The Nrf2/GCH1/BH4 Axis Ameliorates Radiation-Induced Skin Injury by Modulating the ROS Cascade.

Radiation-induced skin injury is a common side effect of radiotherapy and can limit the duration and dose of radiotherapy. Most early work focused on elimination of reactive oxygen species (ROS) after radiation; however, less is known about the mechanisms underlying amplification of ROS and consequent skin injury by radiation. 5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for all nitric oxide synthases. Inadequate availability of BH4 leads to uncoupling of nitric oxide synthases and production of highly oxidative radicals. In this study, we demonstrated that radiation disrupted BH4, which resulted in nitric oxide synthases uncoupling and augmented radiation-induced ROS. Overexpression of GTP cyclohydrolase I (GCH1), the rate-limiting enzyme for BH4 synthesis, restored cellular BH4 levels and nitric oxide production and decreased radiation-induced ROS. GCH1 also protected skin cells and rat skins against radiation-induced damage. We found that GCH1 was regulated by NF-E2-related factor 2, a key mediator of the cellular antioxidant response. Importantly, we identified GCH1 as a key effector for NF-E2-related factor 2-mediated protection against radiation-induced skin injury by inhibiting ROS production. Taken together, the findings of this study illustrate the key role of the NF-E2-related factor 2/GCH1/BH4 axis during radiation-induced skin damage.

Translational effects and coding potential of an upstream open reading frame associated with DOPA Responsive Dystonia.

Upstream open reading frames (uORFs) have emerged as major post-transcriptional regulatory elements in eukaryotic species. In general, uORFs are initiated by a translation start codon within the 5' untranslated region of a gene (upstream ATG; uATG), and they are negatively correlated with translational efficiency. In addition to their translational regulatory role, some uORFs can code for biologically active short peptides. The importance of uATGs/uORFs is further underscored by human diseases associated with single nucleotide polymorphisms (SNPs), which disrupt existing uORFs or introduce novel uORFs. Although several functional proteins translated from naturally occurring uORFs have been described, the coding potential of uORFs created by SNPs has been ignored because of the a priori assumption that these proteins are short-lived with no likely impact on protein homeostasis. Thus, studies on SNP-created uORFs are limited to their translational effects, leaving unexplored the potential cellular consequences of a SNP/uORF-encoded protein. Here, we investigate functionality of a uATG/uORF introduced by a +142C>T SNP within the GCH1 gene and associated with a familial form of DOPA Responsive Dystonia. We report that the +142C>T SNP represses GCH1 translation, and introduces a short, frame shifted uORF that encodes a 73-amino acid peptide. This peptide is localized within the nucleus and compromises cell viability upon proteasome inhibition. Our work extends the list of uATG/uORF associated diseases and advances research on peptides translated from SNP-introduced uORFs, a neglected component of the proteome.

Increasing tetrahydrobiopterin in cardiomyocytes adversely affects cardiac redox state and mitochondrial function independently of changes in NO production.

Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterin™ or Kuvan™) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (<6 months) have lower BH4:BH2 ratios than hearts of older mice while both GTPCH activity and tissue ascorbate levels were higher in hearts of young than older mice. No evident changes in nitric oxide (NO) production assessed by nitrite and endogenous iron-nitrosyl complexes were detected in any of the age groups. Increased BH4 production in cardiomyocytes resulted in a significant loss of mitochondrial function. Diminished oxygen consumption and reserve capacity was verified in mitochondria isolated from hearts of 12-month old compared to 3-month old mice, even though at 12 months an improved BH4:BH2 ratio is established. Accumulation of 4-hydroxynonenal (4-HNE) and decreased glutathione levels were found in the mGCH hearts and isolated mitochondria. Taken together, our results indicate that the ratio of BH4:BH2 does not predict changes in neither NO levels nor cellular redox state in the heart. The BH4 oxidation essentially limits the capacity of cardiomyocytes to reduce oxidant stress. Cardiomyocyte with chronically high levels of BH4 show a significant decline in redox state and mitochondrial function.

Nucleotide Variants of the BH4 Biosynthesis Pathway Gene GCH1 and the Risk of Orofacial Clefts.

