Induction Of Heme Oxygenase-1 Gene Expression Research Articles

Delivery of Hypoxia-Inducible Heme Oxygenase-1 Gene for Site-Specific Gene Therapy in the Ischemic Stroke Animal Model.

To reduce side effects due to non-specific expression, the heme oxygenase-1 (HO-1) gene under control of a hypoxia-inducible erythropoietin (Epo) enhancer (pEpo-SV-HO-1) was developed for site-specific gene therapy of ischemic stroke. pEpo-SV-HO-1 was constructed by insertion of the Epo enhancer into pSV-HO-1. Dexamethasone-conjugated polyamidoamine (PAMAM-Dexa) was used as a gene carrier. In vitro transfection assays were performed in the Neuro2A cells. In vivo efficacy of pEpo-SV-HO-1 was evaluated in the transient middle cerebral artery occlusion (MCAO) model. In vitro transfection assay with the PAMAM-Dexa/pEpo-SV-HO-1 complex showed that pEpo-SV-HO-1 had higher HO-1 gene expression than pSV-HO-1 under hypoxia. In addition, pEpo-SV-HO-1 reduced the level of apoptosis more efficiently than pSV-HO-1 in Neuro2A cells under hypoxia. For in vivo evaluation, the PAMAM-Dexa/pEpo-SV-HO-1 complex was injected into the ischemic brain of the transient MCAO model. pEpo-SV-HO-1 increased HO-1 expression and reduced the number of apoptotic cells in the ischemic brain, compared with the pSV-HO-1 injection group. As a result, the infarct volume was more efficiently decreased by pEpo-SV-HO-1 than by pSV-HO-1. pEpo-SV-HO-1 induced HO-1 gene expression and therapeutic effect in the ischemic brain. Therefore, pEpo-SV-HO-1 may be useful for site-specific gene therapy of ischemic stroke.

Myrosinase-treated glucoerucin is a potent inducer of the Nrf2 target gene heme oxygenase 1 — studies in cultured HT-29 cells and mice

In this study, the effect of myrosinase-treated glucoerucin (GER+MYR), which releases the isothiocyanate (ITC) erucin, on heme oxygenase 1 (HO-1) gene expression and Nrf2 signaling was investigated in vitro in cultured cells and in vivo in mice. Treatment of HT-29 cells with GER+MYR resulted in a significant increase in the mRNA and protein levels of nuclear Nrf2 and HO-1. GER+MYR was more potent at enhancing the nuclear Nrf2 levels than were the following myrosinase-treated glucosinolates: sinigrin, glucoraphanin and gluconasturtiin, which are the precursors of allyl-ITC, R-sulforaphane and 2-phenylethyl ITC, respectively. GER+MYR also significantly induced HO-1 gene expression in the mouse intestinal mucosae and liver but not in the brain. Mechanistic studies suggest that GER+MYR induces Nrf2 via ERK1/2-, p38- and JNK-dependent signal transduction pathways. The GER+MYR-mediated increase in HO-1 expression is primarily attributable to p38 signaling.

β-Cyclodextrin-hemin complex-induced lateral root formation in tomato: involvement of nitric oxide and heme oxygenase 1.

