Hypoxia reduces the expression of heme oxygenase-2 in various types of human cell lines A possible strategy for the maintenance of intracellular heme level Yongzhao Zhang1, Kazumichi Furuyama1, Kiriko Kaneko1, Yuanying Ding1, Kazuhiro Ogawa2,*, Miki Yoshizawa1, Masaki Kawamura1, Kazuhisa Takeda1, Tadashi Yoshida3 and Shigeki Shibahara1 Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Japan Department of Molecular Pharmacology, Tohoku University School of Medicine, Sendai, Japan Department of Biochemistry, Yamagata University School of Medicine, Yamagata, Japan Keywords erythroid cells; heme oxygenase-1; heme oxygenase-2; hemoglobin; hypoxia Correspondence S Shibahara, Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai, Miyagi 980-8575, Japan Fax: +81 22 717 8118 Tel: +81 22 717 8117 E-mail: shibahar@mail.tains.tohoku.ac.jp *Present address Department of Molecular Pharmacology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan (Received 26 January 2006, revised May 2006, accepted 15 May 2006) doi:10.1111/j.1742-4658.2006.05319.x Heme oxygenase consists of two structurally related isozymes, heme oxygenase-1 and and heme oxygenase-2, each of which cleaves heme to form biliverdin, iron and carbon monoxide Expression of heme oxygenase-1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase-2 expression Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase-2 mRNA and protein after 48 h of incubation in human cell lines, including Jurkat T-lymphocytes, YN-1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses In contrast, the expression level of heme oxygenase-1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN-1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells Moreover, heme oxygenase-1 protein was decreased in YN-1 cells under the conditions used, despite the induction of heme oxygenase-1 mRNA under hypoxia The heme oxygenase activity was significantly decreased in YN-1, K562 and HepG2 cells after 48 h of hypoxia To explore the mechanism for the hypoxia-mediated reduction of heme oxygenase-2 expression, we showed that hypoxia shortened the half-life of heme oxygenase-2 mRNA (from 12 h to h) in YN-1 cells, without affecting the half-life of heme oxygenase-1 mRNA (9.5 h) Importantly, the heme contents were increased in YN-1, HepG2 and HeLa cells after 48 h of incubation under hypoxia Thus, the reduced expression of heme oxygenase-2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and cleaves heme to release iron, carbon monoxide and biliverdin at the expense of molecular oxygen and NADPH [1,2] HO consists of two structurally related isozymes, HO-1 and HO-2 [3–5] Characteristically, human HO-1 contains no cysteine residue [6], whereas HO-2 contains at least two copies of a potential heme-binding site, consisting of the cysteine and proline (CP motif) [7,8] Importantly, these CP motifs are not involved in heme breakdown reactions [8], suggesting that HO-2 may sequester heme to maintain the intracellular heme level In addition, expression of HO-1 mRNA is induced by various stimuli, such as hemin and nitric oxide donors, in which expression of Abbreviations HO, heme oxygenase; HRE, hypoxia response element; MARE, Maf recognition element 3136 FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Zhang et al HO-2 mRNA is largely unchanged [9–12] Notably, hypoxia decreases the expression of HO-1 in several types of human cell [13,14], but conversely induces it in cultured human dermal fibroblasts [15] and a retinal pigment epithelial cell line [16] Thus, expression of HO-1 mRNA is differentially regulated in human cells by hypoxia, depending on cell type On the other hand, recent reports have shown that expression of HO-2 is decreased in the placental tissues of abnormal pregnancies [17,18] and in cultured human trophoblast cells [19] However, little is known about the regulation of HO-2 expression Using HO-2-deficient mice [20], we have shown that the mice lacking HO-2 exhibit hypoxemia with normal arterial CO2 tension (Paco2) and attenuated hypoxic ventilatory responses with normal hypercapnic ventilatory responses [21], which led us to propose a novel function of HO-2 as an oxygen sensor Subsequently, it has been shown that HO-2 interacts with the a-subunit of a large-conductance, calcium-sensitive potassium channel (the BK channel) and