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Research Article |
Department of Obstetrics and Gynecology,3
Mount Sinai Hospital, Department of Pediatrics,4
The Hospital for Sick Children, Department of Physiology,5 University of Toronto, Toronto, Ontario, Canada M5G 1X5
ABSTRACT
The human placenta is a unique organ in terms of oxygenation as it undergoes a transition from a low to a more oxygenated environment. This physiological switch in oxygen tension is a prerequisite for proper placental development and involves the hypoxia inducible factor (HIF). HIF is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau tumor suppressor protein (VHL) leads to rapid degradation of the HIF1A subunit. The degradation requires formation of a multiprotein complex (VHLCBC) and hydroxylation of HIF1A proline residues via members of the egg-laying-defective nine (EGLN) family. Herein, we have investigated the regulatory mechanisms of HIF1A expression during human placental development. Expression of HIF1A and VHL was high at 7–9 wk of gestation, when oxygen tension is low, and decreased when placental oxygen tension increases (10–12 wk of gestation). During early placentation, HIF1A localized in cytotrophoblasts, while VHL was present in syncytiotrophoblasts. At 10–12 wk, VHL appeared in cytotrophoblast cells, which coincided with the disappearance of HIF1A. At the same time the association of VHL and Cullin 2 as well as ubiquitination of HIF1A was maximal. EGLN1, EGLN2, and EGLN3 were also temporally expressed in an oxygen-dependent fashion, with greatest mRNA expression at 10–12 wk of gestation. Inhibition of EGLN activity increased HIF1A stability in villous explants and stimulated transforming growth factor beta 3 (TGFB3) expression consistent with promoter analyses showing that HIF1A transactivates TGFB3. These data demonstrate that during placental development, HIF1A is regulated by temporal and spatial changes in expression and association of molecules forming the multi-protein VHLCBC complex as well as prolyl hydroxylase activities.
gene regulation, placenta, trophoblast
INTRODUCTION
Early placental development takes place in a low-oxygen environment, which is vital, as the early conceptus has little protection against oxygen-generated free radicals. In vivo oxygen electrode studies reveal that prior to 10 wk of gestation, O2 tension is 
20 mm Hg (equivalent to 2%–3% O2), while after the intervillous space opens to maternal blood (10–12 wk), O2 levels increase to 55 mm Hg (8%–10% O2) [1, 2]. Increasing evidence indicates that O2 is a key regulator of trophoblast differentiation. In vitro studies show that low pO2 levels, comparable to that found in the early pregnancy uterine environment, support trophoblast proliferation, while increasing O2 levels are associated with the acquisition of an invasive phenotype by trophoblast cells [3–5]. In most mammalian systems, the cellular responses to chronic and acute hypoxia are mediated through a highly conserved hypoxia-inducible factor (HIF) family of transcriptional regulators [6]. The HIF transcriptional complex is a heterodimer composed of one of the three alpha subunits (HIF1A, EPAS1, or HIF3A) and a beta subunit (ARNT). The regulation of HIF by oxygen occurs through modifications of the alpha subunit, whereas the beta subunit is a constitutive nuclear protein and is not affected by hypoxia. Under hypoxic conditions, the alpha subunit is stable, allowing it to accumulate in the nucleus, where, on binding to ARNT, it recognizes HIF-response elements (HRE) within the promoter regions of hypoxia-responsive target genes. Under normoxic conditions, the alpha subunit is rapidly degraded by means of ubiquitination and proteosomal degradation [7, 8]. Recent studies have indicated that the regulation of HIF1A stability and transactivation activity involves several proteins and their well-coordinated interaction, raising the possibility that a wide range of control mechanisms are involved in mediating the physiological responses to O2 availability. The ubiquitination process requires the specific interaction of HIF1A and the product of the von Hippel-Lindau tumor suppressor gene (VHL), known to function as a substrate-recognition component of an E3 ubiquitin protein ligase complex [VHLCBC complex], which includes elongin B, elongin C, and Cullin 2 (CUL2) [9, 10]. Other reports have shown that ubiquitin-like molecule NEDD8 (neural precursor cell expressed, developmentally downregulated 8) binds to CUL2 and that this conjugation is required for VHL-mediated HIF1A degradation [11, 12]. Additionally, it has been shown that HIF1A is hydroxylated in an oxygen-dependent manner on specific key proline residues (Pro402 and Pro564) located in the oxygen-dependent degradation domain by a family of prolyl hydroxylase domain enzymes, termed EGLN1, EGLN2, EGLN3 [13]. The hydroxylation of the two proline residues promotes VHLCBC binding and targets HIF1A for degradation [14]. The intracellular oxygen concentration also plays an important role in regulating the transactivation potency of HIF1A. Under normoxic conditions, hydroxylation of asparigine 803 residue in the C-terminal transactivation domain of HIF1A by another hydroxylase, termed hypoxia-inducible factor 1, alpha subunit inhibitor (HIF1AN), has been shown to inhibit the recruitment of transcriptional coactivator proteins, including CBP/p300 [15]. Thus, numerous independent pathways working in a well-orchestrated manner regulate HIF1A stability and activity.
While numerous studies have investigated specific aspects of HIF1A regulation, the multiple pathways by which HIF activity is regulated under hypoxia and normoxia have never been systematically evaluated within any specific tissue, much less during the development of an organ. We previously demonstrated that HIF1A plays an important role in mediating oxygen-regulated events of early trophoblast differentiation [5, 16] and that the temporal expression of HIF1A correlated with TGFB3, which in turn is an important contributor to normal trophoblast differentiation [17]. However, we did not directly demonstrate that increase in HIF1A was responsible for increased TGFB3 expression, and hence we investigated the mechanisms by which HIF1A stability and transactivation potency, with respect to TGFB3, is regulated. Herein, we demonstrate that the VHLCBC complex is required for HIF1A degradation and that the formation of the VHLCBC complex is spatially and temporally regulated during human placental development. Furthermore, we show that EGLNs are differentially expressed in an O2-dependent fashion. Inhibition of EGLNs in placental explants revealed their importance in regulating HIF1A stability and transactivation of TGFB3 during early placentation. Together, these findings provide novel insight into how trophoblast cells sense and transduce placental oxygen changes via HIF1A.
MATERIALS AND METHODS
Tissue Collection
All tissues were collected after informed consent in accordance with the Ethics Guidelines of the University of Toronto's Faculty of Medicine and Mount Sinai Hospital, Toronto. First- and second-trimester human placental tissues (5–18 wk of gestation, n = 69) were obtained from elective terminations of pregnancies. Term placental tissue (n = 12) was collected from vaginal deliveries at Mount Sinai Hospital, Toronto.
Human Villous Explant Culture
Villous explant cultures were established from first-trimester human placentae (5–10 wk of gestation, n = 19) as previously described [5]. Villous explants were cultured under standard tissue culture conditions of 5% CO2 in 95% air (20% O2 environment) or maintained in an atmosphere of either 3% O2/92% N2/5% CO2 or 8% O2/87% N2/5% CO2. For each treatment, tissue samples from the same placenta were used, and in each experiment, explant cultures were set up in triplicate. The treatment with the prolyl hydroxylase inhibitor 2-dimethyloxalylglycine (DMOG; Frontier Scientific) was performed under standard culture conditions.
RNA Isolation and RT-PCR
Total RNA was treated with DNase I in order to remove genomic DNA contamination. One microgram of total RNA was reverse transcribed in a total volume 50 µl using random hexamers (Applied Biosystems). The resulting templates (50 ng of cDNA for our target genes and 5 ng for 18S) were quantified by RT-PCR (ABI Prism 7700).