A deficiency of GTP cyclohydrolase, encoded by the GCH1 gene, results in two neurological diseases: hyperphenylalaninaemia type HPABH4B and DOPA-responsive dystonia. Genes involved in neurotransmitter metabolism and motor systems may contribute to palatogenesis. The purpose of the study was to analyse polymorphic variants of the GCH1 gene as risk factors for non-syndromic cleft lip with or without cleft palate (NSCL/P). Genotyping of nine polymorphisms was conducted in a group of 281 NSCL/P patients and 574 controls. The GCH1 variant rs17128077 was associated with a 1.7-fold higher risk for NSCL/P (95 %CI = 1.224-2.325; p = 0.001). We also found a significant correlation between the rs8004018 and rs17128050 variants and an increased risk of oral clefts (p trend = 0.003 and 0.004, respectively). The best evidence of the global haplotype association was observed for rs17128050 and rs8004018 (p corr = 0.0152). This study demonstrates that the risk of NSCL/P is associated with variants of the GCH1 gene related to BH4 metabolism and provides some evidence of the relationships between morphological/functional shifts in the central nervous system and orofacial clefts.

Isoflurane favorably modulates guanosine triphosphate cyclohydrolase-1 and endothelial nitric oxide synthase during myocardial ischemia and reperfusion injury in rats.

BACKGROUND: The authors investigated the hypothesis that isoflurane modulates nitric oxide (NO) synthesis and protection against myocardial infarction through time-dependent changes in expression of key NO regulatory proteins, guanosine triphosphate cyclohydrolase (GTPCH)-1, the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin and endothelial nitric oxide synthase (eNOS). METHODS: Myocardial infarct size, NO production (ozone-mediated chemiluminescence), GTPCH-1, and eNOS expression (real-time reverse transcriptase polymerase chain reaction and western blotting) were measured in male Wistar rats with or without anesthetic preconditioning (APC; 1.0 minimum alveolar concentration isoflurane for 30 min) and in the presence or absence of an inhibitor of GTPCH-1, 2,4-diamino-6-hydroxypyrimidine. RESULTS: NO2 production (158 ± 16 and 150 ± 13 pmol/mg protein at baseline in control and APC groups, respectively) was significantly (P < 0.05) increased 1.5 ± 0.1 and 1.4 ± 0.1 fold by APC (n = 4) at 60 and 90 min of reperfusion, respectively, concomitantly, with increased expression of GTPCH-1 (1.3 ± 0.3 fold; n = 5) and eNOS (1.3 ± 0.2 fold; n = 5). In contrast, total NO (NO2 and NO3) was decreased after reperfusion in control experiments. Myocardial infarct size was decreased (43 ± 2% of the area at risk for infarction; n = 6) by APC compared with control experiments (57 ± 1%; n = 6). 2, 4-Diamino-6-hydroxypyrimidine decreased total NO production at baseline (221 ± 25 and 175 ± 31 pmol/mg protein at baseline in control and APC groups, respectively), abolished isoflurane-induced increases in NO at reperfusion, and prevented reductions of myocardial infarct size by APC (60 ± 2%; n = 6). CONCLUSION: APC favorably modulated a NO biosynthetic pathway by up-regulating GTPCH-1 and eNOS, and this action contributed to protection of myocardium against ischemia and reperfusion injury.

Selective depletion of vascular EC-SOD augments chronic hypoxic pulmonary hypertension.