may function as an oxygen sensor for the BK channel [22] Taken together, these results suggest that hypoxia may inhibit the BK channel activity in the carotid body through HO-2, which ultimately enhances ventilation Clinically, hypoxia represents a decrease in O2 pressure in inspired gas and causes hypoxemia, which is a hemodynamic stress and could lead to pulmonary hypertension [23,24] Hypoxemia is a common manifestation of various diseases, such as chronic obstructive pulmonary disease [25], and is also seen in the HO-2-deficient mice [21] Moreover, we have shown that the expression levels of HO-2 protein were decreased by about 40% in the mouse liver after days of normobaric hypoxia (10% oxygen) and returned to the basal level after 14 days of hypoxia [26] It is therefore of significance to study the regulation of HO-2 expression in human cells under hypoxia In the present study, we have analyzed the effect of hypoxia on the expression levels of HO-1 and HO-2 in various types of human cell line, including erythroleukemia and hepatoma cells We have shown that hypoxia reduces the expression of HO-2 in five out of six cell lines examined We suggest that the reduced expression of HO-2 represents an important response during acclimatization to hypoxia Results Effects of hypoxia on HO-1 and HO-2 expression in human cell lines We initially analyzed the effects of hypoxia on the expression of HO-1 and HO-2 in human cell lines of Reduced expression of heme oxygenase-2 A B Fig Effects of hypoxia on expression of heme oxygenase (HO)-1 and HO-2 in human cell lines of bone marrow origin (A) Northern blot analysis of HO-1 and HO-2 mRNA KG1 myeloid cells, Jurkat T-lymphocytes and K562 and YN-1 erythroleukemia cells were cultured under normoxia (N) or hypoxia (H) (5% or 1% oxygen) for 48 h, and harvested Total RNA was extracted from each cell line, and then subjected to northern blot analysis Each lane contains 15 lg of total RNA The bottom panel shows the expression of b-actin mRNA as an internal control (B) Western blot analysis The indicated cells were harvested after cultivation under normoxia (N) or hypoxia (1% oxygen) for 48 h The cell extracts were prepared for western blot analysis of HO-1 and HO-2 The lane labeled h contained cell extracts prepared from untreated cells harvested just before starting the experiment Each lane contains 20 lg of protein To normalize the expression levels, the same filter was reused for a-tubulin monoclonal antibody Note that HO-1 mRNA and protein were undetectable in Jurkat and K562 cells under the conditions used bone marrow origin, including KG1 myeloid cells, Jurkat T-lymphocytes, and K562 and YN-1 erythroid cells YN-1 cells were established from the peripheral blood of a patient with chronic myelogenous leukemia in blastic crisis [27] Each cell line was incubated for 48 h under normoxia or hypoxia (5% or 1% oxygen) HO-1 mRNA expression was substantially increased under 1% oxygen in KG1 and YN-1 cells, whereas HO-1 mRNA was undetectable in Jurkat and K562 cells (Fig 1A) In contrast, expression of HO-2 mRNA was detected in these four cell lines and decreased by hypoxia (1% oxygen) in Jurkat, K562 and YN-1 cells, but remained unchanged in KG1 cells Under hypoxia, expression levels of b-actin mRNA were unchanged We next measured the levels of HO-1 and HO-2 proteins by western blot analysis (Fig 1B) The expression levels of HO-1 protein remained unchanged in KG1 cells and decreased by about 20% in YN-1 cells after FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS 3137 Reduced expression of heme oxygenase-2 Y Zhang et al 48 h of hypoxia (1% oxygen), despite the increased expression of HO-1 mRNA in these two cell lines Consistent with the HO-1 mRNA level, HO-1 protein was undetectable in Jurkat and K562 cells The HO-2 protein levels were noticeably decreased under hypoxia in Jurkat, K562 and YN-1 cells, in which a-tubulin protein levels were not changed The expression levels of HO-2 protein were decreased by about 26% under hypoxia in both K562 and YN-1 cells Thus, hypoxia consistently decreased the expression levels of HO-2 mRNA and protein in Jurkat T-lymphocytes and YN-1 and K562 erythroid cell lines To further analyze the effects of hypoxia on the expression of HO-1 and HO-2, we performed similar analyses in two human cancer cell