REVIEW URRENT C OPINION
Posttranscriptional adaptations of the vascular endothelium to hypoxia Jr Jyun David Ho a,b and Philip A. Marsden c
Purpose of review Remarkable new advances have been made in the field of posttranscriptional gene regulation over recent years. These include the revelation of noncoding RNAs, such as microRNAs, antisense transcripts and their interactions with RNA-binding proteins (RBPs) in the context of both health and disease settings, such as hypoxia. In particular, these discoveries bear much relevance to the field of vascular biology, which historically has focused upon transcriptional processes. Thus, the contributions of these posttranscriptional gene regulatory mechanisms to vascular and endothelial biology represent a newer concept that warrants discussion. Recent findings Recent studies have revealed two emerging themes that are critical to endothelial/vascular biology and function. First is the functional integration between the microRNA pathway and the cellular hypoxic response, which, in addition to specific microRNAs, involves key components of the microRNA biogenesis machinery. A key concept here is the regulation of a master transcriptional programme through posttranscriptional mechanisms. The second major theme involves the dynamic interactions between RBPs, microRNAs and antisense RNAs. The condition-dependent collaborations and competitions between these different classes of posttranscriptional regulators reveal a critical layer of control for gene expression. Summary Taken together, these findings bear significant diagnostic and therapeutic implications for vascular disease. Keywords antisense, endothelial nitric oxide synthase, hypoxia, microRNAs, RNA-binding proteins
INTRODUCTION The vascular endothelium constitutes the innermost lining of the entire circulatory system. Being more than a passive physical barrier between the circulating blood and surrounding tissues, endothelial cells play an active and essential role in physiology. Indeed, endothelial cells are crucial for vasculogenesis [1,2] and angiogenesis [3], and they contribute to the maintenance of vascular homeostasis [1,4]. Recent research has revealed that endothelial cells are actively involved in sensing environmental stresses, including changes in oxygen concentration and bioavailability, as well as in orchestrating coordinated responses to these physiological perturbations [5]. Indeed, the impairment of endothelial cell function has been implicated in numerous cardiovascular diseases (CVDs), including atherosclerosis, stroke and ischaemia-reperfusion injury [6]. Hypoxia (low oxygen bioavailability) is a critical environmental stress that profoundly affects aerobic organisms. It is associated with many diseases,
including CVDs [7–11]. To cope, mammalian cells, including endothelial cells, have evolved a comprehensive cellular response that strives to promote cellular survival during hypoxia [12–18]. In particular, the cell activates a critical transcriptional response pathway, which leads to the activation and/or inhibition of key genes involved in processes such as angiogenesis, glycolysis, cell growth, apoptosis and cell cycle regulation [19–21]; the coordinate regulation of these genes contributes synergistically to cellular survival [9,10,19,22]. At
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Department of Biochemistry and Molecular Biology, bSylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida, USA and cKeenan Research Centre for Biomedical Science in the Li Ka Shing Knowledge Institute, St. Michael’s, Hospital, Department of Medicine, University of Toronto, Toronto, Ontario, Canada Correspondence to Philip A. Marsden, St. Michael’s Hospital, 30 Bond St., Toronto, ON M5B 1W8, Canada. Tel: +1 416 847 1736; e-mail: [email protected] Curr Opin Hematol 2015, 22:243–251 DOI:10.1097/MOH.0000000000000139
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KEY POINTS Remarkable new advances have been made in the field of posttranscriptional gene regulation in recent years, especially in the vascular endothelium. A biologically functional integration exists between the microRNA pathway and the cellular hypoxic response, which, in addition to specific microRNAs, involves key components of the microRNA biogenesis machinery. The dynamic, condition-dependent interactions between RBPs, microRNAs and antisense RNAs play a crucial role in regulating endothelial biology. Changes in posttranscriptional gene regulation offer diagnostic and therapeutic opportunities and nuances for the treatment of vascular disease.
the heart of this transcriptional response is the hypoxia-inducible factor (HIF) [23–29], which is a hetero-dimeric transcription factor comprising a hypoxia-inducible a-subunit (i.e. HIF-1a) and a constitutively expressed nuclear b-subunit (HIF-1b) [27–30]. A second a-subunit isoform, HIF-2a, also exists that can also dimerize with HIF-1b [31]. Initially discovered in the endothelium as endothelial Per-Arnt-Sim domain protein one (EPAS1) [31,32], HIF-2a expression has been observed in a wide variety of cell types/tissues [33,34]. Functionally, HIF-1a and HIF-2a share a number of common targets, but each isoform also has unique targets [35–42]. In healthy cells, HIF-a protein is rapidly and continuously degraded in normoxia via a ubiquitin-proteasome pathway [43] that involves two critical components [9,10,44–47]. The first is a family of oxygen-dependent enzymes known as HIF prolyl4-hydoxylases, also termed prolyl hydroxylase domain containing proteins (PHDs) [48–50]. In the presence of oxygen, PHDs catalyse the hydroxylation of two key proline residues [51] in HIF-1a [52] and HIF-2a proteins [45,53]. These hydroxyproline residues are recognized by the von Hippel–Lindau protein (pVHL) [47,54–58], which, as part of an E3 ubiquition ligase complex, mediates the ubiquitylation and proteasomal degradation of HIF-a [59–67]. In hypoxia, oxygen-dependent HIF-a prolyl hydroxylation does not occur, and HIF-a is not polyubiquitylated by pVHL. Therefore, HIF-a escapes proteasomal degradation, accumulates and translocates into the nucleus to regulate target genes. Posttranscriptional regulation involves a complex network of factors that control transcript modifications, nuclear-cytoplasmic transport, subcellular localization, mRNA stability and translational efficiency [68–74]. These regulatory processes are 244
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largely mediated by cis/trans interactions between cis-elements in the 3’-untranslated region (UTR) of mature mRNAs and trans-factors that mostly fall into one of the three following categories: RNAbinding proteins (RBPs), natural antisense transcripts and noncoding RNAs such as microRNAs and short interfering RNAs [74–82]. This review will focus on the new and emerging concepts of posttranscriptional regulatory mechanisms in the hypoxia endothelium.
FUNCTIONAL INTEGRATION OF THE microRNA AND CELLULAR HYPOXIC RESPONSE PATHWAYS The microRNA pathway is a potent, endogenous regulatory pathway that involves translational inhibition and mRNA degradation by nearly 21nt singlestranded RNA molecules [83–87]. First discovered in 1993 [88,89], microRNAs are highly conserved across evolution, existing in organisms ranging from unicellular green alga [90] to humans [91]. A recent estimate indicates that at least 60% of all human protein-coding genes are subject to microRNA regulation [92], and the ubiquitous influence of microRNAs exerts a widespread impact on mRNA repression and evolution [93], generating thresholds in target gene expression [94] under both basal and stress conditions [95]. Key details regarding the canonical microRNA biogenesis have been elucidated [96,97] (Fig. 1). In particular, the key cytoplasmic enzyme Dicer catalyses the final step in microRNA maturation, producing functional microRNAs that can then be loaded (with the aid of Dicer) onto their downstream effector RNA-induced silencing complexes (RISCs). The biological significance of Dicer is underscored by the observation that Dicer knockout results in embryonic lethality in both mice and zebrafish [98–100]. The existence of a biological relationship between hypoxia and microRNAs was first established in the late 2000s by several independent laboratories [101–109]. Specifically, miR-210 was identified as the first proto-typical hypoxia/HIFinducible microRNA [109] across a variety of cell types, including endothelial and vascular smooth muscle cells [102,104,105]. Since its discovery, many targets of miR-210 have been identified, and they implicate miR-210 in the regulation of cell cycle, angiogenesis, mitochondrial metabolism, DNA damage response and cell survival [110–112]. Specifically in endothelial cells, miR-210 has been shown to target Ephrin-A3, which is a ligand for the ephrin receptor family of receptor tyrosine kinases [102,104]. Ephrins vital in angiogenesis and vascular remodelling [113], and cells expressing Volume 22 Number 3 May 2015
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FIGURE 1. Functional importance of Dicer in the adaptive cellular response to hypoxia. Dicer and an important number of Dicer-dependent microRNAs are downregulated in chronic hypoxia. This is an important adaptive strategy that serves to maintain the cellular hypoxic response through hypoxia-inducible factor-a (HIF-a) and microRNA-dependent mechanisms. A significant number of downregulated microRNAs are predicted regulators of hypoxia-inducible genes, including the critical hypoxia-inducible factor-a (HIF-a) subunits, glucose transporter 1 (GLUT1), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L), vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2. In particular, the hypoxic downregulation of the Dicer-dependent microRNA miR-185 contributes to the hypoxic induction of HIF-2a. Reproduced with permission from [97]. RISC, RNA-induced silencing complexes.
miR-210-resistant Ephrin-A3 mRNAs exhibited impaired tubulogenesis and chemotaxis [102]. In addition, miR-210 also targets the iron–sulphur cluster assembly proteins ISCU1/2 in endothelial cells [105]. ISCU1/2 play crucial roles in mediating electron transport and mitochondrial oxidationreduction reactions [114], and their inhibition by miR-210 results in the impairment of mitochondrial respiration [105], which is believed to be a beneficial adaption that optimizes energy generation and prevents the production of excessive levels of toxic reactive oxygen species [115]. Taken together, these findings suggest that miR-210 exhibits both endothelial cell specific roles and generalized functions that are relevant across cell types. More recently, the potential diagnostic and prognostic value of miR-210 has also been explored. For example, miR-210 was recently reported as a
marker of hypoxia in glioblastoma and renal cell carcinoma [116,117]. Beyond miR-210, however, little else was known regarding the extent of integration between the cellular hypoxic response and the microRNA pathway, such as the effects of hypoxia on microRNA biogenesis, and the biological rationale for such regulation. To address this question, a number of recent studies have revealed that microRNA biogenesis and activity are fundamentally linked to the cellular hypoxic response. The revelation began with the observation that Dicer was downregulated in chronically hypoxic rat lungs in vivo [118], and then in chronically hypoxic vascular smooth muscle cells in vitro [119]. However, the biological significance of this phenomenon remained unknown, until our laboratory recently discovered that the downregulation of Dicer expression in chronic
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hypoxia represents an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-a and microRNA-dependent mechanisms [120] (Fig. 1). Specifically, we found that the hypoxia-mediated decrease in Dicer expression and activity resulted in a global decrease in mature microRNA levels that was accompanied by a corresponding accumulation of precursor microRNAs (Fig. 1). Interestingly, despite this global impairment of microRNA biogenesis in chronic hypoxia, only a subset of microRNAs was significantly downregulated at the steady-state level. Notably, the majority of the downregulated microRNAs were predicted regulators of key hypoxia-inducible genes, including the glucose transporter 1 (GLUT1), BCL2/ adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L), vascular endothelial growth factor (VEGF), VEGF receptors VEGFR1 and VEGFR2, and especially the HIF-a subunits themselves. These findings support the model that microRNAs with elevated target mRNA levels (such as the increased expression of hypoxia-inducible genes in chronic hypoxia) undergo an increased rate of activitydependent microRNA decay [97]. Future studies will be required to elucidate the underlying molecular details of such regulation. Nonetheless, it was not surprising then that the full induction of HIF-1a and HIF-2a, as well as key hypoxia-responsive/HIF target mRNAs in chronic hypoxia, was observed to be dependent upon the hypoxic downregulation of Dicer function. Indeed, when Dicer was overexpressed in hypoxic cells, the induction of HIF-a isoforms and HIF-target genes, including those of particular relevance to endothelial and vascular biology (e.g. VEGF, VEGFR1, VEGFR2), was markedly blunted. In addition, we also identified miR185 as the first microRNA that targets HIF-2a, which is the predominant HIF-a isoform in endothelial cells [31,120]. Thus, the hypoxic downregulation of Dicer maintains the induction of key survival genes not just by attenuating microRNA-mediated suppression of the genes themselves, such as VEGF, but also through the attenuation of the microRNAdependent downregulation of their upstream master transcriptional activator, HIF-a [120]. The initial studies were performed in vascular cell types, including macro and microvascular endothelial cells and vascular smooth muscle cells. Remarkably, this functional interaction between the microRNA and hypoxia response pathways is a broadly relevant mechanism that has been observed across cell types. Indeed, since our initial discovery, this adaptive strategy has been observed in a number of different cell types, including human breast and ovarian cancer cells, whereby the hypoxic downregulation of Dicer was found to 246
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promote tumourigenesis and tumour progression [121 –123 ]. Moreover, the miR-185/ HIF-2a interaction has also been confirmed in colon and ovarian cancer models [122 ]. In addition, the biological significance of hypoxic Dicer downregulation is highlighted by the fact that cells invest significant resources to ensure the suppression of Dicer expression and activity in chronic hypoxia. For instance, we and others have shown that Dicer is downregulated via multiple mechanisms, including decreased mRNA stability, decreased translation, reduced protein stability and epigenetic mechanisms [120,123 ]. Interestingly, Dicer downregulation is von Hippel-Lindau, but not HIF dependent [120,121 ,123 ]. Future studies will be required to examine the biological implications of this observation, as well as whether Dicer is subject to hypoxic posttranslational modification, such as prolyl hydroxylation, as it has been shown for Argonaute 2 (AGO2) [119,124]. Finally, hypoxia significantly attenuates the efficiency of Dicer-dependent, but not Dicer-independent RNA interference (RNAi)based therapeutics [120]. This has clear implications in human disease, wherein tissue hypoxia is often encountered in CVDs, and it is also a critical phenotype of the tumour microenvironment [125,126]. In addition to Dicer, several other components of the microRNA biogenesis machinery are also downregulated by hypoxia in various cell types, including vascular cells such as endothelial cells and vascular smooth muscle cells. These components include Drosha, DGCR8, TRBP and AGO proteins [119,120,121 ,122 ,127 ]. In particular, one study reported that in endothelial cells, AGO1, which is a key component of the RISC complex, is selectively targeted by hypoxia-responsive microRNAs in order to suppress the translational inhibition of VEGF [127 ]. This adaptation likely works in synergy with the hypoxic downregulation of Dicer to maximize the induction of key hypoxiaresponsive genes. Indeed, it is likely that hypoxic cells, especially endothelial cells, adopt the adaptive strategy of maintaining their HIF-mediated hypoxic response by limiting microRNA production and activity through the downregulation of their effector and biogenesis machinery. &
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DYNAMIC INTERACTIONS BETWEEN RNABINDING PROTEINS, microRNAs AND ANTISENSE TRANSCRIPTS Posttranscriptional regulators are mostly trans-factors that fall into these three major categories: RBPs, microRNAs and antisense transcripts. Given that these factors are all involved in the control of mRNA stability and translation, it is not surprising that Volume 22 Number 3 May 2015
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and hypoxia-associated vasoconstriction [129,130]. eNOS mRNAs are highly stable, with a half-life of over 24 h in human endothelial cells [131 ]. In addition, it is well established that decreased eNOS mRNA stability plays a major role in reducing eNOS expression and activity under a variety of pathophysiological conditions, including hypoxia [120,131 ,132]. However, until recently, the molecular basis and rationale for this remarkable stability, and the mechanisms that mediated the hypoxic decrease in eNOS mRNA stability have remained unknown. Recently, studies have revealed a novel role for the RBP hnRNP E1 in mediating eNOS mRNA stability. Specifically, it was found that hnRNP E1 stabilizes eNOS mRNAs by interacting with several
they often act collaboratively or competitively with each other. Indeed, the fate of an mRNA is largely dependent on the net effect of its interactions with these regulatory factors, as ‘naked’ mRNAs do not exist in cells. Rather, they interact dynamically with a myriad of trans-factors and exist virtually exclusively in the context of macromolecular complexes throughout their entire life cycle in the cell [71,128]. To illustrate the complex yet elegant interactions between these molecules [97], and their effect on endothelial cell biology, we will use the endothelial nitric oxide synthase (eNOS) gene as an example. eNOS is a critical endothelial cell enriched gene that synthesizes the vasodilator nitric oxide in the vascular endothelial cell [5]. Its expression is often disrupted in disease, including atherosclerosis
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FIGURE 2. Stabilization of endothelial nitric oxide synthase (eNOS) mRNA by hnRNP E1. (Top) In normal endothelial cells, hnRNP E1 interacts with conserved pyrimidine (C/CU)-rich elements in the eNOS 30 -untranslated region, stabilizing and protecting eNOS mRNAs from the inhibitory effects of microRNAs, such as miR-765. (Bottom) Under chronic hypoxic conditions, hnRNP E1 exhibits increased serine phosphorylation and nuclear sublocalization in an AKT-dependent manner. These events inhibit the interaction between hnRNP E1 and eNOS mRNAs, thus rendering eNOS sensitive to the inhibitory posttranscriptional effects of the hypoxia-inducible natural cis-antisense transcript, sONE. Reproduced with permission from [97]. 1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
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conserved pyrimidine (C/CU)-rich elements in the eNOS mRNA 30 -UTR [131 ] (Fig. 2, top). Functionally, these ribonucleoprotein (RNP) complexes mediate the remarkable stability of eNOS mRNAs in normal endothelial cells by protecting them against the inhibitory actions of endogenous microRNAs, such as miR-765, whose target site overlaps with one of the conserved pyrimidine-rich elements that hnRNP E1 interacts with (Fig. 2, top). This is reminiscent of the relationship reported previously whereby the RBP HuR protects COX-2 mRNAs from miR-16, whose target sites overlaps with the HuRbinding site [133]. In addition, we also found that hnRNP E1 protects eNOS mRNAs from exogenous short interfering RNAs that target eNOS mRNA at 30 UTR sites that are distinct from hnRNP E1 binding sites [131 ]. Again, this observation is conceptually consistent with previous reports that HuR protects CAT-1 mRNAs from distally binding microRNAs [134,135]. Our study demonstrates the complexity of posttranscriptional regulation involving RBPs, microRNAs and antisense transcripts in the field of endothelial biology. Interestingly, we also observed that the formation of the eNOS-stabilizing complex was disrupted in the setting of hypoxia [131 ] (Fig. 2, bottom). Indeed, further examination revealed that hnRNP E1 is unable to engage eNOS mRNAs in hypoxia because of increased AKT-dependent serine phosphorylation and nuclear sublocalization. Notably, the change in subcellular localization is similar to the reported interaction between hnRNP L and VEGF in hypoxic cells [136]. Furthermore, our findings are also consistent with the previous report that the phosphorylation of hnRNP E1 results in increased nuclear retention [137]. Consequently, these modifications render eNOS mRNAs susceptible to degradation and translational inhibition by posttranscriptional inhibitory forces under hypoxic conditions. Specifically, we have previously identified a hypoxia-inducible natural cis-antisense transcript to eNOS, termed sONE, which exhibits significant overlap with the eNOS mRNA in a tailto-tail orientation [138,139]. In the absence of the stabilizing complex, sONE downregulates eNOS mRNA posttranscriptionally, resulting in reduced eNOS expression and activity [138]. Further studies will be required to elucidate the underlying mechanisms by which this occurs. Nevertheless, we now appreciate that the remarkable basal stability of eNOS mRNA is mediated by hnRNP E1 containing stabilizing complexes, which serves to protect them from inhibitory microRNAs. In addition, that the hypoxia-mediated decrease in eNOS mRNA stability is primarily due to impaired stabilizing complex formation as a consequence of changes in hnRNP &
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E1 phosphorylation status and subcellular localization, which renders eNOS mRNAs susceptible to posttranscriptional downregulation by its antisense transcript. This work further underscores the importance of defining why a protein-coding mRNA is not subject to basal microRNA-mediated repression. Some mRNAs are protected from RNAi-mediated repression under basal conditions. Under the appropriate environmental conditions, especially hypoxia, an mRNA can be sensitized to miRNAmediated repression.
CONCLUSION These findings illustrate the complexity and biological significance of posttranscriptional regulation in the vascular endothelium, especially in the setting of hypoxia. The dynamic and conditiondependent interactions between these different regulators, and the net effects on their target mRNAs, represent a critical layer of gene expression control, which is a new and emerging concept in the field of vascular biology. The significant diagnostic and therapeutic implications of these regulatory mechanisms should be the focus of future studies.
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Acknowledgements None. Financial support and sponsorship J.J.D.H. holds a Canadian Institutes of Health Research (CIHR) Fellowship. P.A.M. is a Heart and Stroke Foundation of Canada Career Investigator and is supported by a grant from the Heart and Stroke Foundation of Canada (grant T-6777). Conflicts of interest There are no conflicts of interest.
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105. Chan SY, Zhang YY, Hemann C, et al. MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2. Cell Metab 2009; 10:273–284. 106. Huang X, Ding L, Bennewith KL, et al. Hypoxia-inducible mir-210 regulates normoxic gene expression involved in tumor initiation. Mol Cell 2009; 35:856–867. 107. Zhang Z, Sun H, Dai H, et al. MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT. Cell Cycle 2009; 8:2756–2768. 108. Crosby ME, Kulshreshtha R, Ivan M, Glazer PM. MicroRNA regulation of DNA repair gene expression in hypoxic stress. Cancer Res 2009; 69:1221– 1229. 109. Kulshreshtha R, Ferracin M, Wojcik SE, et al. A microRNA signature of hypoxia. Mol Cell Biol 2007; 27:1859–1867. 110. Chan SY, Loscalzo J. MicroRNA-210: a unique and pleiotropic hypoxamir. Cell Cycle 2010; 9:1072–1083. 111. Magenta A, Greco S, Gaetano C, Martelli F. Oxidative stress and microRNAs in vascular diseases. 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Nat Commun 2014; 5:5202. & & Together with references [122 ] and [123 ], these studies confirm the original finding from reference [120] that the hypoxic downregulation of Dicer represents an important adaptive strategy in the cellular response to hypoxia that is broadly relevant and utilized across cell types. 123. van den Beucken T, Koch E, Chu K, et al. Hypoxia promotes stem cell & phenotypes and poor prognosis through epigenetic regulation of DICER. Nat Commun 2014; 5:5203. Together with references [122&] and [123&], these studies confirm the original finding from reference [120] that the hypoxic downregulation of Dicer represents an important adaptive strategy in the cellular response to hypoxia that is broadly relevant and utilized across cell types. 124. Qi HH, Ongusaha PP, Myllyharju J, et al. Prolyl 4-hydroxylation regulates Argonaute 2 stability. Nature 2008; 455:421–424. 125. Harris AL. Hypoxia – a key regulatory factor in tumour growth. Nat Rev Cancer 2002; 2:38–47. 126. 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Endothelial posttranscri ptional hypoxic responses Ho and Marsden 131. Ho JJ, Robb GB, Tai SC, et al. Active stabilization of human endothelial nitric & oxide synthase mRNA by hnRNP E1 protects against antisense RNA and microRNAs. Mol Cell Biol 2013; 33:2029–2046. This is the first study that demonstrates the dynamic, context-dependent interactions between RBPs, microRNAs and antisense RNA in endothelial cells. 132. McQuillan LP, Leung GK, Marsden PA, et al. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am J Physiol 1994; 267 (5 Pt 2):H1921–H1927. 133. Young LE, Moore AE, Sokol L, et al. The mRNA stability factor HuR inhibits microRNA-16 targeting of COX-2. Mol Cancer Res 2012; 10:167– 180. 134. Bhattacharyya SN, Habermacher R, Martine U, et al. Relief of microRNAmediated translational repression in human cells subjected to stress. Cell 2006; 125:1111–1124.
135. Kundu P, Fabian MR, Sonenberg N, et al. HuR protein attenuates miRNAmediated repression by promoting miRISC dissociation from the target RNA. Nucleic Acids Res 2012; 40:5088–5100. 136. Jafarifar F, Yao P, Eswarappa SM, Fox PL. Repression of VEGFA by CA-rich elementbinding microRNAs is modulated by hnRNP L. Embo J 2011; 30:1324–1334. 137. Meng Q, Rayala SK, Gururaj AE, et al. Signaling-dependent and coordinated regulation of transcription, splicing, and translation resides in a single coregulator, PCBP1. Proc Natl Acad Sci U S A 2007; 104:5866–5871. 138. Fish JE, Matouk CC, Yeboah E, et al. Hypoxia-inducible expression of a natural cis-antisense transcript inhibits endothelial nitric-oxide synthase. J Biol Chem 2007; 282:15652–15666. 139. Robb GB, Carson AR, Tai SC, et al. Posttranscriptional regulation of endothelial nitric-oxide synthase by an overlapping antisense mRNA transcript. J Biol Chem 2004; 279:37982–37996.
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Posttranscriptional adaptations of the vascular endothelium to hypoxia Jr Jyun David Ho a,b and Philip A. Marsden c
Purpose of review Remarkable new advances have been made in the field of posttranscriptional gene regulation over recent years. These include the revelation of noncoding RNAs, such as microRNAs, antisense transcripts and their interactions with RNA-binding proteins (RBPs) in the context of both health and disease settings, such as hypoxia. In particular, these discoveries bear much relevance to the field of vascular biology, which historically has focused upon transcriptional processes. Thus, the contributions of these posttranscriptional gene regulatory mechanisms to vascular and endothelial biology represent a newer concept that warrants discussion. Recent findings Recent studies have revealed two emerging themes that are critical to endothelial/vascular biology and function. First is the functional integration between the microRNA pathway and the cellular hypoxic response, which, in addition to specific microRNAs, involves key components of the microRNA biogenesis machinery. A key concept here is the regulation of a master transcriptional programme through posttranscriptional mechanisms. The second major theme involves the dynamic interactions between RBPs, microRNAs and antisense RNAs. The condition-dependent collaborations and competitions between these different classes of posttranscriptional regulators reveal a critical layer of control for gene expression. Summary Taken together, these findings bear significant diagnostic and therapeutic implications for vascular disease. Keywords antisense, endothelial nitric oxide synthase, hypoxia, microRNAs, RNA-binding proteins
INTRODUCTION The vascular endothelium constitutes the innermost lining of the entire circulatory system. Being more than a passive physical barrier between the circulating blood and surrounding tissues, endothelial cells play an active and essential role in physiology. Indeed, endothelial cells are crucial for vasculogenesis [1,2] and angiogenesis [3], and they contribute to the maintenance of vascular homeostasis [1,4]. Recent research has revealed that endothelial cells are actively involved in sensing environmental stresses, including changes in oxygen concentration and bioavailability, as well as in orchestrating coordinated responses to these physiological perturbations [5]. Indeed, the impairment of endothelial cell function has been implicated in numerous cardiovascular diseases (CVDs), including atherosclerosis, stroke and ischaemia-reperfusion injury [6]. Hypoxia (low oxygen bioavailability) is a critical environmental stress that profoundly affects aerobic organisms. It is associated with many diseases,
including CVDs [7–11]. To cope, mammalian cells, including endothelial cells, have evolved a comprehensive cellular response that strives to promote cellular survival during hypoxia [12–18]. In particular, the cell activates a critical transcriptional response pathway, which leads to the activation and/or inhibition of key genes involved in processes such as angiogenesis, glycolysis, cell growth, apoptosis and cell cycle regulation [19–21]; the coordinate regulation of these genes contributes synergistically to cellular survival [9,10,19,22]. At
a
Department of Biochemistry and Molecular Biology, bSylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida, USA and cKeenan Research Centre for Biomedical Science in the Li Ka Shing Knowledge Institute, St. Michael’s, Hospital, Department of Medicine, University of Toronto, Toronto, Ontario, Canada Correspondence to Philip A. Marsden, St. Michael’s Hospital, 30 Bond St., Toronto, ON M5B 1W8, Canada. Tel: +1 416 847 1736; e-mail: [email protected] Curr Opin Hematol 2015, 22:243–251 DOI:10.1097/MOH.0000000000000139
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Vascular biology
KEY POINTS Remarkable new advances have been made in the field of posttranscriptional gene regulation in recent years, especially in the vascular endothelium. A biologically functional integration exists between the microRNA pathway and the cellular hypoxic response, which, in addition to specific microRNAs, involves key components of the microRNA biogenesis machinery. The dynamic, condition-dependent interactions between RBPs, microRNAs and antisense RNAs play a crucial role in regulating endothelial biology. Changes in posttranscriptional gene regulation offer diagnostic and therapeutic opportunities and nuances for the treatment of vascular disease.
the heart of this transcriptional response is the hypoxia-inducible factor (HIF) [23–29], which is a hetero-dimeric transcription factor comprising a hypoxia-inducible a-subunit (i.e. HIF-1a) and a constitutively expressed nuclear b-subunit (HIF-1b) [27–30]. A second a-subunit isoform, HIF-2a, also exists that can also dimerize with HIF-1b [31]. Initially discovered in the endothelium as endothelial Per-Arnt-Sim domain protein one (EPAS1) [31,32], HIF-2a expression has been observed in a wide variety of cell types/tissues [33,34]. Functionally, HIF-1a and HIF-2a share a number of common targets, but each isoform also has unique targets [35–42]. In healthy cells, HIF-a protein is rapidly and continuously degraded in normoxia via a ubiquitin-proteasome pathway [43] that involves two critical components [9,10,44–47]. The first is a family of oxygen-dependent enzymes known as HIF prolyl4-hydoxylases, also termed prolyl hydroxylase domain containing proteins (PHDs) [48–50]. In the presence of oxygen, PHDs catalyse the hydroxylation of two key proline residues [51] in HIF-1a [52] and HIF-2a proteins [45,53]. These hydroxyproline residues are recognized by the von Hippel–Lindau protein (pVHL) [47,54–58], which, as part of an E3 ubiquition ligase complex, mediates the ubiquitylation and proteasomal degradation of HIF-a [59–67]. In hypoxia, oxygen-dependent HIF-a prolyl hydroxylation does not occur, and HIF-a is not polyubiquitylated by pVHL. Therefore, HIF-a escapes proteasomal degradation, accumulates and translocates into the nucleus to regulate target genes. Posttranscriptional regulation involves a complex network of factors that control transcript modifications, nuclear-cytoplasmic transport, subcellular localization, mRNA stability and translational efficiency [68–74]. These regulatory processes are 244
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largely mediated by cis/trans interactions between cis-elements in the 3’-untranslated region (UTR) of mature mRNAs and trans-factors that mostly fall into one of the three following categories: RNAbinding proteins (RBPs), natural antisense transcripts and noncoding RNAs such as microRNAs and short interfering RNAs [74–82]. This review will focus on the new and emerging concepts of posttranscriptional regulatory mechanisms in the hypoxia endothelium.
FUNCTIONAL INTEGRATION OF THE microRNA AND CELLULAR HYPOXIC RESPONSE PATHWAYS The microRNA pathway is a potent, endogenous regulatory pathway that involves translational inhibition and mRNA degradation by nearly 21nt singlestranded RNA molecules [83–87]. First discovered in 1993 [88,89], microRNAs are highly conserved across evolution, existing in organisms ranging from unicellular green alga [90] to humans [91]. A recent estimate indicates that at least 60% of all human protein-coding genes are subject to microRNA regulation [92], and the ubiquitous influence of microRNAs exerts a widespread impact on mRNA repression and evolution [93], generating thresholds in target gene expression [94] under both basal and stress conditions [95]. Key details regarding the canonical microRNA biogenesis have been elucidated [96,97] (Fig. 1). In particular, the key cytoplasmic enzyme Dicer catalyses the final step in microRNA maturation, producing functional microRNAs that can then be loaded (with the aid of Dicer) onto their downstream effector RNA-induced silencing complexes (RISCs). The biological significance of Dicer is underscored by the observation that Dicer knockout results in embryonic lethality in both mice and zebrafish [98–100]. The existence of a biological relationship between hypoxia and microRNAs was first established in the late 2000s by several independent laboratories [101–109]. Specifically, miR-210 was identified as the first proto-typical hypoxia/HIFinducible microRNA [109] across a variety of cell types, including endothelial and vascular smooth muscle cells [102,104,105]. Since its discovery, many targets of miR-210 have been identified, and they implicate miR-210 in the regulation of cell cycle, angiogenesis, mitochondrial metabolism, DNA damage response and cell survival [110–112]. Specifically in endothelial cells, miR-210 has been shown to target Ephrin-A3, which is a ligand for the ephrin receptor family of receptor tyrosine kinases [102,104]. Ephrins vital in angiogenesis and vascular remodelling [113], and cells expressing Volume 22 Number 3 May 2015
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Endothelial posttranscri ptional hypoxic responses Ho and Marsden
Normoxia
Chronic hypoxia
Transcription Processing
AAA Pri-miRNA
Pre-miRNA
Pre-miRNA
Accumulation of Pre-miRNAs
Decreased Dicer expression and activity
Processing by dicer AAA AAA AAA AAA AAA HIF-a mRNA and HIF-responsive mRNA (VEGF, VEGFR!, GLUT1, BNIP3L)
Mature miRNA
Mature miRNA
RISC
RISC AAA
AAA
Polyribosome AAA
• Translational inhibition and mRNA degradation by RISC • Minimal HIF-α translation
Polyribosome AAA
AAA
• Decreased translation inhibition and mRNA degradation by RISC
High-α protein
• Increased HIF-α translation
HIF-α protein
FIGURE 1. Functional importance of Dicer in the adaptive cellular response to hypoxia. Dicer and an important number of Dicer-dependent microRNAs are downregulated in chronic hypoxia. This is an important adaptive strategy that serves to maintain the cellular hypoxic response through hypoxia-inducible factor-a (HIF-a) and microRNA-dependent mechanisms. A significant number of downregulated microRNAs are predicted regulators of hypoxia-inducible genes, including the critical hypoxia-inducible factor-a (HIF-a) subunits, glucose transporter 1 (GLUT1), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L), vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2. In particular, the hypoxic downregulation of the Dicer-dependent microRNA miR-185 contributes to the hypoxic induction of HIF-2a. Reproduced with permission from [97]. RISC, RNA-induced silencing complexes.
miR-210-resistant Ephrin-A3 mRNAs exhibited impaired tubulogenesis and chemotaxis [102]. In addition, miR-210 also targets the iron–sulphur cluster assembly proteins ISCU1/2 in endothelial cells [105]. ISCU1/2 play crucial roles in mediating electron transport and mitochondrial oxidationreduction reactions [114], and their inhibition by miR-210 results in the impairment of mitochondrial respiration [105], which is believed to be a beneficial adaption that optimizes energy generation and prevents the production of excessive levels of toxic reactive oxygen species [115]. Taken together, these findings suggest that miR-210 exhibits both endothelial cell specific roles and generalized functions that are relevant across cell types. More recently, the potential diagnostic and prognostic value of miR-210 has also been explored. For example, miR-210 was recently reported as a
marker of hypoxia in glioblastoma and renal cell carcinoma [116,117]. Beyond miR-210, however, little else was known regarding the extent of integration between the cellular hypoxic response and the microRNA pathway, such as the effects of hypoxia on microRNA biogenesis, and the biological rationale for such regulation. To address this question, a number of recent studies have revealed that microRNA biogenesis and activity are fundamentally linked to the cellular hypoxic response. The revelation began with the observation that Dicer was downregulated in chronically hypoxic rat lungs in vivo [118], and then in chronically hypoxic vascular smooth muscle cells in vitro [119]. However, the biological significance of this phenomenon remained unknown, until our laboratory recently discovered that the downregulation of Dicer expression in chronic
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Vascular biology
hypoxia represents an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-a and microRNA-dependent mechanisms [120] (Fig. 1). Specifically, we found that the hypoxia-mediated decrease in Dicer expression and activity resulted in a global decrease in mature microRNA levels that was accompanied by a corresponding accumulation of precursor microRNAs (Fig. 1). Interestingly, despite this global impairment of microRNA biogenesis in chronic hypoxia, only a subset of microRNAs was significantly downregulated at the steady-state level. Notably, the majority of the downregulated microRNAs were predicted regulators of key hypoxia-inducible genes, including the glucose transporter 1 (GLUT1), BCL2/ adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L), vascular endothelial growth factor (VEGF), VEGF receptors VEGFR1 and VEGFR2, and especially the HIF-a subunits themselves. These findings support the model that microRNAs with elevated target mRNA levels (such as the increased expression of hypoxia-inducible genes in chronic hypoxia) undergo an increased rate of activitydependent microRNA decay [97]. Future studies will be required to elucidate the underlying molecular details of such regulation. Nonetheless, it was not surprising then that the full induction of HIF-1a and HIF-2a, as well as key hypoxia-responsive/HIF target mRNAs in chronic hypoxia, was observed to be dependent upon the hypoxic downregulation of Dicer function. Indeed, when Dicer was overexpressed in hypoxic cells, the induction of HIF-a isoforms and HIF-target genes, including those of particular relevance to endothelial and vascular biology (e.g. VEGF, VEGFR1, VEGFR2), was markedly blunted. In addition, we also identified miR185 as the first microRNA that targets HIF-2a, which is the predominant HIF-a isoform in endothelial cells [31,120]. Thus, the hypoxic downregulation of Dicer maintains the induction of key survival genes not just by attenuating microRNA-mediated suppression of the genes themselves, such as VEGF, but also through the attenuation of the microRNAdependent downregulation of their upstream master transcriptional activator, HIF-a [120]. The initial studies were performed in vascular cell types, including macro and microvascular endothelial cells and vascular smooth muscle cells. Remarkably, this functional interaction between the microRNA and hypoxia response pathways is a broadly relevant mechanism that has been observed across cell types. Indeed, since our initial discovery, this adaptive strategy has been observed in a number of different cell types, including human breast and ovarian cancer cells, whereby the hypoxic downregulation of Dicer was found to 246
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promote tumourigenesis and tumour progression [121 –123 ]. Moreover, the miR-185/ HIF-2a interaction has also been confirmed in colon and ovarian cancer models [122 ]. In addition, the biological significance of hypoxic Dicer downregulation is highlighted by the fact that cells invest significant resources to ensure the suppression of Dicer expression and activity in chronic hypoxia. For instance, we and others have shown that Dicer is downregulated via multiple mechanisms, including decreased mRNA stability, decreased translation, reduced protein stability and epigenetic mechanisms [120,123 ]. Interestingly, Dicer downregulation is von Hippel-Lindau, but not HIF dependent [120,121 ,123 ]. Future studies will be required to examine the biological implications of this observation, as well as whether Dicer is subject to hypoxic posttranslational modification, such as prolyl hydroxylation, as it has been shown for Argonaute 2 (AGO2) [119,124]. Finally, hypoxia significantly attenuates the efficiency of Dicer-dependent, but not Dicer-independent RNA interference (RNAi)based therapeutics [120]. This has clear implications in human disease, wherein tissue hypoxia is often encountered in CVDs, and it is also a critical phenotype of the tumour microenvironment [125,126]. In addition to Dicer, several other components of the microRNA biogenesis machinery are also downregulated by hypoxia in various cell types, including vascular cells such as endothelial cells and vascular smooth muscle cells. These components include Drosha, DGCR8, TRBP and AGO proteins [119,120,121 ,122 ,127 ]. In particular, one study reported that in endothelial cells, AGO1, which is a key component of the RISC complex, is selectively targeted by hypoxia-responsive microRNAs in order to suppress the translational inhibition of VEGF [127 ]. This adaptation likely works in synergy with the hypoxic downregulation of Dicer to maximize the induction of key hypoxiaresponsive genes. Indeed, it is likely that hypoxic cells, especially endothelial cells, adopt the adaptive strategy of maintaining their HIF-mediated hypoxic response by limiting microRNA production and activity through the downregulation of their effector and biogenesis machinery. &
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DYNAMIC INTERACTIONS BETWEEN RNABINDING PROTEINS, microRNAs AND ANTISENSE TRANSCRIPTS Posttranscriptional regulators are mostly trans-factors that fall into these three major categories: RBPs, microRNAs and antisense transcripts. Given that these factors are all involved in the control of mRNA stability and translation, it is not surprising that Volume 22 Number 3 May 2015
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Endothelial posttranscri ptional hypoxic responses Ho and Marsden
and hypoxia-associated vasoconstriction [129,130]. eNOS mRNAs are highly stable, with a half-life of over 24 h in human endothelial cells [131 ]. In addition, it is well established that decreased eNOS mRNA stability plays a major role in reducing eNOS expression and activity under a variety of pathophysiological conditions, including hypoxia [120,131 ,132]. However, until recently, the molecular basis and rationale for this remarkable stability, and the mechanisms that mediated the hypoxic decrease in eNOS mRNA stability have remained unknown. Recently, studies have revealed a novel role for the RBP hnRNP E1 in mediating eNOS mRNA stability. Specifically, it was found that hnRNP E1 stabilizes eNOS mRNAs by interacting with several
they often act collaboratively or competitively with each other. Indeed, the fate of an mRNA is largely dependent on the net effect of its interactions with these regulatory factors, as ‘naked’ mRNAs do not exist in cells. Rather, they interact dynamically with a myriad of trans-factors and exist virtually exclusively in the context of macromolecular complexes throughout their entire life cycle in the cell [71,128]. To illustrate the complex yet elegant interactions between these molecules [97], and their effect on endothelial cell biology, we will use the endothelial nitric oxide synthase (eNOS) gene as an example. eNOS is a critical endothelial cell enriched gene that synthesizes the vasodilator nitric oxide in the vascular endothelial cell [5]. Its expression is often disrupted in disease, including atherosclerosis
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FIGURE 2. Stabilization of endothelial nitric oxide synthase (eNOS) mRNA by hnRNP E1. (Top) In normal endothelial cells, hnRNP E1 interacts with conserved pyrimidine (C/CU)-rich elements in the eNOS 30 -untranslated region, stabilizing and protecting eNOS mRNAs from the inhibitory effects of microRNAs, such as miR-765. (Bottom) Under chronic hypoxic conditions, hnRNP E1 exhibits increased serine phosphorylation and nuclear sublocalization in an AKT-dependent manner. These events inhibit the interaction between hnRNP E1 and eNOS mRNAs, thus rendering eNOS sensitive to the inhibitory posttranscriptional effects of the hypoxia-inducible natural cis-antisense transcript, sONE. Reproduced with permission from [97]. 1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
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conserved pyrimidine (C/CU)-rich elements in the eNOS mRNA 30 -UTR [131 ] (Fig. 2, top). Functionally, these ribonucleoprotein (RNP) complexes mediate the remarkable stability of eNOS mRNAs in normal endothelial cells by protecting them against the inhibitory actions of endogenous microRNAs, such as miR-765, whose target site overlaps with one of the conserved pyrimidine-rich elements that hnRNP E1 interacts with (Fig. 2, top). This is reminiscent of the relationship reported previously whereby the RBP HuR protects COX-2 mRNAs from miR-16, whose target sites overlaps with the HuRbinding site [133]. In addition, we also found that hnRNP E1 protects eNOS mRNAs from exogenous short interfering RNAs that target eNOS mRNA at 30 UTR sites that are distinct from hnRNP E1 binding sites [131 ]. Again, this observation is conceptually consistent with previous reports that HuR protects CAT-1 mRNAs from distally binding microRNAs [134,135]. Our study demonstrates the complexity of posttranscriptional regulation involving RBPs, microRNAs and antisense transcripts in the field of endothelial biology. Interestingly, we also observed that the formation of the eNOS-stabilizing complex was disrupted in the setting of hypoxia [131 ] (Fig. 2, bottom). Indeed, further examination revealed that hnRNP E1 is unable to engage eNOS mRNAs in hypoxia because of increased AKT-dependent serine phosphorylation and nuclear sublocalization. Notably, the change in subcellular localization is similar to the reported interaction between hnRNP L and VEGF in hypoxic cells [136]. Furthermore, our findings are also consistent with the previous report that the phosphorylation of hnRNP E1 results in increased nuclear retention [137]. Consequently, these modifications render eNOS mRNAs susceptible to degradation and translational inhibition by posttranscriptional inhibitory forces under hypoxic conditions. Specifically, we have previously identified a hypoxia-inducible natural cis-antisense transcript to eNOS, termed sONE, which exhibits significant overlap with the eNOS mRNA in a tailto-tail orientation [138,139]. In the absence of the stabilizing complex, sONE downregulates eNOS mRNA posttranscriptionally, resulting in reduced eNOS expression and activity [138]. Further studies will be required to elucidate the underlying mechanisms by which this occurs. Nevertheless, we now appreciate that the remarkable basal stability of eNOS mRNA is mediated by hnRNP E1 containing stabilizing complexes, which serves to protect them from inhibitory microRNAs. In addition, that the hypoxia-mediated decrease in eNOS mRNA stability is primarily due to impaired stabilizing complex formation as a consequence of changes in hnRNP &
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E1 phosphorylation status and subcellular localization, which renders eNOS mRNAs susceptible to posttranscriptional downregulation by its antisense transcript. This work further underscores the importance of defining why a protein-coding mRNA is not subject to basal microRNA-mediated repression. Some mRNAs are protected from RNAi-mediated repression under basal conditions. Under the appropriate environmental conditions, especially hypoxia, an mRNA can be sensitized to miRNAmediated repression.
CONCLUSION These findings illustrate the complexity and biological significance of posttranscriptional regulation in the vascular endothelium, especially in the setting of hypoxia. The dynamic and conditiondependent interactions between these different regulators, and the net effects on their target mRNAs, represent a critical layer of gene expression control, which is a new and emerging concept in the field of vascular biology. The significant diagnostic and therapeutic implications of these regulatory mechanisms should be the focus of future studies.
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Acknowledgements None. Financial support and sponsorship J.J.D.H. holds a Canadian Institutes of Health Research (CIHR) Fellowship. P.A.M. is a Heart and Stroke Foundation of Canada Career Investigator and is supported by a grant from the Heart and Stroke Foundation of Canada (grant T-6777). Conflicts of interest There are no conflicts of interest.
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Endothelial posttranscri ptional hypoxic responses Ho and Marsden 131. Ho JJ, Robb GB, Tai SC, et al. Active stabilization of human endothelial nitric & oxide synthase mRNA by hnRNP E1 protects against antisense RNA and microRNAs. Mol Cell Biol 2013; 33:2029–2046. This is the first study that demonstrates the dynamic, context-dependent interactions between RBPs, microRNAs and antisense RNA in endothelial cells. 132. McQuillan LP, Leung GK, Marsden PA, et al. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am J Physiol 1994; 267 (5 Pt 2):H1921–H1927. 133. Young LE, Moore AE, Sokol L, et al. The mRNA stability factor HuR inhibits microRNA-16 targeting of COX-2. Mol Cancer Res 2012; 10:167– 180. 134. Bhattacharyya SN, Habermacher R, Martine U, et al. Relief of microRNAmediated translational repression in human cells subjected to stress. Cell 2006; 125:1111–1124.
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