Potential role of increased oxygenation in altering perinatal adrenal steroidogenesis
Vishal Agrawal1, Meng Kian Tee1, Jie Qiao1, Marcus O. Muench2,3 and Walter L. Miller1
BACKGROUND: At birth, the large fetal adrenal involutes rap- idly, and the patterns of steroidogenesis change dramatically; the event(s) triggering these changes remain largely unex- plored. Fetal abdominal viscera receive hypoxic blood having a partial pressure of oxygen of only ~2 kPa (20-23 mm Hg); peri- natal circulatory changes change this to adult values (~20 kPa). We hypothesized that transition from fetal hypoxia to postna- tal normoxia participates in altering perinatal steroidogenesis. METHODS: We grew midgestation human fetal adrenal cells and human NCI-H295A adrenocortical carcinoma cells in 2% O2, then transitioned them to 20% O2 and quantitated ste- roidogenic mRNAs by quantitative PCR and microarrays.
RESULTS: Transitioning fetal adrenal cells from hypoxia to normoxia increased mRNAs for 17a-hydroxylase/17,20 lyase (P450c17), 3ß-hydroxysteroid dehydrogenase (3ßHSD2), and steroidogenic acute regulatory protein (StAR). We repeated the protocol with NCI-H295A cells acclimated to hypoxia for 15 d, quantitating 31,255 transcripts by microarray. Using an arbitrary 1.5-fold difference, 1 d of normoxia increased 4 tran- scripts and decreased 56, whereas 2 d of normoxia increased 62 transcripts and decreased 105. P450c17, 3ßHSD2, and StAR were ranked among the top eight increased transcripts.
CONCLUSION: These data suggest that the hypoxic/nor- moxic transition at birth contributes to perinatal changes in adrenal steroidogenesis.
A t birth, the transition from intrauterine to extrauterine life requires major endocrine adjustments, such as the meta- bolic adjustments following the discontinuation of glucose supplied from cord blood and the rapid shift from producing reverse T3 to T3. Understanding these transitions is essential in the endocrine care of the perinatal patient. Evaluation of newborn adrenal function is complicated by the fact that the fetal and later infant adrenals are very different. The human fetal adrenal has two zones, fetal and definitive, in contrast to the three zones, glomerulosa, fasciculata, and reticularis, of the adult gland. Fetal adrenal steroidogenesis begins at about 7 wk gestation: steroidogenic enzymes are detectable by immu- nocytochemistry in the fetal zone at 50-52 d postconception, and primary cultures of the 8 wk adrenal produce cortisol and
respond to adrenocorticotropic hormone (1). The fetal adrenal transiently expresses 3ß-hydroxysteroid dehydrogenase, type 2 (3ßHSD2, encoded by HSD3B2) at about 8-10 wk, permit- ting fetal adrenal cortisol synthesis at the same time when male genital development occurs, thus helping to prevent the viril- ization of female fetuses by suppressing fetal adrenal andro- gen synthesis (1). The fetal adrenal has relatively little 3ßHSD2 activity after 12 wk (1,2) but has 17a-hydroxylase and robust 17,20 lyase activity (both catalyzed by cytochrome P450c17, encoded by CYP17A1), considerable sulfotransferase activity, and little steroid sulfatase activity accounting for its abun- dant production of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS). DHEAS is secreted, 16a-hydroxylated in the fetal liver by CYP3A7 (3-5), and then acted on by pla- cental 3ßHSD1, 17ßHSD1 (17ß-hydroxysteroid dehydroge- nase type 1, encoded by HSD17B1), and aromatase (P450aro, encoded by CYP19A1) to produce estriol (6,7). Fetal adrenal steroids are the source of about half of the estrone and estradiol and 90% of the estriol in the maternal circulation (8). Despite the large amounts of DHEA and DHEAS produced by the fetal adrenal and their consequent metabolism to estrogens by the placenta, evidence for an essential role for these steroids is scant, as fetuses with genetic disorders of adrenal steroido- genesis develop normally, reach term gestation, and undergo normal parturition (9). Although glucocorticoids can induce premature lung maturation, they do not appear to be needed when human gestation goes to term, as complete absence of the glucocorticoid receptor is compatible with normal term birth and pulmonary function (10).
It has long been known that the adrenal grows rapidly through- out fetal life, reaching a combined weight of 8-9 g at birth (equal to the combined weight of the adult adrenals) (11-13), but within weeks of birth, the fetal adrenals involute to a total weight of about 2g (12,14). This change is mediated by apoptosis of the fetal zone, possibly in response to activin A or transforming growth factor-ß (15). In parallel with the involution of the fetal zone of the adre- nal, secretion of DHEA and DHEAS falls dramatically (16-18). The triggering mechanism for this rapid, profound change in adrenal morphology, cellular architecture, and steroidogenesis is not known. It has been suggested that the involution of the fetal adrenal is more related to gestational age than to timing
The first two authors contributed equally to this work.
1Department of Pediatrics, University of California-San Francisco, San Francisco, California; 2Blood Systems Research Institute, University of California-San Francisco, San Francisco, California; 3Department of Laboratory Medicine, University of California-San Francisco, San Francisco, California. Correspondence: Walter L. Miller (wlmlab@ucsf.edu) Received 9 June 2014; accepted 13 August 2014; advance online publication 24 December 2014. doi:10.1038/pr.2014.194
after birth (19), but more recent studies indicate that parturition itself triggers fetal adrenal involution, which was interpreted as suggesting that the withdrawal of a placental factor stimulated the onset of fetal adrenal apoptosis (20). We hypothesize that the transition from intrauterine hypoxia to extrauterine normoxia is also a key event in triggering the remodeling of the fetal adrenal. Human fetal abdominal viscera receive hypoxic blood having a partial pressure of oxygen (Po2) of only ~2 kPa (1 kPa = 7.5 Torr; 1 Torr = 1 mm Hg); perinatal circulatory changes change this to adult values of ~20 kPa. As parturition itself appears to trigger the changes in fetal adrenal steroidogenesis and architecture, we considered whether the perinatal change in arterial oxygen ten- sion participates in these changes. As a preliminary test of this hypothesis, we grew adrenal cells in long-term hypoxic condi- tions designed to mimic the intrauterine environment and then examined changes in gene expression upon transition to a nor- moxic environment that models extrauterine life.
RESULTS
Human Fetal Adrenal Cells
To study the changes in the human adrenal as it transitions from the hypoxia of fetal life to the normoxia of the extrauterine new- born environment, we first incubated adrenal cells from a single 17-wk human fetus under hypoxic conditions for 1 d, followed by normoxic conditions for 1 or 2 d. Total cellular RNA from these cells was hybridized to Illumina BeadChip microarrays for gene expression analyses. Using an arbitrary cutoff of >1.5-fold change for gene activation or <0.67-fold change for gene repres- sion, the mRNAs encoded by 107 genes were increased, and those for 114 genes were decreased when the cells were shifted from fetal hypoxic conditions to normoxic conditions for 1 d, and 179 mRNAs were increased and 296 genes were decreased after 2 d in normoxic conditions (Supplementary Tables S1
a
Total number of genes activated or inhibited in fetal adrenal cells in normoxia over control in hypoxia: 1 d normoxia, 221 genes (107 1 + 114 4) 2 d normoxia, 475 genes (179 1 + 296 4)
b
Adrenal 1 d
Adrenal 2 d
53 1 34
54 1 111 Į
125 1 185
and S2 online). Of these transcripts, 54 were increased and 111 were decreased on both days (Figure 1).
While this experiment showed that the hypoxic-normoxic transition can change the abundance of many adrenal mRNAs, changes were not seen in the transcripts for any gene encod- ing a steroidogenic enzyme or its electron-transfer cofactor. To examine mRNAs encoding steroidogenic factors more closely, we obtained additional adrenals, incubated primary adrenal cell cultures under hypoxic and normoxic conditions, and mea- sured the relative abundances of selected mRNAs under each condition for each adrenal by reverse transcription followed by quantitative real-time PCR. Consistent with prior observa- tions (21), preliminary experiments showed that the relative abundances of mRNAs for P450scc and P450c17 decreased after 4 d due to the overgrowth of fibroblasts and apoptosis of fetal adrenal cells (data not shown). Thus, we used 2 d of cul- ture for more detailed studies with adrenals from five fetuses (three male and two female; 17-23 wk gestation). We noted no changes in the morphology of the adrenal cells after transition from hypoxia to normoxia for 1-2 d. Under normoxic con- ditions, the abundance of the mRNAs for P450c17, steroido- genic acute regulatory protein (StAR), and 3ßHSD2 increased after 2 d (Figure 2). Consistent with the data from other cell types (22), glyceraldehyde-3-phosphate dehydrogenase gene expression decreased in normoxia compared with hypoxia, but the expression of mRNAs for 3ßHSD2, StAR, and P450c17 increased 2.6-, 2.0-, and 1.6-fold under normoxic conditions, while expression of P450scc barely changed. However, there was a substantial variation with the fetal adrenal cells from dif- ferent fetuses, so that the statistical analyses were of marginal significance.
Human Adrenal NCI-H295A Cells
To avoid differences between individual fetal adrenals, we sought to use the immortalized human adrenal NCI-H295A cell line, in which the patterns of steroidogenesis closely
5
T
mRNA fold change (2-44Ct)
4
3
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1
T
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T
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P450c17
StAR
3฿HSD2
P450scc
GAPDH
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resemble those of the fetal adrenal (23). Because these cells are normally cultured in normoxic conditions, we first acclimated them to the fetal environment by culturing them in hypoxic conditions for 15 d before “delivering” them to extrauterine normoxic conditions for either 1 or 2 d; control cells were maintained in hypoxic conditions throughout the experiment. We noted no changes in cellular morphology when the NCI- H295A cells were transitioned from 15 d of hypoxia to nor- moxia. The resulting mRNAs were analyzed by hybridization to Illumina BeadChip microarrays, thus permitting analysis of the entire transcriptome. In NCI-H295A cells, only 4 mRNAs were increased and 56 were decreased when the cells were shifted from fetal hypoxic conditions to extrauterine normoxic conditions for 1 d, and none of the altered mRNAs appeared to participate in steroidogenesis (Table 1 and Supplementary Table S3 online). By contrast, after the NCI-H295A cells had been returned to normoxia for 2 d, 62 mRNAs were increased and 105 mRNAs were decreased (Table 2 and Supplementary Table S3 online). Among the mRNAs that increased, three encode proteins that participate in steroidogenesis: P450c17 (CYP17A1), increased 1.65-fold; 3ßHSD2 (HSD3B2), increased 1.89-fold; and StAR (STAR), increased 1.78-fold over con- trols. In addition, sterol isomerase (EBP), which participates in cholesterol biosynthesis, increased 1.57-fold. The degree of overlap in these gene populations is shown in Figure 3. These gene expression profiles showed that 46 mRNAs were regu- lated in the same fashion after both 1 and 2 days of normoxia (1 increased and 45 decreased). In addition, the mRNAs for 3 other genes were increased and 11 were decreased after 1 d of normoxia and 61 mRNAs were increased and 60 reduced after 2 d of normoxia.
Among the genes whose mRNAs increased or decreased under normoxic conditions (Tables 1 and 2), ALDOA, ALDOC, BNIP3, BNIP3L, CA9, ENO1, GADPH, HK2, IGFBP2, JMJDIA, LDHA, NDRG1, PKM2, SLC2A1, SLC2A3, and TPI1 are known to be transcriptionally regulated by hypoxia (24). Only four genes, BNIP3, NDRG1, SERPINA3, and SLC2A1 were regulated in common in both NCI-H295A cells and in the primary culture of fetal adrenal cells. The complete expression profile data for all genes in NCI-H295A and in the primary cultures of fetal adrenal cells are shown in Supplementary Tables S1-S4 online.
Gene ontology analyses using Ingenuity Pathway Analysis (https://analysis.ingenuity.com) showed that many of the repressed genes in NCI-H295A incubated for 1 or 2 d in nor- moxic conditions participate in common pathways such as glycolysis, sucrose degradation, vitamin C transport, thyroid hormone receptor/retinoid X receptor activation, and HIFlo signaling (Tables 3-5). In contrast, the activated genes in NCI- H295A are involved in glutathione-mediated detoxification, dendritic-natural killer cells crosstalk, p53 signaling, and, of course, steroidogenesis.
