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Keywords:

  • heme oxygenase-1;
  • Gly139His mutant;
  • Gly143His mutant;
  • mouse

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Heme oxygenase-1 (HO-1), which catalyzes the degradation of heme to iron, carbon monoxide, and biliverdin, performs a cytoprotective function. Previous studies on the crystal structure of the human and rat HO-1 in complex with heme showed that Gly139His (G139H) and Gly143His (G143H) mutants have no HO activity. In the present study, we reported the effect of the G139H, G143H, and Ser142His mutants of mouse HO-1 on the HO reaction in vivo and in vitro. In vitro, of the mutant transfectants, only Ser142His catalyzed degradation of heme, retaining 31.7% of the wild-type mouse HO-1 activity, whereas G139H and G143H mutants exhibited no activity. In vivo, only Tg HO-1 G143H females presented with anemia, enlarged spleen and tissue iron overload, which was similar to HO-1−/− mice. The results suggested the critical role of Gly139 and Gly143 in maintaining HO-1 activity in vitro and the critical role of Gly143 in maintaining HO-1 activity in vivo. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in the oxidative degradation of heme to release iron, carbon monoxide, and biliverdin (Tenhunen et al.,1968). Due to the potent antioxidant activity of biliverdin and bilirubin, and to the cytoprotective actions of carbon monoxide on vascular endothelium and nerve cells, it is widely accepted that upregulation of HO-1 causes beneficial effects (Stocker et al.,1987; Verma et al.,1993; Brouard et al.,2000; Colle et al.,2008; Mancuso and Barone,2009; Ryter and Choi,2009; Gozzelino et al.,2010). In contrast, the lack of, or inhibition of, HO-1 generally accelerates growth retardation, anemia, and tissue iron deposition, as seen in HO-1-deficient humans and mice (Poss and Tonegawa,1997a,b; Kapturczak et al.,2004; Zhao et al.,2009).

There are two isoforms of HO; HO-1 and HO-2. A third isoform, known as HO-3, has been described in the rat brain; however, no homolog of this gene is found in either humans or mice (Hayashi et al.,2004). HO-1 is inducible by various substances and is highly expressed in the spleen and liver. HO-2 is constitutively expressed in the brain, testis, and vascular systems. HO enzymes are membrane-bound proteins but are water soluble. Truncated forms of HO-1, which lack the C-terminal 23-amino acid membrane anchor, have been used to obtain crystal structures of both the human and rat enzymes and their catalytically active versions have been expressed in Escherichia coli (Wilks and Ortiz de Montellano,1993; Liu et al.,2000; Sugishima et al.,2000). The truncated rat and human HO-1 crystal structure shows that the heme pocket is primarily formed by two helices, one on the proximal and the other on the distal side of the heme molecule. The proximal helix consists of Leu13 to Glu29, including the proximal ligand of heme, His25. The distal helix consists of Leu129 to Met155 and bends at Leu141 and Ser142 (Wilks and Ortiz de Montellano,1993; Liu et al.,2000; Sugishima et al.,2000; Lad et al.,2005). In these studies, the crystal structure of the truncated rat and human HO-1 suggested that Gly139 and Gly143 interact directly with heme. Gly139His (G139H) and Gly143His (G143H) mutants of HO-1 bind heme but have no HO activity.

As the amino acid sequence in the distal helix is well conserved among animal species (Maines and Gibbs,2005), we constructed and expressed G139H, G143H, and Ser142His (S142H) mutants of mouse HO-1 (mHO-1) to determine the importance of the three conserved residues in mHO-1-catalyzed heme degradation in vivo and in vitro.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Preparation of Plasmid cDNAs

We prepared pCAGG G139H mutant mouse HO-1 cDNA (pCAGG-mHO-1-G139H), pCAGG S142H mutant mouse HO-1 cDNA (pCAGG-mHO-1-S142H) and pCAGG G143H mutant mouse HO-1 cDNA (pCAGG-mHO-1-G143H) constructs using the QuikChange site-directed mutagenesis kit (Stratagene). In brief, a mHO-1 cDNA (provided by Professor Osamu Nakajima, Yamagata University, Japan) was inserted into the pCAGGS vector (Niwa et al.,1991) at the EcoRI site (pCAGG-mHO-1), which expresses the mHO-1 cDNA under the control of the chicken β-actin promoter. pCAGG-mHO-1 was used as a template to mutate Gly139, Ser142, and Gly143 of mHO-1 protein to His. Transformants were screened by restriction digestion and confirmed by sequence analysis. Plasmid purification, sequencing, subcloning, and bacterial transformations were carried out by standard procedures (Sambrook and Russel,2001).

