Accumulation Of Heme Oxygenase-1 Research Articles

Poly-ubiquitinated p62/SQSTM1-mediated hemeoxygenase-1 stabilization plays a critical role in cadmium-induced apoptosis of mouse monocyte Raw264.7 cells

Cadmium (Cd) is a toxic heavy metal that can affect many organs, leading to serious pathological disorders through immune suppression. Here, we investigated the molecular mechanisms underlying the response of monocytes to Cd exposure. Cd treatment of Raw264.7 cells activated antioxidant enzymes, such as hemeoxygenase-1 (HO-1), superoxide dismutase, and catalase. Cd exposure upregulated p53, p53 phosphorylation, p21, and γH2AX phosphorylation. Cd exposure also induced poly ADP-ribose polymerase 1 (PARP-1) cleavage. These findings indicated that Cd induces apoptosis through oxidative stress-mediated DNA damage. Furthermore, upregulation of microtubule-associated protein 1 light chain 3B-II (LC3B-II), an indicator of autophagy, was found to depend on Cd concentration. Accumulation of an autophagy substrate p62/SQSTM1 in monomeric p62 and polyubiquitinated (polyUb)-p62 forms, was suppressed upon N-acetylcysteine treatment Cd-exposed Raw264.7 cells, indicating an impairment of autophagic degradation during oxidative stress. Knockdown of p62 in Raw264.7 cells using small interfering RNA (siRNA) downregulated HO-1 expression and reduced apoptosis. HO-1 knockdown suppressed apoptosis by decreasing the poly-ubiquitination of p62. Treatment with hemin and MG132 enhanced Cd-mediated increases in HO-1 and polyUb-p62 levels, resulting in increased apoptosis, which indicated that Cd-induced HO-1 accumulation is associated with polyUb-p62 formation. p62 and HO-1 interactions were demonstrated by immunofluorescence and immunoprecipitation assays. Additionally, p62 was downregulated in Raw264.7 cells in response to H2O2 and a low level of HO-1 was induced. Cells that were highly sensitive to Cd did not form polyUb-p62, resulting in insufficient HO-1 accumulation. These results suggest that maintenance of HO-1 stability via poly-ubiquitination of p62 in Cd-exposed monocytes promotes apoptosis, which could be involved in immune suppression.

Effect of hemin, baicalein and heme oxygenase-1 (HO-1) enzyme activity inhibitors on Cd-induced accumulation of HO-1, HSPs and aggresome-like structures in Xenopus kidney epithelial cells

Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl2. In the present study, CdCl2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl2-induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl2-induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures.

Effect of isothiocyanates, BITC and PEITC, on stress protein accumulation, protein aggregation and aggresome-like structure formation in Xenopus A6 kidney epithelial cells

Numerous studies have elucidated the health benefits of organosulfur compounds, known as isothiocyanates (ITCs), derived from cruciferous vegetables. As electrophiles, ITCs have the ability to directly bind and modify thiol-containing compounds such as glutathione and cellular protein, including tubulin. While the biochemical effects of ITCs have been well characterized, less information is available regarding their effects on the accumulation of stress-inducible heme oxygenase-1 (HO-1), heat shock proteins (HSPs) and the possible formation of aggregated protein due to thiol modification. The present study has examined the effect of the ITCs, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), on the accumulation of HO-1, HSP70 and HSP30 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that both BITC and PEITC induced the accumulation of HO-1 and HSP70 whereas HSP30 levels were enhanced only in cells treated with BITC. Immunocytochemistry determined that ITC treatment induced F-actin disorganization and membrane ruffling and enhanced accumulation of HO-1 in the cytoplasm. Additionally, BITC induced enhanced levels of ubiquitinated protein, aggregated protein, and the collapse and fragmentation of microtubules. In comparison, treatment of cells with the proteasomal inhibitor, MG132, induced the accumulation of all three stress proteins, aggregated protein and aggresome-like structures. Finally, cells pretreated with BITC inhibited the formation of MG132-induced aggresome-like structures in the perinuclear region. This latter finding suggests that BITC-induced microtubule fragmentation may impede the movement of aggregated protein via microtubules and their subsequent coalescence into aggresome-like structures in the perinuclear region.

