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      Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

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          Abstract

          Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele ( Gch1 fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes ( Gch1 fl/flTie2cre). Macrophages from Gch1 fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1 fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1 fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1 fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1 fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes ( gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1 fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.

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          Highlights

          • Gch1 −/− macrophages lack GTPCH protein expression, rendering cells BH4 deficient.

          • Gch1 −/− cells express normal iNOS protein levels, but lack nitric oxide production.

          • BH4-deficient cells exhibit higher cellular ROS production.

          • Alterations in NO and ROS production can be reversed by pharmacological BH4 rescue.

          • Stimulated BH4-deficient cells show defective NRF2-dependent gene expression.

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          Most cited references38

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          Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.

          Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.
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            Conditional Vascular Cell Adhesion Molecule 1 Deletion in Mice

            We generated vascular cell adhesion molecule (VCAM)-1 “knock-in” mice and Cre recombinase transgenic mice to delete the VCAM-1 gene (vcam-1) in whole mice, thereby overcoming the embryonic lethality seen with conventional vcam-1–deficient mice. vcam-1 knock-in mice expressed normal levels of VCAM-1 but showed loss of VCAM-1 on endothelial and hematopoietic cells when interbred with a “TIE2Cre” transgene. Analysis of peripheral blood from conditional vcam-1–deficient mice revealed mild leukocytosis, including elevated immature B cell numbers. Conversely, the bone marrow (BM) had reduced immature B cell numbers, but normal numbers of pro-B cells. vcam-1–deficient mice also had reduced mature IgD+ B and T cells in BM and a greatly reduced capacity to support short-term migration of transferred B cells, CD4+ T cells, CD8+ T cells, and preactivated CD4+ T cells to the BM. Thus, we report an until now unappreciated dominant role for VCAM-1 in lymphocyte homing to BM.
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              Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages: activation by oxidatively modified LDL and 4-hydroxynonenal.

              CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase-1 (HO-1), and peroxiredoxin I (Prx I). 4-Hydroxy-2-nonenal (HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2-deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO-1, and Prx I expression. However, treatment of smooth muscle cells with HNE significantly enhanced nuclear accumulation of Nrf2 and increased A170, HO-1, and Prx I protein levels. Because PPAR-gamma can be activated by oxLDLs and controls expression of CD36 in macrophages, our results implicate Nrf2 as a second important transcription factor involved in the induction of the scavenger receptor CD36 and antioxidant stress genes in atherosclerosis.
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                Author and article information

                Contributors
                Journal
                Free Radic Biol Med
                Free Radic. Biol. Med
                Free Radical Biology & Medicine
                Elsevier Science
                0891-5849
                1873-4596
                1 February 2015
                February 2015
                : 79
                : 206-216
                Affiliations
                [a ]Division of Cardiovascular Medicine, British Heart Foundation Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK
                [b ]Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
                [c ]Sir William Dunn School of Pathology University of Oxford, Oxford, UK
                Author notes
                [* ]Corresponding author. Address: Division of Cardiovascular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK. keith.channon@ 123456cardiov.ox.ac.uk
                Article
                S0891-5849(14)01076-4
                10.1016/j.freeradbiomed.2014.10.575
                4344222
                25451639
                2291e440-c1d5-49fa-9664-b07dc135dbb4
                © 2014 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

                History
                : 17 August 2014
                : 20 October 2014
                : 20 October 2014
                Categories
                Original Contribution

                Molecular biology
                as3mt, arsenic(iii)-methyltransferase,bh4, tetrahydrobiopterin,[fe(detc)2], colloid iron (ii) diethyldithiocarbamate,gtpch, gtp cyclohydrolase 1,2-he, 2-hydroxyethidium,inos, inducible nitric oxide synthase,ros, reactive oxygen species.,tetrahydrobiopterin,bh4,gch1,gtpch,macrophage,nos2,inos,nitric oxide,as3mt

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