lines, HeLa cervical cancer and HepG2 hepatoma cells Hypoxia (1% oxygen) decreased the expression levels of HO-1 and HO-2 mRNA after 48 h of incubation in the two cell lines (Fig 2A) Likewise, hypoxia decreased the levels of HO-1 and HO-2 proteins by more than 60% and 30%, respectively, in HeLa cells, and by 90% and 20%, respectively, in HepG2 cells (Fig 2B) Taken together, these results indicate that hypoxia reduces the expression levels of HO-2 mRNA and protein in five out of six cell lines, with the exception of KG1 myeloid cells A Hypoxia decreases the expression levels of HO-2 protein in YN-1 and K562 cells To confirm the hypoxia-mediated reduction of HO-2 expression, we performed a time-course study in YN-1 and K562 erythroleukemia cell lines In YN-1 cells, the expression levels of HO-1 and HO-2 proteins were significantly reduced after 48 h of hypoxia (1% oxygen) (Fig 3A,B) In K562 cells, hypoxia reduced HO-2 protein after 48 h (Fig 3C,D), although the expression of HO-1 protein remained undetectable Hypoxia decreases HO activity in YN-1, K562 and HepG2 cells We next measured HO activity in YN-1, K562, HeLa and HepG2 cells exposed to hypoxia for 48 h, when the expression levels of HO-2 protein were significantly decreased (Figs and 3) HO activity was determined in the microsomal fraction, prepared from normoxia or hypoxia-exposed cells, as described in Experimental procedures It should be noted that sufficient amounts of purified biliverdin reductase and cytochrome P450 reductase were added to the reaction mixture to measure the full HO activity HO activity was decreased in YN-1, K562 and HepG2 cells by hypoxia (Fig 4) In contrast, HO activity was undetectable in HeLa cells treated under normoxia or hypoxia, which is consistent with our previous report that HO activity was undetectable in HeLa cells [9] Thus, HO activity is not proportional to the expression levels of HO-1 and HO-2 proteins, detected by western blot analysis Opposite effects of hypoxia on expression of HO-1 and HO-2 mRNA in YN-1 cells B Fig Decreased expression of heme oxygenase (HO)-1 and HO-2 under hypoxia in human cancer cells (A) Northern blot analysis of HO-1 and HO-2 mRNA HeLa cervical cancer and HepG2 hepatoma cells were cultured under normoxia (N) or hypoxia (H: 1% oxygen) for 48 h, and harvested Each lane contains 15 lg of total RNA The bottom panel shows the expression of 28S rRNA as an internal control (B) Western blot analysis HeLa and HepG2 cells were harvested after cultivation under normoxia (N) or hypoxia (1% oxygen) for 48 h The cell extracts were prepared for western blot analysis of HO-1 and HO-2 Each lane contains 20 lg of protein To normalize the expression levels, the same filter was reused for a-tubulin monoclonal antibody 3138 To explore the mechanism of the hypoxia-mediated reduction of HO-1 and HO-2 protein levels, we analyzed the effects of hypoxia on the expression of HO-1 and HO-2 mRNA in YN-1 erythroleukemia cells, which express detectable levels of both HO-1 and HO-2 mRNA (Fig 1A) The expression levels of HO-1 mRNA were induced after 48 h of hypoxia (1% oxygen) (Fig 5A,B), whereas HO-2 mRNA levels were significantly decreased (Fig 5A,C) In contrast, under mild hypoxia (5% oxygen), the changes in HO-1 and HO-2 mRNA levels were not statistically significant Thus, hypoxia (1% oxygen) decreased the expression levels of HO-1 protein in YN-1 cells (Fig 3A), despite the increased expression of HO-1 mRNA (Fig 5A) On the other hand, the expression levels of HO-2 mRNA and protein were both reduced under hypoxia (1% oxygen) (Figs 3A and 5A) FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Zhang et al Reduced expression of heme oxygenase-2 HO activity (nmol bilirubin/mg protein/h) Fig Time-dependent effects of hypoxia on heme oxygenase (HO)-2 protein levels in two erythroid cell lines YN-1 (A, B) and K562 cells (C, D) were cultured under normoxia (N) or hypoxia (1% oxygen) for the indicated numbers of hours, and the cell extracts were subjected to western blot analysis (A, C) Other conditions are described in Fig 1B The intensities of the signals in (A) and (C) were quantified, and the intensity representing HO-1 or HO-2 protein was normalized with respect to the intensity for the a-tubulin signal Shown are the relative expression levels of HO-1 and HO-2 proteins