Does interplay between nitric oxide and mitochondria affect hypoxia-inducible transcription factor-1 activity?
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ABSTRACT:
SUBMITTER: Murphy MP
PROVIDER: S-EPMC1223796 | biostudies-other | 2003 Dec
REPOSITORIES: biostudies-other
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Project description:Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the alpha-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1alpha is unstable in cells maintained at 21% O(2), but is progressively stabilized as the O(2) concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 microM) stabilize HIF-1alpha at all O(2) concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (rho(0)-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1alpha stabilized by exposure of the cells to 3% O(2). This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1alpha by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance.
| S-EPMC1223794 | biostudies-other
Project description:Background and purposeKinins are vasoactive and pro-inflammatory peptides whose biological effects are mediated by two GPCRs, named B1 and B2 receptors. While the B2 receptor plays a protective role in the cardiovascular system via the activation of endothelial NOS, the B1 receptor is associated with vascular inflammation, insulin resistance and diabetic complications. Because the B1 receptor is a potent activator of the inducible form of NOS (iNOS), this study has addressed the role of iNOS in the deleterious effects of B1 receptors in insulin resistance.Experimental approachMale Sprague-Dawley rats (50-75 g) had free access to a drinking solution containing 10% d-glucose or tap water (control) for 9 weeks. During the last week, a selective iNOS inhibitor (1400W, 1 mg·kg(-1) twice daily) or its vehicle was administered s.c.Key resultsProlonged glucose treatment caused insulin resistance and several hallmarks of type 2 diabetes. Whereas the treatment with 1400W had no impact on the elevated systolic blood pressure and leptin levels in glucose-fed rats, it significantly reversed or attenuated hyperglycaemia, hyperinsulinaemia, insulin resistance (HOMA index), body weight gain, peroxynitrite formation (nitrotyrosine expression) and the up-regulation of biomarkers of inflammation (B1 receptor, carboxypeptidase M, iNOS and IL-1β) in renal cortex and aorta and to some extent in the liver.Conclusions and implicationsPharmacological blockade of iNOS prevents the formation of peroxynitrite, which amplifies the pro-inflammatory effects of B1 receptors through a positive feedback mechanism. Hence, targeting iNOS can prevent the deleterious effects of B1 receptors in insulin resistance and peripheral inflammation.
| S-EPMC4882489 | biostudies-literature
Project description:Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor α (HIF-α) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for therapeutic intervention against tuberculosis.
| S-EPMC3868520 | biostudies-literature
Project description:Photodynamic therapy (PDT) is a cancer therapy that capitalizes on cancer-specific porphyrin accumulation. We have investigated this phenomenon to propose the following three conclusions: 1) the mechanism underlying this phenomenon is closely related to both nitric oxide (NO) and heme carrier protein-1 (HCP-1), 2) NO inactivates ferrochelatase, and thus, the intracellular porphyrin levels in the cells are increased by the administration of an NO donor after 5-aminolevulinic acid treatment, 3) HCP-1 transports not only heme but also other porphyrins. Since NO stabilizes hypoxia-inducible factor (HIF)-1?, resulting in the upregulation of heme biosynthesis, HCP-1 expression can be increased by HIF-1? stabilization. In this study, we determined whether NO regulates HCP-1 expression by stabilizing HIF-1? expression. For this purpose, rat gastric cancer cell line RGK36 was treated with L-arginine or N6-(1-iminoethyl)-L-lysine (L-NIL). L-arginine treatment increased the intracellular NO concentration, and both HCP-1 and HIF-1? expression, while L-NIL treatment decreased them. Cytotoxicity of PDT was enhanced by L-arginine, following intracellular hemato-porphyrin dihydrochloride (HpD) accumulation. Both Cytotoxicity of PDT and HpD accumulation were decreased by L-NIL. The HCP-1 and HIF-1? expression, intracellular HpD accumulation and PDT cytotoxicity were decreased by 2-methoxyestradiol, which is a HIF-1? inhibitor. Moreover, these phenomena were not increased by a combination of both L-arginine and 2-Me. Thus, HCP-1 can be a downstream target of HIF-1?. These effects were also induced in the human gastric cancer cell line MKN45. Taken together, we conclude that HCP-1 expression is regulated by NO via HIF-1? stabilization.
