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Regulation of Heme Oxygenase-1 Protein Expression by miR-377 in Combination with miR-217*

Open AccessPublished:November 24, 2010DOI:https://doi.org/10.1074/jbc.M110.148726
      Heme oxygenase-1 (HO-1) enzyme plays a critical role in metabolizing the excess heme generated during hemolysis. Our previous studies suggested that during intravascular hemolysis the expression of HO-1 protein is not sufficient to reduce the oxidative burden of free heme in the vasculature. This led us to hypothesize that a post-translational mechanism of control exists for HO-1 expression. Micro-RNAs (miRNA) affect gene expression by post-transcriptional gene regulation of transcripts. We performed in silico analysis for the human HMOX1–3′ untranslated region (3′ UTR) and identified candidate miRNA binding sites. Two candidate miRNAs, miR-377 and miR-217, were cloned and co-transfected with a luciferase vector containing the human HMOX1-3′UTR region. The combination of miR-377 and miR-217 produced a 58% reduction in HMOX1–3′UTR luciferase reporter expression compared with controls. The same constructs were then used to assess how overexpression of miR-217 and miR-377 affected HO-1 levels after induction with hemin. Cells transfected with the combination of miR-377 and miR-217 exhibited no change in HMOX1 mRNA levels, but a significant reduction in HMOX1 (HO-1) protein expression and enzyme activity compared with non-transfected hemin-stimulated controls. Transfection with either miR-377 or miR-217 alone did not produce a significant decrease in HO-1 protein expression or enzyme activity. Knockdown of miR-217 and miR-377 in combination leads to up-regulation of HO-1 protein. Exposure to hemin induced a significant reduction in miR-217 expression and a trend toward decreased miR-377 expression in two different cells lines. In summary, these data suggests that the combination of miR-377 and miR-217 help regulate HO-1 protein expression in the presence of hemin.

      Introduction

      During hemolysis, up to 20 μm of free heme can be released leading to the breakdown of heme detoxification systems and subsequent damage to lipids, proteins, and DNA primarily through the ability of heme to generate reactive oxygen species (
      • Kumar S.
      • Bandyopadhyay U.
      ). Free heme is quite hydrophobic in nature and readily enters cell membranes and increases cellular susceptibility to oxidant-mediated killing (
      • Kumar S.
      • Bandyopadhyay U.
      ). Heme oxygenase-1 (HO-1)
      The abbreviations used are: HO-1, HMOX1, heme oxygenase-1; miRNA, microRNA; 3′ UTR, 3′ untranslated region; HUVEC, human umbilical vein cells; SOD, superoxide dismutase; DGCR8, DiGeorge critical region 8; qRT-PCR, quantitative reverse-transcribed polymerase chain reaction.
      is a 32-kDa microsomal/mitochondrial (
      • Sugishima M.
      • Omata Y.
      • Kakuta Y.
      • Sakamoto H.
      • Noguchi M.
      • Fukuyama K.
      ) enzyme, which oxidizes protoheme to biliverdin IXα in a three-step process, which requires oxygen and reducing equivalents from NADPH. In the process, this enzyme releases the antioxidant molecules carbon monoxide and biliverdin (
      • Sugishima M.
      • Omata Y.
      • Kakuta Y.
      • Sakamoto H.
      • Noguchi M.
      • Fukuyama K.
      ). Transcription of HO-1 is induced by a variety of agents, such as heme, oxidants, hypoxia, and cytokines and leads to induction of the enzyme and protection of tissues and cells against ischemia-reperfusion injury and oxidative stress (
      • Otterbein L.E.
      • Soares M.P.
      • Yamashita K.
      • Bach F.H.
      ,
      • Wagener F.A.
      • Volk H.D.
      • Willis D.
      • Abraham N.G.
      • Soares M.P.
      • Adema G.J.
      • Figdor C.G.
      ). As illustrated in patients and HMOX1−/− mice, HO-1 deficiency leads to oxidant-mediated injury, highlighting that the control and regulation of HO-1 expression is critical to protect cells from oxidative stress and damage (
      • Yachie A.
      • Niida Y.
      • Wada T.
      • Igarashi N.
      • Kaneda H.
      • Toma T.
      • Ohta K.
      • Kasahara Y.
      • Koizumi S.
      ,
      • Wagener F.A.
      • Eggert A.
      • Boerman O.C.
      • Oyen W.J.
      • Verhofstad A.
      • Abraham N.G.
      • Adema G.
      • van Kooyk Y.
      • de Witte T.
      • Figdor C.G.
      ,
      • Kawashima A.
      • Oda Y.
      • Yachie A.
      • Koizumi S.
      • Nakanishi I.
      ).
      To date several hundred microRNA (miRNA) genes have been identified in the human genome and it is proposed that at least 50% of all protein-encoding genes are regulated by miRNA (
      • Jackson R.J.
      • Standart N.
      ,
      • Faller M.
      • Matsunaga M.
      • Yin S.
      • Loo J.A.
      • Guo F.
      ). Mature miRNAs are ∼21–22 nucleotides in size and affect post-translational expression of genes by interacting with complementary target sites within the 3′ untranslated region of the messenger RNA (mRNA) (
      • Jackson R.J.
      • Standart N.
      ). The exact molecular mechanisms by which miRNAs mediate translational repression are still under intense study. However, the dogma is that most miRNAs control gene expression post-transcriptionally by regulating mRNA translation or stability in the cytoplasm (
      • Chekulaeva M.
      • Filipowicz W.
      ).
      Our lab and others have demonstrated that in sickle cell disease, HO-1 levels are elevated, particularly during hemolytic crisis (
      • Beckman J.D.
      • Belcher J.D.
      • Vineyard J.V.
      • Chen C.
      • Nguyen J.
      • Nwaneri M.O.
      • O'Sullivan M.G.
      • Gulbahce E.
      • Hebbel R.P.
      • Vercellotti G.M.
      ,
      • Belcher J.D.
      • Mahaseth H.
      • Welch T.E.
      • Otterbein L.E.
      • Hebbel R.P.
      • Vercellotti G.M.
      ,
      • Nath K.A.
      • Grande J.P.
      • Haggard J.J.
      • Croatt A.J.
      • Katusic Z.S.
      • Solovey A.
      • Hebbel R.P.
      ). However, exposure of animals or humans with elevated HO-1 expression to agents that are able to promote HO-1 transcription can lead to additional up-regulation of the protein, providing additional protection against oxidative stress and inflammation (
      • Beckman J.D.
      • Belcher J.D.
      • Vineyard J.V.
      • Chen C.
      • Nguyen J.
      • Nwaneri M.O.
      • O'Sullivan M.G.
      • Gulbahce E.
      • Hebbel R.P.
      • Vercellotti G.M.
      ,
      • Belcher J.D.
      • Mahaseth H.
      • Welch T.E.
      • Otterbein L.E.
      • Hebbel R.P.
      • Vercellotti G.M.
      ,
      • Belcher J.D.
      • Vineyard J.V.
      • Bruzzone C.M.
      • Chen C.
      • Beckman J.D.
      • Nguyen J.
      • Steer C.J.
      • Vercellotti G.M.
      ). These observations lead us to hypothesize that a post-translational mechanism may exist for HO-1 expression. Our results demonstrate that there are at least two mature miRNAs which interact with the HMOX1–3′UTR in regulating HO-1 protein expression and enzymatic activity.

      DISCUSSION

      The results of our studies support our hypothesis that a post-translational mechanism may exist to attenuate HO-1 expression. We demonstrated that miRNAs interact directly with the HMOX1–3′ UTR. We further demonstrate that two miRNAs, miR-217 and miR-377, combine to attenuate HO-1 protein expression, which ultimately results in a significant reduction in HO-1 enzyme activity. In return, we demonstrate that knockdown of both miR-217 and miR-377 increases HO-1 protein expression. Additionally, we demonstrate that exposure to hemin influences the levels of some, but not all miRNAs involved with HO-1 expression. Based on these studies, we propose a model (Fig. 7) in which miRNAs may be serving as a rheostat to titrate the levels HO-1 expression to a physiologic set point. This model illustrates that a decrease in miR-217 and miR-377 leads to increased HO-1 protein (Fig. 7B), and overexpression of the same miRNAs leads to attenuation of protein expression (Fig. 7C).