β-Cyclodextrin-hemin complex-induced tomato lateral root formation was associated with nitric oxide and heme oxygenase 1 by modulating cell cycle regulatory genes. β-Cyclodextrin-hemin complex (β-CDH), a complex by combining β-cyclodextrin (β-CD) with hemin, a heme oxygenase 1 (HO1) inducer, was a trigger of cucumber adventitious root formation by enhancing HO1 gene expression. In this report, our results identified the previously unknown function of β-CDH in plants: the inducer of tomato lateral root (LR) formation. β-CDH-triggered LR formation is hemin-specific, since β-CD failed to induce LR development. Because nitric oxide (NO) is involved in LR formation, the correlation of β-CDH with NO and HO1 was investigated. Our analysis suggested that β-CDH induced an increase in endogenous NO production, followed by up-regulation of tomato HO1 gene and LR formation, all of which were mimicked by hemin and two NO-releasing compounds (SNP and GSNO). The induction of HO1 gene expression and LR formation triggered by β-CDH or hemin were significantly blocked by an inhibitor of HO1. Further results revealed that both β-CDH- and SNP-stimulated HO1 gene expression and thereafter LR formation were sensitive to the removal of NO with a potent NO scavenger, and the responses of SNP were significantly blocked by an inhibitor of HO1. Molecular evidence illustrated that representative cell cycle regulatory genes, including SlCDKA1, SlCYCA3;1, SlCYCA2;1, and SlCYCD3;1, were significantly up-regulated by β-CDH and SNP, but obviously blocked when seedlings were co-treated with the scavenger of NO or the inhibitor of HO1. In summary, our physiological and molecular evidence demonstrated that both NO and HO1 were involved in the β-CDH-induced LR formation with, at least partially, HO1 acting downstream of NO signaling.

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

Xyloketal B Exhibits Its Antioxidant Activity through Induction of HO-1 in Vascular Endothelial Cells and Zebrafish

We previously reported that a novel marine compound, xyloketal B, has strong antioxidative actions in different models of cardiovascular diseases. Induction of heme oxygenase-1 (HO-1), an important endogenous antioxidant enzyme, has been considered as a potential therapeutic strategy for cardiovascular diseases. We here investigated whether xyloketal B exhibits its antioxidant activity through induction of HO-1. In human umbilical vein endothelial cells (HUVECs), xyloketal B significantly induced HO-1 gene expression and translocation of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) in a concentration- and time-dependent manner. The protection of xyloketal B against angiotensin II-induced apoptosis and reactive oxygen species (ROS) production could be abrogated by the HO-1 specific inhibitor, tin protoporphyrin-IX (SnPP). Consistently, the suppressive effects of xyloketal B on NADPH oxidase activity could be reversed by SnPP in zebrafish embryos. In addition, xyloketal B induced Akt and Erk1/2 phosphorylation in a concentration- and time-dependent manner. Furthermore, PI3K inhibitor LY294002 and Erk1/2 inhibitor U0126 suppressed the induction of HO-1 and translocation of Nrf-2 by xyloketal B, whereas P38 inhibitor SB203580 did not. In conclusion, xyloketal B can induce HO-1 expression via PI3K/Akt/Nrf-2 pathways, and the induction of HO-1 is mainly responsible for the antioxidant and antiapoptotic actions of xyloketal B.

Clofibrate Induces Heme Oxygenase 1 Expression through a PPARα-Independent Mechanism in Human Cancer Cells

Background and Aims: Clofibrate, an established PPARα ligand, has recently been shown to have anticancer activity yet its mechanisms of action remain to be characterized. This study examined the effect of clofibrate on heme oxygenase-1 (HO-1) gene expression in A2780 (human ovarian cancer) and DU145 (human prostate cancer) cells. Methods and Results: We demonstrate that clofibrate induces HO-1 expression in a concentration- and time-dependent manner. The induction of HO-1 by clofibrate was detected at both mRNA and protein levels and the HO-1 gene promoter activity was also dramatically induced by clofibrate, indicating that clofibrate up-regulates HO-1 gene transcription. Surprisingly, the induction of HO-1 by clofibrate was mediated by the Nrf2 signaling pathway, not by the PPARα pathway. This was primarily demonstrated by siRNA knockdown of Nrf2 expression that significantly attenuated clofibrate-induced HO-1 gene transcription, and siRNA knockdown of PPARα that had no effect on clofibrate-induced HO-1 promoter activity. Furthermore, deletion of the antioxidant response elements (AREs) in the HO-1 gene promoter diminished clofibrate-induced HO-1 transcription and deletion of the PPAR response elements (PPREs) had no such effect. Likewise, application of PPARα antagonists had no effect on clofibrate-induced HO-1 expression. Conclusion: Clofibrate induces HO-1 gene expression in cancer cells through a PPARα-independent mechanism and the Nrf2 signaling pathway is indispensible for this induction.

Protective Roles of CX3CR1-Mediated Signals in Toxin A-Induced Enteritis through the Induction of Heme Oxygenase-1 Expression

The injection of Clostridium difficile toxin A into the ileal loops caused fluid accumulation with the destruction of intestinal epithelial structure and the recruitment of neutrophils and macrophages. Concomitantly, intraileal gene expression of CX3CL1/fractalkine (FKN) and its receptor, CX3CR1, was enhanced. When treated with toxin A in a similar manner, CX3CR1-deficient (CX3CR1(-/-)) mice exhibited exaggerated fluid accumulation, histopathological alterations, and neutrophil recruitment, but not macrophage infiltration. Mice reconstituted with CX3CR1(-/-) mouse-derived bone marrow cells exhibited exacerbated toxin A-induced enteritis, indicating that the lack of the CX3CR1 gene for hematopoietic cells aggravated toxin A-induced enteritis. A heme oxygenase-1 (HO-1) inhibitor, tin-protoporphyrin-IX, markedly increased fluid accumulation in toxin A-treated wild-type mice, indicating the protective roles of HO-1 in this situation. HO-1 expression was detected mainly in F4/80-positive cells expressing CX3CR1, and CX3CR1(-/-) mice failed to increase HO-1 expression after toxin A treatment. Moreover, CX3CL1/FKN induced HO-1 gene expression by isolated lamina propria-derived macrophages or a mouse macrophage cell line, RAW264.7, through the activation of the ERK signal pathway. Thus, CX3CL1/FKN could induce CX3CR1-expressing macrophages to express HO-1, thereby ameliorating toxin A-induced enteritis.

Up-regulation of heme oxygenase-1 by sevoflurane is not dependent on Kupffer cells and associates with ERK1/2 and AP-1 activation in the rat liver

Sevoflurane is a potent non-toxic inducer of the hepatoprotective enzyme heme oxygenase-1 (HO-1). So far, little is known about the underlying molecular mechanism. Therefore the aim of this study was to characterize the respective signal transduction pathway and in particular to elucidate the role of Kupffer cells in this context. Rats were treated with or without sevoflurane. The effects on hepatic HO-1 gene expression, mitogen-activated protein kinases and transcription factors were studied by Northern and Western blot analyses, immunostaining, electrophoretic mobility shift assays, and enzymatic activity assays. Kupffer cells were depleted by administration of clodronate liposomes in vivo to characterize their role in HO-1 signal transduction. In additional in vitro experiments, HO-1 mRNA expression in primary rat hepatocytes and HepG2 cells was assessed. Sevoflurane up-regulated HO-1 gene expression in pericentral hepatocytes and increased HO enzyme activity in vivo. This was associated with activation of ERK1/2 and activator protein-1. We identified c-jun/AP-1, JunD, c-fos, and Fra-1 as active subunits of the activator protein-1 complex. Administration of clodronate liposomes to rats led to depletion of Kupffer cells without affecting sevoflurane induced HO-1 expression. Moreover, sevoflurane up-regulated HO-1 mRNA in primary rat hepatocytes but not in HepG2 cells. Our results suggest that sevoflurane induced HO-1 gene expression in pericentral hepatocytes does not depend on Kupffer cells and is associated with activation of ERK1/2 and activator protein-1. Since we could recently demonstrate significant hepatoprotective effects of HO-1 induced by isoflurane, the present results may help to establish new concepts in hepatic organ protection.