Excess superoxide has been implicated in pulmonary hypertension (PH). We previously found lung overexpression of the antioxidant extracellular superoxide dismutase (EC-SOD) attenuates PH and pulmonary artery (PA) remodeling. Although comprising a small fraction of total SOD activity in most tissues, EC-SOD is abundant in arteries. We hypothesize that the selective loss of vascular EC-SOD promotes hypoxia-induced PH through redox-sensitive signaling pathways. EC-SOD(loxp/loxp) × Tg(cre/SMMHC) mice (SMC EC-SOD KO) received tamoxifen to conditionally deplete smooth muscle cell (SMC)-derived EC-SOD. Mice were exposed to hypobaric hypoxia for 35 days, and PH was assessed by right ventricular systolic pressure measurements and right ventricle hypertrophy. Vascular remodeling was evaluated by morphometric analysis and two-photon microscopy for collagen. We examined cGMP content and soluble guanylate cyclase expression and activity in lung, lung phosphodiesterase 5 (PDE5) expression and activity, and expression of endothelial nitric oxide synthase and GTP cyclohydrolase-1 (GTPCH-1), the rate-limiting enzyme in tetrahydrobiopterin synthesis. Knockout of SMC EC-SOD selectively decreased PA EC-SOD without altering total lung EC-SOD. PH and vascular remodeling induced by chronic hypoxia was augmented in SMC EC-SOD KO. Depletion of SMC EC-SOD did not impact content or activity of lung soluble guanylate cyclase or PDE5, yet it blunted the hypoxia-induced increase in cGMP. Although total eNOS was not altered, active eNOS and GTPCH-1 decreased with hypoxia only in SMC EC-SOD KO. We conclude that the localized loss of PA EC-SOD augments chronic hypoxic PH. In addition to oxidative inactivation of NO, deletion of EC-SOD seems to reduce eNOS activity, further compromising pulmonary vascular function.

Design of a single AAV vector for coexpression of TH and GCH1 to establish continuous DOPA synthesis in a rat model of Parkinson's disease.

Preclinical efficacy of continuous delivery of 3,4-dihydroxyphenylalanine (DOPA) with adeno-associated viral (AAV) vectors has recently been documented in animal models of Parkinson's disease (PD). So far, all studies have utilized a mix of two monocistronic vectors expressing either of the two genes, tyrosine hydroxylase (TH) and GTP cyclohydrolase-1 (GCH1), needed for DOPA production. Here, we present a novel vector design that enables efficient DOPA production from a single AAV vector in rats with complete unilateral dopamine (DA) lesions. Functional efficacy was assessed with drug-induced and spontaneous motor behavioral tests where vector-treated animals showed near complete and stable recovery within 1 month. Recovery of motor function was associated with restoration of extracellular DA levels as assessed by online microdialysis. Histological analysis showed robust transgene expression not only in the striatum but also in overlying cortical areas. In globus pallidus, we noted loss of NeuN staining, which might be due to different sensitivity in neuronal populations to transgene expression. Taken together, we present a single AAV vector design that result in efficient DOPA production and wide-spread transduction. This is a favorable starting point for continued translation toward a therapeutic application, although future studies need to carefully review target region, vector spread and dilution with this approach.

Induction of vascular GTP-cyclohydrolase I and endogenous tetrahydrobiopterin synthesis protect against inflammation-induced endothelial dysfunction in human atherosclerosis.

BACKGROUND: The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is essential for maintenance of enzymatic function. We hypothesized that induction of BH4 synthesis might be an endothelial defense mechanism against inflammation in vascular disease states. METHODS AND RESULTS: In Study 1, 20 healthy individuals were randomized to receive Salmonella typhi vaccine (a model of acute inflammation) or placebo in a double-blind study. Vaccination increased circulating BH4 and interleukin 6 and induced endothelial dysfunction (as evaluated by brachial artery flow-mediated dilation) after 8 hours. In Study 2, a functional haplotype (X haplotype) in the GCH1 gene, encoding GTP-cyclohydrolase I, the rate-limiting enzyme in biopterin biosynthesis, was associated with endothelial dysfunction in the presence of high-sensitivity C-reactive protein in 440 coronary artery disease patients. In Study 3, 10 patients with coronary artery disease homozygotes for the GCH1 X haplotype (XX) and 40 without the haplotype (OO) underwent S Typhi vaccination. XX patients were unable to increase plasma BH4 and had a greater reduction of flow-mediated dilation than OO patients. In Study 4, vessel segments from 19 patients undergoing coronary bypass surgery were incubated with or without cytokines (interleukin-6/tumor necrosis factor-α/lipopolysaccharide) for 24 hours. Cytokine stimulation upregulated GCH1 expression, increased vascular BH4, and improved vasorelaxation in response to acetylcholine, which was inhibited by the GTP-cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine. CONCLUSIONS: The ability to increase vascular GCH1 expression and BH4 synthesis in response to inflammation preserves endothelial function in inflammatory states. These novel findings identify BH4 as a vascular defense mechanism against inflammation-induced endothelial dysfunction.

Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase.

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.

Differential regulation of dopaminergic gene expression by Er81.

A recent study proposed that differentiation of dopaminergic neurons requires a conserved "dopamine motif" (DA-motif) that functions as a binding site for ETS DNA binding domain transcription factors. In the mammalian olfactory bulb (OB), the expression of a set of five genes [including tyrosine hydroxylase (Th)] that are necessary for differentiation of dopaminergic neurons was suggested to be regulated by the ETS-domain transcription factor ER81 via the DA-motif. To investigate this putative regulatory role of ER81, expression levels of these five genes were compared in both olfactory bulbs of adult wild-type mice subjected to unilateral naris closure and the olfactory bulbs of neonatal Er81 wild-type and mutant mice. These studies found that ER81 was necessary only for Th expression and not the other cassette genes. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) experiments showed that ER81 bound directly to a consensus binding site/DA-motif in the rodent Th proximal promoter. However, the ER81 binding site/DA-motif in the Th proximal promoter is poorly conserved in other mammals. Both ChIP assays with canine OB tissue and EMSA experiments with the human Th proximal promoter did not detect ER81 binding to the Th DA-motif from these species. These results suggest that regulation of Th expression by the direct binding of ER81 to the Th promoter is a species-specific mechanism. These findings indicate that ER81 is not necessary for expression of the OB dopaminergic gene cassette and that the DA-motif is not involved in differentiation of the mammalian OB dopaminergic phenotype.

GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

Superoxide dismutase restores eNOS expression and function in resistance pulmonary arteries from neonatal lambs with persistent pulmonary hypertension.

Endothelial nitric oxide (NO) synthase (eNOS) expression and activity are decreased in fetal lambs with persistent pulmonary hypertension (PPHN). We sought to determine the impact of mechanical ventilation with O(2) with or without inhaled NO (iNO) or recombinant human SOD (rhSOD) on eNOS in the ductal ligation model of PPHN. PPHN lambs and age-matched controls were ventilated with 100% O(2) for 24 h alone or combined with 20 ppm iNO continuously or a single dose of rhSOD (5 mg/kg) given intratracheally at delivery. In 1-day spontaneously breathing lambs, eNOS expression in resistance pulmonary arteries increased relative to fetal levels. eNOS expression increased in control lambs ventilated with 100% O(2), but not in PPHN lambs. Addition of iNO or rhSOD increased eNOS expression and decreased generation of reactive oxygen species (ROS) in PPHN lambs relative to those ventilated with 100% O(2) alone. However, only rhSOD restored eNOS function, increased tetrahydrobiopterin (BH(4)), a critical cofactor for eNOS function, and restored GTP cyclohydrolase I expression in isolated vessels and lungs from PPHN lambs. These data suggest that ventilation of PPHN lambs with 100% O(2) increases ROS production, blunts postnatal increases in eNOS expression, and decreases available BH(4) in PPHN lambs. Although the addition of iNO or rhSOD diminished ROS production and increased eNOS expression, only rhSOD improved eNOS function and levels of available BH(4). Thus therapies designed to decrease oxidative stress and restore eNOS coupling, such as rhSOD, may prove useful in the treatment of PPHN in newborn infants.

2,4-Diamino-6-hydroxypyrimidine (DAHP) suppresses cytokine-induced VCAM-1 expression on the cell surface of human umbilical vein endothelial cells in a BH(4)-independent manner.