| S-EPMC6742216 | biostudies-literature
Project description:Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)(-/-) mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS(-/-) mice. At the cellular level, anemia increased expression of HIF-1? protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS(-/-) mice, despite comparable reductions in tissue PO(2). Paradoxically, nNOS(-/-) mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-? protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-?(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-? protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.
| S-EPMC3198321 | biostudies-literature
Project description:Despite a long history, the clinical efficacy of cupping therapy is still under debate. This is likely due to the lack of direct evidence for the biological actions of cupping, since the short exposure of cells to vacuum condition rarely has affects cellular activity. In this study, the medicinal properties of a recent medical technology, non-thermal plasma, were added to classical cupping and designated as 'plasma cupping' (PC). In our results, the plasma-generating efficacy was increased under a cupping-like semi-vacuum condition (410 Torr) rather than normal atmospheric pressure (760 Torr). Notably, while cupping rarely affects the angiogenic factor vascular-endothelial growth factor (VEGF)-A, the PC treatment on HaCaT human keratinocytes significantly induced the expression of VEGF-A. The increased expression of the VEGF-A gene after the PC treatment was expected to be a result of PC-mediated ERK protein activation. The PC-mediated activation of ERK was essential for the activity of hypoxia inducible factor (HIF) 1 alpha, which is responsible for the PC-mediated expression of VEGF-A. The PC mediated increase of NO in the media was thought as a main reason for the elevated HIF-1 protein activity. In addition to the angiogenesis-promoting action of PC, it also showed anti-inflammatory activity by reducing TNF-α-mediated IL-1β and IL-6 expression. Taken together, this study indicates the potential for PC that could enhance the clinical efficacy of cupping by adding the effects of non-thermal plasma to traditional cupping.
| S-EPMC6405951 | biostudies-literature
Project description:Intrauterine hypoxia impacts fetal growth and organ function. Inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) expression was measured to assess the response of fetal hearts to hypoxic (HPX) stress. Pregnant guinea pigs were housed in a hypoxic chamber (10.5% O2 for 14 d, n = 17) or room air [normoxic (NMX), n = 17]. Hearts of anesthetized near-term fetuses were removed. mRNA [hypoxia-inducible factor, (HIF)-1alpha, 1beta, 2alpha, 3alpha, iNOS, and nNOS] and protein levels (HIF-1alpha, iNOS, and nNOS) of fetal cardiac left ventricles were quantified by real time polymerase chain reaction (PCR) and Western analysis, respectively. Cardiac nitrite/nitrate levels were measured in the presence/absence of L-N6-(1-iminoethyl)-lysine (L-NIL), an iNOS inhibitor, administered to pregnant sows. Hypoxia significantly increased fetal cardiac HIF-1alpha and -2alpha mRNA, HIF-1alpha protein but not HIF-3alpha or -1beta mRNA levels. Hypoxia increased both iNOS mRNA (by 5x) and protein (by 23%) levels but had no effect on nNOS levels. Nitrite/nitrate levels were increased in HPX hearts by 2.5x and decreased with L-NIL by 67 +/- 14%. Thus, up-regulation of iNOS-derived nitric oxide (NO) generation is an important mechanism by which fetal hearts respond to chronic hypoxic stress.