in YN-1 cells (B) and those of HO-2 protein in K562 cells (D) The intensity representing HO-1 or HO-2 protein at the time (0 h) is considered to be 100% The ratio of each normalized value to the time value (indicated by 0) is shown as the relative expression level of HO-1 or HO-2 protein Asterisks represent statistically significant differences compared to h: *P < 0.05; **P < 0.01 unchanged under hypoxia (Fig 6A,B) In contrast, the half-life of HO-2 mRNA was about 12 h under normoxia (Fig 6A,C), and was shortened to h under hypoxia The decreased levels of HO-2 mRNA may be in part due to the enhanced degradation of the HO-2 mRNA N H 1% ND ND ND YN-1 K 56 HepG2 H eL a Fig Hypoxia decreases the heme oxygenase (HO) activity in YN-1, K562 and HepG2 cells.YN-1, K562, HepG2 and HeLa cells were cultured for 48 h under normoxia or hypoxia, and then harvested The microsome fraction was prepared and used for the assay of HO activity The data are means ± SEM of three independent experiments Note that the HO activity was undetectable in YN-1 cells exposed to hypoxia and in HeLa cells exposed to normoxia or hypoxia (shown as ND) Stability of HO-1 and HO-2 mRNA under hypoxia We then analyzed the stability of HO-1 and HO-2 mRNA in YN-1 cells under hypoxia (1% oxygen) In this series of experiments, YN-1 cells were precultured for 12 h under normoxia or hypoxia before addition of actinomycin D The half-life of HO-1 mRNA in YN-1 cells was about 9.5 h under normoxia, and remained Functional analysis of the HO-1 and HO-2 gene promoters under hypoxia To address the question of whether hypoxia influences the promoter activity of the human HO-1 or HO-2 gene, we performed transient expression assays Prior to the functional analysis of the HO-2 gene promoter, we determined its transcription initiation site by 5¢-RACE, and this indicated that transcription is initiated from multiple sites (Fig 7A) The most 5¢-upstream initiation site was identified as the G residue at position + 1, which is located 139 bp upstream of the start ATG codon in exon The presence of the exon sequence was also confirmed by RT-PCR analysis of YN-1 RNA The 5¢-flanking sequence lacks a consensus TATA box but contains several sequence motifs for binding of transcription factors, such as Sp1 Incidentally, the HO-2 gene and the gene encoding HSCARG of unknown function (GenBank acces- FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS 3139 Reduced expression of heme oxygenase-2 Y Zhang et al A B C Fig Differential effects of hypoxia on heme oxygenase (HO)-1 and HO-2 mRNA expression in YN-1 cells (A) Northern blot analysis YN-1 cells were harvested after cultivation under normoxia (N) or hypoxia (5% or 1% oxygen) for the indicated numbers of hours Each lane contains 15 lg of total RNA The lane labeled h contained RNA prepared from untreated cells harvested just before starting the experiment At the bottom of each panel, 28S rRNA of each sample was visualized by ethidium bromide staining The data are from one of three independent experiments with similar results (B, C) Relative expression levels of HO-1 and HO-2 mRNA The intensities of the signals in (A) were quantified, and the intensity representing HO-1 or HO-2 mRNA was normalized with respect to the intensity for the 28S RNA signal The intensity representing HO-1 or HO-2 mRNA at the time (0 h) is considered to be 100% The ratio of each normalized value to the time value (indicated by h) is shown as the relative expression level of HO-1 or HO-2 mRNA Asterisks represent statistically significant differences compared to h: *P < 0.05; **P < 0.01 sion number AAG09721) are located adjacently in a head-to-head orientation, and their transcription start sites are  1.5 kb apart We therefore analyzed the promoter function of the 1.5 kb 5¢-flanking region of the HO-2 gene in the present study YN-1 cells were transfected with each construct of the HO-1 and HO-2 gene promoters (Fig 7B) The reporter plasmids used for the HO-1 gene included phHOLUC45 with a Maf recognition element (MARE) and phHOLUC40 without MARE [16], and those for the HO-2 gene, phHO2(-1492), phHO2(-663), and phHO2(-25) The HO-1 gene promoter contains a putative hypoxiaresponsive element (HRE) sequence CACGTGA (positions ) 44 to ) 39) that overlaps the functional E-box [14,16,28] Hypoxia did not change the