Role of Bach-1 in Regulation of Heme Oxygenase-1 in Human Liver Cells

Heme oxygenase-1 is an antioxidant defense enzyme that converts heme to biliverdin, iron, and carbon monoxide. Bach-1 is a bZip protein that forms heterodimers with small Maf proteins and was reported recently to down-regulate theHO-1gene in mice. Using small interfering RNAs targeted to human Bach-1 mRNA, we investigated whether modulation of human hepatic Bach-1 expression by small interfering (si)RNA technology influences heme oxygenase-1 gene expression. We found that Bach-1 siRNAs transfected into Huh-7 cells significantly reduced Bach-1 mRNA and protein levels ∼80%, compared with non siRNA-treated cells. In contrast, transfection with the same amounts of nonspecific control duplexes or LaminB2-duplex did not reduce Bach-1 mRNA or protein levels, confirming the specificity of Bach-1 siRNA. Expression of the heme oxygenase-1 gene in Bach-1 siRNA-transfected cells was up-regulated 7-fold, compared with cells without Bach-1 siRNA. The effect of increasing concentrations of heme to up-regulate levels of heme oxygenase-1 was more pronounced when Bach-1 siRNA was present. Taken together, these results indicated that Bach-1 has a specific and selective ability to repress expression of human hepatic heme oxygenase-1. Silencing of Bach-1 by siRNAs is a useful method for up-regulatingHO-1gene expression. Exogenous heme produces additional up-regulation, beyond that produced by Bach-1 siRNAs, suggesting that heme does not act solely through its effects on Bach-1.

Download Full-text

Synergistic Up-Regulation of Heme Oxygenase-1 in Human Liver by Heme and siRNA to Bach1:Possible Therapeutic Implications.

Blood ◽

10.1182/blood.v104.11.1633.1633 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1633-1633

Author(s):

Tahereh Ghaziani ◽

Ying Shan ◽

Richard W. Lambrecht ◽

Herbert L. Bonkovsky

Keyword(s):

Gene Expression ◽

Heme Oxygenase ◽

Ischemia Reperfusion Injury ◽

Tissue Injury ◽

Ischemia Reperfusion ◽

Negative Regulator ◽

Heme Oxygenase 1 ◽

Significant Finding ◽

Mrna Levels ◽

Protein Levels

Abstract Background: Heme oxygenase-1 (HO-1) is an antioxidant defense enzyme that converts toxic heme into antioxidants. HO-1 is strongly up-regulated by its physiologic substrate, heme, which is currently the treatment of choice for acute attacks of porphyria and which may have other therapeutic uses, as well (e.g., for cytoprotection or amelioration of ischemia/reperfusion injury by increasing supply of carbon monoxide, biliverdin, or bilirubin). Up-regulation of HO-1 expression has been associated with increased resistance to tissue injury. Bach1 is a bZip protein which forms heterodimers with small Maf proteins. HO-1 is expressed at higher levels in tissues of Bach1-deficient mice, indicating that Bach1 acts as a negative regulator of the mouse HO-1 gene. The molecular mechanism that confers repression of HO-1 by Bach1, and whether there are similar effects in human cells, has remained elusive. The aim of this study was to assess whether modulation of human hepatic Bach1 expression by siRNA technology influences HO-1 gene expression and whether such gene silencing would enhance the inducing effects of heme on HO-1. Methods: siRNAs targeted 4 different positions of human Bach1 mRNA were designed and synthesized. We transfected Bach1-siRNA (25–200 nM) into Huh-7 cells using Lipofectamine for 24–72 h, after which, cells were treated with or without heme. We quantified HO-1 and Bach1 mRNA and protein levels by quantitative RT-PCR and western blotting, respectively. Effects and specificity of Bach1-siRNA were analyzed and compared with those of non-Bach1 related siRNAs (non-specific control-duplex (NSCD) and LaminB2-siRNA). Results: Bach1-siRNAs (25–200 nM) transfected into Huh-7 cells for 24–72 h significantly reduced Bach1 mRNA and protein levels approximately 80%, compared with non siRNA treated cells. In contrast, transfection with same amounts of NSCD or LaminB2 siRNA did not reduce Bach1 mRNA or protein levels, confirming the specificity of Bach1-siRNA in Huh-7 cells. A significant finding of these studies was the 7-fold up-regulation of the HO-1 gene in Bach1-siRNA transfected cells, compared to cells without Bach1-siRNA or those transfected with NSCD or LaminB2. Bach1, NSCD, and LaminB2 siRNAs had no effect on HO-2 or 5-aminolevulinate synthase-1 mRNA levels (two genes that are not induced by heme). The effects of increasing concentrations of heme (up to 10 μM) in the presence or absence of Bach1-siRNA on the levels of HO-1 mRNA expression are shown in the Figure. For all of the heme concentrations tested, the levels of HO-1 mRNA were greater when Bach1 siRNA was present. Conclusions: Bach1 has a specific and selective effect to repress expression of human hepatic HO-1. Silencing of the Bach1 gene by siRNAs may be a useful method for up-regulating HO-1 gene expression. The combination of intravenous heme and Bach1 silencing may be useful for therapy of acute porphyrias in relapse or other conditions in which up-regulation of HO-1 may be beneficial. (Supported by grants from NIH [DK38825] and Ovation Pharmaceuticals, Inc.) Figure Figure

Download Full-text

The Role of Heme Oxygenase 1 in the Protective Effect of Caloric Restriction against Diabetic Cardiomyopathy

International Journal of Molecular Sciences ◽

10.3390/ijms20102427 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2427 ◽

Cited By ~ 8

Author(s):

Maayan Waldman ◽

Vadim Nudelman ◽

Asher Shainberg ◽

Romy Zemel ◽

Ran Kornwoski ◽

...

Keyword(s):

Oxidative Stress ◽

Beneficial Effect ◽

Heme Oxygenase ◽

Diabetic Cardiomyopathy ◽

High Glucose ◽

Heme Oxygenase 1 ◽

Ros Production ◽

Diabetic Mice ◽

Protein Levels

Type 2 diabetes mellitus (DM2) leads to cardiomyopathy characterized by cardiomyocyte hypertrophy, followed by mitochondrial dysfunction and interstitial fibrosis, all of which are exacerbated by angiotensin II (AT). SIRT1 and its transcriptional coactivator target PGC-1α (peroxisome proliferator-activated receptor-γ coactivator), and heme oxygenase-1 (HO-1) modulates mitochondrial biogenesis and antioxidant protection. We have previously shown the beneficial effect of caloric restriction (CR) on diabetic cardiomyopathy through intracellular signaling pathways involving the SIRT1–PGC-1α axis. In the current study, we examined the role of HO-1 in diabetic cardiomyopathy in mice subjected to CR. Methods: Cardiomyopathy was induced in obese diabetic (db/db) mice by AT infusion. Mice were either fed ad libitum or subjected to CR. In an in vitro study, the reactive oxygen species (ROS) level was determined in cardiomyocytes exposed to different glucose levels (7.5–33 mM). We examined the effects of Sn(tin)-mesoporphyrin (SnMP), which is an inhibitor of HO activity, the HO-1 inducer cobalt protoporphyrin (CoPP), and the SIRT1 inhibitor (EX-527) on diabetic cardiomyopathy. Results: Diabetic mice had low levels of HO-1 and elevated levels of the oxidative marker malondialdehyde (MDA). CR attenuated left ventricular hypertrophy (LVH), increased HO-1 levels, and decreased MDA levels. SnMP abolished the protective effects of CR and caused pronounced LVH and cardiac metabolic dysfunction represented by suppressed levels of adiponectin, SIRT1, PPARγ, PGC-1α, and increased MDA. High glucose (33 mM) increased ROS in cultured cardiomyocytes, while SnMP reduced SIRT1, PGC-1α levels, and HO activity. Similarly, SIRT1 inhibition led to a reduction in PGC-1α and HO-1 levels. CoPP increased HO-1 protein levels and activity, SIRT1, and PGC-1α levels, and decreased ROS production, suggesting a positive feedback between SIRT1 and HO-1. Conclusion: These results establish a link between SIRT1, PGC-1α, and HO-1 signaling that leads to the attenuation of ROS production and diabetic cardiomyopathy. CoPP mimicked the beneficial effect of CR, while SnMP increased oxidative stress, aggravating cardiac hypertrophy. The data suggest that increasing HO-1 levels constitutes a novel therapeutic approach to protect the diabetic heart. Brief Summary: CR attenuates cardiomyopathy, and increases HO-1, SIRT activity, and PGC-1α protein levels in diabetic mice. High glucose reduces adiponectin, SIRT1, PGC1-1α, and HO-1 levels in cardiomyocytes, resulting in oxidative stress. The pharmacological activation of HO-1 activity mimics the effect of CR, while SnMP increased oxidative stress and cardiac hypertrophy. These data suggest the critical role of HO-1 in protecting the diabetic heart.

Download Full-text

The role of cyp1a and heme oxygenase 1 gene expression for the toxicity of 3,4-dichloroaniline in zebrafish (Danio rerio) embryos

Aquatic Toxicology ◽

10.1016/j.aquatox.2007.10.007 ◽

2008 ◽

Vol 86 (1) ◽

pp. 112-120 ◽

Cited By ~ 19

Author(s):

Doris Voelker ◽

Nicole Stetefeld ◽

Kristin Schirmer ◽

Stefan Scholz

Keyword(s):

Gene Expression ◽

Heme Oxygenase ◽

Danio Rerio ◽

Heme Oxygenase 1 ◽

Danio Rerio Embryos

Download Full-text

COMPARATIVE ROLE OF ANTIOXIDANTS FOR MODULATION OF AP-1 AND NF-B AND THEIR ROLE FOR HEME OXYGENASE-1 GENE EXPRESSION IN THE LIVER AFTER HEMORRHAGIC SHOCK

Shock ◽

10.1097/00024382-199906001-00216 ◽

1999 ◽

Vol 11 (Supplement) ◽

pp. 62 ◽

Cited By ~ 1

Author(s):

H. Rensing ◽

Bauer ◽

H. Jäschke ◽

M. Bauer

Keyword(s):

Gene Expression ◽

Hemorrhagic Shock ◽

Heme Oxygenase ◽

Heme Oxygenase 1

Download Full-text

Role of heme oxygenase-1 in the regulation of manganese superoxide dismutase gene expression in oxidatively-challenged astroglia

Journal of Cellular Physiology ◽

10.1002/1097-4652(200010)185:1<80::aid-jcp7>3.0.co;2-w ◽

2000 ◽

Vol 185 (1) ◽

pp. 80-86 ◽

Cited By ~ 91

Author(s):

Dov Frankel ◽

Khalil Mehindate ◽

Hyman M. Schipper

Keyword(s):

Gene Expression ◽

Superoxide Dismutase ◽

Heme Oxygenase ◽

Manganese Superoxide Dismutase ◽

Heme Oxygenase 1 ◽

Manganese Superoxide ◽

Superoxide Dismutase Gene

Download Full-text

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

Cellular & Molecular Biology Letters ◽

10.2478/s11658-006-0052-0 ◽

2007 ◽

Vol 12 (1) ◽

Cited By ~ 6

Author(s):

Malte Silomon ◽

Inge Bauer ◽

Michael Bauer ◽

Julia Nolting ◽

Markus Paxian ◽

...

Keyword(s):

Gene Expression ◽

Stress Response ◽

Heat Shock ◽

Heme Oxygenase ◽

Rat Hepatocytes ◽

Glutathione Depletion ◽

Heme Oxygenase 1 ◽

Hsp70 Gene ◽

Stress Response Genes

AbstractStress 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.

Download Full-text

Dysregulated Autophagy and Mitophagy in a Mouse Model of Duchenne Muscular Dystrophy Remain Unchanged Following Heme Oxygenase-1 Knockout

International Journal of Molecular Sciences ◽

10.3390/ijms23010470 ◽

2021 ◽

Vol 23 (1) ◽

pp. 470

Author(s):

Olga Mucha ◽

Katarzyna Kaziród ◽

Paulina Podkalicka ◽

Kinga Rusin ◽

Józef Dulak ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Heme Oxygenase ◽

Mrna Level ◽

Mdx Mice ◽

Heme Oxygenase 1 ◽

Protein Levels ◽

Dystrophic Mice

Dysregulation of autophagy may contribute to the progression of various muscle diseases, including Duchenne muscular dystrophy (DMD). Heme oxygenase-1 (HO-1, encoded by Hmox1), a heme-degrading enzyme, may alleviate symptoms of DMD, inter alia, through anti-inflammatory properties. In the present study, we determined the role of HO-1 in the regulation of autophagy and mitophagy in mdx animals, a commonly used mouse model of the disease. In the gastrocnemius of 6-week-old DMD mice, the mRNA level of mitophagy markers: Bnip3 and Pink1, as well as autophagy regulators, e.g., Becn1, Map1lc3b, Sqstm1, and Atg7, was decreased. In the dystrophic diaphragm, changes in the latter were less prominent. In older, 12-week-old dystrophic mice, diminished expressions of Pink1 and Sqstm1 with upregulation of Atg5, Atg7, and Lamp1 was depicted. Interestingly, we demonstrated higher protein levels of autophagy regulator, LC3, in dystrophic muscles. Although the lack of Hmox1 in mdx mice influenced blood cell count and the abundance of profibrotic proteins, no striking differences in mRNA and protein levels of autophagy and mitophagy markers were found. In conclusion, we demonstrated complex, tissue, and age-dependent dysregulation of mitophagic and autophagic markers in DMD mice, which are not affected by the additional lack of Hmox1.

Download Full-text