Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.     

Water-soluble polymer conjugates of ZnPP for photodynamic tumor therapy

Zn-protoporphyrin (ZnPP) is a promising candidate for cancer therapy. It is known to inhibit heme-oxygenase-1 (HO-1), resulting in suppressed biliverdin/bilirubin production accompanying lowered antioxidative capacity. As a consequence, a significant suppression of tumor growth in vivo was reported. Recent findings also showed that ZnPP efficiently generated reactive singlet oxygen under illumination of visible light. In the present report, we describe the photosensitizing capabilities of water-soluble polymer conjugates of ZnPP as novel compounds for photodynamic therapy against solid tumors. The polymer conjugation made ZnPP water-soluble, thus possible for injection for its aqueous solution. The cellular uptake and photobiological activity of ZnPP derivatives have been tested using a human T-cell leukemia cell line in vitro and demonstrated most potent phototoxic effects of SMA-ZnPP followed by PEG-ZnPP under aerobic conditions.     

Backbone assignments of the apo and Zn(II) protoporphyrin IX-bound states of the soluble form of rat heme oxygenase-1

In nature, heme is a prosthetic group that is universally used as a cofactor for heme proteins. It is necessary for the execution of fundamental biological processes including electron transfer, oxidation and metabolism. However, free heme is toxic to cells, because of its capability to enhance oxidative stress, hence its cellular concentration is strictly regulated through multiple mechanisms. Heme oxygenase (HO) serves as an irreplaceable member in the heme degradation system. It is a ubiquitous protein, existing in many species including mammals, higher plants, and interestingly, certain pathogenic bacteria. In the HO reaction, HO catalyzes oxidative cleavage of heme to generate biliverdin and release carbon monoxide and ferrous iron. Because of the beneficial effects of these heme catabolism products, HO plays a key role in iron homeostasis and in defense mechanism against oxidative stress. HO is composed of an N-terminal structured region and a C-terminal membrane-bound region. Furthermore, the soluble form of HO, which is obtainable by excision of the membrane-bound region, retains its catalytic activity. Here, we present the backbone resonance assignments of the soluble form (residues 1–232) of HO-1 in the free and Zn(II) protoporphyrin IX (ZnPP)-bound states, and analyzed the structural differences between the states. ZnPP is a potent enzyme inhibitor, and the ZnPP-bound structure of HO-1 mimics the heme-bound structure. These assignments provide the structural basis for a detailed investigation of the HO-1 function.     

Heme oxygenase modulates selectin expression in different regional vascular beds

Heme oxygenase (HO) catalyzes the degradation of heme to biliverdin, iron, and CO. The inducible isoform (HO-1) has been implicated as a modulator of the inflammatory response. HO-1 activity can be induced by hemin and inhibited with zinc protoporphyrin IX (ZnPP). Using these reagents, we assessed the possibility that HO-1 modulates the inflammatory response by altering the expression of endothelial cell adhesion molecules. Endotoxin (lipopolysaccharide, LPS)-induced expression of P- and E-selectin expression was quantified in different vascular beds of the rat using the dual radiolabeled monoclonal antibody technique. Pretreatment with hemin attenuated, whereas ZnPP treatment exacerbated, the increased selectin expression normally elicited by LPS. Biliverdin, at an equimolar dosage, was as effective as hemin in attenuating LPS-induced selectin expression in the lung, kidneys, liver, and intestines. These findings indicate that the anti-inflammatory properties of HO-1 may be related to an inhibitory action of P- and E-selectin expression in the vasculature. Biliverdin (or its metabolite, bilirubin), rather than CO, may account for this action of HO-1 on endothelial cell adhesion molecule expression.     

Zinc Protoporphyrin Regulates Cyclin D1 Expression Independent of Heme Oxygenase Inhibition

Zinc protoporphyrin IX (ZnPP), an endogenous heme analogue that inhibits heme oxygenase (HO) activity, represses tumor growth. It can also translocate into the nucleus and up-regulate heme oxygenase 1 (HMOX1) gene expression. Here, we demonstrate that tumor cell proliferation was inhibited by ZnPP, whereas tin protoporphyrin (SnPP), another equally potent HO-1 inhibitor, had no effect. Microarray analysis on 128 tumorigenesis related genes showed that ZnPP suppressed genes involved in cell proliferation and angiogenesis. Among these genes, CYCLIN D1 (CCND1) was specifically inhibited as were its mRNA and protein levels. Additionally, ZnPP inhibited CCND1 promoter activity through an Sp1 and Egr1 overlapping binding site (S/E). We confirmed that ZnPP modulated the S/E site, at least partially by associating with Sp1 and Egr1 proteins rather than direct binding to DNA targets. Furthermore, administration of ZnPP significantly inhibited cyclin D1 expression and progression of a B-cell leukemia/lymphoma 1 tumor in mice by preferentially targeting tumor cells. These observations show HO independent effects of ZnPP on cyclin D1 expression and tumorigenesis.     