Overexpression of HO-1 assisted PM2.5-induced apoptosis failure and autophagy-related cell necrosis

Severe smog/haze events accompanied by extremely high concentrations of airborne fine particulate matter (PM2.5) have emerged frequently in China and the potential health risks have attracted ever-growing attention. During these episodes, a surge in hospital visits for acute respiratory symptoms and respiratory diseases exacerbation has been reported to be associated with acute exposure to high-levels of particulate matters. To investigate cell fate determination and the underlying pathogenic mechanisms during severe haze episodes or smog events, we exposed human lung epithelial cells (BEAS-2B) to PM2.5 (0–400μg/mL) for 24h and found that high doses of PM2.5 caused cell necrosis and autophagy dysfunction, while co-treatment with the autophagy inhibitor 3-MA could partially reduce PM2.5-induced cell necrosis. Exposure to PM2.5 also increased the expression and mitochondrial transposition of heme oxygenase 1 (HO-1), which consequently reduced the release of cytochrome C from mitochondria to cytosol. Knockdown of HO-1 by siRNA attenuated the mitochondrial accumulation of HO-1, reversed HO-1-induced the reduction of cytochrome C release and promoted PM2.5-induced cell apoptosis. In contrast to necrosis, PM2.5-induced autophagy was independent of HO-1. In conclusion, our results demonstrate that acute exposure to high PM2.5 concentrations causes autophagy-related cell necrosis. The decrease in cytochrome C release and apoptosis by upregulation of HO-1 maybe assist PM2.5-induced autophagy-related cell necrosis. Further, this study reveals dual roles for HO-1 in PM2.5-induced cytotoxicity and presents a possible explanation for the onset of acute respiratory symptoms under extreme particulate air pollution.

Extratumoral Heme Oxygenase-1 (HO-1) Expressing Macrophages Likely Promote Primary and Metastatic Prostate Tumor Growth.

Aggressive tumors induce tumor-supporting changes in the benign parts of the prostate. One factor that has increased expression outside prostate tumors is hemoxygenase-1 (HO-1). To investigate HO-1 expression in more detail, we analyzed samples of tumor tissue and peritumoral normal prostate tissue from rats carrying cancers with different metastatic capacity, and human prostate cancer tissue samples from primary tumors and bone metastases. In rat prostate tumor samples, immunohistochemistry and quantitative RT-PCR showed that the main site of HO-1 synthesis was HO-1+ macrophages that accumulated in the tumor-bearing organ, and at the tumor-invasive front. Small metastatic tumors were considerably more effective in attracting HO-1+ macrophages than larger non-metastatic ones. In clinical samples, accumulation of HO-1+ macrophages was seen at the tumor invasive front, almost exclusively in high-grade tumors, and it correlated with the presence of bone metastases. HO-1+ macrophages, located at the tumor invasive front, were more abundant in bone metastases than in primary tumors. HO-1 expression in bone metastases was variable, and positively correlated with the expression of macrophage markers but negatively correlated with androgen receptor expression, suggesting that elevated HO-1 could be a marker for a subgroup of bone metastases. Together with another recent observation showing that selective knockout of HO-1 in macrophages reduced prostate tumor growth and metastatic capacity in animals, the results of this study suggest that extratumoral HO-1+ macrophages may have an important role in prostate cancer.

Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Differential induction of heme oxygenase‐1 against nicotine‐induced cytotoxicity via the PI3K, MAPK, and NF‐kappa B pathways in immortalized and malignant human oral keratinocytes

Heme oxygenase-1 (HO-1) exhibits cytoprotective effects in many different cell types and is induced by nicotine exposure in human gingival fibroblasts. However, the role of HO-1 in cancer cells exposed to nicotine has not previously been described. We investigated the effects of nicotine on HO-1 protein expression and cell viability in immortalized (IHOK) and malignant (HN12) human oral keratinocyte cells using the 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay and Western blotting. We also examined the involvement of the phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and nuclear factor-kappaB (NF-kappaB) signaling pathways in nicotine-induced cytotoxicity and HO-1 levels in IHOK and HN12 cells. Nicotine-induced HO-1 production and had cytotoxic effects on cells in both a concentration- and time-dependent manner. Nicotine-induced cytotoxicity and accumulation of HO-1 were greater in IHOK cells than in HN12 cells. Molecular inhibitors of the ERK, p38 MAP kinase, PI3 K, and NF-kappaB signaling pathways blocked the cytotoxic effects and induction of HO-1 expression by nicotine. Treatment with antioxidants (bilirubin, N-acetylcysteine) protected cells against nicotine-induced cytotoxicity and blocked the upregulation of HO-1, the effects of which were more pronounced in IHOK cells than in HN12 cells. Collectively, these results suggest that HO-1 plays a principal role in the protective response to nicotine in oral cancer and immortalized keratinocytes. Moreover, the addition of exogenous antioxidants may help to protect oral epithelial cells as chemopreventive agents against nicotine-induced oxidative stress.

Inflammatory leukocytes and iron turnover in experimental hemorrhagic lung trauma

To monitor cascade of events following alveolar extravasation of blood due to exposure to shock wave (SW), we conducted spatiotemporal assessment of myeloperoxidase (MPO), heme oxygenase 1 (HO-1), Cu,Zn superoxide dismutase (SOD-1), transferrin (TRF), 3-nitrotyrosine (3NTyr), alveolar endothelial cadherin (VE-CDH), and the CD11b adhesion molecules on leukocytes using electron microscopy, electron paramagnetic resonance spectroscopy, immunofluorescence imaging, and immunoblotting. Accumulation of HO-1, MPO, 3NTyr, and SOD-1 in HIL at the first 12 h was associated with transmigration of inflammatory leucocytes (ILK) into hemorrhagic lesions (HLs). Biodegradation of extravasated hemoglobin (exvHb) and deposition of iron in alveoli occurred at 3–56 h post-exposure and was preceded by LKC degranulation and accumulation of MPO, HO-1, and SOD-1 in HLs. These alterations were accompanied by appearance of heme and non-heme iron complexes in HLs. A significant increase in TRF-bound [Fe 3+] (i.e., 14.6 ± 5.3 μM vs. 4.8 ± 2.1 μM immediately after exposure) and non-TRF complexes of [Fe 3+] (i.e., 4.5 ± 1.8 μM vs. < 0.3 μM immediately after exposure) occurred at 24 h post-exposure. Transmigrations of ILK, nitroxidative stress, and iron deposition in endothelial and epithelial cells were accompanied by destruction of endothelial integrity at 3 h post-exposure, and alveolar capillary network and necrotic changes in the pulmonary epithelial cells at 24–56 h post-exposure.

Dietary restriction attenuates the neuronal loss, induction of heme oxygenase-1 and blood–brain barrier breakdown induced by impaired oxidative metabolism

Experimental thiamine deficiency (TD) is a model of impaired oxidative metabolism associated with region-selective neuronal loss in the brain. Oxidative stress is a prominent feature of TD neuropathology, as evidenced by the accumulation of heme oxygenase-1 (HO-1), ferritin, reactive iron and superoxide dismutase in microglia, nitrotyrosine and 4-hydroxynonenal in neurons, as well as induction of endothelial nitric oxide synthase within the vulnerable areas. Dietary restriction (DR) reduces oxidative stress in several organ systems including the brain. DR increases lifespan and reduces neurodegeneration in a variety of models of neuronal injury. The possibility that DR can protect vulnerable neurons against TD-induced oxidative insults has not been tested. The current studies tested whether approximately 3 months of DR (60% of ad libitum intake) altered the response to TD. Six month-old ad libitum-fed or dietary restricted C57BL/6 mice received a thiamine-deficient diet either ad libitum, or under a DR regimen respectively for eleven days. The TD mice also received daily injections of the thiamine antagonist pyrithiamine. Control ad libitum-fed or DR mice received an unlimited amount, or 60% of ad libitum intake, respectively, of thiamine-supplemented diet. As in past studies, TD produced region-selective neuronal loss (−60%), HO-1 induction, and IgG extravasation in the thalamus of ad libitum-fed mice. DR attenuated the TD-induced neuronal loss (−30%), HO-1 induction and IgG extravasation in the thalamus. These studies suggest that oxidative damage is critical to the pathogenesis of TD, and that DR modulates the extent of free radical damage in the brain. Thus, TD is an important model for studying the relationship between aging, oxidative stress and nutrition.

Nitrosative and oxidative stress induced heme oxygenase-1 accumulation in rat mesangial cells.

The formation of nitric oxide (NO.) and superoxide (O2-) promotes rat mesangial cell death. Apoptotic death is characterized by DNA fragmentation, caspase-3 activation and concomitant poly(ADPribose) polymerase cleavage, as well as accumulation of the tumor suppressor protein p53. In close association with apoptotic parameters we noticed upregulation of heme oxygenase by the NO donor S-nitrosoglutathione (GSNO) and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) in a time- and concentration-dependent manner. In response to the NO. donor, heme oxygenase-1 expression was more easily obtained than initiation of apoptosis. Radical (NO./O2-) cogeneration abrogated DNA fragmentation, suppressed caspase activation and lowered p53 accumulation, thereby promoting cell survival of mesangial cells. In contrast, heme oxygenase-1 expression remained elevated under conditions of GSNO/DMNQ coadministration. Conclusively, heme oxygenase-1 is a stress marker for both nitrosative and oxidative stress. Accumulation of heme oxygenase-1 is found under conditions of both, apoptotic cell death and cell survival, thereby questioning a specific cytoprotective role of heme oxygenase-1 under conditions of NO. and/or O2- formation in rat mesangial cells.