Induction of heme oxygenase-1 and heat shock protein 70 in rat hepatocytes: The role of calcium signaling

Stress response genes including heat shock proteins are induced under a variety of conditions to confer cellular protection. This study investigated the role of calcium signaling in the induction of two stress response genes, heme oxygenase-1/hsp32 and hsp70, in isolated rat hepatocytes. Both genes were induced by cellular glutathione depletion. This induction could be inhibited by BAPTA-AM. Culturing in a calcium-free medium prevented the induction of hsp70 gene expression after glutathione depletion without affecting heme oxygenase-1 gene expression. Thapsigargin increased the gene expression of heme oxygenase-1 but not that of hsp70. Thapsigargin-induced heme oxygenase-1 induction was completely inhibited by BAPTA-AM. Incubation with the Ca²⁺-ionophore A23187 augmented heme oxygenase-1 (two-fold) and hsp70 (5.2-fold) mRNA levels. Our data suggests a significant role of Ca²⁺-dependent pathways in the induction of the two stress genes. An increase in the cytoplasmic Ca²⁺ activity seems to play a key role in the cascade of signaling leading to the induction of the two genes. However, the source of Ca²⁺ that fluxes into the cytoplasm seems to be different. Our data provides evidence for a compartmentalization of calcium fluxes, i.e. the Ca²⁺ flux from intracellular stores (e.g. the endoplasmic reticulum) plays a major role in the induction of heme oxygenase-1. By contrast, Ca²⁺ flux from the extracellular medium seems to be a mechanism initiating the cellular signaling cascade leading to hsp70 gene induction.     

The effect of heme oxygenase-1 induction by octreotide on radiation enteritis

Radiation enteritis occurs as a response to abdominal radiation, which can cause mucosal damage in the gastrointestinal mucosal epithelium. The small intestine is one of the most radiosensitive organs in the abdomen. The present study was undertaken to investigate the effect of octreotide (OCT) administration on heme oxygenase-1 (HO-1) expression of the radiation enteritis model. Rats received 50 mg/kg/day OCT for 4 days before irradiation and continued for 3 days after irradiation. Intestinal myeloperoxidase (MPO) activities, malondialdehyde (MDA) levels are indicators of oxidative damage while caspase-3 activities reveal apoptosis degree of the small intestine. At histological examination, the terminal ileum tissue was analyzed for morphological changes. Irradiation significantly increased the intestinal MPO and caspase-3 activities, MDA levels and HO-1 expression in comparison to sham control group. OCT treatment was associated with increased HO-1 expression and caspase-3 activity, decreased MPO activity and MDA levels. Histological examination revealed that the intestinal mucosal structure was preserved in the OCT treated group. OCT appears to have protective effects against radiation-induced intestinal damage. This protective effect is, in part, mediated by modification of the inflammatory response and the induction of HO-1 expression.     

Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with -754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38alpha, beta, and delta decreased and p38gamma increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (-668/-654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (-47/-42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediates HO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box.     

Effects of heme oxygenase-1 expression on sterol homeostasis in rat astroglia

Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. Central oxidation of cholesterol to oxysterols has been implicated in neuroembryogenesis, synaptic plasticity, and membrane repair. In the current study, we demonstrated that transient transfection of rat astroglia with human (h)ho-1 cDNA for 3 days significantly decreased intracellular cholesterol concentrations and increased levels of four oxysterol species (measured by GC/MS) compared to untreated control cultures and HO-1-transfected cells exposed to the HO inhibitor, tin mesoporphyrin (SnMP). Relative to control preparations, oxidative stress was augmented in mitochondria (isolated by subcellular fractionation) and culture media derived from HO-1-transfected astrocytes, as evidenced by enhanced oxidation of the synthetic reporter molecules, linoleoyl tyrosine (LT), linoleoyl tyrosine cholesterol ester (LTC), or linoleoyl tyrosine deoxyguanosyl ester (LTG; measured by GC/MS and LC/MS/MS). We also observed enhanced oxidation of exogenous LTC in human neuroblastoma (M17) cells exposed for 18 h to conditioned media collected from HO-1-transfected astrocytes relative to control media. In AD and other pathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g., beta-amyloid) into altered patterns of glial sterol metabolism which, in turn, may affect neuronal membrane turnover, survival, and adaptability.     

Up-regulation of heme oxygenase-1 in rat spleen after aniline exposure

The splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis, and the development of a variety of sarcomas in rats. However, the underlying mechanisms by which aniline elicits splenotoxic response are not well understood. Previously we have shown that aniline exposure causes oxidative damage to the spleen. To further explore the oxidative mechanism of aniline toxicity, we evaluated the potential contribution of heme oxygenase-1 (HO-1), which catalyzes heme degradation and releases free iron. Male SD rats were given 1 mmol/kg/day aniline in water by gavage for 1, 4, or 7 days, and respective controls received water only. Aniline exposure led to significant increases in HO-1 mRNA expression in the spleen (2-and 2.4-fold at days 4 and 7, respectively) with corresponding increases in protein expression, as confirmed by ELISA and Western blot analysis. Furthermore, immunohistochemical assessment of spleen showed stronger immunostaining for HO-1 in the spleens of rats treated for 7 days, confined mainly to the red pulp areas. No changes were observed in mRNA and protein levels of HO-1 after 1 day exposure. The increase in HO-1 expression was associated with increases in total iron (2.4-and 2.7-fold), free iron (1.9-and 3.5-fold), and ferritin levels (1.9-and 2.1-fold) at 4 and 7 days of aniline exposure. Our data suggest that HO-1 up-regulation in aniline-induced splenic toxicity could be a contributing pro-oxidant mechanism, mediated through iron release, and leading to oxidative damage.     

Piperine protects cisplatin-induced apoptosis via heme oxygenase-1 induction in auditory cells

Piperine is a major component of black pepper, Piper nigrum Linn, used widely in traditional medicine. In this study, we examined whether piperine could protect House Ear Institute-Organ of Corti 1 (HEI-OC1) cells against cisplatin-induced apoptosis through the induction of heme oxygenase (HO)-1 expression. Piperine (10-100 microM) induced the expression of HO-1 in dose- and time-dependent manners. Piperine also induced antioxidant response element-luciferase and translocated nuclear factor-E2-related factor-2 (Nrf2) to nucleus. Piperine activated the c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK) pathways, and the JNK pathway played an important role in piperine-induced HO-1 expression. Piperine protected the cells against cisplatin-induced apoptosis. The protective effect of piperine was abrogated by zinc protoporphyrin IX, an HO inhibitor, and antisense oligodeoxynucleotides against HO-1 gene. These results demonstrate that the expression of HO-1 by piperine is mediated by both JNK pathway and Nrf2, and the expression inhibits cisplatin-induced apoptosis in HEI-OC1 cells.     

Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis

Heme oxygenase-1 (HO-1) is emerging as an important cytoprotective enzyme system in a variety of injury models. To optimize future therapeutic applications of HO-1, it is necessary to delineate the precise functions and mechanisms as well as modes of externally regulating HO-1 expression. Investigations have been limited by difficulties with the generation of HO-1 null mice and the lack of specific HO-1 inhibitors. Lung ischemia-reperfusion (I-R) injury is the inciting event in acute lung failure following transplantation, surgery, and shock. To study the function of HO-1 in I-R-induced lung injury, we designed small interfering RNA (siRNA) sequences that effectively suppress HO-1 expression both in vitro and in vivo in an organ-specific manner. In this study we show that there is enhanced apoptosis, via increased Fas expression and caspase 3 activity, in the presence of HO-1 siRNA in endothelial cells and mouse lung during I-R injury, whereas HO-1 overexpression attenuates apoptosis. To the best of our knowledge, we are the first to demonstrate that lung-specific siRNA delivery can be achieved by intranasal administration without the need for viral vectors or transfection agents in vivo, thereby obviating potential concerns for toxicity if siRNA technology is to have clinical application in the future.     

Anthraquinone cytotoxicity and apoptosis in primary cultures of rat hepatocytes

We compared three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone), with respect to their toxicity and ability to induce apoptosis in primary cultures of rat hepatocytes. Rhein was the most effective in producing free radicals, and was the only one of the tested anthraquinones that could induce apoptosis. Addition of 50μM rhein to hepatocyte cultures led to depletion of intracellular reduced glutathione (GSH) and ATP and accumulation of lipid peroxidation products. The substances N,N′-diphenyl-p-phenylenediamine (DPPD), dithiothreitol (DTT), nifedipine and desferal all protected the hepatocytes, i.e. prevented viability loss and ATP depletion, and decreased the GSH depletion.

Cultures exposed to rhein for 15min and subsequently rinsed and incubated for 16h under normal culture conditions (complete medium) exhibited apoptosis, as shown by DNA fragmentation, nuclear condensation and positive TUNEL reaction. Pretreatment with the antioxidant DPPD and the iron-chelator desferal gave complete protection against apoptosis.

No signs of oxidative cell damage were detected when the cultures were exposed to danthron or chrysophanol. All three anthraquinones did, however, cause an immediate increase in the intracellular Ca²⁺ concentration.

We conclude that rhein, which contains one carboxyl group, is a suitable substrate for one-electron-reducing enzymes and an effective redox cycler, which leads to the production of oxygen-derived free radicals that eventually induce apoptotic cell death.     

Anthraquinone cytotoxicity and apoptosis in primary cultures of rat hepatocytes

We compared three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone), with respect to their toxicity and ability to induce apoptosis in primary cultures of rat hepatocytes. Rhein was the most effective in producing free radicals, and was the only one of the tested anthraquinones that could induce apoptosis. Addition of 50μM rhein to hepatocyte cultures led to depletion of intracellular reduced glutathione (GSH) and ATP and accumulation of lipid peroxidation products. The substances N,N'-diphenyl-p-phenylenediamine (DPPD), dithiothreitol (DTT), nifedipine and desferal all protected the hepatocytes, i.e. prevented viability loss and ATP depletion, and decreased the GSH depletion.

Cultures exposed to rhein for 15min and subsequently rinsed and incubated for 16h under normal culture conditions (complete medium) exhibited apoptosis, as shown by DNA fragmentation, nuclear condensation and positive TUNEL reaction. Pretreatment with the antioxidant DPPD and the iron-chelator desferal gave complete protection against apoptosis.

No signs of oxidative cell damage were detected when the cultures were exposed to danthron or chrysophanol. All three anthraquinones did, however, cause an immediate increase in the intracellular Ca²⁺ concentration.

We conclude that rhein, which contains one carboxyl group, is a suitable substrate for one-electron-reducing enzymes and an effective redox cycler, which leads to the production of oxygen-derived free radicals that eventually induce apoptotic cell death.     

Characterization of ochratoxin A-induced apoptosis in primary rat hepatocytes

The main target organ of the mycotoxin ochratoxin A (OTA) in mammals is the kidney but OTA has also been shown to be hepatotoxic in rats and to induce tumors in mouse liver. Even at very low concentrations, OTA causes perturbations of cellular signaling pathways as well as enhanced apoptosis. OTA has been extensively studied in kidney cell systems. Since this substance also affects liver health, we focused our work on apoptosis-related events induced by OTA in primary rat hepatocytes. We performed pathway-specific polymerase chain reaction arrays to assess the expression of genes involved in apoptosis. Treatment with 1 μM OTA for 24 h caused marked changes in apoptosis-related gene expression. Genes as apaf1, bad, caspase 7, polb (DNA polymerase beta, performs base excision repair), and p53, which are marker genes for DNA damage, were upregulated. FAS and faslg were also markedly induced by treatment with OTA. Treatment of hepatocytes with OTA led to a concentration-dependent inhibition of protein biosynthesis. Apoptosis-inducing factor was released from mitochondria following OTA treatment; the mycotoxin induced the activity of caspases 8, 9, and 3/7 and caused chromatin condensation and fragmentation. Caspase inhibition led to a significant but not complete reduction of OTA-induced apoptosis. Our data suggest that not only OTA leads to p53-dependent apoptosis in rat hepatocytes but it also hints to other mechanisms, independent of caspase activation or protein biosynthesis, being involved.     

Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia

Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions.     

内毒素引起的乳鼠心肌细胞血红素加氧酶—1基因的表达

为了探讨在内毒素作用下的乳鼠心肌细胞（neonatal rat cardiomyocytes,NRCMs）血红素加氧酶－1（heme oxygenase-1,HO-1）基因的表达及其在细胞损伤中的作用,分别用10、30及50μg/ml的脂多糖（lipopolysaccharide,LPS）,10μg/ml LPS 10μmol/ml锌原卟啉Ⅸ（Zn-protoporphyrin-Ⅸ,ZnPPⅨ）和单纯10μmol/ml ZnPPⅨ与培养的NRCMs共同孵育6h,以及10μg/ml LPS与NRCMs共同孵育9h和18h。分别观察细胞HO－1 mRNA表达、MDA含量、LDH释放量与台盼蓝摄取率的变化。结果显示,同样与细胞孵育6h,LPS10μg/ml时HO－1 mRNA表达比对照组增加81.2％,30μg/ml时表达量增加126.3％,50μg/ml时表达量增加92.8％;LPS为10μg/ml时,孵育9h后HO－1 mRNA的表达量比对照组增加93.6％,孵育18h后一增加105.8％。LPS30、50μg/ml,10μg/ml LPS＋10μmol/ml ZnPPⅨ与细胞孵育6h及LPS 10μg/ml孵育18h后,细胞MDA含量、LDH释放量与台盼蓝摄取率明显增加（P＜0.01）;单纯10μg/ml LPS与单纯10μmol/ml ZnPPⅨ孵育6h后,上述指标均无明显升高。结果表明,LPS可诱导NRCMs HO－1 mRNA的表达,且在较低LPS剂量范围内具有时间依赖性和浓度依赖性;NRCMs HO-1 mRNA的表达可减低LPS引起的细胞损伤,这可能是细胞产生的一种自身保护性反应。     

人体血红素加氧酶-1的研究进展

血红素加氧酶（heme oxygenase,HO)是哺乳动物中血红素代谢的限速酶,HO－1是HO同功酶之一,主要分布在肝、脾、肺等多种脏器,具有调节和保护功能。作者拟从人体HO－1蛋白的晶体结构、HO－1的功能和HO－1表达的诱导因素,以及HO－1基因的表达与调控等研究进展做一综述。     

The effect of prior exercise on ex vivo induction of heme oxygenase-1 in human lymphocytes

It was postulated that prior demanding exercise would suppress the induction of the oxidant-responsive protein Heme Oxygenase-1 (HO-1) in mononuclear cells following subsequent ex vivo H(2)O(2) treatment. Eight male subjects completed two trials in a randomized order (one rest and one exercise) and ex vivo HO-1 protein induction was determined following H(2)O(2) treatment in lymphocytes and monocytes before and after each trial using a newly developed and reproducible assay. Lymphocytes obtained 2 h post-exercise showed a modest reduction in HO-1 protein expression in response to ex vivo treatment with H(2)O(2) (p<0.05). The plasma concentration of the HO-1 suppressor alpha1-antitrypsin increased immediately post-exercise (p<0.05) and it is tentatively suggested that this may explain the modest transient reduction in ex vivo HO-1 protein induction in lymphocytes in response to an independent oxidant challenge following a prior bout of demanding exercise.