Tài liệu Báo cáo khoa học: Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell lines docx

Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell lines Yuanying Ding1, Yong Z Zhang1, Kazumichi Furuyama1, Kazuhiro Ogawa2*, Kazuhiko Igarashi3 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, Tohoku University Graduate School of Medicine, Sendai, Japan Keywords cancer cell lines; heme homeostasis; heme oxygenase; isozymes; short interfering RNA 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, 13-1 Takara-machi, Kanazawa 920-8640, Japan (Received 28 July 2006, revised October 2006, accepted October 2006) doi:10.1111/j.1742-4658.2006.05526.x Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2 Recent studies have identified HO-2 as a potential oxygen sensor To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2 Heme is an invaluable molecule that is essential for life and is involved in many cellular processes that sense or use oxygen The intracellular concentration of heme is maintained by the rate of its synthesis and degradation [1] Many enzymes and their regulators are responsible for heme synthesis [1,2] On the other hand, heme degradation is mediated by two structurally related isozymes, HO-1 and HO-2, to generate biliverdin IXa, carbon monoxide (CO), and ferrous iron [3] Biliverdin IXa is immediately reduced to bilirubin IXa HO-1 has attracted particular attention, because its expression is induced by its substrate, heme, in animals [4,5] and in primary cultures of macrophages [6–9] It has therefore provided a good example of substrate-mediated induction of an enzyme in mammals [10,11] Abbreviations GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO, heme oxygenase; MARE, Maf recognition element; SA, succinylacetone; SnPP, Sn-protoporphyrin FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS 5333 Heme oxygenase-2 down-regulates heme oxygenase-1 Y Ding et al On the other hand, it has been reported that expression of HO-1 is decreased in several types of human cell under various conditions, such as hypoxia [12,13] and treatment with interferon-c [14,15] or desferrioxamine, an iron chelator [12] Likewise, the expression of HO-2 is decreased in the placental tissues of abnormal pregnancies [16,17] and in cultured human trophoblast cells [18] We have recently shown that the expression levels of HO-1 and HO-2 are decreased in several human cell lines under hypoxia [19] In mice, expression of HO-1 and HO-2 proteins is decreased in decidua and placenta during Th1-mediated abortion [20] Moreover, expression of HO-1 and HO-2 proteins is transiently decreased in the liver, but increased in the heart during acclimatization of mice to normobaric hypoxia [21] These results suggest that expression of HO-1 and HO-2 is regulated in a complex manner to maintain intracellular heme concentrations or the availability of free heme for various hemoproteins Free heme is defined as either heme that is newly synthesized but not yet bound to hemoproteins or heme that has been released from hemoproteins [22] HO-2 contains the cysteine and proline (CP) motifs [23], whereas HO-1 lacks a cysteine residue [24,25] Each CP motif of HO-2 may function as a heme-binding site [26], suggesting that HO-2 may sequester heme to maintain the intracellular heme concentrations or ameliorate heme-mediated oxidative stress Moreover, unlike the severe phenotypes of HO-1-deficient mice (HO-1– ⁄ –), including prenatal lethality [27], HO-2– ⁄ – mice are fertile and survive for at least year under basal conditions [28], but show ejaculatory abnormalities [29] and high susceptibility to oxygen toxicity [30] Recent studies of our group [31] and other investigators [32] have shown that HO-2 functions as a potential oxygen sensor In the present study, we show that HO-2 knockdown is associated with the induction of HO-1 expression in human cancer cell lines HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis We provide evidence that HO-2 may modulate the expression level of HO-1 by affecting HO-1 mRNA stability and intracellular heme concentration Results Expression profiles of HO-1 and HO-2 in various human cell types We initially analyzed the expression profiles of HO-1 and HO-2 in eight human cell lines by northern and western blot analyses (Fig 1A,B) Expression of HO-1 5334 A B C Fig Expression profiles of HO-1 and HO-2 in various human cell lines (A) Total RNA and proteins were prepared from the indicated cell lines and subjected to northern blot analysis and (B) western blot analysis (A) Northern blot analysis Each lane contains 15 lg total RNA The bottom panel shows the expression of 18S rRNA as an internal control Note that the blot was exposed to the film for the longest time (1 min) to detect the low expression levels of HO-1 in some cell lines (B) Western blot analysis Each lane contains 20 lg protein The same filter was reused for b-actin expression as an internal control (C) Cellular heme contents Cells were cultured for 48 h, and harvested for the measurement of heme content (ng ⁄ 106 cells) mRNA was detected in five of these cell lines, but hardly at all in the other three (K562 erythroleukemia, Jurkat T cell, and H146 small cell lung cancer), in which HO-1 protein was also undetectable In contrast, HO-2 mRNA and protein were both expressed in all eight cell lines (Fig 1A,B) We also measured the heme content of these cell lines: it was about twofold higher in YN-1 and K562 erythroleukemia cells than in the other cell types (Fig 1C) Higher heme content may reflect hemoglobin production in YN-1 and K562 cells [33,34] In fact, the population of hemoglobin-positive cells was about 4.4% in YN-1 cells and 4.9% in K562 cells under basal conditions [19] Otherwise, there was FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Ding et al Heme oxygenase-2 down-regulates heme oxygenase-1 no apparent correlation between the expression levels of HO-1 and HO-2 and cellular heme content The cellular heme content represents the sum of free heme and bound heme of various hemoproteins for maintenance of cellular heme contents More importantly, these results indicate that measurement of heme content is useful for evaluation of heme dynamics in cultured cells Role of heme metabolism in cellular heme content Regulatory role of free heme in expression of HO-1 To evaluate the contribution of heme synthesis and degradation to cellular heme content, we treated YN-1 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma cells for 48 h with succinylacetone (SA) or Sn-protoporphyrin (SnPP) (Fig 2) These distinctive cell lines were chosen because both HO-1 and HO-2 are expressed at detectable concentrations (Fig 1) [19] SA is a specific inhibitor of d-aminolevulinic acid dehydratase, the second enzyme of the heme biosynthetic pathway SnPP is a competitive inhibitor of HO activity [35] The heme content of all three cell lines was significantly decreased after treatment with SA, but increased after treatment with SnPP (Fig 2A,B) Thus, an appropriate balance between heme synthesis and heme degradation is responsible To explore the role of free heme in HO-1 and HO-2 expression, we treated HeLa and HepG2 cells with SA and determined the expression levels of HO-1 and HO-2 (Fig 3) HO-1 mRNA expression was significantly reduced in HeLa and HepG2 cells after treatment with SA for h, whereas HO-2 mRNA expression was not noticeably changed by SA treatment (Fig 3A,C) Western blot analysis revealed that treatment with SA reduced the expression of HO-1 protein in HeLa and HepG2 cells, but did not change the HO-2 protein concentration (Fig 3B,D) These results suggest that a certain threshold concentration of free heme may determine the basal expression levels of HO-1 Effects of HO-1 or HO-2 short interfering RNA (siRNA) on the expression of HO-1 Fig Effects of SA and SnPP on cellular heme content in human cell lines YN-1 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma cells were treated with mM SA (A) or 50 lM SnPP (B) for 48 h, and the cellular heme contents were measured Heme contents are shown as ng ⁄ 106 cells The data are mean ± SEM from three independent experiments *P < 0.05, **P < 0.01 To explore the functional significance of HO-1 and HO-2, we selectively reduced the expression of HO-1 or HO-2 mRNA with each siRNA HeLa and HepG2 cells were transfected with siRNA targeted to HO-1, HO-2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and total RNA was extracted from cells after 24 h incubation and subjected to northern blot analysis (Fig 4A,C) HO-1 siRNA decreased HO-1 mRNA expression levels by 60%, but exerted no noticeable effects on HO-2 and GAPDH mRNA concentrations in HeLa cells (Fig 4A) Likewise, HO-2 siRNA and GAPDH siRNA specifically decreased the expression of HO-2 mRNA and GAPDH mRNA by more than 90%, respectively Unexpectedly, treatment of HeLa cells with HO-2 siRNA induced the expression of HO-1 mRNA and protein without affecting the concentration of GAPDH mRNA and b-actin (Fig 4A,B) Likewise, the selective reduction of HO-2 mRNA with HO-2 siRNA induced the expression of HO-1 mRNA and protein in HepG2 cells, but did not change the concentrations of GAPDH mRNA and b-actin (Fig 4C,D) Consistent with HO-1 mRNA expression, expression of HO-1 protein was also induced by the treatment with HO-2 siRNA in HepG2 cells These results indicate that the down-regulation of HO-2 expression is associated with induction of HO-1 expression FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS 5335 Heme oxygenase-2 down-regulates heme oxygenase-1 Y Ding et al A C B D Fig Effects of SA on HO-1 and HO-2 expression levels in human cell lines HeLa cells (A and B) and HepG2 cells (C and D) were left untreated or treated with SA (5 mM) for the indicated time and harvested The upper panels in (A) and (C) show the northern blot analysis of HO-1 and HO-2 mRNA in HeLa (A) and HepG2 cells (C) Each lane contains 15 lg total RNA The expression of 18S rRNA is also shown as an internal control The data presented are from one of three independent experiments The lower panels in (A) and (C) show relative expression levels of HO-1 and HO-2 mRNA The intensity of the signals representing HO-1 or HO-2 mRNA was normalized with respect to the intensity of the 18S rRNA signal The ratio of each normalized value to the control value in untreated cells at h is shown as the relative expression level of HO-1 or HO-2 mRNA Asterisks represent significant differences compared with the control at h (*P < 0.05, **P < 0.01) (B and D) Western blot analysis of HO-1 and HO-2 proteins in HeLa (B) and HepG2 cells (D) Each lane contains 10 lg protein The relative expression levels are shown in the lower panels To normalize the expression levels, the same filter was reused for b-actin monoclonal antibody The intensity representing HO-1 or HO-2 protein was normalized with respect to the intensity for the b-actin signal The data are mean ± SEM from three independent experiments *P < 0.05 We then confirmed the effects of HO-2 knockdown using another HO-2 siRNA with a different sequence (HO-2 siRNA1) [32] and a scrambled HO-2 siRNA (siHO-2-R) in HeLa and HepG2 cells (Fig 5) HO-2 siRNA1 efficiently decreased the expression of HO-2 mRNA and protein and induced the expression of HO-1 mRNA and protein In contrast, the scrambled HO-2 siRNA did not affect the expression of HO-1 and HO-2 mRNAs and proteins in HeLa and HepG2 cells (Fig 5) Thus, the induction of HO-1 is due to the selective repression of HO-2 expression achieved with HO-2 siRNA 5336 Knockdown of HO-2 expression causes time-dependent induction of HO-1 mRNA expression We then performed a time course study to confirm the effects of HO-2 siRNA on the expression of HO-1 mRNA in HeLa cells (Fig 6A) HO-2 siRNA efficiently reduced the expression of HO-2 mRNA at h, which was further decreased at 12 h In contrast, expression of HO-1 mRNA was time-dependently increased, reaching a maximum at 24 h (Fig 6A,B) HO-2 siRNA had not noticeably changed the concentrations of GAPDH FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Ding et al Heme oxygenase-2 down-regulates heme oxygenase-1 A C B D Fig Knockdown of HO-1 or HO-2 expression by each siRNA in HeLa and HepG2 cells HeLa cells (A and B) and HepG2 cells (C and D) were treated for 24 h with siHO-1, siHO-2 or siGAPDH as described in Experimental procedures Cells treated with Lipofectamine 2000 transfection reagent alone were included as a control siGAPDH was also used as a control for transfection with siRNA Other methods are same as in Fig (A and C) Northern blot analysis of HO-1, HO-2 and GAPDH mRNAs The intensity of the signals representing HO-1 or HO-2 mRNA was normalized with respect to the intensity of the 18S rRNA signal The ratio of each normalized value to the value in untreated cells is shown as the relative expression level of HO-1 or HO-2 mRNA (**P < 0.01) (B and D) Western blot analysis of HO-1 and HO-2 proteins The intensity representing HO-1 or HO-2 protein was normalized with respect to the intensity of the b-actin signal The ratio of each normalized value to the control value in siRNA-untreated cells (control) is shown as the relative expression level of HO-1 or HO-2 protein (*P < 0.05, **P < 0.01) The data are mean ± SEM from three independent experiments mRNA by 24 h It should be noted that HO-2 siRNA causes late-onset induction of HO-1 mRNA In this context, we have reported that maximum induction of HO-1 mRNA expression was detected within h by treatment with cadmium in HeLa cells, which is due to increased transcription of the HO-1 gene [36] Thus, HO-2 FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS 5337 Heme oxygenase-2 down-regulates heme oxygenase-1 Y Ding et al Fig Induction of HO-1 mRNA expression with knockdown of HO-2 in HeLa and HepG2 cells Cells were treated for 24 h with each of two siRNAs against HO-2 (siHO-2 and HO-2siRNA1), scrambled siHO-2 (scramble) or siGAPDH Other methods are the same as in Fig Expression levels of HO-1, HO-2 and GAPDH mRNAs were determined in HeLa cells (A) and HepG2 cells (C) by northern blot analysis HO-1 and HO-2 proteins were determined in HeLa cells (B) and HepG2 cells (D) by western blot analysis The data are mean ± SEM from three independent experiments *P < 0.05, **P < 0.01 knockdown may evoke a certain metabolic change, which in turn induces HO-1 mRNA expression HO-2 knockdown increases stability of HO-1 mRNA Consequently, we analyzed the stability of HO-1 mRNA in HeLa cells treated with HO-2 siRNA In this series of experiments, HeLa cells were left untransfected or transfected with the indicated siRNA, and cultured for 12 h before addition of actinomycin D The half-life of HO-1 mRNA was about h in untransfected and siGAPDH-treated HeLa cells (Fig 7A,B), which is in good agreement with the halflife of HO-1 mRNA determined in HeLa cells [37] Interestingly, the half-life of HO-1 mRNA was prolonged to about h in HeLa cells transfected with siHO-2 Thus, the induction of HO-1 mRNA with HO-2 knockdown may be in part due to the increased stability of HO-1 mRNA Effects of HO-2 knockdown on HO-1 and HO-2 promoter activities To assess the biochemical consequences of HO-2 knockdown, we analyzed whether HO-2 siRNA affects 5338 the promoter activity of the human HO-1 gene, using reporter constructs (Fig 8) The 4.5-kb promoter region of the HO-1 gene, carried by phHOLUC45, has been shown to be responsive to cadmium [36] and sodium nitroprusside [38], but unresponsive to hemin [9,36] We also included model constructs of pRBGP2 and pRBGP4, containing three copies of the Maf recognition element (MARE) and three copies of the mutated MARE, respectively [39] Co-transfection with siHO-2 significantly increased the promoter activity of phHOLUC45, which contains the MARE, but showed no effect on the promoter activity of phHOLUC40, lacking the MARE, the HO-2 gene promoter of phHO2()1494) and phHO2()663), or a control promoter of pGL3-Basic Likewise, transfection with siHO-2 significantly increased the pRBGP2 promoter activity but not pRBGP4 Knockdown of HO-1 with siHO-1 tended to increase the HO-1 promoter activity, but the degree of activation was not statistically significant Neither siHO-1 nor siGAPDH significantly influenced the promoter activities of the HO-2 gene, pRBGP2 or pRBGP4 We also analyzed the effects of hemin treatment on the HO-1 gene promoter activity (Fig 8B), showing that hemin significantly increased the expression of pRBGP2 but not the expression of phHOLUC45 and pRBGP4 These results suggest that FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Ding et al Heme oxygenase-2 down-regulates heme oxygenase-1 A B C Fig Time-dependent induction of HO-1 mRNA expression with HO-2 knockdown (A) Northern blot analysis HeLa cells were cultured for the indicated time (0, 6, 12, 24 and 48 h) after transfection with HO-2 or GAPDH siRNA Total RNA was extracted and subjected to northern blot analysis Each lane contains 10 lg total RNA Relative expression levels of HO-1 (B) and HO-2 (C) mRNAs are shown The intensity representing HO-1 or HO-2 mRNA was normalized with respect to the intensity of 18 S rRNA The ratio of each normalized value to the control value at time (0 h) is shown as the relative expression level of HO-1 or HO-2 mRNA The data are mean ± SEM from three independent experiments *P < 0.05, **P < 0.01 HO-2 knockdown may transactivate the promoter of phHOLUC45 through a heme-independent mechanism Moreover, HO-2 knockdown may cause a metabolic change similar to that evoked by cadmium [36] or sodium nitroprusside [38], each of which activated the expression of a reporter gene under the regulation of the 4.5-kb HO-1 gene promoter It should be noted, however, that heme activates the HO-1 gene promoter [40], but the relevant cis-acting element is not present in the 4.5-kb promoter region It is therefore conceivable that HO-2 knockdown may induce HO-1 expression through not only the heme-independent mechanism but also the heme-dependent mechanism Heme accumulation caused by knockdown of HO-1 or HO-2 expression To explore the biological implication of the knockdown experiments and to evaluate the relative contribution of HO-1 and HO-2 to the total amount of heme degradation, we measured the heme content of HeLa cells and HepG2 cells after transfection with HO-1 or HO-2 siRNA There were no significant changes in heme content in HeLa cells (Fig 9A) and HepG2 cells (Fig 9B), which were transfected with each HO siRNA Thus, heme content may be maintained by a compensatory mechanism, or the changes in heme content may be below the detectable limit of the assay method used Accordingly, we treated HeLa and HepG2 cells for 12 h with lm hemin, a suboptimal concentration for the induction of HO-1 mRNA, and then measured cellular heme content It should be noted that hemin at this concentration does not noticeably induce HO-1 expression in HeLa and HepG2 cells (data not shown) In HeLa cells that were left untransfected (control) or transfected with siGAPDH or siHO-1, the heme content was twofold higher after treatment with hemin (Fig 9A), whereas the heme content remained unchanged in HepG2 cells treated with hemin (Fig 9B) Thus, heme may be more efficiently incorporated into HeLa cells than HepG2 cells or the incorporated heme may exceed the capacity of heme degradation mediated by HO-1 and HO-2 in HeLa cells In fact, HO activity was detected in FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS 5339 Heme oxygenase-2 down-regulates heme oxygenase-1 Y Ding et al A B Fig HO-2 knockdown increases the stability of HO-1 mRNA (A) Northern blot analysis HeLa cells, which were left untransfected (Control) or transfected with the indicated siRNA, were cultured for 12 h, and then treated with actinomycin D (AMD) (1 lgỈmL)1) for the indicated time (h) Each lane contains 15 lg total RNA The lane labeled h contained RNA prepared from cells harvested just before the addition of AMD (0 h) (B) Relative expression levels of HO-1 mRNA The intensity representing HO-1 mRNA was normalized with respect to the intensity of 18S rRNA The intensity representing HO-1 mRNA at the time of addition of AMD (0 h) under each condition was considered to be One representative of two independent experiments with similar results is shown HepG2 cells, but not in HeLa cells [19] Moreover, heme content was further increased twofold in hemintreated HeLa cells transfected with siHO-2 (Fig 9A), even though HO-2 knockdown with siHO-2 induced HO-1 expression (see Figs and 6) On the other hand, knockdown of either HO-1 or HO-2 expression resulted in the accumulation of heme in the hemintreated HepG2 cells Taken together with the siHO-2mediated induction of HO-1 expression, these results suggest that HO-2 rather than HO-1 may play the predominant role in heme degradation in cultured human cells Discussion We have shown that the selective knockdown of HO-2 expression with each of two different siRNAs is consistently associated with increased expression of HO-1 mRNA and protein Moreover, we provide evidence 5340 that at least three mechanisms may account for the siHO-2-mediated induction of HO-1 expression: increased stability of HO-1 mRNA and heme-dependent and heme-independent transcriptional regulation of the HO-1 gene These metabolic consequences of HO-2 knockdown suggest a regulatory role for HO-2 in the co-ordinated expression of HO-1 and HO-2 We therefore propose that HO-2 may modulate the expression of HO-1 Incidentally, the three cell lines with low HO-1 expression, K562, Jurkat, and H146, were maintained in suspension culture (Fig 1), although HO-1 is expressed in two other cell lines, YN-1 and KG1, that were also maintained in suspension culture These results suggest that the cellular microenvironment, such as cell attachment, may influence the expression of HO-1 The dominant expression of HO-2 protein, in comparison with the low expression of HO-1 protein, in H146 small cell lung cancer cells is of particular interest because small cell lung cancer is derived from the airway neuroepithelial body [41], which functions as an oxygen-sensing organ in the lung The neuroepithelial body is responsible for ventilation-perfusion matching, which may be impaired in HO-2– ⁄ – mice [31] It is noteworthy that HO-2 knockdown increased the heme content of the hemin-treated HeLa and HepG2 cells (Fig 9A,B), despite the induction of HO-1 expression (Figs and 6) These results suggest that the increased HO-1 expression may be insufficient to compensate for a certain degree of reduction in HO-2 protein Taken together with the ubiquitous expression profile of HO-2 in the cell lines examined (Fig 1), we suggest that HO-2 may be a key enzyme responsible for maintaining cellular heme concentrations In this context, HO-2 contains at least two copies of the CP motif, which may be bound by heme but is not present in HO-1 [23,26] Thus, the downregulation of HO-2 may transiently increase the cellular free heme concentration, which in turn increases the expression of HO-1 mRNA The induction of HO-1 expression mediated by HO-2 knockdown may account for the phenotypic differences between HO-1– ⁄ – mice and HO-2– ⁄ – mice Unlike the partial lethality of HO-1– ⁄ – [27], HO-2– ⁄ – mice are able to survive for at least a year under basal conditions [28] These mice can probably compensate for the loss of HO-2 by increasing the expression of HO-1, which is supported by the following observations First, HO-2– ⁄ – mice show no noticeable changes or only a marginal decrease in arterial carboxyhemoglobin, a marker of overall heme degradation [27,28] Secondly, no differences in heme concentration were FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Ding et al Heme oxygenase-2 down-regulates heme oxygenase-1 A B Fig Knockdown of HO-2 expression increases the HO-1 promoter activity HeLa cells were transiently transfected with each reporter construct (3 lg), then incubated for 24 h, and either re-transfected with each siRNA (A) or treated with 50 lM hemin (B), as described in Experimental procedures After 24 h of incubation, luciferase activity was measured The test promoters analyzed are shown on the left Relative luciferase activity is shown as the ratio to the normalized luciferase activity obtained with pGL3Basic; the normalized luciferase activity used was that in cells treated with siGAPDH in (A) and that in cells untreated with hemin in (B) The data are mean ± SEM from three independent experiments **P < 0.01 detected in tissue homogenates prepared from multiple tissues of HO-2– ⁄ – mice [42] Thirdly, HO-1 protein is indeed over-expressed in the lung [30] and pulmonary venous myocardium [31] of HO-2– ⁄ – mice Taken together with our proposal that HO-2 down-regulates HO-1 expression, these results suggest that heme homeostasis is maintained in HO-2– ⁄ – mice through appropriate resetting of HO-1 expression In contrast with HO-1 expression, expression of HO-2 mRNA and protein was not increased in several human cell lines examined [15,19,37] Such a mode of regulation of HO-2 expression may account for the severe phenotype of HO-1– ⁄ – mice [27] In particular, the compensation achieved by HO-2 is not sufficient in the HO-1enriched organs, such as spleen, liver, and bone marrow In fact, HO-1– ⁄ – mice suffer from severe anemia and iron deposits [27] Likewise, human HO-1 deficiency is characterized by hypobilirubinemia, persistent hemolytic anemia, and iron deposits in the liver [43] HO-2 knockdown increased the transient expression of phHOLUC45 through the enhancer region of the human HO-1 gene, located between )4.5 kb and )4 kb The increased expression of phHOLUC45 suggests that the cellular microenvironment generated by HO-2 knockdown may mimic the metabolic change evoked by cadmium [36] or sodium nitroprusside [38], each of which activates the expression of a reporter gene under the regulation of the 4.5-kb HO-1 gene promoter This region contains the cadmium-responsive element [36] and the MARE [44,45], but lacks the element required for full activation by hemin [9,36,40] It is the MARE site that is bound by Nrf2, a transcription activator, or Bach1, a transcription repressor, each of which functions as a heterodimer with a member of the Maf family [46] Bach1 is a heme-responsive repressor, and its repression activity is lost when Bach1 is bound by heme, which in turn leads to transcriptional activation of the HO-1 gene FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS 5341 Heme oxygenase-2 down-regulates heme oxygenase-1 Y Ding et al In summary, HO-2 may determine the expression level of HO-1 by affecting HO-1 mRNA stability and transcription of the HO-1 gene This study also reveals an important regulatory role for HO-2 in the co-ordinated expression of HO-1 and HO-2 and the maintenance of cellular heme concentrations Experimental procedures Materials Hemin and 4,6-dioxoheptanoic acid (SA) were purchased from Sigma Chemical (St Louis, MO, USA) SnPP was from Porphyrin Products (Logan, UT, USA) Cell cultures Fig Heme accumulation after knockdown of HO-1 or HO-2 expression HeLa cells (A) and HepG2 cells (B) were cultured for 12 h after transfection with siHO1, siHO2, or siGAPDH, then treated with lM hemin for 12 h, and harvested for the measurement of heme content Cells were treated for 24 h with Lipofectamine 2000 transfection reagent alone (control) The data are mean ± SEM from three independent experiments **P < 0.01 through the MARE [45–49] The increased concentration of endogenous heme may facilitate the binding of Nrf2, instead of Bach1, to the MARE to activate the MARE-dependent promoter, as reported for the mouse HO-1 gene [47] However, in contrast with the mouse HO-1 gene, the human HO-1 gene promoter is under complex regulation [9,40,50] In fact, both knockdown of HO-2 and hemin treatment resulted in activation of the MARE-dependent promoter, pRBGP2, whereas hemin did not increase the transient expression of phHOLUC45 (Fig 8B) It has been reported that hemin induces HO-1 expression in HeLa cells [36,51], and hemin activates transcription of the human HO-1 gene [40] Taken together with the findings that HO-2 knockdown tends to cause heme accumulation, we suggest that HO-2 may modulate transcription of the HO-1 gene through both heme-dependent and heme-independent mechanisms 5342 Human cell lines used were HeLa cervical carcinoma cells, HepG2 hepatoma cells, K562 and YN-1 erythroleukemia cells, Jurkat T-lymphocyte cells, KG1 myeloid cells, H146 small cell lung cancer cells, and HMV-II melanoma cells H146 small cell lung cancer cells were obtained from ATCC (HTB-173) and cultured in RPMI-1640 medium HMV-II melanoma cells were obtained from Riken Cell Bank and cultured in nutrient mixture Ham’s F12 medium HeLa and HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma) YN-1 cells were maintained in Iscove’s modified Dulbecco’s medium (Sigma), and K562, KG1 and Jurkat cells were maintained in RPMI-1640 medium (Sigma) Each medium contained 10% heat-inactivated fetal bovine serum, penicillin G (100 mL)1), and streptomycin sulfate (100 lgỈmL)1) Cells were incubated at 37 °C under 5% CO2 ⁄ 95% room air, unless otherwise specified HepG2, HeLa and YN-1 cells were treated with 50 lm SnPP or mm SA for up to 48 h SnPP was freshly prepared and added immediately to the culture medium The culture dishes were placed in the incubator Northern and western blot analyses Total RNAs and proteins were extracted from cells, and subjected to northern and western blot analyses [15,19] HO-1 and HO-2 RNA probes were transcribed by SP6 RNA polymerase from pCR-hHO1, carrying the human HO-1 cDNA fragment (positions 81–878), and pCRhHO2, carrying the human HO-2 cDNA fragment (positions 85–939), as described previously [19] mRNA signals were detected with the DIG Northern Starter Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol In western blot analysis, the signals of proteins were detected with the ECL Plus Western Blot Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s protocol The antibody FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Ding et al for HO-1 was a gift from S Taketani (Kyoto Institute of Technology, Japan) [52] Antibodies to HO-2 and b-actin were purchased from Stressgen (Victoria, BC, Canada) and Sigma Chemical, respectively Each blot was exposed to the Fuji medical X-ray film (Fuji Photo Film Co., Tokyo, Japan) for 10–60 s, depending on the experiments Heme content of cultured cells Heme content (expressed as ng ⁄ 106 cells) was determined as described previously [19] Cell pellet was dissolved in 0.5 mL m oxalic acid by shaking vigorously and immediately heated for 30 at 100 °C Mixtures that had not been heated were used as a blank for each measurement of endogenous porphyrins After cooling down, fluorescence was measured in a RF-5300PC spectrofluorometer (Shimadzu Corp., Kyoto, Japan) Under the conditions used, the lowest limit of detection is about ng heme ⁄ assay Cells (1 · 106) were used to determine heme content in all assays In some experiments, HeLa cells and HepG2 cells were cultured for 12 h after the transfection with siHO1, siHO2, or siGAPDH, then treated with lm hemin for 12 h, and harvested for heme measurement SiRNA and expression plasmids of HO-1 and HO-2 A specific siRNA against HO-1, siHO-1, which was reported by Miralem et al [53], was used HO-2 siRNA (target base 248–272), named siHO-2, was designed and synthesized by iGENE Therapeutics (Tsukubu, Japan), and scrambled HO-2 siRNA was used as a negative control: HO-2 siRNA: sense, 5¢-AGGACUUCUUGAAAGGCAA CAUUAAAG-3¢, antisense, 3¢-UAUCCUGAAGAACUU UCCGUUGUAAUU-5¢; scrambled HO-2 siRNA: sense, 5¢-UAUAAGAGUCAGUACACAUCAUGGAAG-3¢, antisense, 3¢-UAAUAUUCUCAGUCAUGUGUAGUACCU-5¢ Another HO-2-specific siRNA, HO-2 siRNA1 (target base 212–232) reported by other investigators [32], was also used GAPDH siRNA, named siGAPDH, was used as a control for siRNA When HeLa or HepG2 cells were 50% confluent, they were treated for 24 h with each siRNA by using Lipofectamine 2000 transfection (Invitrogen, Carlsbad, CA, USA), or treated with Lipofectamine 2000 transfection reagent alone as a control, according to the manufacturer’s protocol The amounts of each siRNA used were 40 pmol for HeLa cells and 80 pmol for HepG2 cells, cultured in a 9-cm dish for the indicated hours of incubation The effects of siRNA were assessed by northern blot and western blot analyses To construct an HO-1 expression vector, the human HO-1 cDNA fragment was amplified from the human HO-1 cDNA pHHO1 [25] by PCR, and subcloned between HindIII and XbaI sites of the pRc ⁄ CMV vector to yield an HO-1 expression vector, pRc ⁄ CMV-hHO-1 The PCR Heme oxygenase-2 down-regulates heme oxygenase-1 primers used for HO-1 were: forward, 5¢-TTAAAAGCTT ATGGAGCGTCCGCAACCCGA-3¢; reverse, 5¢-TTAAT CTAGAAAGAAGGCCTTCCACCGG-3¢ The sequences underlined are HindIII and XbaI sites, which were used for cloning into pRc ⁄ CMV Human HO-2 cDNA was amplified from the pHHO2-1 plasmid [54] by PCR with Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA), and then cloned into pCR-bluntII-TOPO (Invitrogen), yielding pCR-hHO-2-1 The primers used for HO-2 cDNA were: forward, 5¢-AAGCTTCATGTCAGCGGAAGTG GAAAC-3¢; reverse, 5¢-CTGCAGTCACATGTAGTACC AGGCCAA-3¢ The sequence underlined is an artificial HindIII site A full-length HO-2 cDNA fragment was isolated from pCR-hHO-2-1 with EcoR1 and subcloned into the pMACS4-IRES vector (Miltenyi Biotec Inc., BergischGladbach, Germany), generating the HO-2 expression vector, pMACS-hHO-2 Effects of HO-2 siRNA on the stability of HO-1 mRNA To study the effects of HO-2 knockdown on the stability of HO-1 mRNA, HeLa cells were left untrasnfected or transfected with siHO-2, and incubated for 12 h, followed by the addition of actinomycin D (1 lgỈmL)1) [37] The cells were further incubated for up to h, and then harvested at each time point for RNA preparation Luciferase assays The luciferase reporter constructs used were the human HO-1 gene constructs, phHOLUC45 and phHOLUC40 [14,36,55,56], and the human HO-2 gene constructs, phHO2()1494) and phHO2()663) [19] The test plasmids, pRBGP2 and pRBGP4, contain three copies of the MARE and mutated MARE, respectively, in each promoter region linked to the luciferase gene [39] HeLa cells were plated day before transfection and grown to 50–70% confluence in 24-well plates For siRNA experiments, HeLa cells at 50% confluence in 24-well plates were transfected with each luciferase reporter construct (0.3 lg) using FuGENE transfection reagent After 24 h of culture, transfected cells were re-transfected with pmol HO-1, HO-2, or GAPDH siRNA using Lipofectamine 2000 transfection reagent, and then cultured for an additional 24 h Luciferase activity in the transfected cells without siRNA treatment was included as a control For hemin treatment, HeLa cells at 50–80% confluence were transfected with each luciferase reporter (0.3 lg) using FuGENE transfection reagent After 24 h culture, transfected cells were treated with control vehicle or 50 lm hemin and further incubated for 24 h Expression of the reporter gene and pRL-TK (internal control) was determined with the Dual-LuciferaseTM Reporter Assay System (Promega, Madison, WI, USA) FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS 5343 Heme oxygenase-2 down-regulates heme oxygenase-1 Y Ding et al Acknowledgements We thank Professor S Taketani for providing the HO-1 antibody This study 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human heme oxygenase-1 gene expression in cultured cells Biochim Biophys Acta 1447, 231–235 FEBS Journal 273 (2006) 5333–5346 ª 2006 The Authors Journal compilation ª 2006 FEBS ... evaluation of heme dynamics in cultured cells Role of heme metabolism in cellular heme content Regulatory role of free heme in expression of HO-1 To evaluate the contribution of heme synthesis and... HO-1 protein was also induced by the treatment with HO-2 siRNA in HepG2 cells These results indicate that the down-regulation of HO-2 expression is associated with induction of HO-1 expression. .. protein, in comparison with the low expression of HO-1 protein, in H146 small cell lung cancer cells is of particular interest because small cell lung cancer is derived from the airway neuroepithelial