Nitric oxide priming protects nitric oxide-mediated apoptosis via heme oxygenase-1 induction

The role of nitric oxide (NO) as a cytotoxic effector molecule of the immune system is clearly established, but recent studies demonstrate cytoprotective functions of NO at low nontoxic concentrations. However, the mechanism of cytoprotection has not been defined completely. Thus, we investigate the involvement of heme oxygenase-1 (HO-1) in the cytoprotective effects of NO. Exposure of L929 cells to sodium nitroprusside (SNP) resulted in the induction of HO-1 protein expression and heme oxygenase activity. Pretreatment of the cells with a low dose of NO (200 microM SNP) significantly inhibited a high dose of (1000 microM SNP) NO-induced apoptosis in L929 cells. Cytoprotection by a low dose of NO was abrogated in the presence of the heme oxygenase inhibitor zinc protoporphyrin IX. A cytoprotective effect comparable to a low dose of SNP was observed when the cells were transfected with HO-1 gene or preincubated with another HO-1 inducer, hemin. Additional experiments revealed the involvement of carbon monoxide in the cytoprotective effect of SNP/HO-1 in L929 cells. Our results presented here provide evidence to support the essential role of HO-1 in the cytoprotective function of NO priming.     

Role of heme oxygenase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to carbon monoxide, bilirubin, and iron. All three products possess biological functions; bilirubin, in particular, is a potent free radical scavenger of which its antioxidant property is enhanced at low oxygen tension. Here, we investigated the effect of severe hypoxia and reoxygenation on HO-1 expression in cardiomyocytes and determined whether HO-1 and its product, bilirubin, have a protective role against reoxygenation damage. Hypoxia caused a time-dependent increase in both HO-1 expression and heme oxygenase activity, which gradually declined during reoxygenation. Reoxygenation of hypoxic cardiomyocytes produced marked injury; however, incubation with hemin or bilirubin during hypoxia considerably reduced the damage at reoxygenation. The protective effect of hemin is attributable to increased availability of substrate for heme oxygenase activity, because hypoxic cardiomyocytes generated very little bilirubin when incubated with medium alone but produced substantial bile pigment in the presence of hemin. Interestingly, incubation with hemin also maintained high heme oxygenase activity levels during the reoxygenation period. Reactive oxygen species generation was enhanced after hypoxia, and hemin and bilirubin were capable once again to attenuate this effect. These results indicate that the HO-1-bilirubin pathway can effectively defend hypoxic cardiomyocytes against reoxygenation injury and highlight the issue of heme availability in the cytoprotective action afforded by HO-1.     

Activated microglia are less vulnerable to hemin toxicity due to nitric oxide-dependent inhibition of JNK and p38 MAPK activation

In intracerebral hemorrhage, microglia become rapidly activated and remove the deposited blood and cellular debris. To survive in a harmful hemorrhagic or posthemorrhagic condition, activated microglia must be equipped with appropriate self-defensive mechanism(s) to resist the toxicity of hemin, a component released from damaged RBCs. In the current study, we found that activation of microglia by pretreatment with LPS markedly reduced their vulnerability to hemin toxicity in vitro. Similarly, intracorpus callosum microinjection of LPS prior to hemin treatment reduced the brain tissue damage caused by hemin and increased microglial density in the penumbra in rats. LPS induced the expressions of inducible NO synthase (iNOS) and heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation in microglia. The preventive effect by LPS was significantly diminished by an iNOS inhibitor, L-N(6)-(1-iminoethyl)lysine, whereas it was mimicked by a NO donor, diethylamine-NONOate, both suggesting the crucial role of NO in the modulation of hemin-induced toxicity in activated microglia. We further found that NO reduced hemin toxicity via inhibition of hemin-induced activation of JNK and p38 MAPK pathways in microglia. Whereas HO-1 expression in LPS-stimulated microglia was markedly blocked by L-N(6)-(1-iminoethyl)lysine, the HO-1 inhibitor, tin protoporphyrin, increased iNOS expression and decreased the susceptibility of LPS-activated microglia to hemin toxicity. The data indicate that the mutual interaction between NO and HO-1 plays a critical role in modulating the adaptive response of activated microglia to hemin toxicity. Better understanding of the survival mechanism of activated microglia may provide a therapeutic strategy to attenuate the devastating intracerebral hemorrhagic injury.     

Induction of heme oxygenase 1 by nitrosative stress. A role for nitroxyl anion

Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO(+)), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997) J. Biol. Chem. 272, 18411-18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613-13620). Here, we report that Angeli's salt (AS) (0.25-2 mm), a compound that releases nitroxyl anion (NO(-)) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO(-)-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 degrees C (t(12) = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 microm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO(-), not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO(-) oxidation. N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO(-). We conclude that NO(-), in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.     

Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported (34) that chronic hemin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 muM for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at G0/G1 phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, hemin-induced HO-1 expression was further increased in the presence of SnPP. Hemin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of hemin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without hemin for 7 days. The inhibitory effect of hemin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin.     

Endothelial heme oxygenase-1 induction by hypoxia. Modulation by inducible nitric-oxide synthase and S-nitrosothiols

The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411-18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO(2) 相似文献   

Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell lines

Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2.     

Bilirubin and ferritin as protectors against hemin-induced oxidative stress in rat liver.

The in vivo effect of hemin on both hepatic oxidative stress and heme oxygenase induction was studied. A marked increase in lipid peroxidation was observed 1 hr after hemin administration. Heme oxygenase-1 activity and expression appeared 6 hr after treatment, reaching a maximum between 12 and 15 hr after hemin administration. Such induction was preceded by a decrease in the soluble and enzymatic defenses, both effects taking place some hours before induction of heme oxygenase. Ferritin content began to increase 6 hr after heme oxygenase induction, and these increases were significantly higher 15 hr after treatment and remained high for at least 24 hr after hemin injection. Co-administration of tin protoporphyrin IX, a potent inhibitor of heme oxygenase, completely prevented the enzyme induction and the increase in ferritin levels, increasing the appearance of oxidative stress parameters. Administration of bilirubin, prevented the heme oxygenase induction as well as the decrease in hepatic GSH and the increase of lipid peroxidation when it was administered 2 hr before hemin treatment. These results indicate that the induction of heme oxygenase by hemin may be a general response to oxidant stress, by increasing bilirubin and ferritin levels and could therefore provide a major cellular defense mechanism against oxidative damage.     

Hyperglycemia in streptozotocin-induced diabetes leads to persistent inflammation and tissue damage following uveitis due to reduced levels of ciliary body heme oxygenase-1

This study investigated the heme oxygenase-1 (HO-1) and the endotoxin-induced uveitis (EIU) in diabetic streptozotocin (STZ)-hyperglycemic rats. STZ-hyperglycemic rats had impaired levels of the enzyme HO-1 within the ciliary bodies if compared with the nondiabetic rats. STZ-hyperglycemic rats also predisposed the eye to produce high levels of both the cytokines IL-1beta and CXCL8. Subsequent EIU further and significantly (P < .01) increased the cytokines production, an effect partly prevented by hemin treatment. Most importantly, hemin, an inducer of heme oxygenase expression and activity, recovered the huge number of infiltrated polymorphonuclear leukocytes PMN within the ciliary bodies associated with STZ-hyperglycemic state and EIU damage. Impairment of the stress-sensitive enzyme HO-1 in STZ-hyperglycemic rats increases and prolongs the inflammatory response to EIU.     

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.     

Heme oxygenase-1 participates in the anti-inflammatory activity of taurine chloramine

Interleukin-6 (IL-6) and interleukin-8 (IL-8) are implicated in the pathogenesis of rheumatic diseases. In affected joints fibroblast-like synoviocytes (FLS) are the major source of these pro-inflammatory cytokines. We have previously found that production of both cytokines is inhibited in vitro by taurine chloramine (Tau-Cl). Heme oxygenase (HO-1) activity was also reported to restrict synthesis of various inflammatory mediators, including IL-6 and IL-8. The aim of present study was to investigate whether this enzyme activity is implicated in the mechanism of Tau-Cl suppressive effect. We have shown that in rheumatoid FLS both hemin (known HO-1 inducer) and Tau-Cl significantly up-regulate HO-1 expression at the mRNA and protein levels and simultaneously inhibit IL-1β-triggered production of pro-inflammatory cytokines. However, the inhibitory potency of these compounds differs, because hemin is more potent inhibitor of IL-8 than IL-6 production, while Tau-Cl exerts opposite effect. Importantly, pretreatment of the cells with HO-1 inhibitor completely reverses the inhibitory effect of hemin on both cytokines production. However, in Tau-Cl treated cells this inhibitor fully restores only IL-8 secretion but has weaker effect on IL-6 response. Thus, the present results: (i) support HO-1 activity to be relevant to negatively control production of pro-inflammatory cytokines, and (ii) underline implication of HO-1 in mediating Tau-Cl inhibitory action.     

Homocysteine attenuates endothelial haem oxygenase-1 induction by nitric oxide (NO) and hypoxia.

The disrupted metabolism of homocysteine (Hcy) causes hyperhomocysteinemia, a condition associated with the impairment of nitric oxide (NO) bio-availability, tissue hypoxia and increased risk of vascular disease. Here, we examined how Hcy modulates the induction of the stress protein haem oxygenase-1 (HO-1) evoked by NO releasing agents and hypoxia in vascular endothelial cells. We found that Hcy (0.5 mM) markedly reduced the increase in haem oxygenase activity and HO-1 protein expression induced by sodium nitroprusside (SNP, 0.5 mM) but did not affect HO-1 activation mediated by S-nitroso-N-acetyl-penicillamine. Cells pre-treated with Hcy followed by addition of fresh medium containing SNP still exhibited an augmented haem oxygenase activity. Interestingly, high levels of Hcy were also able to abolish hypoxia-mediated HO-1 expression in a concentration-dependent manner. These novel findings indicate that hyperhomocysteinemia interferes with crucial signaling pathways required by cells to respond and adapt to stressful conditions.     

BMP6 attenuates oxidant injury in HK-2 cells via Smad-dependent HO-1 induction

Oxidative stress is involved in a variety of kidney diseases, and heme oxygenase 1 (HO-1) induction is a protective response to oxidative stress. Downregulation of bone morphogenetic protein 6 (BMP6) is associated with renal damage in intrauterine growth-restricted newborns. However, it is unknown whether BMP6 has a renoprotective effect or HO-1 induction property. In this study, we demonstrate that BMP6 effectively protects renal proximal tubule cells (HK-2) against hydrogen peroxide (H₂O₂)-induced cell injury. BMP6 also increased HO-1 gene expression and activity of HO. Inhibition of de novo gene expression, the HO inhibitor ZnPPIX, HO-1 knockdown, or the carbon monoxide (CO) scavenger hemoglobin attenuated the cytoprotective effect of BMP6, whereas HO-1 constitutive expression, the HO-1 inducer hemin, or the hemin metabolites bilirubin and CO ameliorated H₂O₂-induced cell injury. Stimulation of HK-2 cells with BMP6 activated Smad signaling but not mitogen-activated protein kinases. In addition, BMP6-mediated induction of HO-1 expression and increase in HO activity were inhibited by Smad5 knockdown. Furthermore, deletion or mutation of the Smad-binding element in the HO-1 promoter also inhibited BMP6-induced luciferase activity. In summary, these findings suggest that induction of HO-1 through a Smad-dependent mechanism is responsible for the cytoprotective effect of BMP6 in H₂O₂-mediated renal cell injury.     

Pentoxifylline protects L929 fibroblasts from TNF-alpha toxicity via the induction of heme oxygenase-1

Tumor necrosis factor-alpha (TNF-alpha) is recognized as a principal mediator of a variety of inflammatory conditions. Pentoxifylline (PTX), which can inhibit cellular TNF-alpha synthesis, also attenuates the toxic effect of TNF-alpha. However, the mechanism underlying PTX-induced cytoprotection is unknown. Heme oxygenase 1 (HO-1) is an enzyme which degrades heme into biliverdin, free iron, and carbon monoxide (CO). This enzyme has recently been shown to have anti-inflammatory and cytoprotective effects. In this study, we investigated whether protection by PTX against TNF-alpha-mediated toxicity could be related to its ability to induce HO-1 expression and HO activity in L929 cells. PTX in the range of 0.1-1.0mM significantly induced HO-1 expression and the resulting HO activity. Pre-incubation of L929 cells with either PTX or the HO activator hemin resulted in the protection of the cells against TNF-alpha-mediated toxicity. Zinc protoporphyrin, a specific HO competitive inhibitor, abrogated the protective effect of PTX. Hemoglobin, a scavenger of CO, reversed the protective effect of PTX. A cytoprotection comparable to PTX was observed when the cells were treated with the CO-releasing compound tricarbonyldichlororuthenium(II) dimer. These results suggest that HO-1 expression and the ensuing formation of the HO metabolite CO may be a novel pathway by which PTX protects L929 cells from TNF-alpha-mediated toxicity.     

Pro-oxidant role of heme oxygenase in mediating glucose-induced endothelial cell damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors. Human umbilical vein endothelial cells (HUVECs) were exposed to low (5mmol/l) and high (25mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10 micromol/l), HO antagonist (SnPPIX, 10 micromol/l), and NO synthase blocker (L-NAME, 200 micromol/l) with or without NO donor (arginine, 1 mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Anti-inflammatory effect of heme oxygenase 1: glycosylation and nitric oxide inhibition in macrophages

Flavonoids including the aglycones, hesperetin (HT; 5,7,3'-trihydroxy-4'-methoxy-flavanone), and naringenin (NE; 5,7,4'-trihydroxy flavanone) and glycones, hesperidin (HD; 5,7,3'-trihydroxy-4'-methoxy-flavanone 7-rhamnoglucoside) and naringin (NI; 5,7,4'-trihydroxy flavanone 7-rhamno glucoside), were used to examine the importance of rutinose at C7 on the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced nitric oxide production in macrophages. Both HT and NE, but not their respective glycosides HD and NI, induced heme oxygenase 1 (HO-1) protein expression in the presence or absence of LPS and showed time and dose-dependent inhibition of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW264.7, J774A.1, and thioglycolate-elicited peritoneal macrophages. Additive inhibitory effect of an HO-1 inducer hemin and NE or NI on LPS-induced NO production and iNOS expression was identified, and HO enzyme inhibitor tin protoporphyrin (SnPP) attenuated the inhibitory effects of HT, NE, and hemin on LPS-induced NO production. Both NE and HT showed no effect on iNOS mRNA and protein stability in RAW264.7 cells. Removal of rutinose at C7 of HD and NI by enzymatic digestion using hesperidinase (HDase) and naringinase (NIase) produce inhibitory activity on LPS-induced NO production, according to the production of the aglycones, HT and NE, by high-performance liquid chromatography (HPLC) analysis. Furthermore, the amount of NO produced by LPS or lipoteichoic acid (LTA) was significantly reduced in HO-1-overexpressing cells (HO-1/RAW264.7) compared to that in parental cells (RAW264.7). Results of the present study provide scientific evidence to suggest that rutinose at C7 is a negative moiety in flavonoid inhibition of LPS-induced NO production, and that HO-1 is involved in the inhibitory mechanism of flavonoids on LPS-induced iNOS and NO production.