Translocation of heme oxygenase-1 to mitochondria is a novel cytoprotective mechanism against non-steroidal anti-inflammatory drug-induced mitochondrial oxidative stress, apoptosis, and gastric mucosal injury

The mechanism of action of heme oxygenase-1 (HO-1) in mitochondrial oxidative stress (MOS)-mediated apoptotic tissue injury was investigated. MOS-mediated gastric mucosal apoptosis and injury were introduced in rat by indomethacin, a non-steroidal anti-inflammatory drug. Here, we report that HO-1 was not only induced but also translocated to mitochondria during gastric mucosal injury to favor repair mechanisms. Furthermore, mitochondrial translocation of HO-1 resulted in the prevention of MOS and mitochondrial pathology as evident from the restoration of the complex I-driven mitochondrial respiratory control ratio and transmembrane potential. Mitochondrial translocation of HO-1 also resulted in time-dependent inhibition of apoptosis. We searched for the plausible mechanisms responsible for HO-1 induction and mitochondrial localization. Free heme, the substrate for HO-1, was increased inside mitochondria during gastric injury, and mitochondrial entry of HO-1 decreased intramitochondrial free heme content, suggesting that a purpose of mitochondrial translocation of HO-1 is to detoxify accumulated heme. Heme may activate nuclear translocation of NF-E2-related factor 2 to induce HO-1 through reactive oxygen species generation. Electrophoretic mobility shift assay and chromatin immunoprecipitation studies indicated nuclear translocation of NF-E2-related factor 2 and its binding to HO-1 promoter to induce HO-1 expression during gastric injury. Inhibition of HO-1 by zinc protoporphyrin aggravated the mucosal injury and delayed healing. Zinc protoporphyrin further reduced the respiratory control ratio and transmembrane potential and enhanced MOS and apoptosis. In contrast, induction of HO-1 by cobalt protoporphyrin reduced MOS, corrected mitochondrial dysfunctions, and prevented apoptosis and gastric injury. Thus, induction and mitochondrial localization of HO-1 are a novel cytoprotective mechanism against MOS-mediated apoptotic tissue injury.     

Nitric oxide stimulates Nrf2 nuclear translocation in vascular endothelium

Vascular endothelial cells respond to nitric oxide by activating MAPK pathways and upregulating stress-activated proteins such as gamma-glutamylcysteine synthetase (gamma-GCS) and heme oxygenase-1 (HO-1). Since consensus sequences for the antioxidant response element (ARE) are found in the promoters of the gamma-GCS and HO-1 genes, we examined nuclear translocation of Nrf2, a CNC-bZIP protein which binds to and activates the ARE. We found a dramatic increase in Nrf2 nuclear translocation 1-8h following the nitric oxide donor spermine NONOate. Translocation was inhibited by pretreatment of cells with N-acetylcysteine suggesting involvement of an oxidative mechanism in this response. Translocation was also blocked by PD 98059 and SB 203580, inhibitors of ERK and p38 pathways, respectively. In addition to effects on Nrf2 subcellular localization, spermine NONOate increased Nrf2 protein levels by a mechanism which was inhibited by PD 98059. Pretreatment with N-acetylcysteine, PD 98059, and SB 203580 decreased HO-1 upregulation in spermine NONOate-treated cells. These results suggest that ERK and p38 pathways may regulate nitric oxide-mediated adaptive responses in vascular endothelium via translocation of Nrf2 and activation of the ARE.     

Cellular and subcellular localization of heme oxygenase-2 in monkey retina

Carbon monoxide (CO), an activator of soluble guanylate cyclase (SGC) and generated enzymatically by heme oxygenases (HO), is considered to function as an intra- and intercellular neuromodulator or neurotransmitter in the central and peripheral nervous systems. HO-2 is the constitutive isoform of HO and is more prevalent in nervous tissues than in the other peripheral tissues. Because previous studies have demonstrated different distributions of HO-2 in the retina depending on the species of animals, the aim of this study was to identify which cell types of the monkey retina express HO-2. The expression of HO-2 protein was examined in monkey retina by Western blot analysis. Immunoblottings from monkey homogenates revealed a single clear protein band with a molecular mass of 36 kDa that is corresponding to rat HO-2. Immunoreactivity of HO-2 was found in the perikarya of ganglion cells. Density of immunoreactive ganglion cells was higher in the central area of retina than in the peripheral retina, and somata of larger ganglion cells were stained more densely than smaller ones. In electron microscopy, immunoreactivity of HO-2 was localized on the membrane of the endoplasmic reticulum and the nuclear outer membrane of the ganglion cells. By contrast, inner plexiform layer, inner nuclear layer and outer nuclear layer were devoid of HO-2 immunoreactivity. cGMP were strongly localized in all of ganglion cells. Some cells contributed to the relatively faint cGMP staining were seen in the inner nuclear layer. In combination of HO-2 and cGMP immunocytochemistry, the overlap of co-localization of HO-2 and cGMP would suggest that HO-2 in the ganglion cells would serve as a source for CO generation and CO could serve as a gaseous signaling molecule modulator of neural activity in the retina of monkey.     