      Figure thumbnail gr7
      FIGURE 7Proposed model of miRNA regulation of HMOX1. A, at baseline, levels of HMOX1 translation are balanced by expression of miR-217 and miR-377, which interact with the 3′ UTR to attenuate protein expression. B, although heme may increase miRNA biogenesis, our experiments indicate that intracellular levels of mature miR-217 and miR-377 decrease in the presence of heme. This reduces miRNA interaction with the HMOX1–3′ UTR allowing for increased HO-1 protein expression. C, conversely, decreased HO-1 protein expression occurs when miR-217 and miR-377 are overexpressed.
      In this work we selected two candidate miRNAs to validate based on their context score. Additionally, we chose to validate each miRNA separately and in combination. As recent publications have highlighted, many genes have multiple binding sites for a single miRNA within their 3′ UTR, but also binding sites for many other miRNAs (
      • Wu S.
      • Huang S.
      • Ding J.
      • Zhao Y.
      • Liang L.
      • Liu T.
      • Zhan R.
      • He X.
      ,
      • Peter M.E.
      ). Therefore, the combination of multiple miRNAs may determine the level of gene expression; in part, because the combination of multiple miRNAs may overcome the relative weakness of each separate miRNA:mRNA seed match (
      • Peter M.E.
      ). Therefore, as demonstrated in Fig. 2, a weak interaction between miR-217 and the HMOX1–3′ UTR may help strengthen the association that occurs between miR-377 and the HMOX1–3′ UTR. In addition, the expression of each miRNA is also controlled by various factors, such as levels of primary and precursor miRNA transcripts and cell type (
      • Elmén J.
      • Lindow M.
      • Schütz S.
      • Lawrence M.
      • Petri A.
      • Obad S.
      • Lindholm M.
      • Hedtjärn M.
      • Hansen H.F.
      • Berger U.
      • Gullans S.
      • Kearney P.
      • Sarnow P.
      • Straarup E.M.
      • Kauppinen S.
      ). Further validation of other predicted targets, such as the ones surveyed in Fig. 6, may yield a more complex system to coordinate HO-1 protein expression depending on the environment of the cell.
      Interestingly, both miR-377 and miR-217 have been investigated for their role in stress responses during pathological conditions, such as diabetes and aging (
      • Menghini R.
      • Casagrande V.
      • Cardellini M.
      • Martelli E.
      • Terrinoni A.
      • Amati F.
      • Vasa-Nicotera M.
      • Ippoliti A.
      • Novelli G.
      • Melino G.
      • Lauro R.
      • Federici M.
      ,
      • Kato M.
      • Putta S.
      • Wang M.
      • Yuan H.
      • Lanting L.
      • Nair I.
      • Gunn A.
      • Nakagawa Y.
      • Shimano H.
      • Todorov I.
      • Rossi J.J.
      • Natarajan R.
      ,
      • Wang Q.
      • Wang Y.
      • Minto A.W.
      • Wang J.
      • Shi Q.
      • Li X.
      • Quigg R.J.
      ). Using mouse models of diabetic nephropathy, Kato and associates (
      • Kato M.
      • Putta S.
      • Wang M.
      • Yuan H.
      • Lanting L.
      • Nair I.
      • Gunn A.
      • Nakagawa Y.
      • Shimano H.
      • Todorov I.
      • Rossi J.J.
      • Natarajan R.
      ) demonstrated that miR-217, and its clustered counterpart miR-216a, are induced by transforming growth factor-β (TGF-β), leading to an inhibition of PTEN, and ultimately activation of Akt. Wang and associates (
      • Wang Q.
      • Wang Y.
      • Minto A.W.
      • Wang J.
      • Shi Q.
      • Li X.
      • Quigg R.J.
      ) also demonstrated that miR-377 plays a role in the pathogenesis of diabetic nephropathy in both human cell lines and mouse models through its ability to target and translationally repress superoxide dismutase 1 and 2 (SOD1, SOD2) and p21/Cdc42/Rac1-activated kinase 1 (PAK1) leading to increased fibronectin production (
      • Wang Q.
      • Wang Y.
      • Minto A.W.
      • Wang J.
      • Shi Q.
      • Li X.
      • Quigg R.J.
      ). SOD2 is also known as mitochondrial superoxide dismutase or manganese SOD (MnSOD) and is responsible for reducing toxic reactive oxygen species in the mitochondria. Interestingly, carbon monoxide, a by-product of HO-1, has been shown to increase levels of MnSOD (
      • Piantadosi C.A.
      • Carraway M.S.
      • Suliman H.B.
      ). Therefore, in accord with our hypothesis a decrease in miR-377 will lead to an up-regulation of both HO-1 and MnSOD, leading to an antioxidant effect. In our study, the effect of hemin on miR-377 and miR-217 was dependent on the cell line and time period evaluated (Fig. 5). Therefore, the balance between levels of miR-217 and miR-377, along with other potential miRNAs, may act like a rheostat that attenuates pro-apoptotic versus the antioxidant pathways in response to cellular stressors. Concordantly, this analysis also highlights the fact that miRNA:mRNA networks may differ between cell types and disease model systems.
      Previous work investigating miRNAs and HO-1 has focused on the effects of miRNAs binding to Bach1, a potent repressor of HMOX1 transcription (
      • Hou W.
      • Tian Q.
      • Zheng J.
      • Bonkovsky H.L.
      ,
      • Shan Y.
      • Zheng J.
      • Lambrecht R.W.
      • Bonkovsky H.L.
      ). Bach-1 is a member of the bZIP transcription factor family that serves as a potent repressor of HMOX1 transcription through its higher binding affinity for multiple Maf recognition elements regions compared with nuclear factor erythroid 2-related factor 2 (Nrf2) (
      • Tahara T.
      • Sun J.
      • Nakanishi K.
      • Yamamoto M.
      • Mori H.
      • Saito T.
      • Fujita H.
      • Igarashi K.
      • Taketani S.
      ,
      • Yano Y.
      • Ozono R.
      • Oishi Y.
      • Kambe M.
      • Yoshizumi M.
      • Ishida T.
      • Omura S.
      • Oshima T.
      • Igarashi K.
      ,
      • Sun J.
      • Brand M.
      • Zenke Y.
      • Tashiro S.
      • Groudine M.
      • Igarashi K.
      ,
      • Ogawa K.
      • Sun J.
      • Taketani S.
      • Nakajima O.
      • Nishitani C.
      • Sassa S.
      • Hayashi N.
      • Yamamoto M.
      • Shibahara S.
      • Fujita H.
      • Igarashi K.
      ,
      • Oyake T.
      • Itoh K.
      • Motohashi H.
      • Hayashi N.
      • Hoshino H.
      • Nishizawa M.
      • Yamamoto M.
      • Igarashi K.
      ). In two recent papers, Bonkovsky and associates (
      • Hou W.
      • Tian Q.
      • Zheng J.
      • Bonkovsky H.L.
      ,
      • Shan Y.
      • Zheng J.
      • Lambrecht R.W.
      • Bonkovsky H.L.
      ) explored the role of miR-122 and miR-196 in regulating Bach1 in hepatocytes infected with hepatitis C virus. miR-122 is the most abundant miRNA transcript in the liver and necessary for hepatic accumulation of the hepatitis C virus (
      • Lanford R.E.
      • Hildebrandt-Eriksen E.S.
      • Petri A.
      • Persson R.
      • Lindow M.
      • Munk M.E.
      • Kauppinen S.
      • Ørum H.
      ). In fact, recent work using lock-nucleic acid antagonists of miR-122 in chimpanzee models of high cholesterol and hepatitis C virus demonstrated the efficacy and feasibility of miRNA-based therapeutics for human clinical trials (
      • Lanford R.E.
      • Hildebrandt-Eriksen E.S.
      • Petri A.
      • Persson R.
      • Lindow M.
      • Munk M.E.
      • Kauppinen S.
      • Ørum H.
      ,
      • Elmén J.
      • Lindow M.
      • Schütz S.
      • Lawrence M.
      • Petri A.
      • Obad S.
      • Lindholm M.
      • Hedtjärn M.
      • Hansen H.F.
      • Berger U.
      • Gullans S.
      • Kearney P.
      • Sarnow P.
      • Straarup E.M.
      • Kauppinen S.
      ). miR-122 is predicted to bind with the Bach1 3′ UTR, which leads to a decrease in Bach1 protein and a subsequent increase in HO-1 expression. In a separate report linking miRNA levels and HO-1 expression, it was demonstrated that miR-196 interacts with Bach1 via two binding sites within the 3′ UTR, and that overexpression of miR-196 leads to repression of Bach1 protein and subsequent up-regulation of HMOX1 mRNA (
      • Hou W.
      • Tian Q.
      • Zheng J.
      • Bonkovsky H.L.
      ). When we surveyed for expression of mature miRNAs after hemin treatment we did find that in HEK 293 cells, but not HUVEC, treatment with hemin resulted in a decrease (p = 0.09) in miR-196 expression (data not shown). This interaction could lead to an up-regulation in Bach1 in an attempt to balance factors required for the control HO-1 expression in the presence of heme.
      It has been reported that heme may play a significant role in miRNA processing. Mature miRNAs are cleaved from ∼70-nucleotide hairpin structures, called precursor miRNAs (pre-miRNAs), in the cytoplasm by the enzyme dicer. Prior to this final step, pre-miRNAs are excised in the nucleus from a primary miRNA (pri-miRNA) transcript by the RNase III enzyme Drosha and its co-factor DiGeorge Critical Region 8 (DGCR8, also known as Pasha), which are necessary and sufficient for pri-mRNA processing (
      • Faller M.
      • Matsunaga M.
      • Yin S.
      • Loo J.A.
      • Guo F.
      ,
      • Chen K.
      • Rajewsky N.
      ). DGCR8 is a double-stranded RNA-binding protein that may be the only member of the small non-coding RNA processing pathway specific for miRNAs (
      • Wang Y.
      • Medvid R.
      • Melton C.
      • Jaenisch R.
      • Blelloch R.
      ). DGCR8 binds heme, which promotes the homodimerization of DGCR8, forming a complex containing one heme molecule per homodimer (
      • Faller M.
      • Matsunaga M.
      • Yin S.
      • Loo J.A.
      • Guo F.
      ). The heme-free DGCR8 monomer is much less active than the heme-bound dimer, perhaps because the conserved cysteine residue that binds heme prevents autoinhibition of DGCR8. The ability of DGCR8 to function as a heme biosensor provides an elegant mechanism of linking heme levels to global protein synthesis and cellular differentiation. Rapid changes in the availability of heme may lead to significant changes in the ability of the body to process crucial miRNA molecules, such as those regulating a key heme catabolizing enzyme, HO-1. As Fig. 6 demonstrates, some, but not all, of the miRNAs predicted to interact with the HMOX1–3′ UTR were affected by heme. Additionally, the timing of this interaction also varied according to the cell type, which may reflect cell-specific copy number variations of the individual miRNAs (
      • Hinton A.
      • Afrikanova I.
      • Wilson M.
      • King C.
      • Maurer B.
      • Yeo G.
      • Hayek A.
      • Pasquinelli A.
      ).
      A recent study on HO-1 expression in endothelial cells derived from different donors demonstrates a wide range of basal HO-1 levels in human populations. However, when the cells were stimulated with oxidized lipids, all cells reached a similar level of HO-1 mRNA, regardless of basal levels of HMOX1 transcript (
      • Romanoski C.E.
      • Lee S.
      • Kim M.J.
      • Ingram-Drake L.
      • Plaisier C.L.
      • Yordanova R.
      • Tilford C.
      • Guan B.
      • He A.
      • Gargalovic P.S.
      • Kirchgessner T.G.
      • Berliner J.A.
      • Lusis A.J.
      ). We propose that miRNAs may be serving as a rheostat to titrate the levels of HO-1 expression to a physiologic set point (Fig. 7). In this work we have demonstrated that two miRNAs, miR-217 and miR-377, work together and in combination to attenuate HO-1 protein expression and enzyme activity, highlighting that miRNA interactions are involved in the control of HO-1 expression. Further work is warranted to clarify the role of miRNA-mRNA interactions during hemolysis and uncover new potential therapeutic modalities to modulate HO-1 expression.

      Acknowledgments

      We acknowledge the following individuals for their support and assistance during the completion of this project: Dr. John Belcher, Dr. Liming Milbauer, Dr. Yvonne Datta, Carol Bruzzone, Paul Marker, and Jean Herron.

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