2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered a specific inhibitor of BH(4) biosynthesis and is widely used in order to elucidate the possible biological function of BH(4) in various cells. In the present study, we found that both the synthesis of tetrahydrobiopterin (BH(4)) and expression of vascular cell adhesion molecule 1 (VCAM-1) were increased in human umbilical vein endothelial cells (HUVEC) treated with proinflammatory cytokines. Thus we examined the effects of DAHP to clarify whether BH(4) might be involved in the expression of VCAM-1 in HUVEC. DAHP reduced the levels of both BH(4) and VCAM-1 induced by TNF-alpha and IFN-gamma. However, the dose-response curves of DAHP for the suppression of the VCAM-1 level and that of BH(4) level were markedly different. Supplementation with sepiapterin failed to restore the depressed VCAM-1 level, although it completely restored the BH(4) level. Furthermore, DAHP significantly reduced the VCAM-1 level under the experimental conditions using TNF-alpha alone, which failed to induce BH(4) production. Taken together, these results indicate that DAHP inhibited the expression of VCAM-1 in a BH(4)-independent manner in HUVEC. In the present study, we also found that DAHP significantly suppressed the accumulation of cytokine-induced NF-kappaB (p65) in the nucleus as well as the mRNA levels of VCAM-1 and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH(4) synthesis. The data obtained in this study suggest that DAHP reduced VCAM-1 and GTPCH protein synthesis at least partially via suppressing the NF-kappaB level in the nucleus of HUVEC.

Oral administration of tetrahydrobiopterin attenuates testicular damage by reducing nitric oxide synthase activity in a cryptorchid mouse model.

Experimental cryptorchidism has been shown to induce germ cell apoptosis. Nitric oxide (NO), a ubiquitous free radical produced by NO synthases (NOSs), has been associated with apoptosis in a number of cell types. However, the regulation of NOSs in experimental cryptorchid testes remains unknown. Tetrahydrobiopterin (BH4), an essential cofactor of NOS, plays an important role in the generation of NO. It has been reported that activation of the immune system stimulates an increase in endogenous BH4 rate-limiting enzyme GTP cyclohydrolase I (GTPCH I) activity, resulting in an increase in intracellular BH4 levels and BH4-dependent NO synthesis in various cells. We examined the effect of dietary treatment with BH4 on GTPCH I, BH4 synthesis, NO production, and testicular damage in cryptorchid model mice. Male mice were treated with oral BH4 starting from age 4 weeks or received standard diet only, and right cryptorchid testes were created surgically at age 10 weeks. The testes were evaluated 0, 3, 5, 7, and 10 days after surgery by assays of testicular weight, BH4 and dihydrobiopterin (oxidized BH4) levels, GTPCH I mRNA levels, NOS protein expression levels, NO concentration, and nitrotyrosine (product of ONOO(-); determinant of NO-dependent damage) levels. In untreated mice, GTPCH I mRNA and BH4 levels increased and eNOS protein expression, NO concentration, and nitrotyrosine levels increased gradually. BH4 treatment decreased GTPCH I mRNA and BH4 levels, with concomitant reduction of eNOS protein levels, nitrotyrosine levels, and NO concentration, resulting in reduced testicular damage. Our findings demonstrate that supplementation with BH4 could provide a new therapeutic intervention for heat stress-based testicular dysfunction.

Regulation of tetrahydrobiopterin biosynthesis by shear stress.

An essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4B levels and enzymatic activity of GTP cyclohydrolase (GTPCH)-1, the first step of H4B biosynthesis. In contrast, protein levels of GTPCH-1 were not affected by shear. Shear did not change protein expression or activity of the downstream enzymes 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase and decreased protein levels of the salvage enzyme dihydrofolate reductase. Oscillatory shear only modestly affected H4B levels and GPTCH-1 activity. We also demonstrate that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTPCH-1 on serine 81 and that this is mediated by the alpha prime (alpha') subunit of casein kinase 2. The increase in H4B caused by shear is essential in allowing proper function of endothelial NO synthase because GPTCH-1 blockade with 2,4-diamino-6-hydroxypyrimidine during shear inhibited dimer formation of endothelial NO synthase, increased endothelial cell superoxide production, and prevented the increase in NO production caused by shear. Thus, shear stress not only increases endothelial NO synthase levels but also stimulates production of H4B by markedly enhancing GTPCH-1 activity via casein kinase 2-dependent phosphorylation on serine 81. These findings illustrate a new function of casein kinase 2 in the endothelium and provide insight into regulation of GTPCH-1 activity.