DISCUSSION
Little information is available concerning the potential effects of environmental oxygenation on fetal adrenal function, and
most such reports have investigated animal models of high- altitude stress. Thus, when pregnant rats were transitioned to reduced air pressure of ~380 Torr (~50 kPa; Po, ~10 kPa; designed to correspond to 18,000 feet above sea level), the adrenals of fetuses were larger, possibly due to increased adre- nocorticotropic hormone secretion (25). Long-term mainte- nance of pregnant sheep at 3,820 m above sea level (Po2 ~102 Torr; 13.6 kPa) reduced expression of mRNAs and proteins for P450scc, P450c17, and MC2R (adrenocorticotropic hor- mone receptor) but did not alter P450c21 (21-hydroxylase, encoded by CYP21A2), StAR (encoded by STAR), 3ßHSD2, or DAX-1 (26). At birth, the fetal zone of the human adrenal cor- tex involutes rapidly, as evidenced by rapidly declining serum concentrations of DHEA and DHEAS. Furthermore, this rapid decline in DHEA/S is seen in both premature and term infants (27). Therefore, we and others have hypothesized that the involution of the fetal adrenal is not “programmed” but “triggered.” A current view is that the trigger is the loss of pla- cental hormones and growth factors (20). Such a trigger could also be secondary to the profound environmental change that accompanies birth. Such changes initiate the transition from fetal to postnatal circulatory patterns, including closure of the foramen ovale and the ductus arteriosus. Closure of the ductus is directly triggered by increased oxygen tension (via prosta- glandins), and many other events in the newborn are triggered by the transition to normoxia (28). Thus, we hypothesized that the transition from the intrauterine hypoxic environment to the extrauterine normoxic environment might participate in initiating the rapid changes in adrenal steroidogenesis that follow birth. The fetal adrenal and other organs served by the fetal abdominal aorta are bathed in oxygen-poor blood having a partial pressure of oxygen of about 20-22 Torr (2.6-2.9 kPa) (29). Therefore, to model the changes in the adrenal environ- ment that accompany birth, we incubated human fetal adre- nal cells and human adrenal NCI-H295A cells in 2% oxygen (hypoxia, Po2 ~2.0 kPa) followed by incubation in atmospheric oxygen (normoxia, ~20 kPa) for 1 and 2 d.
Results with human fetal adrenals suggested that the hypoxic-normoxic transition increased the mRNAs for StAR, 3ßHSD2, and P450c17 and decreased the mRNA for P450scc, but only the data with StAR reached nominal significance of P < 0.05. There was substantial variation among adrenals from different donors, with no pattern attributable to donor sex or gestational age in the 17-23-wk period used. Therefore, we turned to human adrenocortical carcinoma NCI-H295A cells, which possess features typical of fetal, rather than adult, adre- nal cells (e.g., expression of IGF-2 and P450aro) (23). The cells were propagated in hypoxic conditions for 15 d to acclimate them to this model intrauterine environment before “deliver- ing” them to normoxia. This transition induced changes in the abundance of many mRNAs, with many more changes after 2 d than after 1 d. Not surprisingly, one of the induced genes was HIF1A, which encodes a hypoxia-induced transcription factor (30). Most notably, after 2 d of normoxia, the abundances of the mRNAs for the steroidogenic factors 30HSD2, StAR, and P450c17 increased >1.6-fold. This will change the pattern of
Oxygen-induced perinatal adrenal changes
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| Symbol | Gene | Mean ± SDª | Chromosome | Function |
|---|---|---|---|---|
| MIR1974 | MicroRNA 1974 | 1.68 ±1.26 | MicroRNA | |
| UCK2 | Uridine-cytidine kinase 2 | 1.51 ±0.16 | 1 | Pyrimidine metabolism |
| AP1S1 | Adaptor-related protein complex 1, sigma 1 subunit | 1.50±0.66 | 7 | Protein sorting in the late-Golgi/trans- Golgi network and/or endosomes |
| GINS4 | GINS complex subunit 4 (Sld5 homolog) | 1.50±0.67 | 8 | Initiation of DNA replication and progression of DNA replication forks |
| PEG3 | Paternally expressed 3 | 0.66±0.10 | 19 | Apoptosis |
| LOC100132564 | Hypothetical protein LOC100132564 | 0.66±0.27 | 12 | Unknown |
| RNU1-3 | U1 small nuclear 3 | 0.66±0.22 | 1 | Component of the spliceosome |
| SNORA28 | Small nucleolar RNA, H/ACA box 28 | 0.66±0.07 | 14 | Unknown |
| NARF | Nuclear prelamin A recognition factor | 0.66±0.03 | 17 | Binds to the prenylated prelamin A carboxyl-terminal tail domain |
| VIL2 | Villin 2 | 0.66±0.10 | 6 | Actin cytoskeleton signaling |
| ANKRD37 | Ankyrin repeat domain 37 | 0.65±0.02 | 4 | Unknown |
| MT1H | Metallothionein 1H | 0.65±0.18 | 16 | Protein binding and zinc ion binding |
| LOC728188 | Similar to phosphoglycerate mutase processed protein | 0.65±0.11 | X | Unknown |
| WDR54 | WD repeat domain 54 | 0.65±0.07 | 2 | Unknown |
| PGK1 | Phosphoglycerate kinase 1 | 0.64±0.06 | X ☒ | Glycolysis/gluconeogenesis |
| MTP18 | Mitochondrial protein 18 kDa | 0.64±0.04 | 22 | Mitochondrial division |
| HIG-2 | Hypoxia-inducible lipid droplet-associated | 0.64±0.02 | 7 | Intracellular lipid accumulation |
| SNX26 | Sorting nexin 26 | 0.63±0.03 | 19 | Intracellular trafficking |
| RNU1A3 | RNA, U1A3 small nuclear | 0.63±0.20 | 1 | Component of the spliceosome |
| SLC6A8 | Solute carrier family 6 (neurotransmitter transporter, creatine), member 8 | 0.63±0.10 | X ☒ | Creatine uptake in muscles and brain |
| PGM1 | Phosphoglucomutase 1 | 0.63±0.07 | 1 | Glycolysis/gluconeogenesis |