Carbon monoxide promotes hypoxic pulmonary vascular remodeling

CO is a biologically active gas that produces cellular effects by multiple mechanisms. Because cellular binding of CO by heme proteins is increased in hypoxia, we tested the hypothesis that CO interferes with hypoxic pulmonary vascular remodeling in vivo. Rats were exposed to inspired CO (50 parts/million) at sea level or 18,000 ft of altitude [hypobaric hypoxia (HH)], and changes in vessel morphometry and pulmonary pressure-flow relationships were compared with controls. Vascular cell single strand DNA (ssDNA) and proliferating cell nuclear antigen (PCNA) were assessed, and changes in gene and protein expression of smooth muscle alpha-actin (sm-alpha-actin), beta-actin, and heme oxygenase-1 (HO-1) were evaluated by Western analysis, RT-PCR, and immunohistochemistry. After 21 days of HH, vascular pressure at constant flow and vessel wall thickness increased and lumen diameter of small arteries decreased significantly. The presence of CO, however, further increased both pulmonary vascular resistance (PVR) and the number of small muscular vessels compared with HH alone. CO + HH also increased vascular PCNA and nuclear ssDNA expression compared with hypoxia, suggesting accelerated cell turnover. CO in hypoxia downregulated sm-alpha-actin and strongly upregulated beta-actin. CO also increased lung HO activity and HO-1 mRNA and protein expression in small pulmonary arteries during hypoxia. These data indicate an overall propensity of CO in HH to promote vascular remodeling and increase PVR in vivo.     

Symbiotic association between soybean plants and Bradyrhizobium japonicum develops oxidative stress and heme oxygenase-1 induction at early stages

We have previously demonstrated that the induction of heme oxygenase-1 (HO-1) (EC 1.14.99.3) plays a protective role against oxidative stress in leaves and nodules of soybean plants subjected to cadmium, UV-B radiation, and salt stress. Here, we investigated HO-1, localization and their relationship with oxidative stress in different growth stages of soybean plants roots inoculated with Bradyrhizobium japonicum (3, 5, 7, 10, and 20 days post-inoculation) and nodules. After 7 days of inoculation, we observed a 70% increase in thiobarbituric acid-reactive substances that correlates with an enhancement in the gene expression of HO-1, catalase, and superoxide dismutase. Furthermore, the inhibition of HO-1 activity by Zn-protoporphyrin IX produced an increase in lipid peroxidation and a decrease in glutathione content suggesting that, in this symbiotic process, HO-1 may act as a signal molecule that protects the root against oxidative stress. We determined, for the first time, the tissular localization of HO-1 in nodules by electron-microscope examination. These results undoubtedly demonstrated that this enzyme is localized only in the plant tissue and its overexpression may play an important role as antioxidant defense in the plant. Moreover, we demonstrate that, in roots, HO-1 is induced by oxidative stress produced by inoculation of B. japonicum and exerts an antioxidant response against it.     

Repression of heme oxygenase-1 expression as a defense strategy in humans

Heme oxygenase-1 (HO-1) is an essential enzyme in heme catabolism and is characterized by its inducibility in response to various environmental factors, including its substrate heme. The induction of HO-1 has been established as the defense mechanism against oxidative stress. However, striking interspecies or inter-tissue differences are noted in the regulation of HO-1 expression under hypoxia or heat shock, each of which represses HO-1 expression in many types of human cells but rather induces it in rodent cells. The downregulation of HO-1 expression may reduce energy expenditure and local production of carbon monoxide, iron, and bilirubin and transiently increase intracellular heme pool. Here, we discuss the repression of HO-1 expression as a potential defense strategy in humans by highlighting a regulatory role of HO-1 in its own expression.     

