Mapping of the Chick Heme Oxygenase-1 Proximal Promoter for Responsiveness to Metalloporphyrins

doi:10.1006/abbi.2001.2742

Archives of Biochemistry and Biophysics

Volume 399, Issue 2, 15 March 2002, Pages 159-166

https://doi.org/10.1006/abbi.2001.2742 Get rights and content

Abstract

Heme oxygenase (HO) catalyzes the rate-controlling step of physiologic heme catabolism, namely, the oxidation of the α-methene bridge of the macrocycle with formation of CO, Fe, and biliverdin. HO-1, the first isoform of HO to be identified, is highly inducible by a large number of physical and chemical factors. Many of these factors cause oxidative or other stresses to cells. In this work, we have studied the regulation of the chick HO-1 gene, using selected promoter–reporter constructs of the gene transiently or stably transfected into primary cultures of chick embryo liver cells or into the LMH line of chicken hepatoma cells. By use of deletional and mutational analyses, DNase protection, and electromobility shift DNA-binding assays, we identified a heretofore undefined regulatory region in the 5′-UTR of the chick HO-1 gene which confers up-regulation of reporter gene (luciferase) expression in the presence of heme and other selected metalloporphyrins. This new metalloporphyrin-responsive element (MPRE) was localized to a 200-bp region 3.8 to 3.6 kb upstream of the transcription starting point of the chick HO-1 gene. It responded particularly to heme and cobalt protoporphyrin with maximal inductions at 10–15 μM concentrations and 15–18 h of exposure. In contrast, sodium arsenite, a prototypical stress-type inducer of HO-1, led to down-regulation of the reporter gene down stream of MPRE. DNase analysis identified an 18-mer oligonucleotide that was required for the metalloporphyrin response (5′-⁻³⁷¹¹TATTGCAGCTGTGTGGGG-3′). Mutations at any of four sites within this oligonucleotide abrogated the metalloporphyrin-dependent up-regulation of reporter gene expression. Nuclear protein extracts of cells treated with heme or cobalt protoporphyrin showed specific enhanced binding to this 18-mer. We conclude that the chick HO-1 promoter region contains a unique sequence that subserves up-regulation of the gene by metalloporphyrins and propose the name “metalloporphyrin-responsive element” for this sequence.