Transient Transfection of Wild-Type mHO-1 and/or Mutant mHO-1 in Eukaryotic Cells

QT6 quail cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate (all from Sigma). First, transient transfection was performed in 10-cm dishes by calcium phosphate precipitation with 5 μg of pCAGG-mHO-1 plasmid. HO activity was then determined under conditions of 3, 7.5, 15, and 25 μM heme to determine which final heme concentrations were excessive and insufficient for 5 μg of pCAGG-mHO-1 plasmid transfection. Second, transient transfection was performed in 10-cm dishes by calcium phosphate precipitation with 5 μg of pCAGG-mHO-1-G139H, 5 μg of pCAGG-mHO-1-S142H, or 5 μg of pCAGG-mHO-1-G143H plasmid in the presence or absence of 5 μg of pCAGG-mHO-1 plasmid. HO activities were then determined under conditions of excessive heme. Third, transient cotransfection was performed in 10-cm dishes by calcium phosphate precipitation with 5 μg of pCAGG-mHO-1 plasmid, along with 3, 7.5, 15, or 25 μg of pCAGG-mHO-1-G139H or pCAGG-mHO-1-G143H plasmid. HO activities were then determined under conditions of insufficient heme. The pCAGG plasmid was introduced in some reactions to maintain a constant quantity of DNA. pCAGG-EGFP plasmid (provided by Professor Osamu Nakajima, Yamagata University, Japan) was introduced as an internal control. Each transfection replicated in at least three separate experiments.

Transient transfection of COS7, NIH3T3, and Hepa1 cells was performed in 10-cm dishes using lipofection (Invitrogen) with 5 μg of pCAGG-mHO-1-G139H, 5 μg of pCAGG-mHO-1-S142H, or 5 μg of pCAGG-mHO-1-G143H plasmid in the presence or absence of 5 μg of pCAGG-mHO-1 plasmid. HO activities were then determined under conditions of excessive heme.

HO Activities in the Transformants

HO activities in the transfected cells were determined by measuring the formation of bilirubin as described previously (Lincoln et al.,1988). Briefly, cells were homogenized on ice with 0.25 M sucrose, 20 mM Tris-HCl (pH 7.4) buffer. Homogenates were mixed with 5 mM deferoxamine, 25 μM hemin (in the second transient transfection experiment), or 7.5 μM hemin (in the third transient cotransfection experiment), 15 μM bovine serum albumin, 1 mM NADPH, 0.1 M potassium phosphate (pH 7.4), and purified biliverdin reductase and cytochrome P450 reductase (provided by Professor Tadashi Yoshida, Yamagata University, Japan). After incubation at 37°C for 10 min, any insoluble material was removed by centrifugation and supernatants were analyzed for bilirubin concentration by reverse phase high-performance liquid chromatography.

Animals

Animal experiments were approved by the Animal Care Committee of Harbin Medical University. Care and handling of the animals were in accordance with National Institutes of Health guidelines. Mice housed under identical conditions were allowed free access to a standard diet and to tap water, with a 12-hr light: 12-hr dark cycle.

Generation of G139H, S142H, and G143H Mutant mHO-1 Transgenic Mice

The transgenes, isolated from the pCAGG-mHO-1, pCAGG-mHO-1-G139H, pCAGG-mHO-1-G143H, and pCAGG-mHO-1-S142H were separately injected into C57BL/6 fertilized eggs for the generation of transgenic mice, as described previously (Zhou et al.,1998). The HO-1 transgenic allele was identified by analysis of tail DNA by reverse transcription-polymerase chain reaction (RT-PCR), using an upper primer from the β-actin promoter (5′-GCCTTCTTCTTTTTCCTACAGCTC-3′) and a lower primer from the mHO-1 cDNA (5′-GGCATGCTGTCGGGCTGTGGAC-3′).

Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted from the livers of 3.5-month-old mice using Isogen (WAKO), according to the manufacturer's instructions. RNA concentration and purity was determined spectrophotometrically at 260 and 280 nm. First strand cDNA was synthesized using superscript II (Invitrogen).

Blood Cell Counts and Histology

Blood was obtained by retro-orbital sampling, and blood cell counts were determined using a veterinary hematology analyzer (Kohden, Japan). For histology, tissues were fixed in 10% buffered formalin and paraffin embedded. Sections (4-μm thick) were stained with Prussian blue to detect ferric iron (Nakajima et al.,1999). Counterstaining was performed with nuclear fast red for 10 sec to maximize the detection of iron.

Serum Iron Parameters

Mice were bled retro-orbitally and 200 μL of serum from each animal was used for analysis of iron and total iron-binging capacity (TIBC) using a kit from Sigma.

Statistical Analysis

Results are presented as means ± SEM. Statistical evaluation was performed with an unpaired Student's t test for two groups or one-way ANOVA for multiple groups. Differences were considered as significant at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Activities of the G139H, S142H, and G143H mHO-1 Mutants in QT6 Cells

To examine the activities of G139H, S142H, and G143H mHO-1 mutants in eukaryotic cells, transient transfection of QT6 cells was performed with wild-type or mutant HO-1 cDNA. As shown in Fig. 1A, the parent and mock transfectant cells did not show any notable HO activity. The wild-type HO-1 transfectant exhibited 710.01 pmol bilirubin/mg protein/min enzymatic activity. The S142H mutant transfectant exhibited 224.91 pmol bilirubin/mg protein/min enzymatic activity, while the G139H and G143H mutant transfectants did not show any notable elevation of enzymatic activity. In addition, the cells cotransfected with the wild-type mHO-1 and mutant S142H mHO-1 cDNA displayed 1042.23 pmol bilirubin/mg protein/min enzymatic activity, which roughly equates to a combination of 710.01 and 224.91 pmol bilirubin/mg protein/min. Compared with the wild-type mHO-1 transfectant, the cells cotransfected with wild-type and mutant G139H or G143H mHO-1 cDNA did not show any elevation of HO enzymatic activity. We observed similar results using transient transfection of COS7, NIH3T3, and Hepa 1 cells. The G139H and G143H mutant transfectants did not show any notable elevation of HO enzymatic activity. The S142H mutant transfectant exhibited one-third of the enzymatic activity of the wild-type HO-1 transfectant. COS7, NIH3T3, and Hepa 1 cells cotransfected with wild-type and mutant G139H or G143H mHO-1 cDNA did not show any elevation of HO enzymatic activity compared with the wild-type mHO-1 alone transfectant (data not shown). These results indicated that mutant G139H and G143H mHO-1 lacked catalytic activity in eukaryotic cells, and that mutant S142H mHO-1 had decreased catalytic activity in eukaryotic cells. On the other hand, the results suggested that mutant G139H or G143H mHO-1 and wild-type mHO-1 cotransfectants did not contain elevated HO activities compared with wild-type mHO-1 alone transfectant.

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Figure 1. HO activities in transfected or cotransfected QT6 cells. A: HO activities in QT6 cells transfected with pCAGG-mHO-1-G139H (G139H), pCAGG-mHO-1-S142H (S142H), or pCAGG-mHO-1-G143H (G143H) plasmids in the presence or absence of pCAGG-mHO-1 (HO-1) plasmid under conditions of excess heme. *, P < 0.05 compared with only pCAGG-mHO-1 plasmid transfection. B: HO activities under conditions of 3–25 μM heme. *, P < 0.05 compared with 3 μM heme final concentration; #, P < 0.05 compared with 15 μM heme final concentration. C: HO activities in QT6 cells cotransfected with pCAGG-mHO-1 (HO-1) plasmid and increasing dose of pCAGG-mHO-1-G139H or pCAGG-mHO-1-G143H (mut HO-1) plasmid under conditions of insufficient heme. *, P < 0.05 compared with 5 μg of pCAGG-mHO-1 plasmid and 15 μg of pCAGG plasmid cotransfection.