Heme oxygenase-1: emerging target of cancer therapy

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme catalyzing oxidative degradation of cellular heme to liberate free iron, carbon monoxide (CO) and biliverdin in mammalian cells. In addition to its primary role in heme catabolism, HO-1 exhibits anti-oxidative and anti-inflammatory functions via the actions of biliverdin and CO, respectively. HO-1 is highly induced in various disease states, including cancer. Several lines of evidence have supported the implication of HO-1 in carcinogenesis and tumor progression. HO-1 deficiency in normal cells enhances DNA damage and carcinogenesis. Nevertheless, HO-1 overexpression in cancer cells promotes proliferation and survival. Moreover, HO-1 induces angiogenesis through modulating expression of angiogenic factors. Although HO-1 is an endoplasmic reticulum resident protein, HO-1 nuclear localization is evident in tumor cells of cancer tissues. It has been shown that HO-1 is susceptible to proteolytic cleavage and translocates to nucleus to facilitate tumor growth and invasion independent of its enzymatic activity. HO-1 also impacts cancer progression through modulating tumor microenvironment. This review summarizes the current understanding of the protumorigenic role of HO-1 and its potential as a molecular target for cancer therapy.     

Lipoxin A4-Induced Heme Oxygenase-1 Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury via p38 MAPK Activation and Nrf2/ARE Complex

Objective

To investigate whether lipoxin A₄ (LXA₄) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA₄-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA₄-induced HO-1 induction.

Methods

Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA₄ or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay.

Results

Pretreatment of the cells undergoing H/R lesion with LXA₄ significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA₄ on the cells undergoing H/R lesion. LXA₄ increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure.

Conclusion

The protection role of LXA₄ against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway.     

Differential cellular and subcellular localization of heme-binding protein 23/peroxiredoxin I and heme oxygenase-1 in rat liver.

Heme-binding protein 23 (HBP23), also termed peroxiredoxin (Prx) I, and heme oxygenase-1 (HO-1) are distinct antioxidant stress proteins that are co-ordinately induced by oxidative stress. HBP23/Prx I has thioredoxin-dependent peroxidase activity with high binding affinity for the pro-oxidant heme, while HO-1 is the inducible isoform of the rate-limiting enzyme of heme degradation. We investigated the cellular and subcellular localization of both proteins in rat liver. Whereas by immunohistochemistry (IHC) a uniformly high level of HBP23/Prx I expression was observed in liver parenchymal and different sinusoidal cells, HO-1 expression was restricted to Kupffer cells. By immunoelectron microscopy using the protein A-gold technique, HBP23/Prx I immunoreactivity was detected in cytoplasm, nuclear matrix, mitochondria, and peroxisomes of parenchymal and non-parenchymal liver cell populations. In contrast, the secretory pathway, i.e., the endoplasmic reticulum and Golgi complex, was free of label. As determined by immunocytochemical (ICC) studies in liver cell cultures and by Western and Northern blotting analysis, HBP23/Prx I was highly expressed in cultures of isolated hepatocytes and Kupffer cells. In contrast, HO-1 was constitutively expressed only in Kupffer cell cultures but was also inducible in hepatocytes. These data suggest that HBP23/Prx I and HO-1 may have complementary antioxidant functions in different cell populations in rat liver.     

The role of Bach1 in ultraviolet A-mediated human heme oxygenase 1 regulation in human skin fibroblasts

Up-regulation of heme oxygenase 1 (HO-1) by ultraviolet A (UVA; 320-380 nm) irradiation of human skin cells protects them against oxidative stress. The role of Nrf2 in up-regulation of HO-1 and other phase II genes is well established. The mechanism underlying Bach1-mediated HO-1 repression is less well understood although cellular localization seems to be crucial. Because prolonged HO-1 overexpression is likely to be detrimental, it is crucial that activation of the gene is transient. We now show that UVA irradiation of cultured human skin fibroblasts enhances accumulation of Bach1 mRNA and protein severalfold. Endogenous Bach1 protein accumulates in the nucleus after 8h and may occupy MARE sites after HO-1 activation thus providing a compensatory mechanism to control HO-1 overexpression. Overexpression of Bach1, together with MafK, represses basal and UVA-mediated HO-1 protein expression, whereas silencing of the Bach1 gene by Bach1-specific siRNAs causes robust enhancement of constitutive HO-1 levels. UVA treatment of cells in which Bach1 has been silenced leads to higher levels of induction of the HO-1 protein. Although Bach1 protein is exported from the nucleus 12h after UVA irradiation, the release of free cellular heme from microsomal heme-containing proteins is immediate rather than delayed. Although heme does promote the export of Bach1 via the Crm1/exportin 1 pathway and is involved in the delayed UVA-mediated export of the protein, it is not clear how this occurs.     

An N-terminal segment of the active component of the bacterial genotoxin cytolethal distending toxin B (CDTB) directs CDTB into the nucleus

Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G2/M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3-4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTBGFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48-124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport.