. mTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. It is also a key regulator of intracellular processes in glial cells. However, with respect to iNOS expression, both stimulatory and inhibitory actions involving the mTOR pathway have been described. In this study the effects of mTOR inhibition on iNOS regulation were evaluated in astrocytes. Methods Primary cultures of rat cortical astrocytes were activated with different proinflammatory stimuli, namely a mixture of cytokines (TNFα, IFNγ, and IL-1β) or by LPS plus IFNγ. Rapamycin was used at nM concentrations to block mTOR activity and under these conditions we measured its effects on the iNOS promoter, mRNA and protein levels. Functional experiments to evaluate iNOS activity were also included. Results In this experimental paradigm mTOR activation did not significantly affect astrocyte iNOS activity, but mTOR pathway was involved in the regulation of iNOS expression. Rapamycin did not display any significant effects under basal conditions, on either iNOS activity or its expression. However, the drug significantly increased iNOS mRNA levels after 4 h incubation in presence of pro-inflammatory stimuli. This stimulatory effect was transient, since no differences in either iNOS mRNA or protein levels were detected after 24 h. Interestingly, reduced levels of iNOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability. Conclusion The present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies.</p

Reduced Necrosis and Content of Apoptotic M1 Macrophages in Advanced Atherosclerotic Plaques of Mice With Macrophage-Specific Loss of Trpc3

In previous work we reported that ApoeKO mice transplanted with bone marrow cells deficient in the Transient Receptor Potential Canonical 3 (TRPC3) channel have reduced necrosis and number of apoptotic macrophages in advanced atherosclerotic plaques. Also, in vitro studies with polarized macrophages derived from mice with macrophage-specific loss of TRPC3 showed that M1, but not M2 macrophages, deficient in Trpc3 are less susceptible to ER stress-induced apoptosis than Trpc3 expressing cells. The questions remained (a) whether the plaque phenotype in transplanted mice resulted from a genuine effect of Trpc3 on macrophages, and (b) whether the reduced necrosis and macrophage apoptosis in plaques of these mice was a manifestation of the selective effect of TRPC3 on apoptosis of M1 macrophages previously observed in vitro. Here, we addressed these questions using Ldlr knockout (Ldlr−/−) mice with macrophage-specific loss of Trpc3 (MacTrpc3−/−/Ldlr−/− → Ldlr−/−). Compared to controls, we observed decreased plaque necrosis and number of apoptotic macrophages in MacTrpc3−/−/Ldlr−/− → Ldlr−/− mice. Immunohistochemical analysis revealed a reduction in apoptotic M1, but not apoptotic M2 macrophages. These findings confirm an effect of TRPC3 on plaque necrosis and support the notion that this is likely a reflection of the reduced susceptibility of Trpc3-deficient M1 macrophages to apoptosis.Fil: Solanki, Sumeet. University of Toledo; Estados UnidosFil: Dube, Prabhatchandra R.. University of Toledo; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Vazquez, Guillermo. University of Toledo; Estados Unido

In vitro and in vivo TNFa synthesis modulation by methylguanidine, an uremic catabolyte

Original article can be found at: http://www.sciencedirect.com/science/journal/00243205 Copyright Elsevier Inc.We have investigated whether methylguanidine (MG), an uremic toxin, modulates the expression of the inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release in vitro in LPS-induced J774 macrophages.Peer reviewe

Polyamine regulation of nitric oxide production in LPS-activated macrophages

© European Communities, 1999 Reproduction is authorised provided the source is acknowledgedPolyamines are physiological cellular constituents essential for cell growth and differentiation, and regulate a multitude of cellular functions (1-4). Nitric oxide (NO) is an effector molecule in both the cardiovascular and nervous systems (5,6). Intracellularly, NO and polyamines are derived from arginine, the latter via the rate-limiting enzyme ornithine decarboxylase (ODC; 7). This enzyme, like the inducible nitric oxide synthase (iNOS), is induced by proinflammatory cytokines and bacterial lipopolysaccharide (LPS), resulting in enhanced enzyme activity and increased polyamine biosynthesis (8,9). While the increase in polyamine synthesis would have important implications for cell growth and proliferation, it is not clear how this might affect iNOS pathway. Inhibition of polyamine biosynthesis impairs the phagocytic capacity of macrophages (10) and can block macrophage activation by tumour necrosis factor (11). Recently, exogenous polyamines have been shown to inhibit NO production in LPS-activated J774 cells (12) and by isolated neuronal NO synthase (13). However, these effects required relatively high concentrations of polyamines compared to those found in plasma and in intact cells (14), and appear to be due to aldehyde metabolites resulting from polyamine oxidation by the amine oxidase present in calf serum (15-17). In this study we have explored the effects of both endogenous and exogenous polyamines on the inducible L-arginine-NO pathway by examining whether inhibition of ornithine decarboxylase (ODC) and thus of polyamine biosynthesis (7), regulates NO production in lipopolysaccharide-activated J774 cells, a murine macrophage cell lineFinal Published versio

The key role of nitric oxide in hypoxia: hypoxic vasodilation and energy supply-demand matching

Significance: a mismatch between energy supply and demand induces tissue hypoxia with the potential to cause cell death and organ failure. Whenever arterial oxygen concentration is reduced, increases in blood flow - 'hypoxic vasodilation' - occur in an attempt to restore oxygen supply. Nitric oxide is a major signalling and effector molecule mediating the body's response to hypoxia, given its unique characteristics of vasodilation (improving blood flow and oxygen supply) and modulation of energetic metabolism (reducing oxygen consumption and promoting utilization of alternative pathways). Recent advances: this review covers the role of oxygen in metabolism and responses to hypoxia, the hemodynamic and metabolic effects of nitric oxide, and mechanisms underlying the involvement of nitric oxide in hypoxic vasodilation. Recent insights into nitric oxide metabolism will be discussed, including the role for dietary intake of nitrate, endogenous nitrite reductases, and release of nitric oxide from storage pools. The processes through which nitric oxide levels are elevated during hypoxia are presented, namely (i) increased synthesis from nitric oxide synthases, increased reduction of nitrite to nitric oxide by heme- or pterin-based enzymes and increased release from nitric oxide stores, and (ii) reduced deactivation by mitochondrial cytochrome c oxidase. Critical issues: several reviews covered modulation of energetic metabolism by nitric oxide, while here we highlight the crucial role NO plays in achieving cardiocirculatory homeostasis during acute hypoxia through both vasodilation and metabolic suppression Future directions: we identify a key position for nitric oxide in the body's adaptation to an acute energy supply-demand mismatc

Nonylphenol and octylphenol differently affect cell redox balance by modulating the nitric oxide signaling

Nonylphenol (NP) and octylphenol (OP) are pervasive environmental contaminants belonging to the broader class of compounds known as alkylphenols, with potential human toxic effects. Classified as "xenoestrogens," NP and OP are able to interfere with the cell endocrine physiology via a direct interaction with the estrogen receptors. Here, using HepG2 cells in culture, the changes of the cell redox balance and mitochondrial activity induced by OP and NP have been investigated at mu M concentrations, largely below those provoking acute toxicity, as those typical of environmental contaminants. Following 24 h cell exposure to both OP and NP, ROS production appeared significantly increased (p <= 0.01), together with the production of higher NO oxides (p = 0.003) and peroxynitrated protein-derivatives (NP versus CTR, p = 0.003). The mitochondrial proton electrochemical potential gradient instead was decreased (p <= 0.05), as the oxygen consumption by complex IV, particularly following incubation with NP (NP versus CTR, p = 0.017). Consistently, the RT-PCR and Western blot analyses proved that the OP and NP can modulate to a different extent the expression of the inducible NOS (NP versus CTR, p <= 0.01) and the endothelial NOS (OP versus CTR, p <= 0.05), with a significant variation of the coupling efficiency of the latter (NP versus CTR, p <= 0.05), a finding that may provide a novel clue to understand the specific xenoestrogenic properties of OP and NP