Inhibition of endogenous CO by ZnPP protects against stress-induced gastric lesion in adult male albino rats

Carbon monoxide (CO) has been found to be produced in every living cell in a biochemical reaction catalyzed by heme-oxygenase (HO) enzyme which degrades heme into biliverdin, CO, and iron. Endogenous CO is not a waste product, but acts as a chemical messenger mediating and modulating many intracellular biochemical reactions that regulate physiological functions. This study was designed to investigate the effect of inhibition of endogenous CO production by zinc protoporphyrin (ZnPP), an HO inhibitor, on the gastric secretion and ulceration induced by cold-restraint stress (CRS) in adult male albino rats. Rats were pylorically ligated and divided randomly into the following groups (six rats each): control, ZnPP treated (50 μmol/kg/day, s.c. for 10 days), CRS, and stressed ZnPP treated groups. Blood samples were collected from the retro-orbital sinus of anesthetized rats for determination of CO concentration. We found that ZnPP pretreatment significantly decreased HO-1 level, CO level, and volume of gastric juice as compared to the control non-stressed rats. In the present study, ZnPP pretreatment proved to be protective against development of ulcerative lesions in CRS model as evidenced by reduction of the ulcer index, and this could be mediated through reduction of free and total acidity of gastric secretion and decreased lipid peroxidation but with significantly decreased gastric protective nitric oxide and prostaglandin E(2) levels. In conclusion and according to our results, the protective effect of ZnPP on CRS-induced gastric ulcers despite of inhibition of endogenous CO could be attributed to the presence of zinc which is known to have a protective anti-ulcer effect.     

Protective role of heme oxygenase in the blood vessel wall during atherogenesis.

Several lines of evidence suggest that antioxidant processes and (or) endogenous antioxidants inhibit proatherogenic events in the blood vessel wall. Heme oxygenase (HO), which catabolizes heme to biliverdin, carbon monoxide, and catalytic iron, has been shown to have such antioxidative properties. The HO-1 isoform of heme oxygenase is ubiquitous and can be increased several fold by stimuli that induce cellular oxidative stress. Products of the HO reaction have important effects: carbon monoxide is a potent vasodilator, which is thought to play a role in modulation of vascular tone; biliverdin and its by-product bilirubin are potent antioxidants. Although HO induction results in an increase in catalytic free iron release, the enhancement of intracellular ferritin protein through HO-1 has been reported to decrease the cytotoxic effects of iron. Oxidized LDL has been shown to increase HO-1 expression in endothelial and smooth muscle cell cultures, and during atherogenesis. Further evidence of HO-1 expression associated with atherogenesis has been demonstrated in human, murine and rabbit atherosclerotic lesions. Moreover, genetic models of HO deficiency suggest that the actions of HO-1 are important in modulating the severity of atherosclerosis. Recent experiments in gene therapy using the HO gene suggest that interventions aimed at HO in the vessel wall could provide a novel therapeutic approach for the treatment or prevention of atherosclerotic disease.     

Reduction of the NO-mediated response in the rat aorta by metalloporphyrins

Metalloporphyrins (MPs) have been found to affect the production of carbon monoxide (CO) and nitric oxide (NO). Unlike that for CO, little is known about the mechanism of action of MPs on the NO system. We determined the in vitro ability of ferrous protoporphyrin (heme, FePP), zinc protoporphyrin (ZnPP), and bilirubin (BR) to scavenge NO. Heme and ZnPP were studied in the rat aortic ring system for their ability to affect phenylephrine-induced contraction and methacholine-stimulated relaxation. Heme was found to be a good NO scavenger with a ks = 0.53 +/- 0.19 x 10(4) M(-1)xs(-1) (n = 6). ZnPP and BR did not scavenge NO. Neither heme nor ZnPP treatment affected the phenylephrine response as measured by -logEC50 and the maximal effect. However, heme and ZnPP treatments decreased the -logEC50 and the maximal effects of methacholine, therefore decreasing vasorelaxation. We conclude that when ZnPP is administered in vivo blood pressure should be carefully monitored.     

