REVIEW ARTICLE HIF3a: the little we know Linda Ravenna1, Luisa Salvatori1 and Matteo A. Russo2

1 CNR, Institute of Molecular Biology and Pathology, Rome, Italy 2 Laboratory of Molecular and Cellular Pathology, Consorzio MEBIC, San Raffaele University, Rome, Italy

Keywords Hypoxia-inducible factors (HIFs) are key regulators of the transcriptional HIF3a; hypoxia; hypoxia-inducible factors; response to hypoxic stress. Three inducible isoforms of HIF are present in pathology; splice variants; transcriptional mammals. HIF1a and HIF2a are the best characterized and structurally regulation similar isoforms, while HIF3a is the most distantly related and is less stud- a Correspondence ied. The HIF3 undergoes complex regulation and produces a large L. Ravenna, CNR, Institute of Molecular number of long and short mRNA splice variants, which are translated into Biology and Pathology, c/o Department of different polypeptides. These molecules primarily act as negative regulators Experimental Medicine, Sapienza University of HIF1a and HIF2a activity and transcriptional activators of target , of Rome, viale Regina Elena 324, 00161 according to the variant and the biological context. The present review Rome, Italy provides an overview of the available, fragmented and sometimes contra- Tel: +39 06 49972851 dictory information concerning the structure, expression and distinct roles E-mail: [email protected] of the HIF3a variants, in both hypoxic adaptation and in hypoxia-unre- (Received 7 August 2015, revised 9 October lated activities. The pathological consequences of HIF3a deregulation are 2015, accepted 20 October 2015) also illustrated. doi:10.1111/febs.13572

The hypoxia inducible factors

Gene structure molecules is based on studies concerning HIF1a and, Various animals, from mammals to insects, share a to a lesser degree, HIF2a, which are structurally simi- common pathway that associates pO2 sensing with lar, with high sequence identity [3,4]. HIFs are mem- changes in transcriptional regulation. Hypoxia-induced bers of the bHLH/PAS family, containing one is mediated by a number of N-terminal basic-helix–loop–helix (bHLH) domain and known as hypoxia-inducible factors (HIFs). These pro- two Per–ARNT–Sim (PAS) domains, which mediate teins include five major isoforms: oxygen-sensitive DNA binding and dimerization, respectively [5]. The HIF1a, HIF2a and HIF3a and oxygen-insensitive oxygen-dependent degradation domain (ODDD) is HIF1b and HIF1b2 [1,2] (Fig. 1). HIF3a is the most present only in HIFa isoforms and controls pro- structurally distant factor among HIFs and is the tein stability. The N-terminal transactivation domain object of the following sections of the present review. (NTAD), in HIFa isoforms, and the C-terminal trans- HIFs act as a–b heterodimers. Most of the current activation domain (CTAD), in both HIFa and HIFb knowledge on the structure and regulation of these isoforms, contribute to target gene activation [1].

Abbreviations

CoCl2, cobalt chloride; CTAD, C-terminal transactivation domain; DA2B, distal arthrogryposis type 2B; EDN1, endothelin 1; FIH1, factor inhibiting HIF1; GLUT1, glucose transporter type 1; HIF, hypoxia-inducible factor; HRE, hypoxia response element; IPAS, inhibitory PAS domain protein; NEPAS, neonatal embryonic PAS protein; NF-κB, nuclear factor κ light chain enhancer of activated B cells; NTAD, N-terminal transactivation domain; ODDD, oxygen-dependent degradation domain; PHD, prolyl hydroxylase domain protein; pVHL, von Hippel–Lindau protein; VEGF, vascular endothelial growth factor.

FEBS Journal 283 (2016) 993–1003 ª 2015 FEBS 993 HIF3a L. Ravenna et al.

DNA Protein-protein interaction

Binding Dimerization O2 stability Transactivation

P P N

HIF1α bHLH PAS A PAS B ODDD NTAD CTAD

P P N HIF2α bHLH PAS A PAS B ODDD NTAD CTAD

P

HIF3α bHLH PAS A PAS B ODDD NTAD LZIP

HIF1β bHLH PAS A PAS B CTAD

HIF1β2 bHLH PAS A PAS B CTAD

Fig. 1. Functional domains of HIF isoforms. The HIF system consists of three oxygen-sensitive a subunits, HIF1a, HIF2a and HIF3a, and two oxygen-insensitive b subunits, HIF1b and HIF1b2. Multiple HIF1a and HIF3a variants are generated from alternative splicing or alternative promoter utilization. The depicted HIF1a and HIF3a isoforms represent the long variants of the molecules. The structural motives of each HIF isoform are shown in different colors. All isoforms possess a basic helix–loop–helix (bHLH) domain that binds DNA and a Per– ARNT–Sim (PASA and PASB) domain for heterodimerization. HIFa isoforms contain an oxygen-dependent degradation domain (ODDD) that mediates oxygen stability through the hydroxylation of 2 (in HIF1a and HIF2a) or 1 (in HIF3a) prolyl residues, and an N-terminal transactivation domain (NTAD) within the ODDD. The C-terminal transactivation domain (CTAD), which is not found in HIF3a, contains an asparaginyl residue implicated in transcriptional activation. HIF3a possesses a peculiar C-terminal (LZIP) domain that is involved in protein–protein interaction, rather than the CTAD. HIF1b and HIF1b2 both have a CTAD without an asparaginyl residue, and lack ODDD/NTAD and LZIP domains. P: prolyl residue. N: asparaginyl residue.

Oxygen-dependent transactivation activity Hundreds of genes involved in different biological Both HIFa and HIFb subunits are constitutively processes, including metabolic reprogramming, cell sig- expressed. In the presence of a physiological level of naling in response to tissue and cell damage, erythro- oxygen (normoxia), the two prolyl residues in the poiesis, angiogenesis, cell-cycle regulation, stemness ODDD of the HIF1a and HIF2a subunits are hydrox- and tumor progression, are regulated through HIF1a ylated by prolyl hydroxylase domain proteins (PHDs) and/or HIF2a and contribute to the cell adaptation to [6] (Fig. 2). Prolyl hydroxylation promotes interaction hypoxia [8–16]. In this context, HIF1a and HIF2a with the von Hippel–Lindau protein (pVHL), which play overlapping but not redundant roles through the directs HIF1a and HIF2a poly-ubiquitylation and sub- tissue-specific and temporal patterns of induction of sequent proteolytic inactivation by proteasomal degra- each isoform and different transcriptional targets [3]. dation [7]. The transactivation activity of HIF1a and Indeed, the transcription of genes encoding enzymes of HIF2a is further blocked by factor inhibiting HIF1 the glycolytic pathway, such as phosphofructokinase, (FIH1)-dependent asparaginyl hydroxylation in the C phosphoglycerate kinase 1 and lactate dehydrogenase terminus of these molecules [6]. A, appears to be driven primarily by HIF1a in multi- When the oxygen level declines below the physiolog- ple cell types [17], while the genes encoding stem cell ical level (hypoxia), hydroxylation reactions are inhib- makers, such as NANOG, octamer-binding transcrip- ited. As a result, HIF1a and HIF2a are stabilized, tion factor 4 (OCT4) and sex determining region Y translocate to the nucleus, dimerize with the HIFb (), seem to specifically depend on HIF2a activity subunits, recruit the coactivators P300/CBP and bind [14,18]. A variety of broadly expressed hypoxia-induci- to the hypoxia response element (HRE) core sequence ble genes belonging to different pathways, such as car- [A/G]CGTG in the promoter of target genes, modulat- bonic anhydrase XII, glucose transporter type 1 ing their expression (Fig. 2) [4,6]. (GLUT1), vascular endothelial growth factor (VEGF)

994 FEBS Journal 283 (2016) 993–1003 ª 2015 FEBS L. Ravenna et al. HIF3a

Normoxia Hypoxia HIF3a gene structure and splice variants N P HIFα P Identification of the gene HIF3a was initially identified as a member of the PHDs bHLH-PAS protein family in mice by Gu’s group [22]. FIH1 They cloned a cDNA encoding a 662-amino acid polypeptide with amino acid sequence identity to

N-OH HIF1a and HIF2a (57% and 53%, respectively) in the N-terminal bHLH-PAS domain. The C terminus of OH-P HIFα P-OH CBP the molecule presented a 36-amino acid sequence with HIFβ a N P300 61% identity to the HIF1 ODDD. The unique a P HIFα P HIF3 structural features included the presence of a pVHL single hydroxylatable proline in the ODDD and of a leucin zipper domain (LZIP), which is involved in pro- [A/G]CGTG tein–protein interactions, in place of the CTAD, in the

N-OH C terminus of the molecule (Fig. 1). Similar to HIF1a and HIF2a, HIF3a is able to dimerize with HIFb sub- OH-P HIFα P-OH units, and the resulting heterodimer recognizes the HRE on the promoter of target genes (Fig. 2). More- over, the HIF3a–HIFb interaction occurs in vivo, where the activity of HIF3a is upregulated in response

to low oxygen tension and cobalt chloride (CoCl2), a hypoxia-mimicking agent [22]. Proteasomal degradation Target genes transactivation Human HIF3a cDNA has been subsequently cloned [23], and the 667 amino acid sequence of this molecule Fig. 2. Schematic diagram of the HIF pathway. Under normoxic was 81.9% homologous and structurally similar to conditions, the prolyl hydroxylase domain proteins (PHDs) a hydroxylate HIF1a, HIF2a and HIF3a at the prolyl residues (P-OH) that of mouse HIF3 . a in the ODDD, whereas factor inhibiting HIF1 (FIH1) hydroxylates The phylogenetic tree of the HIF3 proteins from a HIF1a and HIF2a at the asparaginyl residue (N-OH) in the CTAD. select list of animal species was generated, showing sig- The von Hippel–Lindau protein (pVHL) binds to HIFa subunits nificant evolutionary conservation among the amino containing hydroxylated prolines, mediating polyubiquitination and acid sequences in typical HIF domains [24–26]. subsequent proteasomal degradation. Under hypoxic conditions, The genomic organization of the HIF3a locus a PHDs and FIH1 activities are inhibited and HIF subunits involves a variable number of exons, depending on the accumulate in the cytoplasm, translocate to the nucleus, heterodimerize with HIFb subunits and recruit the coactivator of species. In particular, the mouse locus in target gene transcription p300/CBP. The HIFa/b transcription 7 consists of 18 exons [27]. Two exons (1 and 1a) con- complexes bind to the hypoxia-response elements [A/G]CGTG in tain alternative start sites. Similarly, the human locus in the target genes’ promoters and control the expression of these consists of 19 exons, and three alterna- genes. tive start sites are located in exons 1a, 1b and 1c [28]. As reported for HIF1a [1], multiple splice variants have been characterized for HIF3a. These variants A and interleukin-6, are common transcriptional tar- were derived from different transcription start sites gets of both HIF1a and HIF2a [17,19]. and through alternative splicing, resulting in long and As new insights are gained, the complexity of the short HIF3a polypeptides, with peculiar biological HIF1a/HIF2a pathway becomes increasingly evident. properties (Figs 3 and 4). However, previous studies, Six alternative HIF1a mRNA isoforms have been frequently do not specify the particular molecule identified, resulting in shorter polypeptides with altered examined, and the authors refer to long HIF3a vari- biological properties, which have not been well studied ants simply as HIF3a. Thus, the function of each vari- to date [1]. Furthermore, in addition to oxygen, HIFa ant in the complex regulation of hypoxia-inducible subunits are intricately responsive to numerous factors genes by the HIF family remains under debate. To including reactive oxygen species, sirtuins and metabo- date, the mouse and human variants have been the lites [20,21]. most structurally and functionally studied.

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HIF3α bHLH PAS A PAS B ODDD NTAD LZIP Human variants The human HIF3a locus undergoes even more NEPAS extensive alternative splicing compared with that of mice, generating a large number of variants [28,31] IPAS (Fig. 4). All the variants utilize three alternative transcription start sites in exons 1a, 1b and 1c as well Fig. 3. Multiple splice variants of the mouse HIF3a gene. as two and three alternative forms of exons 13 and 14. Comparison of the protein domain organization of mouse HIF3a Database analyses predicted the generation of 10 and the splicing variants NEPAS and IPAS. The structural motifs variants: HIF3a1, HIF3a2, HIF3a3, HIF3a4, HIF3a5 bHLH (DNA binding), PASA and PASB (dimerization), ODDD/NTAD and HIF3a6 [24], and HIF3a7, HIF3a8, HIF3a9 and – (oxygen sensing/transactivation activity), and LZIP (protein protein HIF3a10 [28]. While full length cDNA corresponding interaction) are indicated for each variant in different colors. The replacement of the first HIF3a exon, 1b (orange), with the exon 1a to the majority of the predicted isoforms was obtained, a a (yellow) in both NEPAS and IPAS is also shown. cDNA corresponding to HIF3 3 and HIF3 5 could not be cloned and the HIF3a6 DNA sequence did not correspond to the predicted domain structure [31]. Mouse variants HIF3a10 harbors an in-frame stop codon, which leads to the production of a seven-amino-acid peptide. All Neonatal embryonic PAS (NEPAS) and inhibitory variants, except HIF3a10, retain the PASA and PASB PAS domain (IPAS) proteins have been identified in dimerization domains, while the DNA-binding domain mice (Fig. 3). NEPAS is a polypeptide consisting of bHLH is lost in HIF3a7 and HIF3a8, and the LZIP a 664 amino acids, in which the first HIF3 exon, 1b, is domain is lost in HIF3a2, HIF3a4 and HIF3a7. The a replaced with the exon 1a [29]. IPAS is a short HIF3 ODDD domain is missing only in HIF3a4. Hence, all variant consisting of 307 amino acids that lacks part of the molecules, with the exception of HIF3a4 and of the PASB and the entire NTAD, ODDD and LZIP HIF3a10, are potential targets for the pVHL complex. domains. Similar to NEPAS, IPAS is generated from Notably, HIF3a4 has a domain structure similar to exon 1a and undergoes complex splicing involving IPAS, but unlike IPAS, retains the full PASB dimer- exons 4a and 16, which are not expressed in the other ization domain. variants [27,30]. Regulation of the expression of HIF3a splice variants HIF3α1 bHLH PAS A PAS B ODDD NTAD LZIP Normoxic expression HIF3α2 Studies in multiple tissues, cancer cell lines and whole organisms (humans, rats, mice and fish) have suggested HIF3α4 that the mRNA expression of HIF3a variants is tissue and cell specific, with large differences in expression – HIF3α7 patterns and levels [31 37]. Moreover, variants and levels of expression may change depending on the stage of development of the organism [29]. In humans, HIF3α8 HIF3a transcripts are present in a large number of tis- sues (heart, brain, placenta, lung, liver, kidney pan- HIF3α9 creas, skeletal muscle and cartilage) in various patterns [31,38] and their levels are generally higher in fetal tis- HIF3α10 sues than in adult tissues [31]. HIF3a4 and HIF3a7 are the variants that are preferentially expressed in Fig. 4. Multiple splice variants of the human HIF3a gene. most adult tissues, while HIF3a4 is the primary HIF3a Comparison of the protein domain organization of the cloned transcript present in fetal tissues [31]. To date, HIF3a human HIF3a1, HIF3a2, HIF3a4, HIF3a7, HIF3a8, HIF3a9 and HIF3a10 variants. The structural motifs bHLH (DNA binding), PASA variants have been detected in only a few human can- and PASB (dimerization), ODDD/NTAD (oxygen sensing/ cer cell lines, such as Ad4 and Caki-1 (renal), SKOV-3 transactivation) and LZIP (protein–protein interaction) are indicated (ovarian), HeLa (uterine), DU145 (prostate) [31,35], for each variant in different colors. MSTO and MTT (mesothelioma) [36], and A549

996 FEBS Journal 283 (2016) 993–1003 ª 2015 FEBS L. Ravenna et al. HIF3a

(lung) [32]. In rats, HIF-3a levels are higher in the cor- upstream of exon 1b of the HIF3a gene, and the tran- tex, lungs, hippocampus and kidneys [39]. In mice, scription of HIF3a2 and HIF3a4 was induced through HIF3a mRNA is readily detected in the heart, pla- HIF1a but not through HIF2a in Caki-1 carcinoma centa, lungs and muscle [22]. However, the specific renal cells [33]. Moreover, the silencing of HIF1a sig- mouse variant IPAS was predominantly observed in nificantly impairs both hypoxic transcription from all Purkinje cells, cerebellum and corneal epithelium as three alternative transcription initiation sites in Hep3B well as heart and lungs [27], while NEPAS is predomi- hepatoma cells [31] and HIF3a nuclear protein translo- nantly expressed during embryonic and neonatal stages cation in DU145 prostate tumor and MSTO-211H in the lungs and heart [29]. In zebrafish embryos and mesothelioma cell models [35,36]. The silencing of both larvae, HIF3a mRNA is detected in all tissues, with HIF1a and HIF2a is needed to decrease HIF3a expres- higher levels observed in the brain and other anterior sion in HUVEC under hypoxia [40]. HIF1a also plays tissues. In the adult stage, the basal levels of HIF3a an essential role in the transactivation of the mouse mRNA vary in different tissues. The highest expres- IPAS gene through the binding to an HRE-like sion was observed in the ovaries, followed by the motif located in the IPAS promoter [42]. In contrast, kidneys, gills, brain and heart [37]. HIF3a is not regulated through HIF1a in zebrafish embryos [37]. Hypoxic expression Role of NF-jB and microRNAs HIF3a activity is extensively regulated. Hypoxia and hypoxia mimetic chemicals are the primary stimuli for Nuclear factor j light chain enhancer of activated B mRNA transcription, protein synthesis and nuclear cells (NF-jB), the master regulator of the inflamma- translocation of most HIF3a variants [31,32,35–37,39]. tory response, and a number of microRNAs are addi- Treatments with actinomycin D and cycloheximide tional cell-specific factors that contribute to HIF3a prior to hypoxia indicate that increased protein expres- expression modulation. Data obtained from human sion is primarily caused by mRNA synthesis rather vascular, prostate cancer and mesothelioma cell lines than mRNA stabilization and requires active protein suggest that NF-jB inhibition does not interfere with synthesis [40]. Most HIF3a variants contain a con- the normoxic mRNA and nuclear protein levels of served ODDD with a single hydroxylatable proline. HIF3a but rather strongly reduces the hypoxic induc- Therefore, similar to HIF1a and HIF2a, these mole- tion of these molecules [35,36,40]. However, in PC12 cules are potential targets for pVHL and subsequent adrenal cells cultured in medium containing low mag- proteasomal degradation in a PHD-dependent manner, nesium and treated with CoCl2 or exposed to intermit- [28,37,41]. However, in numerous models, HIF3a long tent hypoxia, NF-jB activation upregulated IPAS isoforms are present in the cytoplasm and nucleus expression [44]. In the same cell model, but not in under normoxia, indicating that the hydroxylation of a human hepatoma Hep3B cells, tumor necrosis factor a single ODDD is not sufficient for complete proteaso- activated the expression of IPAS in an NF-jB-depen- mal degradation [31,35,36]. In oxygen-deprived envi- dent manner [45]. ronments, the levels of long HIF3a variants increase in Recently, 11 potential binding sites for miR-485-5p the nucleus, following mRNA induction, and show dif- and a single putative binding site for miR-210-3p, two ferences in kinetics, according to cell type, animal hypoxia-induced microRNAs, have been identified in model and experimental conditions. This expression the 30-UTR of the HIF3a gene in human soft tissue slowly declines to a basal level [32,35,36], consistent sarcoma cell lines. The overexpression of miR-485-5p with the long half-lives of the molecules [32]. The tran- and miR-210-3p mimics reduced the induction of scription of the mouse short variant IPAS also HIF3a under hypoxic conditions, highlighting an addi- increases in response to hypoxia [42], whereas the tional level of control in HIF3a expression [46]. expression of the human ortholog HIF3a4 is both upregulated [33] and downregulated [43]. HIF3a transcriptional activity

Role of HIF1a and/or HIF2a in HIF3a Inhibition of HIF1a and/or HIF2a transcriptional regulation The function of HIF3a in hypoxia adaptation and in HIF1a and/or HIF2a are strongly involved in HIF3a specific gene expression regulation is under debate and transcriptional regulation in human cells. In fact, a depends on both the variant and the biological model functional HRE was identified in the 50 flanking region studied. The absence of the CTAD, and the NTAD in

FEBS Journal 283 (2016) 993–1003 ª 2015 FEBS 997 HIF3a L. Ravenna et al. some variants, impairs the transactivation potential of regulated overlapping, yet distinct, sets of genes HIF3a [23,41]. Indeed, collectively, overexpression in vivo. HIF3a, but not HIF1a, increased the expres- experiments using in vitro systems primarily showed sion of the genes involved in nitrogen and methane that long HIF3a variants, the short mouse IPAS and metabolism and in the Jak-STAT and NOD-like recep- the human ortholog HIF3a4 counteract hypoxic tor signaling pathways. The expression of the genes HIF1a and HIF2a transcriptional activity but do not associated with gluconeogenesis, arachidonic acid induce a specific transcriptional response [23,41,43]. metabolism, glycosphingolipid biosynthesis, the zinc Coimmunoprecipitation studies highlighted that mouse response, inositol phosphate metabolism, peroxisomes, and human HIF3a variants interact with HIF1b, lysosomes, and vascular endothelial growth factor, HIF1a and HIF2a. When the amount of HIF1b is insulin and mitogen-activated protein kinase signaling limiting, several human long HIF3a variants associate was found enriched through HIF1a. Both HIF3a and with HIF1a and/or HIF2a, thus preventing nuclear HIF1a contributed to the expression of the genes translocation of these molecules and thereby acting as involved in glucose and amino acid metabolism, apop- dominant negative transcriptional regulators [41]. tosis, proteolysis, and peroxisome proliferator-acti- NEPAS represses HIF1a and HIF2a activity during vated signaling. Therefore, full-length HIF3a embryonic and neonatal development through compe- in zebrafish acts as an oxygen-dependent transcription tition for HIF1b recruitment [29]. In contrast, IPAS activator that is capable of upregulating unique target directly interacts with HIF1a, and HIF3a4 interacts gene expression. with HIF1a, HIF2a and HIF1b to form inactive com- plexes in the cytoplasm [30,41,43]. Although these Induction of hypoxia-unrelated genes mechanisms do not consistently overlap, taken together these data indicate that the prevailing action In transgenic mice carrying the HIF3a transgene of HIF3a variants in hypoxia adaptation is inhibitory. specifically expressed in lung epithelium, HIF3a can As described in the previous section, the transcription directly induce the transcription of SOX2, forkhead of most HIF3a variants is induced by HIF1a and/or box P2 (FOXP2) and b, which HIF2a in oxygen deprivation. Accordingly, the upreg- are involved in epithelial cell differentiation [48]. Inter- ulation of HIF3a constitutes a negative feedback regu- estingly, in this experimental model, HIF3a affected latory loop of HIF1a/HIF2a-dependent gene genes not typically involved in hypoxic response, pro- regulation that fine-tunes the hypoxic response. viding evidence for a novel function for this molecule. This last finding is supported by a study showing that HIF3a expression upregulates several adipocyte-related Induction of hypoxia-related genes genes in mouse 3T3-L1 cells, thereby enhancing A number of recent reports challenge the consensus adipogenic differentiation [49]. that HIF3a functions only as a negative regulator of HIF1a and HIF2a. When HIF1b is not limiting, cer- Emerging roles tain hypoxia-stimulated long HIF3a variants upregu- late selected HIF target genes, such as erythropoietin, A novel role has recently been suggested for IPAS. GLUT1 and ANGPTL4 (angiopoietin-like 4) [41]. A This mouse isoform also possesses nucleocytoplasmic recent study on zebrafish embryos showed that full- shuttling properties and acts as a pro-apoptotic factor length HIF3a binds to the promoter sequences of in mitochondria through direct binding to the pro- REDD1 (regulated in development and DNA damage survival protein Bcl-xL and its related proteins [50,51]. responses 1), MLP3C (microtubule-associated proteins 1A/1B light chain 3C) and SQRDL (sulfide quinone HIF3a in pathology reductase-like) genes and induces expression of these genes under hypoxic conditions [47]. Moreover, NEPAS/HIF3a in lung remodeling SQRDL and REDD1 were also induced in hypoxic human embryonic kidney HEK293 and U2OS The generation of transgenic mice with a targeted dis- osteosarcoma cells following HIF3a9 stabilization, ruption of the NEPAS/HIF3a locus has facilitated an suggesting that the transcriptional activity of HIF3a is examination of the role of these two variants in the evolutionarily conserved. In the same study, the tran- embryo cardiorespiratory system [29]. Knockdown scriptional program induced by HIF3a and HIF1a in mice were viable but displayed impaired lung remodel- zebrafish embryos was characterized and compared ing. Specifically, alveolar formation was more rapid in using microarray analyses. HIF3a and HIF1a the transgenic mice than in the lungs of wild-type

998 FEBS Journal 283 (2016) 993–1003 ª 2015 FEBS L. Ravenna et al. HIF3a mice, and incomplete alveolar spaces were observed at Nevertheless, the proliferative and angiogenic abilities the late embryonic stage. This abnormality resulted in of the cells were reduced [52]. The mechanism underly- an enlarged right ventricle in the hearts of adult ani- ing this apparent contradiction was ascribed to the mals and was associated with the enhanced expression upregulation of the vascular-endothelial protein cad- of endothelin-1 (EDN1) in both the lungs and heart, herin, an inhibitor of FLK1/phosphoinositide 3- reflecting the absence of the inhibitory action of kinase/Akt signaling. Collectively, these results have NEPAS on the binding of HIF1a and HIF2a to the revealed a role for HIF3a in angiogenic gene regula- HRE sequence of the EDN1 gene. The critical role of tion in pulmonary endothelial cells. HIF3a in lung development was supported also through additional evidence obtained in transgenic Tumor suppressive activity of HIF3a variants mice that expressed HIF3a in airway epithelial cells during gestation [48]. The animals were born alive and In contrast to HIF1a and HIF2a, a specific role for appeared normal, but the lungs showed clear abnor- HIF3a in carcinogenesis has not yet been established. malities, including branching morphogenesis defects However, the control of the transactivation activity of and a decreased number of alveoli. Moreover, it has HIF1a and HIF2a exerted by several variants suggests recently been reported that in endothelial cells that HIF3a could be involved in cancer biology. A obtained from HIF3a knockout mice, the expression number of studies on HIF3a4 have demonstrated of representative angiogenic factors, including VEGF tumor suppressive activity in several models, such as receptor-2 (FLK1), angiopoietin-2 and TIE2 (tyrosine meningioma and kidney [53,54]. In malignant menin- kinase with immunoglobulin-like and EGF-like gioma, the HIF3a4 transcription was silenced through domains 2), was markedly increased by HIF3a disrup- promoter DNA methylation, while HIF3a4 overex- tion under both normoxic and hypoxic conditions. pression reduced neovascularization and glucose meta-

Table 1. Interactions and functions of HIF3a splice variants. The table summarizes the currently available information in the literature on the transcriptional inducers and repressors, the physically interacting molecules and the activities of HIF3a splice variants. The long variants, with the exception of NEPAS, are grouped together. References are also indicated.

HIF3a variant Induced by Repressed by Interacts with Activities

Long variants HIF1a/2a MicroRNA [45] HIF1/2a, HIF1b Inhibition of HIF1/2a mediated gene expression through the [33,39] [40] formation of an abortive transcriptional complex, when HIF1b is limiting [23, 40] Induction of hypoxia-related and unrelated specific gene expression [40, 46–48] Differentiation of pulmonary epithelium and in alveologenesis [47] Angiogenic gene regulation in pulmonary endothelial cells [51] Role in the abnormal bone development in the genetic disease arthrogryposis type 2B [58] Potential causal relation between DNA methylation of HIF3a promoter and adiposity [54–57] NEPAS Hypoxia [29] HIF1b [29] Inhibition of HIF1/2a activity through competition for HIF1b (mouse) recruitment [29] Development of the embryonic and neonatal cardiorespiratory system [29] IPAS Hypoxia [41] HIF1a [41] Dominant-negative inhibition of HIF1a through the formation (mouse) HIF1a [41] of an abortive transcriptional complex [30]. NF-jB [44] Proapoptotic action through binding to Bcl-xL in mitochondria [50] Nucleocytoplasmatic shuttling [49] HIF3a4 Hypoxia [33] Hypoxia [42] HIF1/2a, HIF1b Dominant-negative inhibition of HIF1a through the formation (human) [42] of an abortive transcriptional complex [41] Antiangiogenic action in hypervascular malignant meningioma [52] Suppression of VHL-null CC-RCC [53]

FEBS Journal 283 (2016) 993–1003 ª 2015 FEBS 999 HIF3a L. Ravenna et al. bolism as well as decreasing the proliferation and inva- suggested that the abnormal bone development associ- sion of the cells. Moreover, HIF3a4 is dramatically ated with the TNNI2 mutation might partially depend downregulated in the majority of clear-cell renal cell on deregulated HIF3a and VEGF signaling [60]. carcinoma (CC-RCC) patient samples [43]. Loss-of- function mutations in the VHL gene are the most Conclusions prevalent mutations associated with the development of CC-RCC. These mutations lead to the inappropri- Studies on HIF3a have confirmed the growing com- ate accumulation of HIF2a and increase HIF2a-driven plexity in the modulation of hypoxia-inducible genes endogenous gene expression, which cannot be through the HIF family. The observation that HIF3a counteracted by low level of HIF3a4, a critical event is evolutionarily conserved suggests that the function in the development of CC-RCC. Therefore, HIF3a4 of this gene is important in cell homeostasis. However, also acts as a naturally occurring tumor suppressor in the multiple splice variants of HIF3a, indicate the renal tumorigenesis. Moreover, recent immunohisto- potential for a plurality of cell- and tissue-specific chemical analyses have shown an increased expression actions. Collectively, the currently available data show of long HIF3a variants in malignant mesothelioma that most variants act as HIF1a and HIF2a antago- human tissues compared with non-tumor tissues, and a nists and have identified a number of HIF3a target borderline correlation between HIF3a expression and genes, providing evidence that HIF3a also functions as patient survival. The relevance of HIF3a in mesothe- an oxygen-dependent transcriptional activator. There- lioma progression remains unknown, but these obser- fore, HIF3a plays an important regulatory role in the vations suggest that HIF3a could become a new physiological and pathological processes associated potential prognostic marker for this disease [36]. with oxygen deprivation. However, increasing studies suggest additional roles, unrelated to responses to hypoxic stress, for several HIF3a variants. The known HIF3a promoter methylation interactions and functions of the HIF3a variants stud- Surprisingly, a potential causal relationship between ied thus far are summarized in Table 1. Additional the DNA methylation of the HIF3a promoter and research is warranted to explore the importance of adiposity was recently suggested. Epigenome-wide each variant in the control of the hypoxic response association studies have shown that DNA methyla- and other physiological processes to obtain further tion at the HIF3a locus of whole blood cells, adipose insight into the pathological consequences of HIF3a tissue and skin is associated with body mass index deregulation. [55–57]. In addition, the methylation patterns in DNA extracted from umbilical cords showed that Author contributions higher methylation levels at three CpGs in the HIF3a first were associated with increased infant L.R. conceived and wrote the review; L.S. made the weight and adiposity [58]. HIF3a gene hypermethyla- figures and participated in writing the article and tion was also observed in prostate tumor versus adja- revising it critically. M.A.R. gave final approval of the cent histologically benign tissue [59]. The potential version to be submitted. clinical utility of these observations in relation to the diagnosis and cure of these diseases needs further References investigation. 1 Prabhakar NR & Semenza GL (2012) Adaptive and maladaptive cardiorespiratory responses to continuous HIF3a in genetic diseases and intermittent hypoxia mediated by hypoxia-inducible factors 1 and 2. Physiol Rev 92, 967–1003. a HIF3 deregulation also plays a role in distal arthrogry- 2 Hankinson O (2008) Why does ARNT2 behave posis type 2B (DA2B), a human genetic disease charac- differently from ARNT? Toxicol Sci 103,1–3. terized by hand and/or feet contracture and shortness 3 Semenza GL (2012) Hypoxia-inducible factors in of stature. Mutations in the TNNI2 gene, required for physiology and medicine. Cell 148, 399–408. fast twitch muscle fiber contraction, partially reflects 4 Ratcliffe PJ (2007) HIF-1 and HIF-2: working familial incidences of DA2B. Knock-in mice carrying a alone or together in hypoxia?. J Clin Invest 117, deletion in the TNNI2 gene present a typical phenotype 862–865. of the disease. In these animals, the mutant TNNI2 pro- 5 Bersten DC, Sullivan AE, Peet DJ & Whitelaw ML tein increased HIF3a and IPAS transactivation and (2013) bHLH-PAS proteins in cancer. Nat Rev Cancer reduced VEGF expression. Therefore, it has been 13, 827–841.

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