Rottlerin induces heme oxygenase-1 (HO-1) up-regulation through reactive oxygen species (ROS) dependent and PKC δ-independent pathway in human colon cancer HT29 cells

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by its substrate heme and diverse stimuli. The induction of HO-1 gene expression is one of the important events in cellular response to pro-oxidative and pro-inflammatory insults. In this study, the effect of rottlerin, a putative PKC δ inhibitor, on HO-1 expression in HT29 human colon cancer cells was investigated. Rottlerin-induced HO-1 at both protein and mRNA levels in a dose- and time-dependent manner. Rottlerin-mediated HO-1 induction was abrogated in the presence of N-acetylcysteine (NAC) or glutathione (GSH). Rottlerin induced nuclear translocation of NF-E2-related factor 2 (Nrf2) and increased antioxidant response element (ARE)-driven transcriptional activity. Additionally, rottlerin activated p38 mitogen-activated protein kinase (MAPK) and ERK. The pharmacological inhibition of ERK and p38 MAPK inhibited rottlerin-induced HO-1 up-regulation. However, suppression of protein kinase C δ (PKC δ) expression by siRNA or overexpression of WT-PKC δ did not abrogate the rottlerin-mediated induction of HO-1. These results suggest that rottlerin induces up-regulation of HO-1 via PKC δ-independent pathway. Taken together, the present study identified rottlerin as a novel inducer of HO-1 expression and identified the mechanisms involved in this process.

Diabetes Impairs the Vascular Recruitment of Normal Stem Cells by Oxidant Damage, Reversed by Increases in pAMPK, Heme Oxygenase-1, and Adiponectin

BackgroundAtherosclerosis progression is accelerated in diabetes mellitus (DM) by either direct endothelial damage or reduced availability and function of endothelial progenitor cells (EPCs). Both alterations are related to increased oxidant damage.AimWe examined if DM specifically impairs vascular signaling, thereby reducing the recruitment of normal EPCs, and if increases in antioxidant levels by induction of heme oxygenase-1 (HO-1) can reverse this condition.MethodsControl and diabetic rats were treated with the HO-1 inducer cobalt protoporphyrin (CoPP) once a week for 3 weeks. Eight weeks after the development of diabetes, EPCs harvested from the aorta of syngenic inbred normal rats and labeled with technetium-99m-exametazime were infused via the femoral vein to estimate their blood clearance and aortic recruitment. Circulating endothelial cells (CECs) and the aortic expression of thrombomodulin (TM), CD31, and endothelial nitric oxide synthase (eNOS) were used to measure endothelial damage.ResultsDM reduced blood clearance and aortic recruitment of EPCs. Both parameters were returned to control levels by CoPP treatment without affecting EPC kinetics in normal animals. These abnormalities of EPCs in DM were paralleled by reduced serum adiponectin levels, increased numbers of CECs, reduced endothelial expression of phosphorylated eNOS, and reduced levels of TM, CD31, and phosphorylated AMP-activated protein kinase (pAMPK). CoPP treatment restored all of these parameters to normal levels.ConclusionType II DM and its related oxidant damage hamper the interaction between the vascular wall and normal EPCs by mechanisms that are, at least partially, reversed by the induction of HO-1 gene expression, adiponectin, and pAMPK levels.

Molecular mechanism and functional consequences of lansoprazole-mediated heme oxygenase-1 induction

To investigate the molecular mechanism and functional consequences of heme oxygenase-1 (HO-1) activation by lansoprazole in endothelial cells and macrophages. Expression of HO-1 mRNA was analyzed by Northern blotting. Western blotting was used to determine the HO-1 and ferritin protein levels. NADPH-dependent reactive oxygen species (ROS) formation was measured with lucigenin-enhanced chemiluminescence. HO-1 promoter activity in mouse fibroblasts, stably transfected with a 15-kb HO-1 gene that drives expression of the reporter gene luciferase, was assessed using in vivo bioluminescence imaging. Lansoprazole increased HO-1 mRNA levels in endothelial cells and HO-1 protein levels in macrophages. In addition, lansoprazole-induced ferritin protein levels in both cell systems. Moreover, induction of the antioxidant proteins HO-1 and ferritin by lansoprazole was followed by a decrease in NADPH-mediated ROS formation. The radical scavenging properties of lansoprazole were diminished in the presence of the HO inhibitor, chromium mesoporphyrin IX. Induction of HO-1 gene expression by lansoprazole was not related to oxidative stress or to the activation of the mitogen-activated protein kinase pathway. However, the phosphatidylinositol 3-kinase inhibitor LY294002 showed a concentration-dependent inhibition of HO-1 mRNA and promoter activity. Activation of HO-1 and ferritin may account for the gastric protection of lansoprazole and is dependent on a pathway blocked by LY294002.

Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.

Heme Oxygenase-1 Inhibits the Proliferation of Pancreatic Stellate Cells by Repression of the Extracellular Signal-Regulated Kinase1/2 Pathway

Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.

Enzymatic metabolites of lycopene induce Nrf2‐mediated expression of phase II detoxifying/antioxidant enzymes in human bronchial epithelial cells

Lycopene can be cleaved by carotene 9',10'-oxygenase at its 9',10' double bond to form apo-10'-lycopenoids, including apo-10'-lycopenal, -lycopenol and -lycopenoic acid. The latter has been recently shown to inhibit lung carcinogenesis both in vivo and in vitro, however, the mechanism(s) underlying this protection is not well defined. In the present study, we report that treatment with apo-10'-lycopenoic acid, in a time- and dose-dependent manner, results in the nuclear accumulation of transcription factor Nrf2 (nuclear factor E(2)-related factor 2) protein in BEAS-2B human bronchial epithelial cells. The activation of Nrf2 by apo-10'-lycopenoic acid is associated with the induction of phase II detoxifying/antioxidant enzymes including heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate-cysteine ligases in BEAS-2B cells. Furthermore, apo-10'-lycopenoic acid treatment increased total intracellular glutathione levels and suppressed both endogenous reactive oxygen species generation and H(2)O(2)-induced oxidative damage in BEAS-2B cells. In addition, both apo-10'-lycopenol and apo-10'-lycopenal induced heme oxygenase-1 gene expression in BEAS-2B cells. These data strongly suggest that the anti-carcinogenic and antioxidant functions of lycopene may be mediated by apo-10'-lycopenoids via activating Nrf2 and inducing phase II detoxifying/antioxidant enzymes.

Heme oxygenase-1 is a novel target and antioxidant mediator of S-adenosylmethionine

The sulfur compound and dietary supplement S-adenosylmethionine (SAM) has been reported to have cytoprotective and antioxidant properties. However, the underlying mechanisms remain unresolved. The present study investigates the effect of SAM on the expression of the antioxidant stress proteins heme oxygenase-1 (HO-1) and ferritin in endothelial cells. Induction of the HO-1/ferritin-system leads to protection of tissues against several inflammatory stimuli. SAM increased the protein and mRNA levels of HO-1 in cultured endothelial cells. Induction of HO-1 gene expression was associated with elevated ferritin protein levels and regulated at the transcriptional level via increased promoter activity. HO-1 upregulation by SAM was causally related to a decrease in NADPH-mediated production of oxygen radicals. Our results demonstrate that the HO-1/ferritin-system is a novel target of the antioxidant compound SAM.

Baicalein inhibition of hydrogen peroxide-induced apoptosis via ROS-dependent heme oxygenase 1 gene expression