| PGAM1 | Phosphoglycerate mutase 1 (brain) | 0.63±0.03 | 10 | Glycolysis/gluconeogenesis |
| C4orf3 | Chromosome 4 open reading frame 3 | 0.62±0.05 | 4 | Unknown |
| EZR | Ezrin | 0.62±0.09 | 6 | Actin cytoskeleton signaling |
| PGAM1P8 | Phosphoglycerate mutase 1 pseudogene 8 | 0.62±0.06 | 11 | Unknown |
| SLC2A3b | Solute carrier family 2 (facilitated glucose transporter), member 3 | 0.62±0.03 | 12 | Glucose transporter |
| ORM1 | Orosomucoid 1 | 0.62±0.03 | 9 | Transport protein in the blood stream |
| GPI | Glucose phosphate isomerase | 0.61±0.06 | 19 | Glycolysis/gluconeogenesis |
| LOC732165 | Similar to triosephosphate isomerase (TIM) (triose- phosphate isomerase) | 0.61 ±0.07 | 1 | Unknown |
| ADSSL1 | Adenylosuccinate synthase like 1 | 0.60±0.07 | 14 | Component of the purine nucleotide cycle |
| MGC16121 | Hypothetical protein MGC16121 | 0.60±0.07 | X ☒ | Unknown |
| GAPDHb | Glyceraldehyde-3-phosphate dehydrogenase | 0.60±0.06 | 12 | Glycolysis/gluconeogenesis |
| EFHD2 | EF-hand domain family, member D2 | 0.59±0.04 | 1 | Calcium ion binding, protein binding |
| TPI1b | Triosephosphate isomerase 1 | 0.59±0.09 | 12 | Glycolysis/gluconeogenesis |
| PNCK | Pregnancy upregulated nonubiquitously expressed CaM kinase | 0.59±0.12 | X ☒ | Calcium signaling |
| TMEM45A | Transmembrane protein 45A | 0.58±0.07 | 3 | Unknown |
| RN7SK | RNA, 7SK small nuclear | 0.57 ±0.19 | 6 | Unknown |
| LOC732007 | Similar to phosphoglycerate mutase 1 (phosphoglycerate mutase isozyme B) (PGAM-B) (BPG-dependent PGAM 1) | 0.57 ±0.08 | 12 | Unknown |
Table 1. Continued on next page
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| Symbol | Gene | Mean ± SDª | Chromosome | Function |
|---|---|---|---|---|
| PLOD1 | Procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1 | 0.56±0.09 | 1 | Lysine degradation |
| JMJD1Ab | Jumonji domain containing 1A | 0.55±0.06 | 2 | Histone demethylation |
| ALDOAb | Aldolase A, fructose-bisphosphate | 0.55±0.06 | 16 | Glycolysis and gluconeogenesis |
| TPI1P2 | Triosephosphate isomerase 1 pseudogene 2 | 0.54±0.01 | 7 | Unknown |
| PFKP | Phosphofructokinase, platelet | 0.53±0.08 | 10 | Fructose and mannose metabolism; galactose metabolism; glycolysis/ gluconeogenesis |
| SLC6A10P | Solute carrier family 6 (neurotransmitter transporter, creatine), member 10 | 0.52±0.10 | 16 | Unknown |
| ENO1b | Enolase 1, (alpha) | 0.51 ±0.04 | 1 | Glycolysis/gluconeogenesis |
| TPI1P1 | Triosephosphate isomerase 1 pseudogene 1 | 0.50±0.06 | 1 | Unknown |
| PKM2b | Pyruvate kinase, muscle | 0.49±0.08 | 15 | Glycolysis/gluconeogenesis |
| BNIP3b | BCL2/adenovirus E1B 19 kDa interacting protein 3 | 0.49±0.08 | 10 | Nuclear gene encoding mitochondrial protein |
| SERPINA3 | Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 | 0.49±0.11 | 14 | Plasma protease inhibitor |
| BNIP3Lb | BCL2/adenovirus E1B 19 kDa interacting protein 3-like | 0.47±0.01 | 8 | Apoptosis |
| LOC644774 | Similar to phosphoglycerate kinase 1 | 0.45±0.02 | X | Unknown |
| SPRR2F | Small proline-rich protein 2F | 0.45±0.11 | 1 | Structural constituent of cytoskeleton |
| DDIT4 | DNA-damage-inducible transcript 4 | 0.45±0.09 | 10 | Inhibits cell growth by regulating the mTOR signaling pathway |
| SLC2A1b | Solute carrier family 2 (facilitated glucose transporter), member 1 | 0.44±0.03 | 1 | Glucose transporter |
| ALDOCb | Aldolase C, fructose-bisphosphate | 0.41±0.07 | 17 | Glycolysis and gluconeogenesis |
| LDHAb | Lactate dehydrogenase A | 0.39±0.04 | 11 | Glycolysis/gluconeogenesis |
| NDRG1b | N-myc downstream regulated gene 1 | 0.36±0.08 | 8 | p53-mediated caspase activation and apoptosis |
| BHLHB2 | Basic helix-loop-helix domain containing, class B, 2 | 0.35±0.08 | 3 | Transcriptional factor modulating chondrogenesis in response to the cAMP pathway |
| CYP2J2 | Cytochrome P450, family 2, subfamily J, polypeptide 2 | 0.29±0.06 | 1 | Arachidonic acid metabolism; drug metabolism |
| PFKFB4 | 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 | 0.18±0.05 | 3 | Fructose and mannose metabolism |
CAMP, cyclic adenosine monophosphate; mTOR, mammalian target of rapamycin. aThe data are expressed as mean fold change in normoxic conditions over control in hypoxic conditions ± SD. “Positive control genes known to be transcriptionally regulated by hypoxia.
steroidogenesis from 45 to 44 steroids, as is seen following birth.
Our study emphasizes analysis of mRNAs and did not mea- sure the abundances of steroidogenic enzyme proteins or the steroid products of our adrenal cell systems; such measure- ments will be of interest in future studies. In addition, our experimental design only examined events in the first 2 d fol- lowing the transition to normoxia, yet the involution of the fetal adrenal and the transition from fetal to newborn pattern of ste- roidogenesis takes several weeks, so that future studies may also examine a broader time frame. However, it seems likely that the rapid changes in oxygenation at delivery would constitute an acute trigger to change the adrenal’s transcriptional program- ming and that such acute changes would subsequently affect adrenal morphology and steroid secretory patterns over the first weeks of life, so we would expect that the changes in mRNA
abundances that we have measured would precede changes in adrenal morphology and steroid secretion. In this context, it may be important to add tropic activators of the protein kinase A pathway (adrenocorticotropic hormone to adrenal cells and 8-Br-cAMP to NCI-H295A cells), to mimic conditions in vivo.
Our data are consistent with our hypothesis that the change in oxygenation that follows birth is a key factor in determin- ing the change in the patterns of adrenal steroidogenesis that follow birth. However, no aspect of our data is inconsistent with the hypothesis that withdrawal of placental factors also plays a role, especially in the rapid involution of adrenal size, as known adrenal growth factors (IGF-2, EGF, FGF) (31) were not among the factors dramatically changed by the hypoxic- normoxic transition in our studies. Thus, we propose that both the hypoxic-normoxic transition and the potential with- drawal of placental factors are required to initiate the anatomic
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Oxygen-induced perinatal adrenal changes
| Symbol | Gene | Mean ± SDª | Chromosome | Function |
|---|---|---|---|---|
| PTMA | Prothymosin, alpha | 2.31 ± 1.78 | 2 | Mediate immune function |
| RPS2P28 | Ribosomal protein S2 pseudogene 28 | 2.18±0.64 | 6 | Unknown |
| RPS3P3 | Ribosomal protein S3 pseudogene 3 | 2.07 ±0.86 | 3 | Unknown |
| FLJ40504 | Hypothetical protein FLJ40504 | 1.98±0.40 | 17 | Unknown |
| ID3 | Inhibitor of DNA binding 3, dominant negative helix- loop-helix protein | 1.91 ±0.31 | 1 | bHLH transcription factor binding |
| HSD3B2 | Hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 | 1.89±0.57 | 1 | Steroidogenesis |
| IGFBP2b | Insulin-like growth factor binding protein 2, 36 kDa | 1.83±0.28 | 2 | IGF-1 signaling |
| StAR | Steroidogenic acute regulatory protein | 1.78±0.25 | 8 | Steroidogenesis |
| PL6P10 | Ribosomal protein L6 pseudogene 10 | 1.77 ±1.09 | 4 | Unknown |
| GSTA5 | Glutathione S-transferase alpha 5 | 1.77±0.13 | 6 | Conjugation of reduced glutathiones and electrophiles |
| LOC651816 | Similar to ubiquitin-conjugating enzyme E2S (ubiquitin-conjugating enzyme E2-24 kDa) (ubiquitin- protein ligase) (ubiquitin carrier protein) (E2-EPF5) | 1.76±0.52 | Unknown | |
| HLA-G | HLA-G histocompatibility antigen, class I, G | 1.75±0.76 | 6 | Allograft rejection signaling; antigen presentation pathway |
| LOC644936 | Cytoplasmic beta-actin pseudogene | 1.75±0.87 | 5 | Unknown |
| LOC647169 | Glutathione S-transferase alpha 3 pseudogene | 1.74±0.19 | 6 | Unknown |
| FLJ20489 | Hypothetical protein FLJ20489 | 1.72±0.45 | 12 | Unknown |
| KRT18P31 | Keratin 18 pseudogene 31 | 1.70±0.12 | 5 | Unknown |
| RPS8P10 | Ribosomal protein S8 pseudogene 10 | 1.70±0.61 | 10 | Unknown |
| FLJ43681 | Ribosomal protein L23a pseudogene | 1.68±0.24 | 17 | Unknown |
| KLK1 | Kallikrein 1 | 1.65±0.28 | 19 | Serine protease |
| CYP17A1 | Cytochrome P450, family 17, subfamily A, polypeptide 1 | 1.65±0.32 | 10 | Steroidogenesis |
| RPL23AP64 | Ribosomal protein L23a pseudogene 64 | 1.64±0.24 | 11 | Unknown |
| CD9 | CD9 molecule | 1.64±0.09 | 12 | Platelet activation and aggregation, differentiation, adhesion, and signal transduction |
| PPPDE2 | PPPDE peptidase domain containing 2 | 1.63±0.40 | 22 | Desumoylating isopeptidase |
| KRT18P59 | Keratin 18 pseudogene 59 | 1.62±0.28 | 11 | Unknown |
| CCDC124 | Coiled-coil domain containing 124 | 1.61 ±0.48 | 19 | DNA binding |
| KRT8 | Keratin 8 | 1.60±0.16 | 12 | Cellular structural integrity, signal transduction, and cellular differentiation |
| MICA | MHC class I polypeptide-related sequence A | 1.60±0.18 | 6 | Antigen processing and presentation |
| PERP | PERP, TP53 apoptosis effector | 1.60±0.46 | 6 | p53 signaling |
| LOC728602 | Ornithine decarboxylase antizyme 1 pseudogene | 1.60±0.48 | 1 | Unknown |
| GSTA2 | Glutathione S-transferase alpha 2 | 1.60±0.25 | 6 | Detoxification of electrophilic compounds |
| KRT18P13 | Keratin 18 pseudogene 13 | 1.60±0.17 | 9 | Unknown |
| RPS2P51 | Ribosomal protein S2 pseudogene 51 | 1.59±0.32 | 19 | Unknown |
| RPL18AP3 | Ribosomal protein L18a pseudogene 3 | 1.59±0.23 | 12 | Unknown |
| RPL28P5 | Ribosomal protein L28 pseudogene 5 | 1.58±0.18 | 19 | Unknown |
| UCK2 | Uridine-cytidine kinase 2 | 1.58±0.35 | 1 | Pyrimidine metabolism |
| RPL7P32 | Ribosomal protein L7 pseudogene 32 | 1.58±0.02 | 7 | Unknown |
| KRT8P47 | Keratin 8 pseudogene 47 | 1.58±0.07 | 1 | Unknown |
| EBP | Emopamil-binding protein (sterol isomerase) | 1.57 ±0.54 | X | Cholesterol biosynthesis |
Table 2. Continued on next page
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Agrawal et al.
| Symbol | Gene | Mean ± SDª | Chromosome | Function |
|---|---|---|---|---|
| CKB | Creatine kinase, brain | 1.57±0.29 | 14 | Transfer of phosphate between ATP and phosphogens |
| LOC730323 | Hypothetical LOC730323 | 1.57±0.70 | 7 | Unknown |
| LOC389672 | Similar to 40S ribosomal protein SA (p40) (34/67 kDa laminin receptor) (colon carcinoma laminin-binding protein) (NEM/1CHD4) (multidrug resistance- associated protein MGr1-Ag) | 1.56±0.47 | 8 | Unknown |
| LOC649555 | Similar to eukaryotic translation initiation factor 4E | 1.56±0.19 | 17 | Unknown |
| CATSPER2 | Cation channel, sperm associated 2 | 1.56±0.77 | 15 | Voltage-gated calcium channel |
| NOP16 | NOP16 nucleolar protein homolog (yeast) | 1.55±0.16 | 5 | Unknown |
| LOC100134053 | Similar to POLR2J4 protein | 1.55±0.24 | 7 | Unknown |
| XBP1 | X-box binding protein 1 | 1.55±0.18 | 22 | Endoplasmic reticulum stress pathway |
| LOC100128098 | Hypothetical protein LOC100128098 | 1.54±0.64 | 10 | Unknown |
| LOC642502 | Succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa pseudogene | 1.54±0.37 | 17 | Unknown |
| HMGN2P25 | High mobility group nucleosomal binding domain 2 pseudogene 25 | 1.53±0.25 | 3 | Unknown |
| PPIB | Peptidylprolyl isomerase B (cyclophilin B) | 1.53±0.20 | 15 | Folding of proteins |
| LOC100129502 | Hypothetical protein LOC100129502 | 1.53±0.42 | 15 | Unknown |
| SDF2L1 | Stromal cell-derived factor 2-like 1 | 1.53±0.21 | 22 | Unknown |
| DCXR | Dicarbonyl/L-xylulose reductase | 1.52±0.15 | 17 | Uronate cycle of glucose metabolism; osmoregulation in renal tubules |
| RPS26P35 | Ribosomal protein S26 pseudogene 35 | 1.52±0.24 | 8 | Unknown |
| LOC651149 | Similar to 60S ribosomal protein L3 (L4) | 1.52±0.09 | 10 | Unknown |
| ADI1 | Acireductone dioxygenase 1 | 1.52±0.46 | 2 | Catalyzes the formation of formate and 2-keto-4-methylthiobutyrate from 1,2-dihydroxy-3-keto-5-methylthiopentene |
| RPSAP56 | Ribosomal protein SA pseudogene 56 | 1.51 ±0.20 | 16 | Unknown |
| RPS2P29 | Ribosomal protein S2 pseudogene 29 | 1.51 ±0.27 | 6 | Unknown |
| CCT3 | Chaperonin containing TCP1, subunit 3 (gamma) | 1.51 ±0.23 | 1 | Molecular chaperone; assists the folding of proteins upon ATP hydrolysis |
| CDK5 | Cyclin-dependent kinase 5 | 1.51±0.31 | 7 | Cell cycle |
| RPL29P15 | Ribosomal protein L29 pseudogene 15 | 1.50±0.32 | 5 | Unknown |
| HIF1A | Hypoxia-inducible factor 1, alpha subunit | 1.50±0.45 | 14 | Basic helix-loop-helix transcription factor activated in response to reduced oxygen availability in the cellular environment |
| SNORD13 | Small nucleolar RNA, C/D box 13 | 0.66±0.11 | 8 | Unknown |
| MTMR11 | Myotubularin-related protein 11 | 0.66±0.06 | 1 | Probable pseudophosphatase |
| MXD4 | MAX dimerization protein 4 | 0.66±0.04 | 4 | Transcriptional repressor |
| KANK4 | KN motif and ankyrin repeat domains 4 | 0.66±0.14 | 1 | Control of cytoskeleton formation by regulating actin polymerization |
| WDR54 | WD repeat domain 54 | 0.66±0.19 | 2 | Unknown |
| RNU1G2 | RNA, U1G2 small nuclear | 0.65±0.22 | 1 | Unknown |
| MYLK4 | Myosin light chain kinase family, member 4 | 0.65±0.07 | 6 | Protein serine/threonine kinase activity and ATP binding |
| HOXA5 | Homeobox A5 | 0.65±0.15 | 7 | Transcription factor |
| MT1H | Metallothionein 1H | 0.65±0.06 | 16 | Protein binding and zinc ion binding |
| RHBDL3 | Rhomboid, veinlet-like 3 (Drosophila) | 0.65±0.05 | 17 | Intramembrane proteolysis |
| GOLGA8B | Golgin A8 family, member B | 0.64±0.11 | 15 | Maintaining Golgi structure |
| UBA7 | Ubiquitin-like modifier activating enzyme 7 | 0.64±0.04 | 3 | Activates ubiquitin |
Table 2. Continued on next page
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| Symbol | Gene | Mean ± SDª | Chromosome | Function |
|---|---|---|---|---|
| C15orf52 | Chromosome 15 open reading frame 52 | 0.64±0.05 | 15 | Unknown |
| PLOD2 | Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 | 0.64±0.02 | 3 | Lysine degradation |
| LOC286016 | Triosephosphate isomerase 1 pseudogene | 0.64±0.14 | 7 | Unknown |
| TAF1C | TATA box binding protein (TBP)-associated factor, RNA polymerase I, C, 110 kDa | 0.64±0.02 | 16 | Component of the transcription factor SL1/ TIF-IB complex |
| PHKA2 | Phosphorylase kinase, alpha 2 (liver) | 0.64±0.04 | X | Catalyzes serine phosphorylation |
| IL11RA | Interleukin 11 receptor, alpha | 0.64±0.03 | 9 | Receptor for interleukin-11 |
| SLC6A8 | Solute carrier family 6 (neurotransmitter transporter, creatine), member 8 | 0.64±0.10 | X | Creatine uptake in muscles and brain |
| TPI1P1 | Triosephosphate isomerase 1 pseudogene 1 | 0.64±0.10 | 1 | Unknown |
| ALDOAb | Aldolase A, fructose-bisphosphate | 0.64±0.07 | 16 | Glycolysis and gluconeogenesis |
| GPI | Glucose phosphate isomerase | 0.64±0.03 | 19 | Glycolysis/gluconeogenesis; pentose phosphate pathway; starch and sucrose metabolism |
| SNORD3A | Small nucleolar RNA, C/D box 3A | 0.63±0.11 | 17 | Unknown |
| KLHL3 | Kelch-like 3 (Drosophila) | 0.63±0.02 | 5 | Substrate-specific adapter of a BTB-CUL3- RBX1 E3 ubiquitin ligase complex |
| DDIT4L | DNA-damage-inducible transcript 4-like | 0.63±0.07 | 4 | Inhibits cell growth by regulating the mTOR signaling pathway |
| DPYSL4 | Dihydropyrimidinase-like 4 | 0.63±0.03 | 10 | Signaling by class 3 semaphorins and subsequent remodeling of the cytoskeleton |