Effect of peripherally administered lipopolysaccharide (LPS) on GTP cyclohydrolase I, tetrahydrobiopterin and norepinephrine in the locus coeruleus in mice.

Lipopolysaccharide (LPS), an endotoxin released from the outer membranes of Gram-negative bacteria, triggers cells to synthesize and release inflammatory cytokines that may progress to septic shock in vivo. We found that LPS enhances tetrahydrobiopterin (BH4) biosynthesis by inducing the biosynthetic enzyme GTP cyclohydrolase I (GCH) in vitro in the mouse neuroblastoma cell line N1E-115. Furthermore, we observed that gene expression of GCH in the locus coeruleus (LC) in mice was enhanced by peripheral administration of LPS, resulting in increased concentrations of BH4, and norepinephrine, and its metabolite 4-hydroxy-3-methoxyphenylglycol (MHPG). These results suggest that tyrosine hydroxylase (TH) activity is increased by increased content of BH4 due to enhanced mRNA expression of GCH in the LC resulting in the increase in norepinephrine in the LC during endotoxemia. LPS in blood may act as a stressor to increase norepinephrine biosynthesis in the mouse LC.

Reduced tyrosine hydroxylase and GTP cyclohydrolase mRNA expression, tyrosine hydroxylase activity, and associated neurochemical alterations in Nurr1-null heterozygous mice.

The nuclear receptor Nurr1 is essential for the development of midbrain dopamine neurons and appears to be an important regulator of dopamine levels as adult Nurr1-null heterozygous (+/-) mice have reduced mesolimbic/mesocortical dopamine levels. The mechanism(s) through which reduced Nurr1 expression affects dopamine levels has not been determined. Quantitative real-time PCR revealed a significant reduction in tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA in ventral midbrain of +/- mice as compared to wild-type mice (+/+). The effect on TH expression was only observed at birth, while reduced GTP cyclohydrolase was also observed in the adult ventral tegemental area. No differences in dopamine transporter, vesicular monoamine transporter, dopamine D2 receptor or aromatic amino acid decarboxylase were observed. Since TH and GTPCH are both involved in dopamine synthesis, regulation of in vivo TH activity was measured in these mice. In vivo TH activity was reduced in nucleus accumbens and striatum of the +/- mice (24.7% and 15.7% reduction, respectively). In the striatum, gamma-butyrolactone exacerbated differences on +/- striatal TH activity (29.8% reduction) while haloperidol equalized TH activity between the +/+ and +/-. TH activity in the nucleus accumbens was significantly reduced in all conditions measured. Furthermore, dopamine levels in the striatum of +/- mice were significantly reduced after inhibition of dopamine synthesis or after haloperidol treatment but not under basal conditions while dopamine levels in the nucleus accumbens were reduced under basal conditions. Based on these data the +/- genotype results in changes in gene expression and impairs dopamine synthesis which can affect the maintenance of dopamine levels, although with differential effects between mesolimbic/mesocortical and nigrostriatal dopamine neurons. Together, these data suggest that Nurr1 may function to modify TH and GTPCH expression and dopamine synthesis.

[The mechanisms of extracellular-signal regulated protein kinase pathway in biopterin induction in rats with endotoxic shock].