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Previous studies on the crystal structure of the truncated rat and human HO-1 suggested that G139H and G143H mutants of HO-1 had no activity but did bind heme. In the above experiments, HO activities were measured in a common reaction buffer containing excessive heme. Under these conditions, it was unclear whether G139H and G143H mHO-1 mutants bound to heme or not. To clarify this, we first demonstrated that for 5 μg of wild-type mHO-1 DNA transfection, 7.5 μM hemin in the reaction buffer of HO activity was insufficient to allow maximum HO activity (Fig. 1B). The cells were then cotransfected with the wild type and increasing doses of mutant G139H or G143H HO-1 cDNA and HO activities were measured under conditions of 7.5 μM heme. As shown in Fig. 1C, cotransfectants displayed decreasing HO activity in a dose-dependent manner. As compared with mutant G139H and wild-type HO-1 cotransfectants, the decrease in HO activity in mutant G143H and wild-type HO-1 cotransfectants was more obvious. These results suggested that G139H and G143H mutant mHO-1 could bind heme, and that cotransfection with mutant G139H or G143H mHO-1 and wild-type mHO-1 might decrease HO activity under conditions of insufficient heme.

Generation of G139H, S142H, and G143H mHO-1 Mutant Transgenic Mice

In vivo heme concentration is insufficient for HO activity reaction. According to the in vitro results, the overexpression of G139H and G143H mHO-1 mutants in vivo was expected to reduce endogenous HO activity. Thus, the phenotype of the mutant HO-1 overexpressing mice might resemble HO-1-deficient mice. Therefore, we generated four founder lines of G139H, three founder lines of S142H, and three founder lines of G143H mutant HO-1 transgenic mice (Tg HO-1 G139H, Tg HO-1 S142H, and Tg HO-1 G143H). RT-PCR analyses of the transgene-derived HO-1 mRNA expression in the livers among the established lines of Tg HO-1 G143H mice showed extremely high expression in two lines (H1 and H2 lines) and low expression in one line (Fig. 2A). RT-PCR analyses of the transgene-derived HO-1 mRNA expression in the livers among the established lines of Tg HO-1 G139H and Tg HO-1 S142H mice showed high expression in two lines and low expression in the other two lines, and high expression in one line and low expression in two lines, respectively (data not shown). No increase in levels of HO-1 mRNA was detected in the livers of the H1 line of Tg HO-1 G143H mice compared with high-expression lines of Tg HO-1 G139H and S142H mice (Fig. 2B).

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Figure 2. RT-PCR analysis of transgene expression. RT-PCR analysis was performed with a primer set specific for transgenic (Trans) HO-1 cDNA. A: RT-PCR analysis for the mutant G143H mHO-1 transgene expression. Total RNA samples were extracted from livers of HO-1 transgenic females among different lines (H1, H2, two high-expression lines; L, one low-expression line). *, P < 0.05 compared with mice of the low-expression line; #, P > 0.05 compared with mice of H1 line. B: RT-PCR analysis for the expression of mutant G143H, G139H, and S142H mHO-1 transgenes from the high line, respectively. HPRT mRNA levels were examined to confirm equal loading. *, P > 0.05 compared with mutant G143H.

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Partial Prenatal and Natal Lethality Among Tg HO-1 G143H Males From H1 and H2 Lines, and Splenomegaly, Anemia, Serum Iron Reduction in Tg HO-1 G143H Females From H1 and H2 Lines

Of 50 offspring, 12 male and 13 female pups from the low line expressed Tg HO-1 G143H, which was in accordance with the Mendelian ratio (Table 1). Of 48 offspring, five male pups from the H1 line and four male pups from the H2 line expressed Tg HO-1 G143H (Table 1). Unexpectedly, these nine Tg males had died within 1 day (Table 1). The birthrate of Tg HO-1 G143H males in two high-expression lines was significantly lower than that in the low-expression line, whereas the birthrate of Tg HO-1 G143H females in the two high- and low-expression lines was in accordance with the Mendelian ratio. Tg mice mated between Tg HO-1 G139H/Tg HO-1 S142H and wild-type mice yield the expected Mendelian ratio. This remainder of the report focuses on the phenotype of the mutant Tg mice, while the basis of the prenatal and natal lethality of Tg HO-1 G143H males will be described in detail elsewhere.

