How Heme Oxygenase-1 Prevents Heme-Induced Cell Death

Earlier observations indicate that free heme is selectively toxic to cells lacking heme oxygenase-1 (HO-1) but how this enzyme prevents heme toxicity remains unexplained. Here, using A549 (human lung cancer) and immortalized human bronchial epithelial cells incubated with exogenous heme, we find knock-down of HO-1 using siRNA does promote the accumulation of cell-associated heme and heme-induced cell death. However, it appears that the toxic effects of heme are exerted by “loose” (probably intralysosomal) iron because cytotoxic effects of heme are lessened by pre-incubation of HO-1 deficient cells with desferrioxamine (which localizes preferentially in the lysosomal compartment). Desferrioxamine also decreases lysosomal rupture promoted by intracellularly generated hydrogen peroxide. Supporting the importance of endogenous oxidant production, both chemical and siRNA inhibition of catalase activity predisposes HO-1 deficient cells to heme-mediated killing. Importantly, it appears that HO-1 deficiency somehow blocks the induction of ferritin; control cells exposed to heme show ~10-fold increases in ferritin heavy chain expression whereas in heme-exposed HO-1 deficient cells ferritin expression is unchanged. Finally, overexpression of ferritin H chain in HO-1 deficient cells completely prevents heme-induced cytotoxicity. Although two other products of HO-1 activity–CO and bilirubin–have been invoked to explain HO-1-mediated cytoprotection, we conclude that, at least in this experimental system, HO-1 activity triggers the induction of ferritin and the latter is actually responsible for the cytoprotective effects of HO-1 activity.     

血红素加氧酶-1在缺血/再灌注损伤中的保护作用

血红素加氧酶-1(Heme Oxygenase-1,HO-1)是催化血红素分解的关键酶。近年来,人们对血红素降解产物的抗氧化、抗炎症等功能的认识推动了对HO酶系的研究。缺血／再灌注损伤(IRI)是一个重要的临床问题,而临床上对IRI的防治尚缺乏有效的方法。目前发现HO-1过表达具有抗IRI的作用,其保护作用的可能机制有：抗氧化作用、调节微循环、调节细胞周期和抗炎症作用。     

Bradykinin Postconditioning Protects Pyramidal CA1 Neurons Against Delayed Neuronal Death in Rat Hippocampus

Aims The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat. Methods Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 μg/kg) applied 48 h after ischemia. Results We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation. Conclusions Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.     

Prion Protein Fragment 106–126 Differentially Induces Heme Oxygenase-1 mRNA in Cultured Neurons and Astroglial Cells

Abstract: Heme oxygenase (HO), which catalyzes the degradation of heme, has two isozymes (HO-1 and HO-2). In brain the noninducible HO-2 isoform is predominant, whereas the inducible HO-1 is a marker of oxidative stress. Because brain oxidative stress might be present in prion-related encephalopathies (PREs), as in other neurodegenerative diseases, we investigated whether HO-1 mRNA was induced in neuronal and astroglial cell cultures by a peptide corresponding to residue 106–126 of human prion protein (PrP). This peptide is amyloidogenic, and when added in vitro to cultured cells it reproduces the neuronal death and astroglial proliferation and hypertrophy occurring in PREs. HO-1 mRNA did not accumulate in rat cultured neurons from hippocampus or cortex exposed to PrP 106–126 (50 µ M for 5 days). PrP 106–126 induced HO-1 mRNA accumulation in rat astroglial cultures depending on the exposure time and concentration, being maximal (33-fold) after 7 days of exposure at 50 µ M . The nonamyloidogenic amidated or amidated-acetylated PrP 106–126 was ineffective, as was a scrambled peptide used as control. N -Acetylcysteine reduced (50%) the accumulation of HO-1 mRNA in astroglial cells after PrP 106–126 (25 µ M ) given for 5 days. Thus, oxidative stress is apparently a feature of the toxicity of PrP 106–126, and it might also occur in PREs; induction of HO-1 could contribute to the greater resistance of astrocytes compared with neurons to PrP 106–126 toxicity.     

1-甲基-4-苯基-吡啶盐致多巴胺能细胞系MES23.5的死亡机制

通过测定环境毒素1-甲基-4-苯基-吡啶盐(MPP )作用于多巴胺能细胞系MES23．5后细胞存活率的变化及细胞线粒体膜电位(△ψM)、活性氧(ROS)、羟自由基、超氧化物岐化酶(SOD)的变化,发现MPP^ 作用于多巴胺能细胞系MES23．5,可导致细胞存活率显著性减少,浓度达到200mol／L以上后,细胞存活率的下降呈时间与MPP^ 浓度依赖;以200μmol/L MPP^ 作用细胞6∽48h后,△ψM逐渐下降、ROS、羟自由基逐渐增加,48h后SOD开始显著性减少。结果表明早期线粒体能量代谢障碍和膜电位变化导致ROS(尤其是羟自由基)含量增加是MPP^ 导致多巴胺能细胞氧化应激的原因,而细胞内自由基的清除机制受损,则最终导致细胞变性死亡。     

Heme Oxygenase-1 Protects Corexit 9500A-Induced Respiratory Epithelial Injury across Species

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.     