OBJECTIVE: To observe the influence of treatment with the inhibitor of extracellular-signal regulated protein kinase (ERK) signal transduction pathway on the expression of biopterin/nitric oxide (NO) as well as the activation of nuclear factor-kappaB (NF-kappaB), and to clarify the potential cross-talk regulation mechanisms between ERK and NF-kappaB pathway in biopterin-mediated NO induction in rats with endotoxic shock. METHODS: Using an endotoxic shock model, 60 male Wistar rats were randomly divided into normal controls (n = 8), endotoxic shock group (n = 32) and PD98059 treatment group (n = 20). At serial time points animals in each group were sacrificed, and tissue samples from liver, lungs as well as kidneys were harvested to detect NF-kappaB activity, guanosine triphosphate-cyclohydrolase (GTP-CHI) and inducible nitric oxide synthase (iNOS) mRNA expression. Biopterin and NO levels in plasma and tissues were also assayed. RESULTS: It was found that after lipopolysaccharide (LPS) challenge, GTP-CHI mRNA expression and biopterin levels significantly elevated in liver, lungs and kidneys, keeping at high values up to 24 h, so did the values of iNOS mRNA expression and NO levels. NF-kappaB DNA binding activity was enhanced rapidly in various tissues, peaking at 2 h after LPS challenge. Treatment with PD98059, an inhibitor of ERK signal transduction pathway, could significantly inhibit GTP-CHI mRNA expression in kidneys, and GTP-CHI mRNA expression in liver and lungs showed certain down-regulation tendency. At the same time, biopterin level was significantly decreased in plasma, liver and kidneys at 12 h. Similarly, iNOS/NO induction at early stage markedly decreased in various tissues. In addition, treatment with PD98059 reduced NF-kappaB DNA binding activity in liver, lungs, as well as kidneys at 2-6 h, 2 h, 24 h and 24 h after LPS challenge, respectively. CONCLUSIONS: Inhibition of ERK pathway could partially inhibit the production of biopterin/NO as well as the activation of NF-kappaB pathway, which indicated that cross-talk regulation seems to be existed between ERK and NF-kappaB pathway, and they might be involved in the regulatory process of biopterin-mediated nitric oxide induction in rats with endotoxic shock.

Regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper transcription factors.

Tetrahydrobiopterin is an essential cofactor for the phenylalanine, tyrosine and tryptophan hydroxylases, and the family of nitric oxide synthases. The initial and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin is GTP cyclohydrolase I. The proximal promoter of the human GTP cyclohydrolase I gene contains the sequence motif 5'-TGACGCGA-3', resembling a cAMP response element (CRE). The objective of this study was to analyze the regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper (bZIP) transcription factors. A constitutively active mutant of the cAMP response element binding (CREB) protein strongly stimulated GTP cyclohydrolase I promoter activity, indicating that the CRE in the context of the GTP cyclohydrolase I gene is functional. Likewise, GTP cyclohydrolase I promoter/luciferase gene transcription was stimulated following nuclear expression of the catalytic subunit of cAMP-dependent protein kinase. Constitutively active mutants of activating transcription factor 2 (ATF2) and c-Jun additionally stimulated GTP cyclohydrolase I promoter activity, but to a lesser extent than the constitutively active CREB mutant. The fact that stress-activated protein kinases target the GTP cyclohydrolase I gene was corroborated by expression experiments involving p38 and MEKK1 protein kinases. We conclude that signaling pathways involving either the cAMP-dependent protein kinase or stress-activated protein kinases converge to the GTP cyclohydrolase I gene. Hence, enzymatic reactions that require tetrahydrobiopterin as cofactor are therefore indirectly controlled by signaling cascades involving the signal-responsive transcription factors CREB, c-Jun, and ATF2.

Nerve growth factor-induced expression of the GTP cyclohydrolase I gene via Ras/MEK pathway in PC12D cells.

Neurotrophins are essential for the development and survival of the catecholaminergic neurons. GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme in the biosynthesis of 5,6,7,8-tertahydrobiopterin (BH4), the required cofactor for tyrosine hydroxylase. Previously, we reported that TH requires the Ras/mitogen-activated protein kinase kinase (MEK) pathway for its induction by nerve growth factor (NGF). Here, we examined intracellular signals required for NGF-induced expression of the GCH gene in PC12D cells. The activity of GCH was increased up to 5-fold after the NGF treatment, and the increase was repressed by pretreatment with U0126, an MEK1/2 inhibitor, but not with protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) inhibitors. Induction of GCH mRNA by NGF was also abolished by pretreatment with U0126. The human GCH promoter activity was significantly enhanced by NGF treatment. Deletion analysis showed that the 465-bp 5'-flanking region is responsible for NGF-enhanced promoter activity. These data suggest that the Ras-MEK pathway is required for coordinate expression of the GCH and TH genes induced by neurotrophins.