expression of phHOLUC45, phHOLUC40 or phHOLUC(-58) in YN-1 cells (Fig 7B), despite the fact that a putative HRE sequence is present in phHOLUC45 and phHOLUC40 Likewise, hypoxia did not influence the expression of HO-2 promoter constructs in YN-1 cells (Fig 7B) In contrast, hypoxia consistently increased the promoter activity of a construct, HRESV40, which contains four copies of HRE, but showed only marginal effects on the promoter activity of NHRESV40, a negative control for hypoxic induction 3140 Hypoxia increases cellular heme contents in human cell lines To explore the implication for the reduced expression levels of HO-1 and HO-2 proteins under hypoxia, we studied whether hypoxia influences the cellular heme contents in YN-1, HepG2 and HeLa cells (Fig 8) Heme contents were measured in each cell line after incubation under normoxia or hypoxia for 48 h Heme contents were increased in the three cell lines after 48 h of culture under hypoxia (Fig 8) The degree of increase was small but statistically significant Discussion We have hypothesized that a certain degree of reduction in heme degradation is probably important in the preservation of intracellular heme, an essential component of various hemoproteins [5] The present study has shown that hypoxia consistently reduces the expression levels of HO-2 mRNA and protein in five out of six human cell lines: Jurkat, YN-1, K562, HeLa and HepG2 In this context, Newby et al [29] described decreased levels of HO-2 protein in placentas of women who reside at high altitude and are thus FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Zhang et al Reduced expression of heme oxygenase-2 A B C Fig Effects of hypoxia on the stability of heme oxygenase (HO)-1 and HO-2 mRNAs (A) Northern blot analysis YN-1 cells were cultured for 12 h under normoxia or hypoxia (1% oxygen), and then treated with or without actinomycin D (AMD) (1 lgỈmL)1) for the indicated number of hours Each lane contains 15 lg of total RNA The lane labeled h contained RNA prepared from precultured cells harvested just before the addition of actinomycin D (0 h) (B, C) Relative expression levels of HO-1 and HO-2 mRNA under normoxia or hypoxia The intensity representing HO-1 or HO-2 mRNA was normalized with respect to the intensity of b-actin mRNA The intensity representing HO-1 or HO-2 mRNA at the time of addition of actinomycin D (0 h) under each condition is considered to be 100% exposed to chronic hypoxia Moreover, we have shown that the expression levels of HO-2 protein were transiently decreased in the mouse liver after days of normobaric hypoxia [26] These results suggest that the reduced expression of HO-2 protein may be an important hypoxic response in certain cell types Hypoxia exerted differential effects on the expression of HO-1, depending on the cell line (Figs and 2) It should be noted that HO-1 protein levels remained unchanged in KG1 myeloid cells and were significantly reduced in YN-1 erythroleukemia cells after 48 h of hypoxia (1% oxygen), despite the increased level of HO-1 mRNA These results suggest that certain mechanisms, such as the active degradation of HO-1 protein, might ensure constant or reduced expression levels of HO-1 protein in these cell lines under hypoxia In contrast, the expression levels of HO-1 mRNA and protein were consistently decreased in HeLa and HepG2 cells Interestingly, hypoxia tends to increase the cellular heme contents in YN-1, HepG2 and HeLa cells, which might be a consequence of reduced heme degradation and ⁄ or enhanced heme synthesis [30,31] In the present study, we focused on YN-1 erythroleukemia cells to investigate the hypoxia-mediated reduction of HO-2 expression It is tempting to speculate that the decreased heme degradation may contribute in part to the maintenance of the heme supply for hemoglobin production in erythroid cells In fact, it has been reported that chemically induced erythroid differentiation is associated with a reduction of HO-1 expression in MEL mouse erythroleukemia cells [32] Moreover, heme induces the expression of the a-globin gene in K562 human erythroleukemia cells [33] and the number of hemoglobin-producing cells in YN-1 cells [27,34] Conversely, the deficiency of heme in erythroid cells causes differentiation arrest in mice [35] These results indicate that heme is essential