The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties

The heme biosynthetic and catabolic pathways generate pro- and antioxidant compounds, and consequently, influence cellular sensitivity to oxidants. Heme precursors (delta-aminolevulinic acid, porphyrins) generate reactive oxygen species (ROS), from autoxidation and photochemical reactions, respectively. Heme, an essential iron chelate, serves in respiration, oxygen transport, detoxification, and signal transduction processes. The potential toxicity of heme and hemoproteins points to a critical role for heme degradation in cellular metabolism. The heme oxygenases (HOs) provide this function and participate in cellular defense. This hypothesis emerges from the observation that the activation of HO-1 is an ubiquitous cellular response to oxidative stress. The reaction products of HO activity, biliverdin, and its subsequent metabolite bilirubin, have antioxidant properties. Furthermore, iron released from HO activity stimulates ferritin synthesis, which ultimately provides an iron detoxification mechanism that may account for long-term cytoprotection observed after HO induction. However, such models have overlooked potential pro-oxidant consequences of HO activity. The HO reaction releases iron, which could be involved in deleterious reactions that compete with iron reutilization and sequestration pathways. Indeed, the induction of HO activity may have both pro- and antioxidant sequelae depending on cellular redox potential, and the metabolic fate of the heme iron.     

内源性一氧化碳减轻大鼠双侧后肢缺血再灌注所致的肺损伤

通过观察血红素氧化酶（HO）阻断剂－－锌原卟啉（ZnPP)对肺组织、肺泡间质多形核白细胞数目肺组织丙二醛含量和湿重干重之比的影响,并对肺组织HO活性和血内碳氧血红蛋白水平（COHb）进行检测,以探讨内源性HO／一氧化碳（CO）在肢体缺血再灌注（I／R）所致肺损伤中的作用。结果发现,大鼠双侧后肢I／R可导致急性肺损伤,同时使肺组织中HO活性和血内COHb水平显著升高;应用ZnPP预处理可使HO活性和COHb水平显著降低,但肺损伤却进一步加重。上述实验结果表明,肢体I／R致肺损伤时,肺组织中HO活性和内源性CO生成增多或减轻大鼠肢体I／R所致的肺损伤。     

Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer's disease

Heme oxygenase (HO) is the rate-limiting enzyme for the degradation of heme, a prooxidant, coming from a multitude of heme-containing proteins/enzymes. With the action of cytochrome P₄₅₀ reductase, HO cleaves the heme ring into biliverdin which is converted into bilirubin, both have been shown to have intrinsic radical scavenger activities. Iron is also released from the heme core and in its free form can act as a catalyst for oxidative stress damage or can be sequested by several iron-binding proteins. Under physiological conditions, the newly generated iron can be neutralized within the cell. The third product of the opening of the porphyrin ring is carbon monoxide, which role has been puzzling. It has been reported as a potential neuromodulator, it modulates guanylate cyclase activity and has vasodilation, anti-inflammatory and antiapoptotic effects. In the brain, HO2 accounts for the vast majority of HO activity. By decreasing HO2 activity, one would expect more neuronal damage after oxidative stress injury with possible direct implications to acute and chronic neurodegenerative disorders. Pharmacological ways to increase neuronal HO activity is likely to have therapeutic applications.     

Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1–2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-B nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 µM), and by bilirubin (1 µM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium. endothelium; carbon monoxide; bilirubin; injury; reactive oxygen species; heme oxygenase     

Heme oxygenase activity causes transient hypersensitivity to oxidative ultraviolet A radiation that depends on release of iron from heme

Heme oxygenase (HO) breaks down heme to iron, biliverdin, and carbon monoxide, and activity of this enzyme increases in many tissues and cell types after exposure to oxidative stress. There is evidence that increased HO activity is involved in long-term protective mechanisms against oxidative stress. We studied the effect of artificially overexpressed HO activity on the cytotoxicity of oxidative ultraviolet A (UVA) radiation after loading human cells with the HO substrate ferric heme (hemin). In contrast to the reported long-term protection attributed to HO activity, cells overexpressing HO activity were hypersensitive to UVA radiation shortly after heme treatment when compared with control cells. Cells overexpressing HO activity showed an increased rate of heme consumption and a higher level of accumulated free chelatable iron when compared with control cells. The hypersensitivity of cells overexpressing HO to UVA radiation after heme treatment was apparently caused by the increased accumulation of chelatable iron, because the iron chelator desferrioxamine strongly reduced the hypersensitivity. One day after the heme treatment, cells overexpressing HO activity were no longer hypersensitive to UVA radiation. We conclude that increased HO activity can temporarily increase the sensitivity of cells to oxidative stress by releasing iron from heme.     

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.     

Carbon monoxide protects PC12 cells from peroxynitrite-induced apoptotic death by preventing the depolarization of mitochondrial transmembrane potential

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in catalyzing heme degradation into biliverdin, free iron, and carbon monoxide (CO), serves as a protective enzyme against oxidative and nitrosative stresses. In the present study, we investigated the cytoprotective effects of HO-1 upregulation and its product CO against the peroxynitrite-induced PC12 cell death. PC12 cells treated with 3-morphoinosydonimine (SIN-1), a generator of peroxynitrite (ONOO-), underwent apoptotic cell death as evidenced by dissipation of mitochondrial transmembrane potential (DeltaPsim), release of mitochondrial cytochrome c into cytoplasm, cleavage of poly(ADP-ribose)polymerase and fragmentation of internucleosomal DNA. Pretreatment of PC12 cells with a low non-toxic concentration of SIN-1 (0.5 mM) induced HO-1 expression and abrogated the cell death caused by subsequent challenge with high dose SIN-1 (2.5 mM). Furthermore, pretreatment of PC12 cells with SnCl2, a potent inducer of HO-1 expression, increased endogenous production of CO (HO activity) and rescued the PC12 cells from peroxynitrite-induced apoptosis. The cytoprotective effect of SnCl2 was abolished when the HO activity was inhibited by zinc protoporphyrin IX (ZnPP IX). PC12 cells treated directly with the CO-releasing molecule, tricarbonyldichlororuthenium (II) dimer ([Ru(CO)3Cl2]2) became tolerant to the depolarization of DeltaPsim and apoptosis induced by high dose peroxynitrite. Taken together, these data demonstrate that the adaptive protection against peroxynitrite-induced apoptotic death in PC12 cells is mediated by CO formed as a consequence of HO-1 induction.     

大鼠肢体缺血/再灌注后肝脏 HO-1表达的变化及其意义

目的:探讨肢体缺血/再灌注(I/R)致肝脏损伤时肝组织诱导型血红素氧合酶(HO-1)表达的变化及其意义.方法:夹闭大鼠双侧股动脉根部4 h、开放2～24 h,制备肢体I/R模型.RT-PCR检测肝组织HO-1 mRNA表达的变化,免疫组化染色法观察HO-1蛋白在肝内的生成与分布.对肢体I/R大鼠应用锌原卟啉抑制其体内HO-1活性后,光镜观察其肝组织的病理变化.结果:肢体I/R后肝组织HO-1 mRNA的表达水平显著高于各对照组,再灌12 h表达至峰值,至再灌24 h仍显著高于各对照组(P＜0.01).肢体I/R组肝组织内出现大量弥散分布的HO-1阳性肝细胞,抑制HO-1活性,使肢体I/R组肝组织损伤明显加重.结论:肢体I/R损伤可诱导肝细胞HO-1基因表达上调,所诱生的HO-1对肝细胞具有保护效应.     

Induction of heme oxygenase 1 by Ginkgo biloba in neuronal cultures and potential implications in ischemia.

Ginkgo biloba extract (EGb 761) is a standardized extract originating in traditional Chinese medicine. Ginkgo biloba dried leaves have been used for centuries to treat various neurological conditions. The constituents from the extract are likely to have synergistic effects that have been shown to be protective against oxidative stress injury. However, the cellular mechanisms of protection afforded by Ginkgo biloba are still unclear. The cascade leading to neuronal cell death in acute and chronic neurodegenerative conditions, such as cerebral ischemia and Alzheimer's disease, has been postulated to be mediated by free radical damage. We tested the hypothesis that the neuroprotective action of EGb 761 could be due partially to an induction of heme oxygenase I (HO1). We and others have previously reported that modulation of HO total activity may well have direct physiological implications in stroke and in Alzheimer's disease. Heme oxygenase acts as an antioxidant enzyme by degrading heme into iron, carbon monoxide, and biliverdin which is rapidly converted into bilirubin. Through the use of primary neuronal cultures, we demonstrated that EGb 761 induces HO1 in a dose-dependent manner (0, 10, 50, 100 and 500 microg/ml) and time-dependent manner with a maximal induction at 8 hr. We are proposing that several of the protective effects of EGb 761 in ischemia could be mediated through beneficial actions of heme degradation and its metabolites.