In the present study, baicalein (BE) but not its glycoside, baicalin (BI), induced heme oxygenase-1 (HO-1) gene expression at both the mRNA and protein levels, and the BE-induced HO-1 protein was blocked by adding cycloheximide (CHX) or actinomycin D (Act D). Activation of ERK, but not JNK or p38, proteins via induction of phosphorylation in accordance with increasing intracellular peroxide levels was detected in BE-treated RAW264.7 macrophages. The addition of the ERK inhibitor, PD98059, (but not the p38 inhibitor, SB203580, or the JNK inhibitor, SP600125) and the chemical antioxidant, N-acetyl cysteine (NAC), significantly reduced BE-induced HO-1 protein expression by respectively blocking ERK protein phosphorylation and intracellular peroxide production. Additionally, BE but not BI effectively protected RAW264.7 cells from hydrogen peroxide (H 2O 2)-induced cytotoxicity, and the preventive effect was attenuated by the addition of the HO inhibitor, SnPP, and the ERK inhibitor, PD98059. H 2O 2-induced apoptotic events including hypodiploid cells, DNA fragmentation, activation of caspase 3 enzyme activity, and a loss in the mitochondrial membrane potential with the concomitant release of cytochrome c from mitochondria to the cytosol were suppressed by the addition of BE but not BI. Blocking HO-1 protein expression by the HO-1 antisense oligonucleotide attenuated the protective effect of BE against H 2O 2-induced apoptosis by suppressing HO-1 gene expression in macrophages. Overexpression of the HO-1 protein inhibited H 2O 2-induced apoptotic events such as DNA fragmentation and hypodiploid cells by reducing intracellular peroxide production induced by H 2O 2, compared with those events in neo-control (neo-RAW264.7) cells. In addition, CO, but not bilirubin and biliverdin, addition inhibits H 2O 2-induced cytotoxicity in macrophages. It suggests that CO can be responsible for the protective effect associated with HO-1 overexpression. The notion of induction of HO-1 gene expression through a ROS-dependent manner suppressing H 2O 2-induced cell death is identified in the present study.

Nitric oxide stimulates heme oxygenase-1 gene transcription via the Nrf2/ARE complex to promote vascular smooth muscle cell survival

Previous studies from our laboratory and others found that NO is a potent inducer of heme oxygenase-1 (HO-1) gene transcription in vascular smooth muscle cells (SMC), however, the mechanism responsible for the induction of HO-1 gene expression has not been elucidated. In the present study, we determined the signaling pathway responsible for the induction of HO-1 and its biological significance. Cultured rat aortic SMC were exposed to nitrosative stress by treating cells with various NO donors or with inflammatory cytokines. Nitrosative stress stimulated an increase in HO-1 mRNA expression and promoter activity in vascular SMC. However, mutation of the antioxidant response element (ARE) in the HO-1 promoter or overexpression of a dominant-negative mutant of NF-E2-related factor-2 (Nrf2) abrogated the activation by NO. Electromobility shift assays using an ARE probe detected a complex that was significantly increased in intensity by NO. In addition, the migration of this complex was retarded by using an antibody directed against Nrf2. NO also increased Nrf2 mRNA expression, total and nuclear Nrf2 levels, and the binding of Nrf2 to the HO-1 promoter. Finally, treatment of SMC with NO stimulated apoptosis that was increased by HO-1 inhibition. These results demonstrate that nitrosative stress induces HO-1 gene transcription through the activation of the Nrf2/ARE complex to counteract NO-induced apoptosis of vascular SMC. The capacity of nitrosative stress to activate Nrf2 and stimulate HO-1 gene transcription may represent a critical adaptive response to maintain cell viability at sites of vascular inflammation and atherosclerosis.

Adrenomedullin induces heme oxygenase-1 gene expression and cGMP formation in rat vascular smooth muscle cells

Adrenomedullin (ADM) is a potent vasodilatory peptide. It regulates blood pressure by increasing cyclic adenosine monophosphate (cAMP) and guanosine-3′,5′-monophosphate (cGMP). We sought to investigate the effect of ADM on heme oxygenase-1 (HO-1) gene expression and cGMP formation in cultured rat vascular smooth muscle cells (VSMCs). ADM treatment, 10 −9 and 10 −8 mol/L, increased cGMP production, and it increased the intracellular cGMP content of platelets coincubated with VSMCs. It increased cGMP content by 158.8% and 273.5%, respectively; increased HO-1 activity by 49.5% and 87%, respectively; augmented HO-1 protein levels by 66% and 126%, respectively; upregulated the steady-state level of HO-1 mRNA by 73% and 159%, respectively, and increased HO-1 mRNA transcription synthesis by four- and seven-fold, respectively. These results suggest that ADM induces HO-1 gene expression and cGMP formation in rat VSMCs.

15-Deoxy-Δ12,14-prostaglandin J2 Induces Heme Oxygenase-1 Gene Expression in a Reactive Oxygen Species-dependent Manner in Human Lymphocytes

15-Deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2) has been recently proposed as a potent anti-inflammatory agent. However, the mechanisms by which 15dPGJ(2) mediates its therapeutic effects in vivo are unclear. We demonstrate that 15dPGJ(2) at micromolar (2.5-10 microm) concentrations induces the expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, at both mRNA and protein levels in human lymphocytes. In contrast, troglitazone and ciglitazone, two thiazolidinediones that mimic several effects of 15dPGJ(2) through their binding to the peroxisome proliferator-activated receptor (PPAR)-gamma, did not affect HO-1 expression, and the positive effect of 15dPGJ(2) on this process was mimicked instead by other cyclopentenone prostaglandins (PG), such as PGD(2) (the precursor of 15dPGJ(2)) and PGA(1) and PGA(2) which do not interact with PPAR-gamma. Also, 15dPGJ(2) enhanced the intracellular production of reactive oxygen species (ROS) and increased xanthine oxidase activity in vitro. Inhibition of intracellular ROS production by N-acetylcysteine, TEMPO, Me(2)SO, 1,10-phenanthroline, or allopurinol resulted in a decreased 15dPGJ(2)-dependent HO-1 expression in the cells. Furthermore, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe(2+)/Cu(2+) ions enhanced the positive effect of 15dPGJ(2) on HO-1 expression. On the other hand, the inhibition of phosphatidylinositol 3-kinase or p38 mitogen-activated protein kinase, or the blockade of transcription factor NF-kappaB activation, hindered 15dPGJ(2)-elicited HO-1 expression. Collectively, the present data suggest that 15dPGJ(2) anti-inflammatory actions at pharmacological concentrations involve the induction of HO-1 gene expression through mechanisms independent of PPAR-gamma activation and dependent on ROS produced via the xanthine/xanthine oxidase system and/or through Fenton reactions. Both phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways also appear implicated in modulation of HO-1 expression by 15dPGJ(2).

Oxidized Phospholipids Induce Expression of Human Heme Oxygenase-1 Involving Activation of cAMP-responsive Element-binding Protein

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, protects against oxidative stress, and shows potent anti-inflammatory effects. Oxidized phospholipids, which are generated during inflammation and apoptosis, modulate the inflammatory response by inducing the expression of several genes including HO-1. Here we investigated the signaling pathways and transcriptional events involved in the induction of HO-1 gene expression by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in human umbilical vein endothelial cells. OxPAPC up-regulated HO-1 mRNA and protein in a time- and concentration-dependent manner, whereas pro-inflammatory agents like TNF-alpha and lipopolysaccharide did not significantly induce HO-1 expression in human umbilical vein endothelial cells. Signaling pathways involved in the OxPAPC-mediated HO-1 induction included protein kinases A and C, as well as the mitogen-activated protein kinases p38 and ERK. The cAMP-responsive element-binding protein (CREB) was phosphorylated via these pathways in response to OxPAPC treatment and expression of a dominant-negative mutant of CREB inhibited OxPAPC-induced activity of a human heme oxygenase-1 promoter-driven luciferase reporter construct. We identified a cAMP-responsive element and a Maf recognition element to be involved in the transcriptional activation of the HO-1 promoter by OxPAPC. In gel shift assays we observed binding of CREB to the cAMP-responsive element after OxPAPC treatment. Induction of HO-1 expression by lipid oxidation products via CREB may represent a feedback mechanism to limit inflammation and associated tissue damage.