Heme Oxygenase-1 Protects Neurons from Ischemic Damage by Upregulating Expression of Cu,Zn-Superoxide Dismutase,Catalase, and Brain-Derived Neurotrophic Factor in the Rabbit Spinal Cord

Neurochemical Research - In the present study, we investigated the protective effects of heme oxygenase (HO-1) against ischemic damage in motor neurons of the rabbit spinal cord. A PEP-1-HO-1...     

Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes

Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22^phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.     

血红素氧合酶-1 与肺纤维化

血红素氧合酶-1(Hene Oxygenase-1)是一种氧化应激反应蛋白,广泛存在全身各组织器官。HO-1及催化产物组成了重要的内源性保护系统,具有调控炎症、抗氧化损伤及抗细胞凋亡等作用,对于组织器官具有保护作用。肺纤维化发病机制复杂,氧化应激是肺纤维化的致病机制之一。HO-1是一种重要的抗氧化剂,其通过多种途径参与致病,在肺纤维化致病过程中发挥重要作用。     

Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1^+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1^+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE^-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.     

血红素氧合酶-1与肺纤维化

血红素氧合酶-1（Hene Oxygenase-1）是一种氧化应激反应蛋白,广泛存在全身各组织器官。HO-1及催化产物组成了重要的内源性保护系统,具有调控炎症、抗氧化损伤及抗细胞凋亡等作用,对于组织器官具有保护作用。肺纤维化发病机制复杂,氧化应激是肺纤维化的致病机制之一。HO-1是一种重要的抗氧化剂,其通过多种途径参与致病,在肺纤维化致病过程中发挥重要作用。     

DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4 weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3‐kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62-positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12 h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD.     

Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation

Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

莱茵衣藻血红素加氧酶1过表达载体的构建和遗传转化

莱茵衣藻(Chlamydomonas reinhardti)是一种3套基因组都能进行遗传转化的真核生物,作为一种模式生物,它被用于生物学研究的各个领域。目前,发现血红素加氧酶具有多种功能活性,但关于它的作用机理还不是很清楚。本研究利用分子生物学技术,构建了莱茵衣藻HO-1过表达载体,用SpeI和BglII双酶切,DNA测序,GUS染色,PCR检测证明表达载体构建成功。将此构建通过农杆菌介导法导入莱茵衣藻细胞中,获得了能够稳定遗传的转化子。上述结果为后续进一步的功能研究奠定基础。     

The Nonfibrillar Amyloid β-Peptide Induces Apoptotic Neuronal Cell Death

The toxicity of the nonaggregated amyloid beta-peptide (1-40) [A beta(1-40)] on the viability of rat cortical neurons in primary culture was investigated. We demonstrated that low concentrations of A beta peptide, in a nonfibrillar form, induced a time- and dose-dependent apoptotic cell death, including DNA condensation and fragmentation. We compared the neurotoxicity of the A beta(1-40) peptide with those of several A beta-peptide domains, comprising the membrane-destabilizing C-terminal domain of A beta peptide (e.g., amino acids 29-40 and 29-42). These peptides reproduced the effects of the (1-40) peptide, whereas mutant nonfusogenic A beta peptides and the central region of the A beta peptide (e.g., amino acids 13-28) had no effect on cell viability. We further demonstrated that the neurotoxicity of the nonaggregated A beta peptide paralleled a rapid and stable interaction between the A beta peptide and the plasma membrane of neurons, preceding apoptosis and DNA fragmentation. By contrast, the peptide in a fibrillar form induced a rapid and dramatic neuronal death mainly through a necrotic pathway, under our conditions. Taken together, our results suggest that A beta induces neuronal cell death by either apoptosis and necrosis and that an interaction between the nonfibrillar C-terminal domain of the A beta peptide and the plasma membrane of cortical neurons might represent an early event in a cascade leading to neurodegeneration.     

Carbon Monoxide Induces Heme Oxygenase-1 to Modulate STAT3 Activation in Endothelial Cells via S-Glutathionylation

IL-6/STAT3 pathway is involved in a variety of biological responses, including cell proliferation, differentiation, apoptosis, and inflammation. In our present study, we found that CO releasing molecules (CORMs) suppress IL-6-induced STAT3 phosphorylation, nuclear translocation and transactivity in endothelial cells (ECs). CO is a byproduct of heme degradation mediated by heme oxygenase (HO-1). However, CORMs can induce HO-1 expression and then inhibit STAT3 phosphorylation. CO has been found to increase a low level ROS and which may induce protein glutathionylation. We hypothesized that CORMs increases protein glutathionylation and inhibits STAT3 activation. We found that CORMs increase the intracellular GSSG level and induce the glutathionylation of multiple proteins including STAT3. GSSG can inhibit STAT3 phosphorylation and increase STAT3 glutathionylation whereas the antioxidant enzyme catalase can suppress the glutathionylation. Furthermore, catalase blocks the inhibition of STAT3 phosphorylation by CORMs treatment. The inhibition of glutathione synthesis by BSO was also found to attenuate STAT3 glutathionylation and its inhibition of STAT3 phosphorylation. We further found that HO-1 increases STAT3 glutathionylation and that HO-1 siRNA attenuates CORM-induced STAT3 glutathionylation. Hence, the inhibition of STAT3 activation is likely to occur via a CO-mediated increase in the GSSG level, which augments protein glutathionylation, and CO-induced HO-1 expression, which may enhance and maintain its effects in IL-6-treated ECs.