Table 1. Prenatal and natal lethality in Tg HO-1-G143H males from H1 and H2 lines
 Tg♂(Tg♀)/Total
LH1H2
Birth12 (13)/505 (12)/484 (13)/48
1 Day12 (13)/500 (12)/430 (13)/48
1 Year12 (13)/500 (12)/430 (13)/48

Tg HO-1 G143H females were slightly smaller than sex-matched wild-type littermates from 24 weeks-of-age to middle age (Fig. 3A) but were otherwise indistinguishable. To assess the possible pathological consequence associated with the wasting in Tg HO-1 G143H females, we first anatomized some 12-month-old mice from the H1 and H2 lines. We observed enlarged spleens in all examined Tg HO-1 G143H females (Fig. 3B). Associated with the splenomegaly was an anemia, as shown in the analyses of blood hemoglobin concentration and mean cell volume, but no inflammatory disease, as shown in the analysis of white blood cell counts (Table 2). This anemia was microcytic and hypochromic, similar to hypoferremia caused by iron deficiency. We then analyzed serum iron levels and TIBC in 12-month-old Tg HO-1 G143H females from the H1 line. As predicted, the serum iron value was reduced and the TIBC was elevated in Tg HO-1 G143H females compared with sex-matched wild-type littermates (Table 3). These results indicated that heme deficiency was caused by transgenic overexpression of G143H mutant mHO-1. That is, mutant G143H mHO-1 bound heme, which caused endogenous heme deficiency, but mutant G143H mHO-1 lacked enzymatic activity according to the results in QT6 cells, which caused serum iron reduction. We did not observe any wasting, enlarged spleen, anemia, and reduction of serum iron levels in Tg HO-1 S142H mice or Tg HO-1 G139H mice, which suggested Gly143 was critical in interacting directly with heme in vivo.

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Figure 3. Wasting, splenomegaly, and iron loading in Tg G143H mHO-1 females from the H1 line. A: Tg G143H mHO-1 females were consistently smaller than wild-type littermates. The number of mice per group was 8. B: Splenomegaly in 12-month-old Tg G143H mHO-1 females from the H1 and H2 lines. Upper panel, significant differences in the spleens of Tg G143H mHO-1 females compared with sex-matched wild-type littermates were observed; lower panel, significant difference (P < 0.05) of spleen weight. Values in parentheses are the number of mice per group. CF: Iron deposition was detectable by Prussian blue staining in the livers (D) and the kidneys (F) of 12-month-old Tg G143H mHO-1 females. Counterstaining was performed with nuclear fast red for 10 sec to maximize detection of iron. C, wild-type liver; D, Tg liver; E, wild-type kidney; F, Tg kidney; arrow, iron deposition in hepatocytes in the liver, in tubular cells in the kidney. Magnification ×100.

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Table 2. WBC, RBC, Hb, and MCV analyzed in 12-month-old Tg HO-1 G143H and WT littermates from the H1 line
 WBC (103/μL)RBC (106/μL)Hb (g/dL)MCV (fL)
  • Values in parentheses are the number of mice per group.

  • WBC, white blood cell counts; RBC, red blood cell counts; Hb, blood hemoglobin concentration; MCV, mean cell volume.

  • a

    P > 0.05 when compared with sex-matched wild-type mice.

  • b

    P < 0.05 when compared with sex-matched wild-type mice.

WT♀ (N = 12)7.49 ± 0.948.49 ± 1.0314.5 ± 5.6951.93 ± 5.13
Tg♀ (N = 10)6.31 ± 1.27a8.78 ± 0.7611.9 ± 1.71b43.03 ± 4.62b
Table 3. Serum iron and TIBC in 12-months-old Tg HO-1 G143H and WT littermates from the H1 line
 Serum iron (μg/dL)TIBC (μg/dL)
  • Values in parentheses are the number of mice per group.