| S-EPMC6314287 | biostudies-literature
Project description:Methylobacter species, members of the Methylococcales, have recently emerged as some of the globally widespread, cosmopolitan species, appearing to play a key role in consumption of methane in a variety of environments and across gradients of dioxygen tensions. We approached the question of how Methylobacter copes with low dioxygen partial pressures that it encounters in its natural habitats, via laboratory manipulation. Through comparative transcriptomics of cultures grown under high dioxygen partial pressure and cultures starved for dioxygen, we identified a gene cluster encoding a hybrid cluster protein along with sensing and regulatory functions. Through mutant analysis we demonstrated that this gene cluster is involved in both oxidative and nitrosative stress responses and in NO reduction, while being responsive to NO, likely through direct sensing. Through additional transcriptomic analyses, we further uncovered a complex interconnection between the NO-mediated stress response and quorum sensing in turn controlling synthesis of the secondary metabolite tundrenone, these two systems being inversely regulated. Some of the key methylotrophy functions, such as the two alternative methanol dehydrogenases seem to be parts of the stress response regulatory cascade. One intriguing scenario that emerged from our analyses is that the main role of the respiratory denitrification pathway may be in producing the signaling molecule, NO, in response to hypoxia, rather than in serving as an energy-generating pathway. This novel and complex hypoxia stress response system is unique to Methylobacter tundripaludum, and it may play a role in the environmental fitness of these organisms and in their cosmopolitan environmental distribution.
2019-10-01 | GSE138276 | GEO
Project description:Methylobacter species, members of the Methylococcales, have recently emerged as some of the globally widespread, cosmopolitan species that play a key role in the environmental consumption of methane across gradients of dioxygen tensions. In this work, we approached the question of how Methylobacter copes with hypoxia, via laboratory manipulation. Through comparative transcriptomics of cultures grown under high dioxygen partial pressure versus cultures exposed to hypoxia, we identified a gene cluster encoding a hybrid cluster protein along with sensing and regulatory functions. Through mutant analysis, we demonstrated that this gene cluster is involved in the hypoxia stress response. Through additional transcriptomic analyses, we uncovered a complex interconnection between the NO-mediated stress response, quorum sensing, the secondary metabolite tundrenone, and methanol dehydrogenase functions. This novel and complex hypoxia stress response system is so far unique to Methylobacter species, and it may play a role in the environmental fitness of these organisms and in their cosmopolitan environmental distribution.IMPORTANCE Here, we describe a novel and complex hypoxia response system in a methanotrophic bacterium that involves modules of central carbon metabolism, denitrification, quorum sensing, and a secondary metabolite, tundrenone. This intricate stress response system, so far unique to Methylobacter species, may be responsible for the persistence and activity of these species across gradients of dioxygen tensions and for the cosmopolitan distribution of these organisms in freshwater and soil environments in the Northern Hemisphere, including the fast-melting permafrosts.
| S-EPMC6977074 | biostudies-literature
Project description:Mitochondria are an important source of reactive oxygen species (ROS), implicated in ischemia/reperfusion injury. When isolated from ischemic myocardium, mitochondria demonstrate increased ROS production as a result of damage to electron transport complexes. To investigate the mechanisms, we studied effects of hypoxia/reoxygenation on ROS production by isolated energized heart mitochondria. ROS production, tracked using Fe(2+)-catalyzed, H(2)O(2)-dependent H(2)DCF oxidation or Amplex Red, was similar during normoxia and hypoxia but markedly increased during reoxygenation, in proportion to the duration of hypoxia. In contrast, if mitochondria were rapidly converted from normoxia to near-anoxia ([O(2)], <1 micromol/L), the increase in H(2)DCF oxidation rate during reoxygenation was markedly blunted. To elicit the robust increase in H(2)DCF oxidation rate during reoxygenation, hypoxia had to be severe enough to cause partial, but not complete, respiratory chain inhibition (as shown by partial dissipation of membrane potential and increased NADH autofluorescence). Consistent with its cardioprotective actions, nitric oxide ( O) abrogated increased H(2)DCF oxidation under these conditions, as well as attenuating ROS-induced increases in matrix [Fe(2+)] and aconitase inhibition caused by antimycin. Collectively, these results suggest that (1) hypoxia that is sufficient to cause partial respiratory inhibition is more damaging to mitochondria than near-anoxia; and (2) O suppresses ROS-induced damage to electron transport complexes, probably by forming O-Fe(2+) complexes in the presence of glutathione, which inhibit hydroxyl radical formation.
| S-EPMC2755534 | biostudies-literature