for differentiation of erythroid cells In this context, our preliminary data suggest that treatment for 48 h under hypoxia may increase the proportion of hemoglobin-positive YN-1 cells (from 4.4 ± 0.3% under normoxia to 9.6 ± 1.0% under hypoxia) and K562 cells (from 4.9 ± 0.7% to 6.9 ± 0.5%); this was measured by staining cells with o-dianisidine This method was based on the peroxidase activity of hemo- FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS 3141 Reduced expression of heme oxygenase-2 Y Zhang et al Fig Characterization of the human heme oxygenase (HO)-2 gene promoter (A) Identification of the transcription start sites The upstream region of the HO-2 gene is shown Exon encodes the untranslated region, and exon encodes the protein-coding region (closed box), including the ATG translation–initiation codon The nucleotide sequences of the proximal promoter and exon are shown Major transcription start sites, identified by 5¢-RACE, are indicated in bold Residue represents the 5¢ end of exon A newly identified 5¢-untranslated region is shown as a stippled box, and underlined is the exon region (from 58 to 98), based on the NCBI Blast database (accession number NT_010552) The 5¢ end of intron is also shown in lower case (B) Promoter activities of HO-1 and HO-2 genes under hypoxia YN-1 cells were transfected with each reporter construct and then incubated under normoxia or hypoxia for 48 h The two constructs, named HRESV40 and N-HRESV40, represent positive and negative controls for hypoxia Relative luciferase activity under normoxia or hypoxia is shown as the ratio to the normalized luciferase activity obtained with pGL3-basic under normoxia or hypoxia, respectively The data are means ± SEM of three independent experiments; **P < 0.01 globin [36] However, the increase in the hemoglobinpositive cells could be a result of a hypoxia-mediated increase of transferrin receptors [37] and ⁄ or erythroidspecific 5-aminolevulinate synthase [30] Further experi- 3142 ments are required to address the role of heme degradation in the population of hemoglobin-positive cells To the best of our knowledge, there has been no report on the promoter function of the human HO-2 FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Zhang et al Reduced expression of heme oxygenase-2 (100 mL)1), and streptomycin sulfate (100 lgỈmL)1) For hypoxia experiments, cells were cultured for the indicated time at 37 °C in a chamber with 5% CO2 ⁄ 94% N2 ⁄ 1% O2 [13] In some experiments, cells were incubated under mild hypoxia (5% CO2 ⁄ 90% N2 ⁄ 5% O2) The cells were harvested for total RNA extraction and protein extraction Northern blot analysis Fig Increased heme contents under hypoxia in YN-1, HepG2 and HeLa cells YN-1, HepG2 and HeLa cells were cultured under normoxia or hypoxia (1% oxygen) for 48 h, and cellular heme contents were measured, as described in Experimental procedures The data are from three independent experiments Asterisks represent statistically significant differences compared to normoxia control: *P < 0.05; **P < 0.01 gene We have identified the multiple transcription initiation sites of the HO-2 gene and confirmed that the HO-2 gene promoter is juxtaposed to the HSCARG gene in the opposite direction Thus, the HO-2 gene and the HSCARG gene share a common promoter region, known as a bidirectional promoter The bidirectional promoters are sometimes found in mammalian genes, and belong to the family of TATA-less and GC-rich promoters [38,39] Such features are consistent with the HO-2 gene promoter In the present study, hypoxia did not influence the expression of a reporter gene, carrying the 1.5 kb bidirectional promoter region Taken together with the shortened half-life of HO-2 mRNA under hypoxia, we suggest that the reduced expression of HO-2 mRNA may be achieved at least in part by post-transcriptional mechanisms, such as enhanced degradation of HO-2 mRNA However, functional studies with further upstream regions or downstream regions including a large intron of the HO-2 gene are required In summary, the present study has suggested that the reduced expression of HO-2 protein may contribute to the maintenance of intracellular heme level in certain human cell types under hypoxia Experimental procedures Cell culture and hypoxia study The human cell lines used were KG1 myeloid cells, K562 erythroid cells, and Jurkat T-lymphocyte cells and were maintained in RPMI-1640 medium (Sigma, St Louis, MO, USA) YN-1 human erythroid cells [27,34] were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma) HeLa and HepG2 cells were maintained in DMEM Each medium contains 10% heat-inactivated FBS, penicillin G Total RNA was extracted from cultured cells and subjected to northern blot analysis, as detailed previously [16] The signals for HO-1, HO-2 and b-actin mRNA were detected with the DIG Northern Starter Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol For preparation of HO-1 and HO-2 RNA probes, the human HO-1 cDNA of positions 81–878 [6] (GenBank accession number X06985) and the human HO-2 cDNA fragment (nucleotide positions 85–939) [7,9] (GenBank accession number P30519) were amplified by PCR using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA), and then cloned into pCR-bluntII-TOPO (Invitrogen, Carlsbad, CA, USA), and named pCR-hHO1 and pCR-hHO2, respectively SP6 RNA polymerase was used for transcription of RNA probe from pCR-hHO1 and pCR-hHO2 Western blot analysis Harvested cells were lysed in the lysis buffer (20 mm Hepes (pH 7.5), 150 mm NaCl, mm EDTA, 0.01 mgỈmL)1 aprotinin, 0.01 mgỈmL)1 antipain, 0.01 mgỈmL)1 pepstatin, 0.1 mgỈmL)1 leupeptin, 1.0% Triton X-100, and mm phenylmethylsulfonyl fluoride), as detailed previously [9,16] The cell lysates were centrifuged at 15 000 g for 10 (KUBOTA RA-50J1 fix-angle rotor, KUBOTA, Tokyo, Japan), and the supernatant (10 mg of protein) was analyzed on a 10% SDS-polyacrylamide gel The proteins in the gel were electrophoretically transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corporation, Billerica, MA, USA) The membranes were treated for h at room temperature (20–22 °C) with anti-HO-1 (a gift from Shigeru Taketani) [40] or anti-HO-2 antibody (Stressgene Canada, Victoria, Canada) HO-1 and HO-2 proteins were detected with ECL Plus western blot kit (Amersham Biosciences, Piscataway, NJ, USA) Expression of a-tubulin was examined as an internal control using a-tubulin monoclonal antibody (NeoMarkers, Fremont, CA, USA) Assay for HO catalytic activity YN-1, K562, HeLa and HepG2 cells (2 · 107) were incubated for 48 h under normoxia or hypoxia (1% oxygen), and harvested for the assay of HO activity, as described previously [2,41,42] Cells were suspended in mL of 20 mm FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS 3143 Reduced expression of heme oxygenase-2 Y Zhang et al potassium phosphate buffer (KPB) (pH 7.4) containing 134 mm KCl, and disrupted by sonication The microsome fraction was prepared by two steps of centrifugation and was suspended by sonication in 150 lL of 50 mm KPB containing 0.1% Triton X-100 Each sample (300 lg of protein) was added to the standard reaction mixture of 200 lL, which contained 0.1 m KPB (pH 7.4), 15 lm hemin, 100 lgỈmL)1 BSA, 30 lg of biliverdin reductase, and 15 lg of cytochrome P450 reductase After of preincubation at 37 °C, the reaction was started by addition of 10 lL of NADPH (8.4 lgỈmL) or 10 lL of water as a blank mixture The reaction mixture was incubated at 37 °C for 20 After centrifugation, the supernatant was used to measure the absorbance at 468 nm The amounts of bilirubin formed in the reaction system were calculated using a value of 43.5 mm)1Ỉcm)1 HO activity was expressed as nmol bilirubinỈmg protein)1Ỉh)1 YN-1 cells were also treated with lm CdCl2 for h as a positive control, because CdCl2 induced HO-1 expression [10,43] Effects of actinomycin D on the expression of HO-1 and HO-2 mRNA To study the effects of hypoxia on the stability of HO-1 and HO-2 mRNA, YN-1 cells were incubated for 12 h in fresh medium under normoxia or hypoxia, followed by the addition of actinomycin D (1 lgỈmL)1) (CalbiochemBehring, La Jolla, CA) [16,44] The cells were further incubated for 2, or 12 h under normoxia or hypoxia, and then harvested at each time point for RNA extraction Identification of 5¢ end of HO-2 mRNA and the promoter region of the HO-2 gene To amplify the 5¢ end of HO-2 cDNA, nested PCR was carried out using the BD Marathon-Ready human testis cDNA library (BD Biosciences Clontech, Palo Alto, CA, USA) and FailSafeTM PCR PreMix Selection Kit (Epicentre, Madison, WI) Human testis cDNA was used, because HO-2 protein is enriched in the testis [21,45] The sense PCR primers were an adapter primer and its nested (downstream) primer, which is located upstream of the cDNA, and the antisense primers for HO-2 cDNA were a gene-specific primer-1, 5¢-CAGGTCCAGGGCGTTCATCCTGGC CCGG-3¢, located in exon-4, and its nested (upstream) primer, 5¢-CCCCCCGAGAGATCCCCCATGTAGCGGG-3¢, located in exon-4 The two steps of PCR were performed according to the supplier’s protocol The nested PCR products, extracted from a gel by using MinEluteTM Gel Extraction Kit (Qiagen, Tokyo, Japan), were cloned into pCRÒIITOPO vector (Invitrogen) DNA sequencing analysis (Applied Biosystems, Foster City, CA, USA) was performed to confirm the nucleotide sequence The tfsearch program on the TRANSFAC databases [46] was 3144 used to identify potential cis-elements in the 5¢-flanking region of the HO-2 gene Luciferase reporter constructs The 1.5 kb 5¢-flanking region of the HO-2 gene was amplified by PCR using human genomic DNA as a template and a primer set designed from a published sequence (GenBank accession number P30519) (sense, 5¢-AGATCTATCCCTT GAGGCCTTGTCCGCTTG-3¢; antisense, 5¢-AAGCTTG CC GCAGGTCGCTGTCGCCTG-3¢; these contain a BglII site and a HindIII site, respectively) The genomic fragment was cloned into the BglII ⁄ HindIII-digested pGL3-basic vector (Promega, Madison, WI, USA) containing luciferase as a reporter gene The cloned 1.5 kb promoter region was used as a template to generate deletions in the HO-2 promoter All PCR products (1494, 663 and 25 bp) were purified and subcloned in the pGL3-basic vector, yielding phHO2(-1492), phHO2(-663), and phHO2(-25) The identity of each construct was confirmed by sequencing The human HO-1 gene–luciferase constructs, phHOLUC45 [16], phHOLUC40, and phHOLUC(-58) [47], carry the 4.5 kb, 4.0 kb and 58 bp fragments of the human HO-1 gene [10,48], respectively Reporter plasmids, HRESV40 containing four copies of HRE and NHRESV40 lacking HRE [49], were used as a positive and a negative control for hypoxia, respectively Transient transfection assays Transfection was performed with DMRIE-C reagent (Invitrogen), following the supplier’s protocol YN-1 cells (4 · 105) were cotransfected with each promoter–reporter fusion plasmid (0.784 lg) and pRL-TK vector (16 ng) (Promega), and incubated for 4.5 h Then, the transfected YN-1 cells were incubated for 48 h under normoxia or hypoxia (1% oxygen), and harvested Luciferase activity was measured with a luminometer using the Dual Luciferase Assay System (Promega), as detailed previously [14,16] A promoterless construct, pGL3-basic, was used as a control The data are means ± SEM of three independent experiments with each plasmid DNA preparation Fluorometric assay of heme Heme contents in cells (expressed as ng per 106 cells) were determined as described previously [50] Cell suspensions were centrifuged at 800 g for at °C using an Eppendorf 5415 R benchtop refrigerated centrifuge (rotor type fix-angle rotor 1-16, 100 · g; Eppendorf AG, Hamburg, Germany), and 0.5 mL of m oxalic acid was added to the pellet The mixtures were shaken vigorously and immediately heated for 30 at 100 °C The mixtures without heating were used as a blank for measurement of FEBS Journal 273 (2006) 3136–3147 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Zhang et al endogenous porphyrins After cooling, fluorescence was determined in an RF-5300PC spectrofluorometer (Shimadzu Corp., Kyoto, Japan) The excitation wavelength was 400 nm, and the fluorescence emission was determined at 662 nm Hemin solutions, containing 0, 1, 10, 50 or 100 ng of hemin, were prepared in 0.5 mL of oxalic acid, and used as standards In all assays, · 106 cells 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Effects of hypoxia on HO-1 and HO-2 expression in human cell lines We initially analyzed the effects of hypoxia on the expression of HO-1 and HO-2 in human cell lines of Reduced expression of heme oxygenase-2. .. we have analyzed the effect of hypoxia on the expression levels of HO-1 and HO-2 in various types of human cell line, including erythroleukemia and hepatoma cells We have shown that hypoxia reduces. .. R, Takahashi K, Takeda K, Furuyama K, Kaneko K, Takahashi S, Tamai M & Shibahara S (2004) Expression of heme oxygenase-1 is repressed by interferon-gamma and induced by hypoxia in human retinal