Heme oxygenase-1 protects the heart.
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Animals↗
Biomedical subjects
A M Choi
Publications and source records attributed to A M Choi.
Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation.
Nrf2 regulates expression of genes encoding enzymes with antioxidant (e.g. heme oxygenase-1 (HO-1)) or xenobiotic detoxification (e.g. NAD(P)H:quinone oxidoreductase, glutathione S-transferase) functions via the stress- or antioxidant-response elements (StRE/ARE). Nrf2 heterodimerizes with small Maf proteins, but the role of such dimers in gene induction is controversial, and other partners may exist. By using the yeast two-hybrid assay, we identified activating transcription factor (ATF) 4 as a potential Nrf2-interacting protein. Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation and mammalian two-hybrid assays. Furthermore, Nrf2.ATF4 dimers bound to an StRE sequence from the ho-1 gene. CdCl(2), a potent inducer of HO-1, increased expression of ATF4 in mouse hepatoma cells, and detectable induction of ATF4 protein preceded that of HO-1 (30 min versus 2 h). A dominant-negative mutant of ATF4 inhibited basal and CdCl(2)-stimulated expression of a StRE-dependent/luciferase fusion construct (pE1-luc) in hepatoma cells but only basal expression in mammary epithelial MCF-7 cells. A dominant mutant of Nrf2 was equally inhibitory in both cell types in the presence or absence of CdCl(2). These results indicate that ATF4 regulates basal and CdCl(2)-induced expression of the ho-1 gene in a cell-specific manner and possibly in a complex with Nrf2.
Activating Transcription Factor 3↗
Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants.
Mouse-to-rat cardiac transplants survive long term after transient complement depletion by cobra venom factor and T cell immunosuppression by cyclosporin A. Expression of heme oxygenase-1 (HO-1) by the graft vasculature is critical to achieve graft survival. In the present study, we asked whether this protective effect was attributable to the generation of one of the catabolic products of HO-1, carbon monoxide (CO). Our present data suggests that this is the case. Under the same immunosuppressive regimen that allows mouse-to-rat cardiac transplants to survive long term (i.e., cobra venom factor plus cyclosporin A), inhibition of HO-1 activity by tin protoporphyrin, caused graft rejection in 3--7 days. Rejection was associated with widespread platelet sequestration, thrombosis of coronary arterioles, myocardial infarction, and apoptosis of endothelial cells as well as cardiac myocytes. Under inhibition of HO-1 activity by tin protoporphyrin, exogenous CO suppressed graft rejection and restored long-term graft survival. This effect of CO was associated with inhibition of platelet aggregation, thrombosis, myocardial infarction, and apoptosis. We also found that expression of HO-1 by endothelial cells in vitro inhibits platelet aggregation and protects endothelial cells from apoptosis. Both these actions of HO-1 are mediated through the generation of CO. These data suggests that HO-1 suppresses the rejection of mouse-to-rat cardiac transplants through a mechanism that involves the generation of CO. Presumably CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction. Additional mechanisms by which CO overcomes graft rejection may involve its ability to suppress endothelial cell apoptosis.
Acute Disease↗
Exhaled monoxides as a pulmonary function test: use of exhaled nitric oxide and carbon monoxide.
Although there has been tremendous improvement in the technologic ability to measure exhaled gases and monitor biologic processes in the lung, it has not yet found a clinical role outside the research laboratory. Common themes seem to be significant overlap in the amount of exhaled gases in clinically distinct populations, confounding variables such as infection, smoking, and environmental exposure, and lack of consistent change with disease management. If these tests are ever to be used by the general pulmonologist, consistent links between the measurements and the response to disease modification will need to be demonstrated at the very least and, ideally, the clinician would like to see improved outcomes when these noninvasive tests are employed regularly.
Breath Tests↗
Carbon monoxide attenuates aeroallergen-induced inflammation in mice.
Carbon monoxide (CO) generated by catalysis of heme by heme oxygenase is increased in the exhaled air of asthmatic patients. Based on recent studies demonstrating that asthma is an inflammatory disease associated with increased oxidants and that CO confers cytoprotection in oxidant-induced lung injury and inflammation, we sought to better understand the functional role of CO in asthma by using an aeroallergen model. Mice were sensitized to ovalbumin, challenged with aerosolized ovalbumin, and maintained in either CO (250 parts/million) or room air for 48 h. The differential effects of CO on bronchoalveolar lavage (BAL) fluid cell types were observed, with a marked attenuation of BAL fluid eosinophils in the CO-treated animals at 24 and 48 h. A marked reduction of the proinflammatory cytokine interleukin-5 was observed in the CO-treated mice, with no significant changes for other proinflammatory cytokines. These differential effects of CO were also observed with leukotrienes (LTs) and prostaglandins in that CO significantly decreased BAL fluid PGE2, and LTB4 but exerted negligible effect on thromboxane B2 or LTC4/D4/E4. Our data suggest a putative immunoregulatory role for CO in aeroallergen-induced inflammation in mice.
Allergens↗
Stimulation of pro-alpha(1)(I) collagen by TGF-beta(1) in mesangial cells: role of the p38 MAPK pathway.
Transforming growth factor-beta(1) (TGF-beta(1)) is a potent inducer of extracellular matrix protein synthesis and a key mediator of renal fibrosis. However, the intracellular signaling mechanisms by which TGF-beta(1) stimulates this process remain incompletely understood. In this report, we examined the role of a major stress-activated intracellular signaling cascade, belonging to the mitogen-activated protein kinase (MAPK) superfamily, in mediating TGF-beta(1) responses in rat glomerular mesangial cells, using dominant-negative inhibition of TGF-beta(1) signaling receptors. We first stably transfected rat glomerular mesangial cells with a kinase-deleted mutant TGF-beta type II receptor (TbetaR-II(M)) designed to inhibit TGF-beta(1) signaling in a dominant-negative fashion. Next, expression of TbetaR-II(M) mRNA was confirmed by Northern analysis. Cell surface expression and ligand binding of TbetaR-II(M) protein were demonstrated by affinity cross-linking with (125)I-labeled-TGF-beta(1). TGF-beta(1) rapidly induced p38 MAPK phosphorylation in wild-type and empty vector (pcDNA3)-transfected control mesangial cells. Interestingly, transfection with dominant-negative TbetaR-II(M) failed to block TGF-beta(1)-induced p38 MAPK phosphorylation. Moreover, dominant-negative TbetaR-II(M) failed to block TGF-beta(1)-stimulated pro-alpha(1)(I) collagen mRNA expression and cellular protein synthesis, whereas TGF-beta(1)-induced extracellular signal-regulated kinase (ERK) 1/ERK2 activation and antiproliferative responses were blocked by TbetaR-II(M). In the presence of a specific inhibitor of p38 MAPK, SB-203580, TGF-beta(1) was unable to stimulate pro-alpha(1)(I) collagen mRNA expression in the control and TbetaR-II(M)-transfected mesangial cells. Finally, we confirmed that both p38 MAPK activation and pro-alpha(1)(I) collagen stimulation were TGF-beta(1) effects that were abrogated by dominant-negative inhibition of TGF-beta type I receptor. Thus we show first demonstration of p38 MAPK activation by TGF-beta(1) in mesangial cells, and, given the rapid kinetics, this TGF-beta(1) effect is likely a direct one. Furthermore, our findings suggest that the p38 MAPK pathway functions as a component in the signaling of pro-alpha(1)(I) collagen induction by TGF-beta(1) in mesangial cells.
Animals↗
"Haemoxygenase-1 induction and exhaled markers of oxidative stress in lung diseases", summary of the ERS Research Seminar in Budapest, Hungary, September, 1999.
In recent years, there has been increasing interest in noninvasive monitoring of airway inflammation and oxidative stress. Several volatile and nonvolatile substances can be measured in exhaled breath and have been suggested as potential biomarkers of these events. Exhaled gases, including carbon monoxide (CO), alkanes (ethane, pentane), and substances measured in breath condensate, such as hydrogen peroxide (H2O2) and isoprostanes were all suggested as potential markers of oxidative stress in the lung. A European Respiratory Society (ERS) International Research Seminar entitled "Haemoxygenase-1 induction and exhaled markers of oxidative stress in lung diseases" was organized by the Airway Regulation and Provocation Group of the Clinical Allergy and Immunology Assembly in Budapest, Hungary in September, 1999 to integrate the latest knowledge on these issues and accelerate further improvement in this area. During this 2-day event several issues were raised about: the use and standardization of measurements in exhaled breath; problems of measuring expired H2O2 and other mediators in breath condensate; role and regulation of haemoxygenase (HO)-1 in the lung; and conditions and factors influencing exhaled CO. This report is a summary of the main presentations at the seminar, together with the current areas of research in this rapidly expanding field.
Antioxidants↗
Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis.
Heme oxygenase 1 (HO-1) inhibits apoptosis by regulating cellular prooxidant iron. We now show that there is an additional mechanism by which HO-1 inhibits apoptosis, namely by generating the gaseous molecule carbon monoxide (CO). Overexpression of HO-1, or induction of HO-1 expression by heme, protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked by tin protoporphyrin (SnPPIX) or the action of CO is inhibited by hemoglobin (Hb), HO-1 no longer prevents EC apoptosis while these reagents do not affect the antiapoptotic action of bcl-2. Exposure of ECs to exogenous CO, under inhibition of HO-1 activity by SnPPIX, substitutes HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling transduction pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhanced p38 MAPK activation by TNF-alpha. Specific inhibition of p38 MAPK activation by the pyridinyl imidazol SB203580 or through overexpression of a p38 MAPK dominant negative mutant abrogated the antiapoptotic effect of HO-1. Taken together, these data demonstrate that the antiapoptotic effect of HO-1 in ECs is mediated by CO and more specifically via the activation of p38 MAPK by CO.
Animals↗
Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor.
The mouse heme oxygenase-1 (HO-1) gene, ho-1, contains two inducible enhancers, E1 and E2. Of several cell lines tested, induction of an E1/luciferase fusion construct, pE1-luc, by CdCl(2) is most pronounced in MCF-7 cells. In these cells, E1, but not E2, is necessary and sufficient for ho-1 gene activation. Exposure of MCF-7 cells to 10 micrometer CdCl(2) stimulates phosphorylation of ERK, JNK, and p38 mitogen-activated protein kinases, implicating one or more of these signaling pathways in ho-1 gene induction. SB203580, an inhibitor of p38, diminishes cadmium-stimulated pE1-luc expression and HO-1 mRNA levels by up to 70-80%. PD098059, an ERK pathway inhibitor, does not affect HO-1 mRNA induction at the highest concentration (40 micrometer) tested. Similarly, co-expression of a dominant-negative mutant of p38alpha, but not of ERK1, ERK2, JNK1, or JNK2, reduces basal and cadmium-induced pE1-luc activity. E1 contains binding sites for the activator protein-1 (Fos/Jun), Cap'n'Collar/basic leucine zipper (CNC-bZIP), and CCAAT/enhancer-binding protein (C/EBP) families of transcription factors. A dominant-negative mutant of Nrf2 (a CNC-bZIP member), but not of c-Jun or C/EBPbeta, inhibits pE1-luc activation by cadmium. Induction of the endogenous ho-1 gene is also inhibited by the Nrf2 mutant. Mutations of E1 that inhibit cadmium inducibility also suppress the trans-activation and DNA binding activities of Nrf2, and SB203580, but not PD098059, attenuates Nrf2-mediated trans-activation of pE1-luc. Taken together, these results indicate that cadmium induces ho-1 gene expression via sequential activation of the p38 kinase pathway and Nrf2.
Animals↗
Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.
The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti-inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and macrophage inflammatory protein-1beta and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses.
Animals↗
Heme oxygenase-1 inhibits TNF-alpha-induced apoptosis in cultured fibroblasts.
Heme oxygenase (HO)-1 catalyzes the oxidative cleavage of heme to yield equimolar amounts of biliverdin, iron, and carbon monoxide. HO-1 is a stress response protein, the induction of which is associated with protection against oxidative stress. The mechanism(s) of protection is not completely elucidated, although it is suggested that one or more of the catalytic by-products provide antioxidant functions either directly or indirectly. The involvement of reactive oxygen species in apoptosis raised the question of a possible role for HO-1 in programmed cell death. Using the tetracycline-regulated expression system, we show here that conditional overexpression of HO-1 prevents tumor necrosis factor-alpha-induced apoptosis in murine L929 fibroblasts. Inhibition of apoptosis was not observed in the presence of tin protoporphyrin, a specific inhibitor of HO activity, and in cells overexpressing antisense HO-1. Interestingly, exogenous administration of a low concentration of carbon monoxide also prevented tumor necrosis factor-alpha-induced apoptosis in L929 fibroblasts. Inhibition of tumor necrosis factor-alpha-induced apoptosis by HO-1 overexpression was reversed by 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of guanylate cyclase, which is a target enzyme for carbon monoxide. Taken together, our data suggest that the antiapoptotic effect of HO-1 may be mediated via carbon monoxide.
Animals↗
AP-1 and STAT mediate hyperoxia-induced gene transcription of heme oxygenase-1.
We have previously shown marked induction of the stress-inducible gene heme oxygenase-1 (HO-1) in vivo and in vitro after hyperoxia. In RAW 264.7 cells, HO-1 induction is transcriptionally regulated and dependent on cooperation between the HO-1 gene promoter and the 5' distal enhancer element SX2. In our present study, further deletional and mutational analyses demonstrate that signal transducer and activator of transcription (STAT) DNA binding sites located in the promoter of HO-1 and activator protein (AP)-1 DNA binding sites in the distal enhancer element SX2 are necessary for optimal HO-1 gene activation after hyperoxia. Interestingly, a second 5' distal enhancer element, AB1, located 10 kb upstream from the HO-1 promoter, alone is activated after hyperoxia but cannot confer maximal hyperoxia-induced HO-1 gene transcription. Mutational analysis of the AB1 enhancer shows that AP-1 is essential for AB1-mediated HO-1 gene transcription after hyperoxia. Electromobility shift assays show increased STAT1, STAT3, STAT5, and AP-1 DNA binding activity in RAW 264.7 cells after hyperoxia. Taken together, our data suggest that the 5' distal enhancer elements of the HO-1 gene in concert with the promoter regulate HO-1 gene induction and highlight the complexity of HO-1 gene transcription in response to hyperoxia.
Animals↗
Lung redox homeostasis: emerging concepts.
This symposium was organized to present some aspects of current research pertaining to lung redox function. Focuses of the symposium were on roles of pulmonary endothelial NADPH oxidase, xanthine oxidase (XO)/xanthine dehydrogenase (XDH), heme oxygenase (HO), transplasma membrane electron transport (TPMET), and the zinc binding protein metallothionein (MT) in the propagation and/or protection of the lung or other organs from oxidative injury. The presentations were chosen to reflect the roles of both intracellular (metallothionein, XO/XDH, and HO) and plasma membrane (NADPH oxidase, XO/XDH, and unidentified TPMET) redox proteins in these processes. Although the lung endothelium was the predominant cell type under consideration, at least some of the proposed mechanisms operate in or affect other cell types and organs as well.
Animals↗
Regulation of plasminogen activator inhibitor-1 and urokinase by hyaluronan fragments in mouse macrophages.
Pulmonary inflammation and fibrosis are characterized by increased turnover and production of the extracellular matrix as well as an impairment of lung fibrinolytic activity. Although fragments of the extracellular matrix component hyaluronan induce macrophage production of inflammatory mediators, the effect of hyaluronan on the fibrinolytic mediators plasminogen activator inhibitor (PAI)-1 and urokinase-type plasminogen activator (uPA) is unknown. This study demonstrates that hyaluronan fragments augment steady-state mRNA, protein, and inhibitory activity of PAI-1 as well as diminish the baseline levels of uPA mRNA and inhibit uPA activity in an alveolar macrophage cell line. Hyaluronan fragments alter macrophage expression of PAI-1 and uPA at the level of gene transcription. Similarly, hyaluronan fragments augment PAI-1 and diminish uPA mRNA levels in freshly isolated inflammatory alveolar macrophages from bleomycin-treated rats. These data suggest that hyaluronan fragments influence alveolar macrophage expression of PAI-1 and uPA and may be a mechanism for regulating fibrinolytic activity during lung inflammation.
Amiloride↗
Heme oxygenase: colors of defense against cellular stress.
The discovery of the gaseous molecule nitric oxide in 1987 unraveled investigations on its functional role in the pathogenesis of a wide spectrum of biological and pathological processes. At that time, the novel concept that an endogenous production of a gaseous substance such as nitric oxide can impart such diverse and potent cellular effects proved to be very fruitful in enhancing our understanding of many disease processes including lung disorders. Interestingly, we have known for a longer period of time that there exists another gaseous molecule that is also generated endogenously; the heme oxygenase (HO) enzyme system generates the majority if not all of the endogenously produced carbon monoxide. This enzyme system also liberates two other by-products, bilirubin and ferritin, each possessing important biological functions and helping to define the uniqueness of the HO enzyme system. In recent years, interest in HO has emerged in numerous disciplines including the central nervous system, cardiovascular physiology, renal and hepatic systems, and transplantation. We review the functional role of HO in lung biology and its real potential application to lung diseases.
Acute Disease↗
Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene.
Stress response elements, which mediate induction of the mouse heme oxygenase-1 (HO-1) gene by several agents, resemble the binding site for the activator protein-1 (Jun/Fos), Maf, and Cap'n'Collar/basic leucine zipper (CNC-bZIP) families of proteins. In L929 fibroblasts, significant activation of an HO-1 enhancer-reporter fusion gene was observed only with the CNC-bZIP class of proteins with Nrf2 exhibiting the highest level of trans-activation, between 25- and 30-fold. To further examine the role of this factor in HO-1 gene regulation, a dominant-negative mutant, Nrf2M, was generated and conditionally expressed in L929 cells. The mutant protein was detected in cytoplasmic and nuclear fractions but did not affect cell growth. Under conditions of Nrf2M overexpression, HO-1 mRNA accumulation in response to heme, cadmium, zinc, arsenite, and tert-butylhydroquinone was inhibited by 85-95%. In contrast, overexpression of a dominant-negative mutant of c-Jun decreased L929 cell growth but did not inhibit HO-1 gene activation. Nrf2 does not homodimerize, but CNC-bZIP.small Maf protein heterodimers and Nrf2. Jun protein complexes are proposed to function as trans-activators. Co-expression of Jun proteins or p18, however, had no significant affect or inhibited Nrf2-mediated trans-activation. Taken together, these results implicate Nrf2 in the induction of the HO-1 gene but suggest that the Nrf2 partner in this function is a factor other than p18 or Jun proteins.
Animals↗
Transforming growth factor beta1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages.
Cell death and cell survival are central components of normal development and pathologic states. Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine that regulates both cell growth and cell death. To better understand the molecular mechanisms that control cell death or survival, we investigated the role of TGF-beta1 in the apoptotic process by dominant-negative inhibition of both TGF-beta1 and mitogen-activated protein kinase (MAPK) signaling pathways. Murine macrophages (RAW 264.7) undergo apoptosis following serum deprivation, as determined by DNA laddering assay. However, apoptosis is prevented in serum-deprived macrophages by the presence of exogenous TGF-beta1. Using stably transfected RAW 264.7 cells with the kinase-deleted dominant-negative mutant of TbetaR-II (TbetaR-IIM) cDNA, we demonstrate that this protective effect by TGF-beta1 is completely abrogated. To determine the downstream signaling pathways, we examined TGF-beta1 effects on the MAPK pathway. We show that TGF-beta1 induces the extracellular signal-regulated kinase (ERK) activity in a time-dependent manner up to 4 h after stimulation. Furthermore, TGF-beta1 does not rescue serum deprivation-induced apoptosis in RAW 264.7 cells transfected with a dominant-negative mutant MAPK (ERK2) cDNA or in wild type RAW 264.7 cells in the presence of the MAPK kinase (MEK1) inhibitor. Taken together, our data demonstrate for the first time that TGF-beta1 is an inhibitor of apoptosis in cultured macrophages and may serve as a cell survival factor via TbetaR-II-mediated signaling and downstream intracellular MAPK signaling pathway.
Animals↗
Carbon monoxide provides protection against hyperoxic lung injury.
Findings in recent years strongly suggest that the stress-inducible gene heme oxygenase (HO)-1 plays an important role in protection against oxidative stress. Although the mechanism(s) by which this protection occurs is poorly understood, we hypothesized that the gaseous molecule carbon monoxide (CO), a major by-product of heme catalysis by HO-1, may provide protection against oxidative stress. We demonstrate here that animals exposed to a low concentration of CO exhibit a marked tolerance to lethal concentrations of hyperoxia in vivo. This increased survival was associated with highly significant attenuation of hyperoxia-induced lung injury as assessed by the volume of pleural effusion, protein accumulation in the airways, and histological analysis. The lungs were completely devoid of lung airway and parenchymal inflammation, fibrin deposition, and pulmonary edema in rats exposed to hyperoxia in the presence of a low concentration of CO. Furthermore, exogenous CO completely protected against hyperoxia-induced lung injury in rats in which endogenous HO enzyme activity was inhibited with tin protoporphyrin, a selective inhibitor of HO. Rats exposed to CO also exhibited a marked attenuation of hyperoxia-induced neutrophil infiltration into the airways and total lung apoptotic index. Taken together, our data demonstrate, for the first time, that CO can be therapeutic against oxidative stress such as hyperoxia and highlight possible mechanism(s) by which CO may mediate these protective effects.
Animals↗