TaqMan probes for human HIF1A EPAS1, ARNT, VHL, EGLN1 EGLN2, EGLN3, HIF1AN, and TGFB3 were purchased from ABI. Primers were obtained from the oligosynthesis service at the Hospital for Sick Children, Toronto, Ontario, Canada. Probes and primers for ribosomal 18S and TGFB3 were purchased from ABI as Assays-on-Demand for human genes. For each probe, a dilution series determined the efficiency of amplification of each primer/probe set, and the relative quantification method was employed [18]. For the relative quantitation, PCR signals were compared between groups after normalization using 18S as an internal reference. Briefly, relative expression was calculated as 2–(Ctgene of interest–Ct18S). Fold change was calculated according to Livak et al. [18].
DNA Plasmids
The human full-length HIF1A cDNA, a gift of Dr. Semenza (Johns Hopkins University), was subcloned in the pcDNA3 expression vector. The chloroamphenicol acetyltransferase (CAT) reporter constructs pB-1387-CAT and pB3–499-CAT, containing either 1387 or 499 bp upstream of the transcription start site of TGFB3, respectively, were kindly provided by Dr. Seong-Jin Kim (National Cancer Institute). Additional reporter constructs containing promoter regions of TGFB3 were generated by PCR and cloned into P basic vector (Promega).
Transfection Experiments
COS7 cells (75% confluency) were transfected with TGFB3 promoter-CAT reporter constructs DNA using lipofectamine 2000 (Invitrogen). Cells were cotransfected with pCMV-ßGal to correct for transfection efficiency. A promoterless CAT construct was included in each experiment as control. Transfected cells were maintained at 20% or 2% O2. At 24 h after transfection, cells were harvested and lysed, and CAT and galactoside activities were measured (Promega). In separate experiments, cells were transfected with either empty pcDNA3 vector or pcDNA3-HIF1A plus pB3–499-CAT, maintained at 20% O2, and analyzed 48 h after transfection. CAT activities were normalized to ß-galactosidase activity.
Western Blot Analysis
Western blot analyses were performed as previously described [16]. Primary antibodies were mouse monoclonal anti-human HIF1A (mgc3, 1:250, ABR; Affinity Bioreagents Inc.), mouse monoclonal anti-human VHL (clone Ig33, 1:250; Oncogene), rabbit polyclonal anti-human CUL2 (1:250; Neomarkers), rabbit polyclonal ARNT (1:2000; Novus Biologicals), rabbit polyclonal EPAS1 (1:1000; Novus Biologicals), rabbit polyclonal anti-human EGLN1, EGLN2 and EGLN3 (1:1000; Novus Biologicals), and goat polyclonal anti-human TGFB3 (1:500, R&D System Inc.). Horseradish peroxidase-conjugated secondary antibodies (1:10 000) were rabbit anti-mouse for HIF1A and VHL; donkey anti-rabbit for CUL2, ARNT, and EGLNs; and donkey anti-goat for TGFB3 (Santa Cruz Biotechnology).
Immunoprecipitation
Immunoprecipitations were performed as previously described [16]. Antisera (1 µg) were added to precleared samples and incubated overnight. Immunoprecipitates were then collected, subjected to SDS-PAGE, and analyzed by immunoblotting using either mouse monoclonal anti-human VHL (1:250), rabbit polyclonal antibodies NEDD8 (1:500; Alexis Biochemicals), or mouse monoclonal anti-human ubiquitin (1:500; Covance Research Products). A 1:10 000 dilution of anti-mouse-Ig-horseradish peroxidase was used as secondary antibody.
Immunohistochemistry
Immunohistochemical analyses were performed as previously described [5]. Mouse monoclonal antibodies against HIF1A and VHL were used at 1:50 dilution, whereas rabbit polyclonal antibodies against ARNT, CUL2, and NEDD8 were used at 1:100, 1:50, and 1:500, respectively. Secondary antibodies (1:300) were either biotinylated goat anti-mouse or goat anti-rabbit IgG. Control experiments were carried out by replacing the primary antibody with normal goat serum.
Statistical Analysis
All data are represented as mean ± SEM of at least three separate experiments carried out in triplicate. For comparison of data between multiple groups, we used Kruskal-Wallis one-way ANOVA with post hoc Dunn test. For comparison between two groups, we used paired and unpaired Student t-test as appropriate. Statistical tests were carried out using Prism statistical software, and P-values <0.05 were considered significant.
RESULTS
Expression of HIF1A , ARNT, and TGFB3
We first determined the HIF1A, EPAS1, ARNT, and TGFB3 mRNA levels in human placental tissues throughout gestation. As previously reported [19], we found that placental TGFB3 showed a parallel developmental pattern of mRNA expression to that of HIF1A. Both transcripts were present throughout gestation, but their expression was gestational age specific. During the first trimester (5–12 wk), HIF1A and TGFB3 mRNA expression peaked at 7–10 wk of gestation and decreased afterward (11–13 wk; Fig. 1, A and B). A second peak was observed during the second trimester at 14–18 wk. The mRNA expression patterns of HIF1A and TGFB3 were confirmed at the protein level (Fig. 1, C–E). As anticipated, neither mRNA nor protein content of ARNT changed during pregnancy (Fig. 1, A and C); therefore, we normalized the HIF1A and TGFB3 protein values to that of ARNT (Fig. 1, D and E). EPAS1 mRNA and protein expression did not significantly alter during placental development (Fig. 1, A and C) and was not further investigated.
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HIF1A Transactivates TGFB3
COS-7 cells were transiently transfected with TGFB3 promoter-CAT (either pB-1387-CAT or pB-499-CAT) constructs and maintained at either 2% or 20% O2. Cells kept at 2% O2 exhibited significantly greater CAT activity when compared to cells transfected with a promoterless CAT or cells maintained at 20% O2 (Fig. 2A), consistent with a functional HRE being present in the proximal TGFB3 promoter. In order to determine whether HIF1 directly transactivates TGFB3, COS-7 cells were cotransfected with pB-499-CAT and a pcDNA3 expression vector containing the full-length human HIF1A. Cells transfected with the TGFB3 promoter construct alone as well as cells cotransfected with HIF1A displayed significantly greater CAT activities compared to cells transfected with the (control) promoterless CAT construct (Fig. 2B). However, cells cotransfected with HIF1A exhibited greater TGFB3 promoter activity than cells transfected with the TGFB3 promoter alone. These findings corroborate recent observations that HIF1 transactivates TGFB3 in trophoblast cell lines [20, 21] and prompted us to further investigate the regulation of HIF1 stability and activity during placental development.
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VHLCBC Complex Formation
We first studied the VHLCBC complex formation during placental development. During the first trimester, VHL mRNA expression was maximal at 7–10 wk and significantly declined thereafter (Fig. 3A, left panel). Western blot analysis using a VHL antibody revealed two protein bands, corresponding to a molecular mass of 19 and 30 kDa, respectively, previously identified as being two distinct biologically active isoforms of VHL [22]. In line with the VHL transcript profile, the protein content of both VHL isoforms increased at around 7–10 wk of gestation and rapidly declined thereafter (Fig. 3A, right panel). We then investigated the spatial and temporal localization of HIF1A, ARNT, and VHL in sections of anchoring villi during the first trimester of pregnancy. Immunohistochemical analysis of placental tissue sections of 6–7 wk of gestation showed strong positive nuclear and cytoplasmic immunoreactivity for HIF1A in cytotrophoblast and extravillous trophoblast cells (EVT) within the proximal part of the invading columns, while HIF1A was absent in syncytiotrophoblasts (Fig. 4E). At 12 wk of gestation, HIF1A was virtually undetectable in all cell layers, including EVTs (Fig. 4F). At 6–7 wk of gestation, strong positive immunoreactivity for VHL was noted in syncytiotrophoblast and EVT cells within the distal part of the invading column (Fig. 4H). VHL protein localized to both trophoblast cell layers and EVT cells at 12 wk of gestation (Fig. 4I). Intense nuclear immunoreactivity for ARNT, uniformly distributed in all cell layers, was observed in placental tissue across the first trimester of gestation (Fig. 4, B and C). No immunoreactivity was observed in control sections in which primary antibodies were omitted (Fig. 4, A, D, and G). Next we determined the temporal and spatial protein expression of CUL2 and NEDD8 in placental tissue throughout gestation. At 6–7 wk of gestation, positive immunoreactivity for NEDD8 and CUL2 was detected in villous cytotrophoblast cells and EVTs within the distal part of the column (Fig. 4, K and N). Low/absent positive immunoreactivity for NEDD8 was noted in the syncytium. At 12 wk of gestation, both trophoblast cell layers showed strong positive immunoreactivity for both NEDD8 and CUL2 (Fig. 4, L and O). No immunoreactivity was observed in control sections in which primary CUL2 and NEDD8 antibodies were omitted (Fig. 4, J and M). Western blot analysis showed that the amount of CUL2 protein, identified as a 76 Mr x 10–3 band, did not change throughout gestation (Fig. 3B). CUL2 immunoblotting also revealed a second band of 84 kDa, the content of which peaked at 8–12 wk of gestation (Fig. 3B). In subsequent experiments, we investigated the interaction of the various proteins of the VHLCBC complex. Immunoprecipitation of placental lysates with anti-VHL antibody followed by SDS-PAGE and immunoblotting with CUL2 showed that the interaction of CUL2 with VHL was maximal at 7–10 wk of gestation (Fig. 3C, top panel). To determine whether changes in VHL association to CUL2 were due to changes in VHL content, the blots were also probed with anti-VHL antibody. As anticipated, VHL content peaked at 7–10 wk of gestation (Fig. 3C, second panel). Additional coimmunoprecipitation experiments demonstrated that HIF1A binds to VHL throughout gestation; however, their association peaked at 12–15 wk of gestation (Fig. 3C, third panel). Immunoblotting of the same samples with HIF1A confirmed high levels of HIF1A at 5–8 and 15 wk of gestation (Fig. 3C, bottom panel). In order to determine when HIF1A gets ubiquitinylated, HIF1A was immunoprecipitated from placental lysates with anti-HIF1A antibody, and immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with an antiubiquitin antibody. Ubiquitinylated HIF1A, identified as a ubiquitin-poly tail, was markedly increased at 10–12 wk of gestation (Fig. 3D, upper panel). Again, immunoblot analysis of the HIF1A immunoprecipitated samples revealed high HIF1A expression at 6–8 and 14 wk of gestation (Fig. 3D, lower panel).
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Hydroxylases Expression
Since the EGLN activities are oxygen sensitive [23] and trophoblast cells in vivo are exposed to different oxygen tension, we investigated the expression of EGLN1, EGLN2, and EGLN3 during early placental development. EGLN1, EGLN2, and EGLN3 showed unique patterns of expression throughout pregnancy (Fig. 5A, upper panels). While EGLN2 mRNA expression significantly increased at 10–12 wk and remained elevated throughout pregnancy, EGLN1 and EGLN3 transcript levels peaked around 11–12 wk. The EGLN2 and EGLN3 protein patterns were consistent with the mRNA patterns (Fig. 5A, lower panels). Only EGLN1 protein expression differed somewhat from its mRNA expression by having high levels at 5–7 wk of gestation. We next examined the mRNA expression of HIF1AN, an asparagyl hydroxylase enzyme known to negatively regulate HIF1A-mediated gene transcription by an oxygen-sensitive mechanism. RT-PCR showed that HIF1AN mRNA expression increased with advancing gestation, reaching a peak at 11–12 wk of gestation (Fig. 5C, left panel) and declining thereafter (14 wk to term). Importantly, EGLNs and HIF1AN mRNA expression levels inversely correlated with that of HIF1A.
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Effect of Varying Oxygen Tension on EGLNs and HIF1AN Expression
Next, we investigated the effect of oxygen on the expression of hydroxylases. Exposure of first-trimester villous explants to various oxygen tensions—3% O2 (physiological <10 wk), 8% O2 (physiological >10 wk), and 20% O2 (standard condition)—resulted in a remarkable oxygen-dependent regulation of EGLN expression (Fig. 5B). Low oxygen (3% O2) induced a significant increase in EGLN1, EGLN2, and EGLN3 mRNA expression when compared to standard conditions (20% O2). The highest induction was observed for EGLN3. While the mRNA expression of EGLN3 did not significantly change between 3% and 8% O2, EGLN2 and EGLN1 transcript levels were significantly higher at 8% O2 when compared to 3% O2 and standard condition (Fig. 5B, left panel). Exposure of explants to 3% and 8% O2 increased EGLN1 and EGLN3 but not EGLN2 protein content when compared to standard (20% O2) condition (Fig. 5B, right panel). These data suggest that the expression of EGLNs is oxygen regulated and is maximal at 8% O2, which corresponds to the pO2 level of the placenta after 10 wk, when expression of EGLNs is also maximal (Fig. 5A, upper panel). Although HIF1AN displayed a temporal regulated pattern of mRNA expression (Fig. 5C, left panel), its expression appeared to be oxygen independent under the conditions studied (Fig. 5C, right panel).
EGLNs Regulate HIF1A Stability
To determine the role of EGLNs in regulating placental HIF1A stability, first-trimester villous explants were maintained overnight at 3% O2 and then transferred to 20% O2 in the presence or absence of 1 mM DMOG, a pharmacological inhibitor of EGLN activity [24]. Morphological examination of explants demonstrated that cultures maintained at 20% O2 in the presence of DMOG showed the typical low oxygen-induced EVT outgrowth seen in explants kept at 3% O2 (Fig. 6A) [3–5]. Immunohistochemical analysis of explants maintained at either 3% O2 or 20% O2 plus DMOG showed strong positive immunoreactivity for HIF1A in cytotrophoblast and EVT cells (Fig. 6B). Weak immunoreactivity for HIF1A was seen in syncytiotrophoblasts. In contrast, control explants maintained at 20% O2 showed low/positive immunoreactivity for HIF1A, restricted mostly to villous cytotrophoblast cells. Western blot analysis of villous explant lysates showed that HIF1A expression in DMOG-treated explants kept at 20% O2 was indeed comparable to that observed in explants cultured at 3% O2 without DMOG (Fig. 6C). These results indicate that during placental development, EGLNs regulate HIF1A stability.
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Hydroxylases Regulate TGFB3 Expression
As placental TGFB3 expression is mediated via HIF (Fig. 2), we investigated the role of EGLNs in regulating oxygen-dependent TGFB3 expression by examining the effect of DMOG on TGFB3 expression in villous explants cultured at 3% or 20% O2. As anticipated, TGFB3 mRNA and protein expression was increased in explants maintained at 3% O2 when compared to 20% O2 explants (Fig. 6D). DMOG treatment of explants kept at 20% O2 significantly increased TGFB3 mRNA and protein expression, suggesting that inhibition of hydroxylase activity promotes TGFB3 transcription via upregulation or maintenance of HIF1A activity.
DISCUSSION
The present study provides new insight into the mechanisms by which HIF1A expression is regulated during human placental development. Our data are the first to demonstrate that placental regulation of HIF1A activity and stability is a multistep process involving several proteins whose expression and function are temporally and spatially regulated. In particular, we provide evidence that placental degradation of HIF1A takes place after 10 wk of gestation at the time when placental oxygenation increases and that this process is dependent on proper assembly/function of VHLCBC multiprotein complex and hydroxylase activities. Moreover, we demonstrate that EGLNs are involved in regulating HIF1A-induced transactivation of TGFB3, a molecule previously shown to be important for placental development [17].
It is generally believed that the hypoxia-induced HIF1A expression is due to protein stabilization, although low oxygen can also increase HIF1A mRNA expression [25]. To our knowledge, this is the first study demonstrating that in human placenta, HIF1A is regulated at both the transcriptional and the posttranslational level. The observation of an elevated HIF1A expression at 14–18 wk of gestation implies that factors other than oxygen may be involved in regulating placental HIF1A expression, possibly oxidative stress-related proteins. Placental oxygen levels rapidly increase at the end of the first trimester of pregnancy, and this is associated with a transient period of placental oxidative stress leading to the release of inflammatory cytokines. Interleukin 1, alpha (IL1A), and tumor necrosis factor have been shown to influence HIF1A stabilization [26], and, as such, these cytokines could also contribute to HIF1A regulation in the first-trimester human placenta. Studies conducted with cell lines have shown that under nonhypoxic conditions, HIF1A expression is induced by growth factors via a mechanism that involves receptor tyrosine kinases [27, 28]. This receptor-mediated activation of HIF1A involves the phosphatidylinositol-3 kinase (PI-3K) and MAPK pathways [29, 30].
We found that at 6–7 wk of gestation, VHL expression was restricted to syncytiotrophoblasts and EVT in the distal part of the invading column, while at 12 wk, VHL was also detected in cytotrophoblast cells and EVTs of the proximal column, in agreement with previous observations [31]. In contrast, HIF1A was expressed only in the cytotrophoblast cells and EVTs of the proximal column. The appearance of VHL in the cytotrophoblast cells at 10–12 wk of gestation coincided with the appearance of CUL2 and NEDD8, both of which are known to be required for proper HIF1A/VHL association. The appearances of these proteins and the corresponding disappearance of HIF1A within the same cell layer suggest a unique spatial control of HIF1A degradation during early trophoblast differentiation. The observation that VHL expression during early gestation (<10 wk) is restricted to syncytiotrophoblast cells is of potential interest, as recent studies have shown that VHL is involved in several other aspects of cell biology, independent of its action on HIF1A regulation. For example, VHL is involved in the control of cellular differentiation in a variety of systems [32, 33].
The present study demonstrates that 10–12 wk of gestation is a critical window for the expression of molecules involved in the formation and activation of the VHLCBC complex, which would be required to inactivate HIF in the face of the steep increase in O2 tension. Reports have indicated that NEDD8 conjugation to CUL2 is critical for the ubiquitin ligase activity of the VHLCBC complex. Although neddylation is not essential for the VHLCBC complex formation itself, the NEDD8 modification is required for the activity of the ubiquitin-conjugating enzymes [34]. In mammalian cells, CUL2 is modified by a single molecule of NEDD8 on Lys689 [11], and this association is visualized as a product of 84 Mr x 10–3. It is likely that this band observed in placental lysates at around 8–12 wk of gestation after immunoblotting with anti-CUL2 antibody represents neddylated CUL2. Thus, we speculate that in normal placentation, this modification is required to efficiently ubiquitinate HIF1A after oxygen tension rises following the onset of blood flow into the intervillous space.
The current data show that proteolysis of placental HIF1A is linked to the activity of a well-conserved family of prolyl hydroxylase enzymes. While EGLN-mediated regulation of HIF1A has been investigated in various tumor cell lines, their physiological role in a developing organ, such as the human placenta, has never been reported. To our knowledge, this is the first study demonstrating that EGLNs are expressed in the human placenta, where they play a dual role as oxygen sensor and regulators of HIF1A expression in trophoblast cells. The expression of the various EGLNs and their relative abundance are tissue specific [35]. Our data demonstrate that in human placental tissue, all three EGLN isoforms are present. Their temporal expression profile, with high levels at the time when trophoblast cells experience the rapid increase in oxygenation, is suggestive of their ability to sense oxygen changes. The observation that EGLN expression in the first-trimester placenta is inversely correlated with HIF1A further supports their importance in the regulation of this transcription factor. Recent reports indicate that the role of individual EGLNs as oxygen sensor varies within different systems and/or conditions tested [36]. In rat C6 glioma cells, hypoxia stimulates the expression of EGLN12, while exposure of human cardiac myocytes, smooth muscle cells, and endothelial cells to hypoxia or pharmacological conditions that mimic hypoxia increases the mRNA expression of EGLN3 but not that of EGLN1 or EGLN2 [23, 37]. On the other hand, hypoxia has been found to increase EGLN1 and EGLN3 mRNA levels in human osteosarcoma cells [38]. Overall, these studies indicate that hypoxia is an important regulator of EGLNs mRNA expression, suggesting a feedback regulatory mechanism of EGLN transcription via HIF. In agreement with these published observations, we demonstrate that exposure of first-trimester placental explants to low oxygen tension increases EGLN expression, specifically EGLN1 and EGLN3. All three EGLNs, but specifically EGLN3, had a high expression at 8% O2, an oxygen concentration similar to the physiological pO2 environment at 11–12 wk of gestation and throughout the remainder of pregnancy. Thus, our findings corroborate previous reports [39] showing that under conditions of reoxygenation, EGLN1 and EGLN3 activities are increased, leading to an efficient hydroxylation of HIF1A and consequently reduction of its half-life.
Inhibition of EGLN activity by DMOG treatment resulted in typical low oxygen-induced morphological changes in villous explants and increased HIF1A expression. Although the action of DMOG is not specific with respect to any of the three EGLNs, our data strongly support the idea that EGLNs regulate the stability of HIF1A in human placenta and, thereby, TGFB3 expression. Finally, our data suggest that in human placenta, HIF1AN may play a role in regulating HIF1A activity. During placental development, HIF1AN mRNA expression peaked at the time when oxygen tension increased. HIF1AN expression inversely correlates with that of TGFB3, suggesting that HIF1AN may also play a role in modulating the HIF1A transcriptional properties, in particular, the expression of TGFB3. However, our placental explant studies indicate that HIF1AN mRNA expression is not affected by changes in oxygen tension. Other studies have also found that HIF1AN expression is oxygen independent [23]. However, HIF1AN is active at lower O2 concentrations than other prolyl hydroxylases, suggesting that it may act as a fine-tuning oxygen sensor [40]. The mechanism of increased HIF1AN mRNA expression at 10–12 wk of placental gestation remains to be elucidated.
Summarizing, the current data demonstrate for the first time that HIF1A stability and activity, as a result of changes in placental oxygenation, is regulated by temporal and spatial changes in expression and association of molecules of the multiprotein VHLCBC complex as well as the activity of prolyl hydroxylase enzymes.
ACKNOWLEDGMENTS
We would like to thank Dr. Ljiljiana Petkovic for providing the placental samples. We would also like to thank Drs. Knox Ritchie and Alan Bocking for the constant support and Dr. Stacy Zamudio for carefully reading the manuscript. I.C. is recipient of an Ontario Women's Health CIHR/IGH Mid-Career Award. M.P. is the holder of a Canadian Research Chair (tier 1) in Fetal, Neonatal, and Maternal Health.
FOOTNOTES
1 Supported by Canadian Institutes of Health Research (CIHR) grant MT1406 to I.C. 
2 Correspondence: Isabella Caniggia, Mount Sinai Hospital, Samuel Lunenfeld Research Institute, 600 University Ave., Room 871, Toronto, ON, Canada M5G 1X5. FAX: 416 586 8588; caniggia{at}mshri.on.ca
Received: 8 February 2006.
First decision: 7 March 2006.
Accepted: 6 April 2006.
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