  • TIBC, total iron-binding capacity.

  • a

    P < 0.05 when compared with sex-matched wild-type mice.

WT♀ (N = 8)153.67 ± 25.33337.23 ± 14.62
Tg♀ (N = 8)136.73 ± 23.91a397.61 ± 24.11a

We noticed that the phenotype of Tg HO-1 G143H females from the H1 line was similar to that of HO-1 knockout mice, which developed anemia, low serum iron levels, but with high iron loading in tissues (Poss and Tonegawa,1997b). Examination of organs for iron by Prussian blue staining also showed hepatic and renal iron deposition in 12-month-old Tg HO-1 G143H females (Fig. 3C–F).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The crystal structure of the human and rat HO-1 in complex with heme has been solved. The possible role of G139 and G143 in the HO reaction is described in these studies. Of the G139 and G143 mutants investigated, G139H and G143H mutants have no HO activity, which suggest the importance of Gly139 and Gly143 in maintaining the appropriate environment for the HO reaction. In the present study, we did not determine the crystal structures of mHO-1-heme complexes, but we report the effect of the G139H, G143H, and S142H mHO-1 mutants on the HO reaction in vivo and in vitro.

In vitro, only S142H catalyzed degradation of heme to biliverdin and retained 31.7% of the wild-type HO activity, whereas the G139H and G143H mutants exhibited no activity, which demonstrated that Gly139 and Gly143 are necessary for HO activity. The results in eukaryotic cells are comparable with those derived from the study of the crystal structure. Under conditions of insufficient heme, cotransfection of wild-type and mutant G139H or G143H mHO-1 showed HO deficiency, which suggested that G139H and G143H mutants were in competition with wild-type mHO-1 to bind heme. We therefore expected that mutant mHO-1 G143H or G139H overexpression in vivo might reduce endogenous heme, mimicking HO deficiency.

In our studies, Tg HO-1 G143H females are presented with anemia, enlarged spleens, and iron overload. We did not observe such a phenotype in Tg HO-1 G139H mice or Tg HO-1 S142H mice, which suggested that Gly143 was more critical in maintaining the HO reaction in vivo. The phenotype shown in Tg HO-1 G143H females constituted the most obvious similarity with HO-1−/− mice (Poss and Tonegawa,1997b). Thus, the Tg HO-1 G143H females described here might represent an animal model of HO deficiency disorders. However, HO-1−/− mice contracted a progressive chronic inflammation, as demonstrated by high peripheral white blood cell counts and lymph node CD4+:CD8+ T-cell ratios, unlike Tg HO-1 G143H females who had normal white blood cell counts. The simplest explanation for these results is that HO-1−/− mice have a defect in the protective effects caused by the products of heme degradation, while Tg HO-1 G143H females have deficient HO activity whilst retaining some protective effects. Interestingly, the appearance of pathological iron overload in the liver and kidney of Tg HO-1 G143H females coincided with the diminishing of serum iron levels, similar to HO-1−/− mice. The explanation for tissue iron retention in the HO-1−/− studies relies on the requirement of HO-1-mediated heme degradation as a part of physiological iron homeostasis, that is, HO-1 activity not only contributes to release of iron but is also required for iron reutilization, although it is still unclear how iron is reutilized by HO-1. We also observed that iron loading in livers and kidneys of HO-1−/− mice, which showed no HO activity, was conspicuous compared with Tg HO-1 G143H females, which showed HO deficiency. This demonstrated that HO has an important role in iron reutilization. Thus, Tg HO-1 G143H females appear to provide another useful model for the study of mammalian iron metabolism, especially for the study of the metabolism of iron reutilization involving HO-1.

Tg HO-1 S142H and Tg HO-1 G139H mice did not show any wasting, enlarged spleen, anemia, or reduction of serum iron levels, which suggested that Gly143 was more critical in maintaining the HO reaction in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The authors thank Osamu Nakajima and Tadashi Yoshida for their help with valuable advice concerning the selection of mutations.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED