Induced Heme Oxygenase-1 mRNA Research Articles

Heme Oxygenase-1 Exerts Antiviral Activity against Hepatitis A Virus In Vitro.

Hepatitis A virus (HAV), the causative pathogen of hepatitis A, induces severe acute liver injuries in humans and is a serious public health concern worldwide. However, appropriate therapeutics have not yet been developed. The enzyme heme oxygenase-1 (HO-1) exerts antiviral activities in cells infected with several viruses including hepatitis B and C viruses. In this study, we demonstrated for the first time the suppression of virus replication by HO-1 in cells infected with HAV. Hemin (HO-1 inducer) induced HO-1 mRNA and protein expression, as expected, and below 50 mM, dose-dependently reduced the viral RNA and proteins in the HAV-infected cells without cytotoxicity. Additionally, HO-1 protein overexpression using a protein expression vector suppressed HAV replication. Although ZnPP-9, an HO-1 inhibitor, did not affect HAV replication, it significantly inhibited hemin-induced antiviral activity in HAV-infected cells. Additionally, FeCl3, CORM-3, biliverdin, and the HO-1 inducers andrographolide and CoPP inhibited HAV replication in the HAV-infected cells; andrographolide and CoPP exhibited a dose-dependent effect. In conclusion, these results suggest that HO-1 effectively suppresses HAV infection in vitro, and its enzymatic products appear to exert antiviral activity. We expect that these results could contribute to the development of a new antiviral drug for HAV.

Free Heme Induces a Heme Importer, Heme Catabolism and an Angiogenic Factor in Multiple Organs in Mice: Implications for Organ-Specific Response to Intravascular Hemolysis

Heme (iron protoporphyrin IX) is a major toxic component of hemoglobin released from red cell turnover especially during intravascular hemolysis such as in sickle cell disease, malaria and paroxysmal nocturnal hemoglobinuria. Heme oxygenase 1 (HO-1) is an enzyme that degrades heme, primarily generated by damaged or senescent red blood cells, releasing iron from protoporphyrin and generating equimolar amounts of biliverdin and carbon monoxide. The HO-1 heme catabolic pathway provides an important defense from oxidant and inflammatory consequences of exposure to free heme. HO-1 induction occurs as a transcriptional response to heme through the NRF2 oxidant-sensing transcription factor. Placental Growth Factor (PGF) is an angiogenic growth factor secreted by proliferating erythroblasts during normal development, which we previously have found is also activated by heme, secondarily regulating expression of the vasoconstrictor endothelin-1 that promotes pulmonary hypertension. We assessed the transcriptional response of HO-1 and PGF in different organs in C57BL/6J wild type (WT) and Nrf2 null mice following acute exposure to heme.WT and Nrf2 null mice were injected intravenously with saline or hemin (120 µmoles/kg body weight), followed by harvest of the bone marrow, spleen, liver, kidney, lung and heart 3 hours after injection. Real time PCR was used to quantify transcripts and relative changes in expression. Heme oxygenase-1 mRNA levels rose sharply in organs from WT mice that received hemin compared with saline control: liver (207-fold), heart (201-fold), kidney (101-fold), lung (69-fold), bone marrow (13-fold) and spleen (7-fold). In Nrf2 null mice, induction of HO-1 mRNA expression by heme was blunted as expected, with the greatest HO-1 mRNA induction in the kidney, followed by the heart, liver, lung, bone marrow and spleen. Our findings suggest that PGF expression is Nrf2-dependent primarily in the bone marrow, lung and kidney (4-fold, 1.65-fold and 1.89-fold, respectively, WT compared to Nrf2 null). Interestingly, in some of the organs, we observed an inverse relationship between HO-1 inducibility and PGF expression in organs from Nrf2 null mice injected with hemin, suggesting that reduced clearance of heme promotes more robust PGF induction. We observed the highest basal expression of PGF (1.23 and 2.21 DeltaCt compared to GUSB) in the heart, rising even higher with heme exposure (0.51 and 0.80 DeltaCt) in WT and Nrf2 null mice respectively. This translates to 70% and 57% respectively of the level of the “housekeeping gene” GUSB, which constitutes relatively high-level expression. In WT mice injected with hemin, the heme importer Hrg1 mRNA induction was mild but significant (heart 2.5 fold, kidney 2.1-fold, and lung 2.2-fold), which are also the organs with highest HO-1 inducibility (all p<0.05). Hrg1 mRNA induction was strongly dependent on Nrf2. Hrg1 activation in the liver increased by 1.82-fold in WT mice injected with hemin (p=0.097).The liver has been shown by others to take up free heme avidly, but these are the first data to demonstrate widely ranging differences in Nrf2-dependent heme response among other organs, implying their uptake of heme. We hypothesize that these differences in heme-induced HO-1 expression may reflect proportional differences in heme import, potentially via the Hrg1 heme importer that we find to be heme-inducible. There is likely also a role for the CD93 receptor for heme-hemopexin complexes and the FLVCR2 heme importer. This means that many organs may have some ability to take up free heme. Also, the ability of many organs to import extracellular heme might potentially play a role in diseases with robust release of heme into the circulation, such as β-thalassemia, sickle cell disease, malaria and other hemolytic anemias. PGF expression in the heart has previously shown by others to play a role in cardiac development and hypertrophy, and our new results point to cardiac PGF as a new hypothetical target of heme-induced dysregulation. Understanding the mechanism of organ-specific response to heme and PGF induction could expand the range of heme-mediated toxicity and pathophysiology of the heme-Nrf2-PGF-endothelin axis in sickle cell disease. [Display omitted] DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties. Kato:Global Blood Therapeutics: Consultancy; Bayer: Research Funding; MAST Therapeutics: Research Funding; Novartis: Consultancy.

Short ELF-EMF Exposure Targets SIRT1/Nrf2/HO-1 Signaling in THP-1 Cells.

Extremely low frequency electromagnetic fields (ELF-EMFs) have been known to modulate inflammatory responses by targeting signal transduction pathways and influencing cellular redox balance through the generation of oxidants and antioxidants. Here, we studied the molecular mechanism underlying the anti-oxidative effect of ELF-EMF in THP-1 cells, particularly with respect to antioxidant enzymes, such as heme oxygenase-1 (HO-1), regulated transcriptionally through nuclear factor E2-related factor 2 (Nrf2) activation. Cells treated with lipopolysaccharides (LPS) were exposed to a 50 Hz, 1 mT extremely low frequency electromagnetic fields for 1 h, 6 h and, 24 h. Our results indicate that ELF-EMF induced HO-1 mRNA and protein expression in LPS-treated THP-1 cells, with peak expression at 6 h, accompanied with a concomitant migration to the nucleus of a truncated HO-1 protein form. The immunostaining analysis further verified a nuclear enrichment of HO-1. Moreover, ELF-EMF inhibited the protein expressions of the sirtuin1 (SIRT1) and nuclear factor kappa B (NF-kB) pathways, confirming their anti-inflammatory/antioxidative role. Pretreatment with LY294002 (Akt inhibitor) and PD980559 (ERK inhibitor) inhibited LPS-induced Nrf2 nuclear translocation and HO-1 protein expression in ELF-EMF-exposed cells. Taken together, our results suggest that short ELF-EMF exposure exerts a protective role in THP-1 cells treated with an inflammatory/oxidative insult such as LPS, via the regulation of Nrf-2/HO-1 and SIRT1 /NF-kB pathways associated with intracellular glutathione (GSH) accumulation.

Ammonia promotes endothelial cell survival via the heme oxygenase-1-mediated release of carbon monoxide

Although endothelial cells produce substantial quantities of ammonia during cell metabolism, the physiologic role of this gas in these cells is not known. In this study, we investigated if ammonia regulates the expression of heme oxygenase-1 (HO-1), and if this enzyme influences the biological actions of ammonia on endothelial cells. Exogenously administered ammonia, given as ammonium chloride or ammonium hydroxide, or endogenously generated ammonia stimulated HO-1 protein expression in cultured human and murine endothelial cells. Dietary supplementation of ammonia also induced HO-1 protein expression in murine arteries. The increase in HO-1 protein by ammonia in endothelial cells was first detected 4h after ammonia exposure and was associated with the induction of HO-1 mRNA, enhanced production of reactive oxygen species (ROS), and increased expression and activity of NF-E2-related factor-2 (Nrf2). Ammonia also activated the HO-1 promoter and this was blocked by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. The induction of HO-1 expression by ammonia was dependent on ROS formation and prevented by N-acetylcysteine or rotenone. Finally, prior treatment of endothelial cells with ammonia inhibited tumor necrosis factor-α-stimulated cell death. However, silencing HO-1 expression abrogated the protective action of ammonia and this was reversed by the administration of carbon monoxide but not bilirubin or iron. In conclusion, this study demonstrates that ammonia stimulates the expression of HO-1 in endothelial cells via the ROS-Nrf2 pathway, and that the induction of HO-1 contributes to the cytoprotective action of ammonia by generating carbon monoxide. Moreover, it identifies ammonia as a potentially important signaling gas in the vasculature that promotes endothelial cell survival.

Omeprazole induces heme oxygenase-1 in fetal human pulmonary microvascular endothelial cells via hydrogen peroxide-independent Nrf2 signaling pathway

Omeprazole (OM) is an aryl hydrocarbon receptor (AhR) agonist and a proton pump inhibitor that is used to treat humans with gastric acid related disorders. Recently, we showed that OM induces NAD (P) H quinone oxidoreductase-1 (NQO1) via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent mechanism. Heme oxygenase-1 (HO-1) is another cytoprotective and antioxidant enzyme that is regulated by Nrf2. Whether OM induces HO-1 in fetal human pulmonary microvascular endothelial cells (HPMEC) is unknown. Therefore, we tested the hypothesis that OM will induce HO-1 expression via Nrf2 in HPMEC. OM induced HO-1 mRNA and protein expression in a dose-dependent manner. siRNA-mediated knockdown of AhR failed to abrogate, whereas knockdown of Nrf2 abrogated HO-1 induction by OM. To identify the underlying molecular mechanisms, we determined the effects of OM on cellular hydrogen peroxide (H2O2) levels since oxidative stress mediated by the latter is known to activate Nrf2. Interestingly, the concentration at which OM induced HO-1 also increased H2O2 levels. Furthermore, H2O2 independently augmented HO-1 expression. Although N-acetyl cysteine (NAC) significantly decreased H2O2 levels in OM-treated cells, we observed that OM further increased HO-1 mRNA and protein expression in NAC-pretreated compared to vehicle-pretreated cells, suggesting that OM induces HO-1 via H2O2-independent mechanisms. In conclusion, we provide evidence that OM transcriptionally induces HO-1 via AhR - and H2O2 - independent, but Nrf2 - dependent mechanisms. These results have important implications for human disorders where Nrf2 and HO-1 play a beneficial role.

Involvement of Heme Oxygenase-1 in particulate matter-induced impairment of NO-dependent relaxation in rat intralobar pulmonary arteries

Particulate air pollution exerts deleterious effects on cardiovascular system. We previously described that exposure to urban particulate matter (SRM1648) impairs nitric oxide (NO, a major vasculoprotective factor) responsiveness in intrapulmonary arteries. As Heme Oxygenase-1 (HO-1) is induced by urban particles in some cell types and is known to alter NO-dependent signaling pathway, the objective was to characterize HO-1 involvement in SRM1648-induced impairment of NO-dependent relaxation in intrapulmonary arteries.Rat intrapulmonary artery rings were exposed or not to Co (III) Protoporphyrin IX Chloride (HO-1 inducer) or SRM1648 in the absence or presence of Cr (III) Mesoporphyrin IX Chloride (HO-1 activity inhibitor). NO-dependent relaxation was assessed with DEA-NOnoate (DEA-NO) on pre-contracted arteries. HO-1 and soluble guanylyl-cyclase (sGC) mRNA and protein expressions were assessed by qRT-PCR and Western blotting, respectively.SRM1648 or Co (III) Protoporphyrin IX Chloride exposure (24) impaired DEA-NO-dependent relaxation. The SRM-induced alteration of DEA-NO responsiveness was partially prevented by Cr (III) Mesoporphyrin IX Chloride. Co (III) Protoporphyrin IX Chloride induced HO-1 mRNA and protein expressions, whereas SRM1648 only induced HO-1 protein expression without affecting its mRNA level. Exposure to either SRM1648 or to Co (III) Protoporphyrin IX Chloride did not affect the expression levels of sGC.In conclusion, this study provides some evidence that impairment of NO signaling pathway in intrapulmonary arteries involves HO-1. Therefore it highlights the role of HO-1 in particulate matter-induced detrimental effects in pulmonary circulation.

Constitutive Effects of Lead on Aryl Hydrocarbon Receptor Gene Battery and Protection by β-carotene and Ascorbic Acid in Human HepG2 Cells.

Lead (Pb) is an environmental pollutant that can get entry into human body through contaminated foods, drinks, and inhaled air leading to severe biological consequences, and has been responsible for many deaths worldwide. The objectives of this study were 1st to investigate the modulatory effects of environmentally relevant concentrations of Pb on AhR gene battery, which is controlling xenobiotics metabolism. 2nd, trials to reduce Pb-induced adverse effects were done using some phytochemicals like β-carotene or ascorbic acid. Human hepatoma (HepG2) cell lines were exposed to a wide range of Pb concentrations varying from physiological to toxic levels (0 to 10 mg/L) for 24 h. High Pb concentrations (1 to 10 mg/L) significantly reduced phase I (CYP1A1 and 1A2) and phase II (UGT1A6 and NQO1) xenobiotic metabolizing enzyme mRNA expression in a mechanistic manner through the AhR regulation pathway. Additionally, these Pb concentrations induced oxidative stress in HepG2 cells in terms of production of reactive oxygen species (ROS) and induced heme oxygenase-1 mRNA expression in a concentration-dependent phenomenon. Coexposure of HepG2 cells to physiological concentrations of some micronutrients, like β-carotene (10 μM) or ascorbic acid (0.1 mM), along with Pb (1 mg/L) for 24 h significantly reduced the levels of ROS production and recovered AhR mRNA expression into the normal levels. Thus, consumption of foods rich in these micronutrients may help to reduce the adverse effects of lead in areas with high levels of pollution.

Inhibiting mast cell degranulation by HO-1 affects dendritic cell maturation in vitro

Mast cell (MC) degranulation can break peripheral immune tolerance. However, its mechanism remains unclear. Our goal was to study the stabilization of MC membranes by heme oxygenase-1 (HO-1) in order to influence dendritic cell (DC) function. Mast cells and dendritic cells were prepared from 8-week-old to 10-week-old C57BL/6 mice; spleen mononuclear cells (SMCs) were prepared from 8-week-old to 10-week-old C57BL/6 and Balb/c mice. Mast cells were pretreated with PBS, DMSO, Hemin (50 μl/ml), and Znpp (50 μl/ml) for 8 h. Real-time PCR and western-blot tested the HO-1 of MC mRNA and protein. The co-stimulatory molecules of DCs (CD80, CD86, CD40) were measured by flow cytometry, and levels of TNF-α, IL-6, and IFN-γ were measured by ELISA. We set up a one-way mixed lymphocyte reaction (MLR) model to test the proliferation of SMCs after MC/DC interaction. *P < 0.05 (t test) was taken as the level of statistical significance. MCs pretreated with hemin induced HO-1 mRNA and protein expression, then interacted with DCs; expression of the co-stimulatory molecules was attenuated. The TNF-α, IL-6, and IFN-γ levels in the co-culture system were decreased. These DCs couldn't stimulate the proliferation of SMCs. Inhibiting MC degranulation by HO-1 restrained DC maturation and attenuated the proliferation of SMCs.

Heme Oxygenase in Neonatal Lung Injury and Repair

Premature and sick neonates are often exposed to high concentrations of oxygen, which results in lung injury and long-term adverse consequences. Nevertheless, neonates are more tolerant to hyperoxia than are adults. This may be, in part, explained by the high lung content of heme oxygenase-1 (HO-1), the rate-limiting enzyme in the degradation of heme and an important stress protein. The abundance of HO-1 dictates its cytoprotective and deleterious effects. Interestingly, in response to hyperoxia, lung HO-1 mRNA is not further up-regulated in neonates, suggesting that lung HO-1 gene expression is tightly regulated so as to optimize cytoprotection when faced with an oxidative stress such as hyperoxia. In addition to the lack of induction of HO-1 mRNA, neonatal lung HO-1 protein is observed in the nucleus in neonatal mice exposed to hyperoxia but not in adults, which is further evidence for the developmental regulation of HO-1. Nuclear HO-1 had unique properties independent of its enzymatic activity. In addition, there has been increasing evidence that nuclear HO-1 contributes to cellular proliferation and malignant transformation in several human cancers. Since HO-1 has dual effects in cytoprotection and cellular proliferation, the titration of HO-1 effects is critical to ensure beneficial actions against oxidative stress. Much more has to be understood about the specific roles of HO-1 so as to manipulate its abundance and/or nuclear migration to maximize the therapeutic benefit of this pleiotropic protein in the neonatal lung.

Mechanistic study on the biological effects of silver and gold nanoparticles in Caco-2 cells – Induction of the Nrf2/HO-1 pathway by high concentrations of silver nanoparticles

The most commonly used metal nanoparticles (NPs) across diverse applications, including in agro-food applications, include silver (AgNPs) and gold (AuNPs). In the present study, we aimed to investigate the biological responses and possible toxicological effects of AgNPs and AuNPs in the Caco-2 cells as an in vitro human GI tract model. Both AgNPs and AuNPs were internalized into the cytoplasm of Caco-2 cells, but not within the nucleus and only exposure to high concentrations of AgNPs, but not AuNPs, caused acute cytotoxicity and depolarization of the mitochondrial membrane potential. In addition, only AgNPs significantly depleted the total intracellular glutathione level, induced the activation of the stress-responsive gene, Nrf2, and dramatically increased the expression of heme oxygenase-1 (HO-1). Furthermore, siRNA silencing of Nrf2 transcripts significantly reduced the AgNP-induced HO-1 mRNA induction, suggesting a key role for Nrf2 in the control of HO-1 expression. Taken together, AgNPs but not AuNPs induced acute cytotoxicity and cellular responses via the oxidative stress-related activation of Nrf2/HO-1 signaling pathway in Caco-2 cells. The expression of HO-1 transcripts may be useful as a sensitive marker for safety evaluation of AgNPs in the GI tract of humans.

Inhibition of fatty acid amide hydrolase activates Nrf2 signalling and induces heme oxygenase 1 transcription in breast cancer cells

Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation and apoptosis. Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Here, we investigated whether FAAH or AEA are involved in NF (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway. The aim of this study was to analyse the effects of AEA or FAAH inhibition by the URB597 inhibitor or FAAH/siRNA on the activation of Nrf2-ARE signalling pathway and heme oxygenase-1 (HO-1) induction and transcription. Endogenous AEA was detected in the immortalized human mammary epithelial MCF-10A cells (0.034 ng per 10(6) cells) but not in MCF-7 or MDA-MB-231 breast cancer cells. Because breast tumour cells express FAAH abundantly, we examined the effects of FAAH on Nrf2/antioxidant pathway. We found that inhibition of FAAH by the URB597 inhibitor induced antioxidant HO-1 in breast cancer cells and MCF-10A cells. RNAi-mediated knockdown of FAAH or treatment with AEA-activated ARE-containing reporter induced HO-1 mRNA and protein expression, independent of the cannabinoid receptors, CB1, CB2 or TRPV1. Furthermore, URB597, AEA and siRNA-FAAH treatments induced the nuclear translocation of Nrf2, while siRNA-Nrf2 treatment and Keap1 expression blocked AEA, URB597 and si-FAAH from activation of ARE reporter and HO-1 induction. siRNA-HO-1 treatment decreased the viability of breast cancer cells and MCF-10A cells. These data uncovered a novel mechanism by which inhibition of FAAH or exposure to AEA induced HO-1 transcripts and implicating AEA and FAAH as direct modifiers in signalling mediated activation of Nrf2-HO-1 pathway, independent of cannabinoid receptors.

Genipin Inhibits TNF-α-Induced Vascular Smooth Muscle Cell Proliferation and Migration via Induction of HO-1

Vascular smooth muscle cell (VSMC) proliferation and migration triggered by inflammatory stimuli contributes importantly to the pathogenesis of atherosclerosis and restenosis. On the other hand, genipin, an aglycon of geniposide, exhibits diverse pharmacological functions such as antitumor and anti-inflammatory effects. The protective effects of genipin on the cardiovascular system have also been reported. However, the molecular mechanism involved remains unknown. This study aimed to elucidate the precise function of genipin in VSMCs, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. We found that pretreatment of genipin induced HO-1 mRNA and protein levels, as well as its activity in VSMCs. Genipin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. At the molecular level, genipin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Genipin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 siRNA partially abolished the beneficial effects of genipin on VSMCs. These results suggest that genipin may serve as a novel drug in the treatment of these pathologies by inducing HO-1 expression/activity and subsequently decreasing VSMC proliferation and migration.

The generation of 4-hydroxynonenal, an electrophilic lipid peroxidation end product, in rabbit cornea organ cultures treated with UVB light and nitrogen mustard

The cornea is highly sensitive to oxidative stress, a process that can lead to lipid peroxidation. Ultraviolet light B (UVB) and nitrogen mustard (mechlorethamine) are corneal toxicants known to induce oxidative stress. Using a rabbit air-lifted corneal organ culture model, the oxidative stress responses to these toxicants in the corneal epithelium was characterized. Treatment of the cornea with UVB (0.5J/cm2) or nitrogen mustard (100nmol) resulted in the generation of 4-hydroxynonenal (4-HNE), a reactive lipid peroxidation end product. This was associated with increased expression of the antioxidant, heme oxygenase-1 (HO-1). In human corneal epithelial cells in culture, addition of 4-HNE or 9-nitrooleic acid, a reactive nitrolipid formed during nitrosative stress, caused a time-dependent induction of HO-1 mRNA and protein; maximal responses were evident after 10h with 30μM 4-HNE or 6h with 10μM 9-nitrooleic acid. 4-HNE and 9-nitrooleic acid were also found to activate Erk1/2, JNK and p38 MAP kinases, as well as phosphoinositide-3-kinase (PI3)/Akt. Inhibition of p38 blocked 4-HNE- and 9-nitrooleic acid-induced HO-1 expression. Inhibition of Erk1/2, and to a lesser extent, JNK and PI3K/Akt, suppressed only 4-HNE-induced HO-1, while inhibition of JNK and PI3K/Akt, but not Erk1/2, partly reduced 9-nitrooleic acid-induced HO-1. These data indicate that the actions of 4-HNE and 9-nitrooleic acid on corneal epithelial cells are distinct. The sensitivity of corneal epithelial cells to oxidative stress may be an important mechanism mediating tissue injury induced by UVB or nitrogen mustard.

Cigarette smoke induces nuclear translocation of heme oxygenase 1 (HO-1) in prostate cancer cells: Nuclear HO-1 promotes vascular endothelial growth factor secretion

Prostate cancer is the second leading cause of male-cancer related death in the United States. Despite a number of evidence-based studies which strongly suggest an association between cigarette smoking and prostate cancer, the underlying biological mechanism is largely unknown. Heme oxygenase 1 (HO-1) has been implicated in maintaining cellular homeostasis, but also in tumor angiogenesis. Nuclear HO-1 protein expression has been observed in various types of tumors including prostate cancer. These studies, however, were reported as clinical and pathological observations, and failed to investigate nuclear HO-1 at the molecular level in cancer. The present study explores the relationship between cigarette smoke and nuclear HO-1-modulated promotion of vascular endothelial growth factor (VEGF) secretion. We have demonstrated that cigarette smoke medium (SM)-induced HO-1 mRNA expression and upregulated HO-1 protein levels in the prostate cancer cell lines DU145 and PC3. We also observed that SM significantly induced nuclear expression of HO-1, and enhanced secretion of VEGF in cells. Nuclear-directed expression of HO-1 activated the transcriptional activity of VEGF and promoted VEGF secretion in prostate cancer cells. This study provides new insights into the molecular mechanism by which cigarette smoke-induced nuclear translocation of HO-1 promotes VEGF secretion in prostate cancer cells. Nuclear HO-1 may, therefore, constitute an attractive therapeutic target to inhibit angiogenesis and the progression of prostate cancer.

Mycoplasma fermentans MALP-2 induces heme oxygenase-1 expression via mitogen-activated protein kinases and Nrf2 pathways to modulate cyclooxygenase 2 expression in human monocytes.

Heme oxygenase-1 (HO-1) is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury and performs a vital function in the maintenance of cell hemostasis. Increasing numbers of reports have indicated that mycoplasma-derived membrane lipoproteins/lipopeptides, such as macrophage-activating lipopeptide-2 (MALP-2), function as agents that stimulate the immune system by producing various inflammatory mediators, such as cytokines and cyclooxygenase 2 (COX-2), which play roles in the pathogenesis of inflammatory responses during mycoplasma infection. Here, we report that MALP-2 induced HO-1 mRNA and protein expression and upregulated HO-1 enzyme activity in THP-1 cells. Specific inhibitors of mitogen-activated protein kinases (MAPKs), SB203580, PD98059, and SP600125, significantly abolished HO-1 expression. In addition, MALP-2 also induced NF-E2-related factor 2 (Nrf2) translocation, and the silencing of Nrf2 expression in THP-1 cells decreased the levels of MALP-2-mediated HO-1 expression. Furthermore, COX-2 protein expression levels were upregulated in THP-1 cells in response to MALP-2, and transfection with small interfering RNAs of HO-1 significantly increased COX-2 accumulation. These results demonstrate that MALP-2 induces HO-1 expression via MAPKs and Nrf2 pathways and, furthermore, that MALP-2-induced COX-2 expression was modulated by HO-1 in THP-1 cells.

Zinc Protoporphyrin Upregulates Heme Oxygenase-1 in PC-3 Cells via the Stress Response Pathway

Zinc protoporphyrin IX (ZnPP), a naturally occurring molecule formed in iron deficiency or lead poisoning, is a potent competitive inhibitor of heme oxygenase-1 (HO-1). It also regulates expression of HO-1 at the transcriptional level. However, the effect of ZnPP on HO-1 expression is controversial. It was shown to induce HO-1 expression in some cells, but suppress it in others. The objective of this study is to investigate the effect of ZnPP on HO-1 expression in prostate cancer PC-3 cells. Incubation of PC-3 cells with 10 μM ZnPP for 4 h showed only a slight induction of HO-1 mRNA and protein, but the induction was high after 16 h and was maintained through 48 h of incubation. Of all the known responsive elements in the HO-1 promoter, ZnPP activated mainly the stress response elements. Of the various protein kinase inhibitors and antioxidant tested, only Ro 31-8220 abrogated ZnPP-induced HO-1 expression, suggesting that activation of HO-1 gene by ZnPP may involve protein kinase C (PKC). The involvement of PKC α, β, δ, η, θ, and ζ isoforms was ruled out by the use of specific inhibitors. The isoform of PKC involved and participation of other transcription factors remain to be studied.

Review of Needling- or Moxibustion-prohibited Points in the Illustrated Manual for the Practice of Acupuncture and Moxibustion with the Help of a Bronze Figure bearing Acupuncture Points and in The Classic of Supplementing Life with Acupuncture and Moxibustion

Kang-Pa Lee, Jin-Young MoonDivision of Bio Science, College of Science and Technology, Dongguk UniversityDept. of Meridian & Acupoint, College of Oriental Medicine, Dongguk UniversityAbstractObjectives : The gnarl of Pinus densiflora, called Songjeol in Korea, has been used as a medicinal herb for the treatment of inflammatory-related diseases such as arthralgia, myalgia and bruise. However, the molecular actions and mechanisms have not been clearly investigated. The aim of this study was to clarify the anti-inflammatory activity of Pinus densiflora gnarl pharmacopuncture (PDGP) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.Methods : Cytotoxicity was assessed by XTT assay. The amount of nitric oxide (NO) production was determined by nitrite assay. The mRNA expressions of interleukin-1β (IL-1β), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) were analyzed by RT-PCR. Reactive oxidative species (ROS) generation was measured using the fluorescence microscopy. In addition, inducible nitric oxide synthase (iNOS) and redox factor-1 (Ref-1) protein expressions were detected by Western blotting. Results : PDGP inhibited NO production and ROS generation in LPS-stimulated RAW264.7 cells. At the mRNA level, PDGP suppressed IL-1β, IL-6 and COX-2 expression. On the other hand, PDGP induced HO-1 mRNA expression. Furthermore, PDGP suppressed iNOS and Ref-1 protein expression.Conclusions : This result suggests that PDGP can act as a suppressor agent on NO and iNOS through induction of HO-1, and play an useful role in blocking inflammatory responses.Key words : Pinus densiflora gnarl, pharmacopuncture, nitric oxide, heme oxygenase-1

Naringenin inhibits TNF-α induced VSMC proliferation and migration via induction of HO-1

Vascular smooth muscle cell (VSMC) proliferation and migration, which is triggered by various inflammatory stimuli, contributes importantly to the pathogenesis of atherosclerosis and restenosis. Naringenin is a citrus flavonoid with both lipid-lowering and insulin-like properties. Here, we investigated whether naringenin affects TNF-α-induced VSMC proliferation and migration and if so, whether heme oxygenase-1 (HO-1) is involved. Rat VSMCs were treated with naringenin alone or in combination of TNF-α stimulation. We found that naringenin induced HO-1 mRNA and protein levels, as well as its activity, in VSMCs. Naringenin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. Mechanistic study demonstrated that naringenin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Naringenin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 inhibitor ZnPP IX or HO-1 siRNA partially abolished the beneficial effects of naringenin on VSMCs. These results suggest that naringenin may serve as a novel drug in the treatment of these pathologies by inducing HO-1 expression/activity and subsequently decreasing VSMC proliferation and migration.

Oxidative Stress Induced by Pure and Iron-Doped Amorphous Silica Nanoparticles in Subtoxic Conditions

Amorphous silica nanoparticles (SiO₂-NPs) have found broad applications in industry and are currently intensively studied for potential uses in medical and biomedical fields. Several studies have reported cytotoxic and inflammatory responses induced by SiO₂-NPs in different cell types. The present study was designed to examine the association of oxidative stress markers with SiO₂-NP induced cytotoxicity in human endothelial cells. We used pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and a positive control, iron-doped nanosilica (16 nm; SFe), to study the generation of hydroxyl radicals (HO·) in cellular-free conditions and oxidative stress in cellular systems. We investigated whether SiO₂-NPs could influence intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, increase lipid peroxidation (malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) concentrations), and up-regulate heme oxygenase-1 (HO-1) mRNA expression in the studied cells. None of the particles, except SFe, produced ROS in cell-free systems. We found significant modifications for all parameters in cells treated with SFe nanoparticles. At cytotoxic doses of S16 (40-50 μg/mL), we detected weak alterations of intracellular glutathione (4 h) and a marked induction of HO-1 mRNA (6 h). Cytotoxic doses of S60 elicited similar responses. Preincubation of cells being exposed to SiO₂-NPs with an antioxidant (5 mM N-acetylcysteine, NAC) significantly reduced the cytotoxic activity of S16 and SFe (when exposed up to 25 and 50 μg/mL, respectively) but did not protect cells treated with S60. Preincubation with NAC significantly reduced HO-1 mRNA expression in cells treated with SFe but did not have any effect on HO-1 mRNA level in cell exposed to S16 and S60. Our study demonstrates that the chemical composition of the silica nanoparticles is a dominant factor in inducing oxidative stress.

Curcumin Ameliorates Hydrogen Peroxide-Induced Epithelial Barrier Disruption by Upregulating Heme Oxygenase-1 Expression in Human Intestinal Epithelial Cells

Disruption of epithelial tight junctions (TJ) followed by loss of barrier function is of crucial importance in the pathogenesis of a variety of gastrointestinal disorders. Heme oxygenase-1 (HO-1), which can be induced by curcumin (Cur), provides protection against various forms of oxidative stress. The protective effect of Cur on oxidative stress-induced intestinal barrier disruption in human intestinal epithelial cells was elucidated in this study. H(2)O(2)-induced Caco-2 enterocytic monolayers were incubated in the presence or absence of Cur and/or zinc protoporphyrin (ZnPP). The trans-epithelial electrical resistance (TEER) and the flux of sodium fluorescein in the filter-grown Caco-2 cell monolayers were measured. The expression and localization of the TJ protein occludin and zonula occluden-1 (ZO-1) were evaluated by western blot and immunofluorescence microscopy. The mRNA and protein levels of HO-1 were analyzed by real-time PCR and western blot. Cur attenuated H(2)O(2)-induced disruption of paracellular permeability (TEER 52.02 ± 10.15% vs 22.71 ± 3.11%; sodium fluorescein flux 12.41 ± 2.19% vs 32.00 ± 4.97%, P < 0.05) and induced HO-1 mRNA (6.64 ± 0.48 vs 3.22 ± 0.28, P < 0.05) and protein (291.00 ± 9.17% vs 99.00 ± 10.00%, P < 0.05) expression in Caco-2 cells. After administration of H(2)O(2), occludin and ZO-1 proteins were restored by Cur (occludin 175.67 ± 29.50% vs 53.67 ± 24.19%, P < 0.05; ZO-1 139.67 ± 33.71% vs 36.00 ± 15.88%, P < 0.05) and this effect was blocked by HO-1 inhibitor, ZnPP (occludin 54.67 ± 10.02% vs 168.33 ± 36.47%, P < 0.05; ZO-1 50.00 ± 15.13% vs 117.67 ± 38.81%, P < 0.05). Cur protects human intestinal epithelial cells against H(2)O(2)-induced disruption of TJ and barrier dysfunction via the HO-1 pathway.