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Citation Excerpt :
Among these isoforms of HO, HO-1 is the only high inducible isoform. Recent studies have been revealed that HO-1 could be up-regulated noticeably by a huge variety of stressful impetus, such as heme or certain other metalloporphyrins (Lu et al., 2000; Shan et al., 2000; Shan et al., 2002). This is the enzyme of the heme degradation and catalyzes the breakdown of heme to biliverdin, iron, and carbon monoxide (CO).
BACH1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1) is a transcriptional factor and a member of cap ‘n’ collar (CNC) and basic region leucine zipper factor family. In contrast to other bZIP family members, BACH1 appeared as a comparatively specific transcription factor. It acts as transcription regulator and is recognized as a recently hypoxia regulator and functions as an inducible repressor for the HO-1 gene in many human cell types in response to stress oxidative. In regard to studies lately, although, BACH1 has been related to the regulation of oxidative stress and heme oxidation, it has never been linked to invasion and metastasis. Recent studies have showed that BACH1 is involved in bone metastasis of breast cancer by up-regulating vital metastatic genes like CXCR4 and MMP1. This newly discovered aspect of BACH1 gene provides new insight into cancer progression study and stands on its master regulator role in metastasis process, raising the possibility of considering it as a potential target for cancer therapy.
Sirt1 mediates the effect of the heme oxygenase inducer, cobalt protoporphyrin, on ameliorating liver metabolic damage caused by a high-fat diet
2015, Journal of Hepatology
Citation Excerpt :
Three HO isozymes, HO-1, HO-2, and HO-3, have been identified; among them, HO-1 has been demonstrated to exert multiple biological effects, including anti-inflammatory, antiapoptotic and antiproliferative actions [15–17]. Many stressful stimuli are known to increase HO-1 expression, including heme or certain other metalloporphyrins, particularly CoPP [18]. CoPP is known to be a potent and effective inducer of HO-1 activity in many tissues [19,20].
Heme oxygenase 1 (HO-1)-mediated increases in adiponectin, ameliorate the deleterious effects of obesity and metabolic syndrome; however, the effect of HO-1 on hepatic lipid metabolism remains elusive. The aim of this study is to evaluate the role of HO-1 in hepatic lipid metabolism.
Functional studies were performed using C57BL/6J (WT) mice and Sirt1 liver specific mutant (Sirt1-deficient) mice. The molecular mechanism was explored in primary hepatocytes and mouse liver.
Chronic exposure to high-fat diet (HFD) induced hepatic steatosis in WT mice. Treatment of WT mice on HFD with cobalt protoporphyrin (CoPP), an inducer of HO-1 activity, decreased body weight and visceral fat content, reduced intracellular hepatic triglyceride and serum total cholesterol concentrations, and decreased liver lipid droplet formation. Compared with WT mice, the administration of CoPP to Sirt1-deficient mice on HFD increased visceral fat content, and slightly promoted liver lipid droplet formation. CoPP improved glucose tolerance and insulin sensitivity in WT mice on HFD, but compromised insulin sensitivity in Sirt1-deficient mice on HFD. Furthermore, CoPP-induced Sirt1 expression and decreased sterol regulatory element binding protein 1c (SREBP-1c) expression in WT mice on HFD. However, CoPP promoted SREBP-1c expression in Sirt1-deficient hepatocytes, which was reversed by a protein tyrosine phosphatase 1b inhibitor. Additionally, while the administration of CoPP to WT mice on HFD improved antioxidant and anti-inflammatory states, these CoPP-mediated effects were abolished in Sirt1-deficient mice.
Sirt1 mediates the effect of CoPP on ameliorating liver metabolic damage caused by HFD.
Cytoprotective responses in HaCaT keratinocytes exposed to high doses of curcumin
2015, Experimental Cell Research
Citation Excerpt :
Thus, although CoPP-treated cells have higher HO-1 protein expression levels, heme-degradation by HO-1 will be lower in CoPP-treated cells because of the limited availability of the FePP. Although CoPP is often used as a strong HO-1 inducer in vivo [22], it acts in vitro as an inhibitor of HO [38,39]. However, in contrast to FePP, CoPP is not degraded by HO-1 [40].
Wound healing is a complex process that involves the well-coordinated interactions of different cell types. Topical application of high doses of curcumin, a plant-derived polyphenol, enhances both normal and diabetic cutaneous wound healing in rodents. For optimal tissue repair interactions between epidermal keratinocytes and dermal fibroblasts are essential. We previously demonstrated that curcumin increased reactive oxygen species (ROS) formation and apoptosis in dermal fibroblasts, which could be prevented by pre-induction of the cytoprotective enzyme heme oxygenase (HO)-1. To better understand the effects of curcumin on wound repair, we now assessed the effects of high doses of curcumin on the survival of HaCaT keratinocytes and the role of the HO system. We exposed HaCaT keratinocytes to curcumin in the presence or absence of the HO-1 inducers heme (FePP) and cobalt protoporphyrin (CoPP). We then assessed cell survival, ROS formation, and caspase activation. Curcumin induced caspase-dependent apoptosis in HaCaT keratinocytes via a ROS-dependent mechanism. Both FePP and CoPP induced HO-1 expression, but only FePP protected against curcumin-induced ROS formation and caspase-mediated apoptosis. In the presence of curcumin, FePP but not CoPP induced the expression of the iron scavenger ferritin. Together, our data show that the induction of ferritin, but not HO, protects HaCaT keratinocytes against cytotoxic doses of curcumin. The differential response of fibroblasts and keratinocytes to high curcumin doses may provide the basis for improving curcumin-based wound healing therapies.
Nrf2-mediated haeme oxygenase-1 up-regulation induced by cobalt protoporphyrin has antinociceptive effects against inflammatory pain in the formalin test in mice
2008, Pain
This study investigated the effect of haeme oxygenase-1 (HO-1) in nociception induced by formalin injection in the mice hind paw. Intraperitoneal (i.p.) administration of cobalt protoporphyrin (CoPP, an HO-1 inducer, 5 mg/kg) 24 h before the test, inhibited the nociceptive response during the second phase, but not during the first phase of the formalin test. The effect of CoPP was prevented by treatment with tin protoporphyrin (SnPP, an inhibitor of HO-1 activity) administered either by i.p. (25 mg/kg, 30 min before the test) or intraplantar (400 nmol/paw, 5 min before the test) routes. Human embryonic kidney (HEK) 293T cells treated with 10 μM CoPP expressed 20-fold higher HO-1 levels when compared to controls; this effect was suppressed by transfection with the dominant negative for the nuclear factor-erythroid 2-related factor 2 (Nrf2). Western blot analysis also revealed that CoPP treatment induced a similar 20-fold increase in HO-1 expression in the paw; this effect was attenuated in knockout mice for Nrf2. CoPP treatment of wild-type, but not in Nrf2 knockout mice, resulted in a striking increase of HO-1 stained cells surrounding the muscular tissues of the hind limbs. HO-1 positive cells were scarce in wild-type and in Nrf2 knockout untreated mice. CoPP-induced HO-1 expression in Nrf2 knockout mice was lost and correlated with the loss of antinociceptive effects. In conclusion, Nrf2-mediated HO-1 expression induced an antinociceptive effect at peripheral sites. These results suggest that HO-1 modulates the inflammatory pain pathways. Hence, the development of drugs that could raise peripheral HO-1 could be relevant in inflammatory pain treatment.
Reciprocal Effects of Micro-RNA-122 on Expression of Heme Oxygenase-1 and Hepatitis C Virus Genes in Human Hepatocytes
2007, Gastroenterology
Background & Aims: Heme oxygenase-1 (HO-1) is an antioxidant defense and key cytoprotective enzyme, which is repressed by Bach1. Micro-RNA-122 (miR-122) is specifically expressed and highly abundant in human liver and required for replication of hepatitis C virus (HCV) RNA. This study was to assess whether a specific miR-122 antagomir down-regulates HCV protein replication and up-regulates HO-1. Methods: We transfected antagomir of miR-122, 2’-O-methyl-mimic miR-122, or nonspecific control antagomir, into wild-type (WT) Huh-7 cells or Huh-7 stably replicating HCV subgenomic protein core through nonstructural protein 3 of HCV (NS3) (CNS3 replicon cells) or NS3-5B (9-13 replicon cells). Results: Antagomir of miR-122 reduced the abundance of HCV RNA by 64% in CNS3 and by 84% in 9-13 cells. Transfection with 2’-O-methlyl-mimic miR-122 increased HCV levels up to 2.5-fold. Antagomir of miR-122 also decreased Bach1 and increased HO-1 mRNA levels in CNS3, 9-13, and WT Huh-7 cells. Increasing HO-1 by silencing Bach1 with 50 nmol/L Bach1-short interfering RNA or by treatment with 5 μmol/L cobalt protoporphyrin or heme (known inducers of HO-1) decreased HCV RNA and protein by 50% in HCV replicon cells. Conclusions: Down-regulation of HCV replication using an antagomir targeted to miR-122 is effective, specific, and selective. Increasing HO-1, by silencing the Bach1 gene or by treatment with cobalt protoporphyrin or heme, decreases HCV replication. Thus, miR-122 plays an important role in the regulation of HCV replication and HO-1/Bach1 expression in hepatocytes. Down-regulation of miR-122 and up-regulation of HO-1 may be new strategies for anti-HCV intervention and cytoprotection.
Role of Bach-1 in regulation of heme oxygenase-1 in human liver cells: Insights from studies with small interfering RNAs
2004, Journal of Biological Chemistry
Citation Excerpt :
We have previously demonstrated heme-dependent inductions of HO-1 gene expression in primary cultures of chick embryo liver cells (29, 30). Recently, we identified four HeREs and one metalloporphyrin response element within the HO-1 promoter region that mediated heme-dependent activation of the HO-1 gene in chick LMH cells (12, 13). To characterize the mechanism whereby heme up-regulates HO-1 gene expression in human liver cells, we first examined HO-1 mRNA and protein levels after treatment with heme in Huh-7 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 the HO-1 gene 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-regulating HO-1 gene 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.

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^☆: This work was supported by United States Public Health Service Grant DK-RO1-38825 and Contract DK-NO1-92326 (to H.L.B.). The opinions expressed herein are those of the authors. They do not necessarily reflect the official views of the USPHS or The University of Massachusetts.

²: To whom correspondence and reprint requests should be addressed at Room 270G, 364 Plantation Street, Worcester, MA 01655. Fax: (508) 856-5971. E-mail: [email protected].

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Regular ArticleMapping of the Chick Heme Oxygenase-1 Proximal Promoter for Responsiveness to Metalloporphyrins☆

Abstract

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Proc. Natl. Acad. Sci. USA

Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2

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Science

Regular Article
Mapping of the Chick Heme Oxygenase-1 Proximal Promoter for Responsiveness to Metalloporphyrins☆