| KLF2 | Kruppel-like factor 2 (lung) | 0.63±0.04 | 19 | Binds CACCC box in the beta-globin gene promoter and activates transcription |
| RNU6-1 | RNA, U6 small nuclear 1 | 0.63±0.17 | 15 | Unknown |
| COL11A1 | Collagen, type XI, alpha 1 | 0.63±0.08 | 1 | Fibrillogenesis |
| APBB3 | Amyloid beta (A4) precursor protein-binding, family B, member 3 | 0.63±0.04 | 5 | Internalization of beta-amyloid precursor protein |
| ZSWIM8 | ZSWIM8 zinc finger, SWIM-type containing 8 | 0.63±0.04 | 10 | Unknown |
| PAM | Peptidylglycine alpha-amidating monooxygenase | 0.63±0.02 | 5 | Catalyzes C-terminal alpha-amidation of peptides |
| MLLT6 | Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 6 | 0.62±0.07 | 17 | Unknown |
| ANKRD37 | Ankyrin repeat domain 37 | 0.62±0.05 | 4 | Unknown |
| ZNF395 | Zinc finger protein 395 | 0.62±0.04 | 8 | Unknown |
| SNORD113-5 | Small nucleolar RNA, C/D box 113-5 | 0.62±0.05 | 14 | Unknown |
| ABP1 | Amiloride binding protein 1 (amine oxidase (copper- containing) | 0.62±0.14 | 7 | Degradation of putrescine, histamine, spermine, and spermidine |
| ZBTB40 | Zinc finger and BTB domain containing 40 | 0.62±0.07 | 1 | Unknown |
| TMEM123 | Transmembrane protein 123 | 0.61 ±0.17 | 11 | Oncotic cell death |
| RN5S9 | RNA, 5S ribosomal 9 | 0.61 ±0.27 | 1 | Unknown |
| RNU1F1 | RNA, U1F1 small nuclear | 0.61 ±0.22 | 14 | Component of the spliceosome |
| WDR90 | WD repeat domain 90 | 0.61 ±0.07 | 16 | Unknown |
| RNU6-15 | RNA, U6 small nuclear 15 | 0.61±0.15 | - | Unknown |
| CLCNKA | Chloride channel Ka | 0.60±0.01 | 1 | Voltage-gated chloride channel |
| SNORD3C | Small nucleolar RNA, C/D box 3C | 0.60±0.12 | 17 | Unknown |
| SIRPA | Signal-regulatory protein alpha | 0.60±0.07 | 20 | Immunoglobulin-like cell surface receptor for CD47 |
| TPI1b | Triosephosphate isomerase 1 | 0.60±0.04 | 12 | Unknown |
| SLC27A3 | Solute carrier family 27 (fatty acid transporter), member 3 | 0.60±0.09 | 1 | Acyl-CoA ligase activity |
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Agrawal et al.
| Symbol | Gene | Mean ± SDª | Chromosome | Function |
|---|---|---|---|---|
| CLCN7 | Chloride channel 7 | 0.60±0.07 | 16 | Antiporter; contributes to the acidification of the lysosome lumen |
| TMEM45A | Transmembrane protein 45A | 0.60±0.12 | 3 | Unknown |
| COL3A1 | Collagen, type III, alpha 1 | 0.60±0.09 | 2 | Regulation of cortical development |
| RNU1A3 | RNA, U1A3 small nuclear | 0.59±0.26 | 1 | Component of the spliceosome |
| JMJD1Ab | Jumonji domain containing 1A | 0.59 ±0.08 | 2 | Histone demethylation |
| SNORD3D | Small nucleolar RNA, C/D box 3D (SNORD3D) | 0.59±0.19 | 17 | Unknown |
| PPP1R3C | Protein phosphatase 1, regulatory (inhibitor) subunit 3C | 0.59±0.12 | 10 | Glycogen-targeting subunit for protein phosphatase 1 |
| COL7A1 | Collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive) | 0.59±0.03 | 3 | Stratified squamous epithelial basement membrane protein that forms anchoring fibrils |
| LOC100132564 | Hypothetical protein LOC100132564 | 0.59±0.23 | 12 | Unknown |
| PGK1 | Phosphoglycerate kinase 1 | 0.58±0.11 | X | Glycolysis/gluconeogenesis |
| LOC100132394 | Hypothetical protein LOC100132394 | 0.58±0.22 | X | Unknown |
| CYP1A1 | Cytochrome P450, family 1, subfamily A, polypeptide 1 | 0.58±0.04 | 15 | Xenobiotic metabolism |
| RAP1GAP | RAP1 GTPase activating protein | 0.58±0.03 | 1 | GTPase activator |
| ALKBH5 | AlkB, alkylation repair homolog 5 (E. coli) | 0.57±0.02 | 17 | Demethylation of RNA by oxidative demethylation |
| SVEP1 | Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 | 0.57±0.06 | 9 | Cell attachment process |
| SCGB1D2 | Secretoglobin, family 1D, member 2 | 0.57±0.07 | 11 | Bind androgens and other steroids |
| TMEM145 | Transmembrane protein 145 | 0.56±0.10 | 19 | Unknown |
| SLC2A3b | Solute carrier family 2 (facilitated glucose transporter), member 3 | 0.56±0.05 | 12 | Glucose transporter |
| FAM62B | Family with sequence similarity 62 (C2 domain containing) member B | 0.56±0.00 | 7 | Calcium-regulated intrinsic membrane protein |
| LOC644774 | Similar to phosphoglycerate kinase 1 | 0.56±0.04 | X | Unknown |
| RNU1-3 | RNA, U1 small nuclear 3 | 0.56±0.24 | 1 | Component of the spliceosome |
| SLC6A10P | Solute carrier family 6 (neurotransmitter transporter, creatine), member 10 (pseudogene) | 0.56±0.09 | 16 | Unknown |
| RNU1-5 | RNA, U1 small nuclear 5 | 0.56±0.24 | 1 | Component of the spliceosome |
| bHK2 | Hexokinase 2 | 0.56±0.03 | 2 | Aminosugars metabolism; fructose and mannose metabolism; galactose metabolism; glycolysis/gluconeogenesis |
| MT1F | Metallothionein 1F | 0.56±0.08 | 16 | Cadmium ion binding and copper ion binding |
| STK36 | Serine/threonine kinase 36, fused homolog (Drosophila) | 0.56±0.02 | 2 | Sonic hedgehog signaling |
| COL22A1 | Collagen, type XXII, alpha 1 | 0.55±0.06 | 8 | Cell adhesion ligand for skin epithelial cells and fibroblasts |
| MST1 | Macrophage stimulating 1 (hepatocyte growth factor- like) | 0.55±0.03 | 3 | IL-12 signaling and production in macrophages; MSP-RON signaling pathway |
| PKM2b | Pyruvate kinase, muscle | 0.54±0.05 | 15 | Glycolysis/gluconeogenesis |
| PFKP | Phosphofructokinase, platelet | 0.54±0.07 | 10 | Fructose and mannose metabolism; galactose metabolism; glycolysis/ gluconeogenesis |
| ARHGEF16 | Rho guanine exchange factor (GEF) 16 | 0.54±0.05 | 1 | Guanyl-nucleotide exchange factor activity |
| PNCK | Pregnancy upregulated nonubiquitously expressed CaM kinase | 0.54±0.08 | X | Calcium signaling |
| ADSSL1 | Adenylosuccinate synthase like 1 | 0.54±0.07 | 14 | Alanine and aspartate metabolism; purine metabolism |
| ENO1b | Enolase 1, (alpha) | 0.53±0.04 | 1 | Glycolysis/gluconeogenesis |
Table 2. Continued on next page
| Symbol | Gene | Mean ± SDª | Chromosome | Function |
|---|---|---|---|---|
| SSPO | SCO-spondin homolog (Bos taurus) | 0.53±0.05 | 7 | Modulation of neuronal aggregation |
| PGM1 | Phosphoglucomutase 1 | 0.51 ±0.05 | 1 | Glycolysis/gluconeogenesis |
| SNX26 | Sorting nexin 26 | 0.51 ±0.09 | 19 | Intracellular trafficking |
| MGC16121 | Hypothetical protein MGC16121 | 0.51 ±0.08 | X | Unknown |
| VIL2 | Villin 2 (ezrin) | 0.51 ±0.07 | 6 | Actin cytoskeleton signaling |
| EFHD2 | EF-hand domain family, member D2 | 0.51±0.04 | 1 | Calcium ion binding, protein binding |
| RFTN1 | Raftlin, lipid raft linker 1 | 0.51 ±0.02 | 3 | Formation and/or maintenance of lipid rafts |
| EZR | Ezrin | 0.50±0.11 | 6 | Actin cytoskeleton signaling |
| DDIT4 | DNA-damage-inducible transcript 4 | 0.49±0.09 | 10 | Inhibits cell growth by regulating the mTOR signaling pathway |
| SLC2A1b | Solute carrier family 2 (facilitated glucose transporter), member 1 | 0.47±0.03 | 1 | Glucose transporter |
| PLOD1 | Procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1 | 0.47 ±0.05 | 1 | Lysine degradation |
| BNIP3b | BCL2/adenovirus E1B 19 kDa interacting protein 3 | 0.47±0.08 | 10 | Apoptosis |
| RN7SK | RNA, 7SK small nuclear | 0.43±0.18 | 6 | Pre-mRNA splicing and processing |
| LDHAb | Lactate dehydrogenase A | 0.43±0.02 | 11 | Glycolysis/gluconeogenesis |
| BNIP3Lb | BCL2/adenovirus E1B 19 kDa interacting protein 3-like | 0.41 ±0.02 | 8 | Apoptosis |
| RNU4-2 | RNA, U4 small nuclear 2 | 0.41 ±0.19 | Pre-mRNA splicing and processing | |
| ALDOCb | Aldolase C, fructose-bisphosphate | 0.41±0.12 | 17 | Glycolysis and gluconeogenesis |
| ORM1 | Orosomucoid 1 | 0.39±0.09 | 9 | Transport protein in the blood stream |
| SERPINA3 | Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 | 0.33±0.08 | 14 | Plasma protease inhibitor |
| BHLHB2 | Basic helix-loop-helix domain containing, class B, 2 | 0.32±0.09 | 3 | Transcriptional factor modulating chondrogenesis in response to the cAMP pathway |
| NDRG1b | N-myc downstream regulated gene 1 | 0.26±0.05 | 8 | p53-mediated caspase activation and apoptosis |
| CYP2J2 | Cytochrome P450, family 2, subfamily J, polypeptide 2 | 0.23±0.06 | 1 | Arachidonic acid metabolism; drug metabolism |
| PFKFB4 | 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 4 | 0.16±0.05 | 3 | Fructose and mannose metabolism |
CAMP, cyclic adenosine monophosphate; mTOR, mammalian target of rapamycin.
aThe data are expressed as mean fold change in normoxic conditions over control in hypoxic conditions ± SD. bPositive control genes known to be transcriptionally regulated by hypoxia.
| Canonical pathways | P value | Molecules |
|---|---|---|
| Glycolysis | 2.51 × 10-23 | PGK1, ENO1, GPI, TPI1, PGAM1, PKM2, GAPDH, ALDOA, PFKP, ALDOC |
| Sucrose degradation | 3.55×10-7 | TPI1, ALDOA, ALDOC |
| Vitamin C transport | 2.29×10-3 | SLC2A1, SLC2A3 |
| TR/RXR activation | 3.80×10-3 | ENO1, SLC2A1, PFKP |
| HIF1 a signaling | 5.50×10-3 | SLC2A1, LDHA, SLC2A3 |
TR/RXR, thyroid hormone receptor/retinoid X receptor.
remodeling of the fetal adrenal and its change in steroidogenic patterns as the fetus transitions to extrauterine life. While there may be differences among expression levels of mRNAs, their encoded proteins, and downstream steroids, our preliminary
data suggest that the hypoxic/normoxic transition at birth is likely to be an important component of the perinatal changes in adrenal architecture and steroidogenesis.
METHODS
Cells
We used two cell systems. First, we used primary cultures from human fetal adrenals obtained with written consent from women undergoing elective procedures at San Francisco General Hospital. This research was performed with Institutional Review Board approval from the University of California San Francisco’s Committee on Human Research. All specimens were anonymous. The gestational age of the fetal specimens was estimated based on foot length. Second, we used the NCI-H295A human adrenocortical cell line (32) that expresses all adrenal steroidogenic enzymes (23) and has been selected to grow in monolayer (33). All cells were grown in Roswell Park Memorial Institute (RPMI) medium (UCSF cell culture facility) with 2% fetal bovine serum. All experiments were performed in triplicate. Hypoxic conditions consisted of an atmosphere of 2% oxygen, 93% nitro- gen, and 5% CO2 in the XVIVO hypoxia tissue culture hood from
a Total number of genes activated or inhibited in NCI-H295A cells in normoxia over control in hypoxia: 1 d normoxia, 60 genes (4 1 + 56 4)
2 d normoxia, 167 genes (62 1 + 105 4)
b
NCI 1 d
NCI 2 d
31
1 1
611 60 Į
11Į
45 ↓
| Canonical pathways | P value | Molecules |
|---|---|---|
| Glutathione-mediated detoxification | 2.04×10-7 | GSTA2, GSTA5 |
| Steroidogenesis | 8.51×10-7 | CYP17A1, EBP, HSD3B2, StAR |
| Crosstalk between dendritic cells and natural killer cells | 3.63×10-3 | HLA-G, MICA |
| p53 signaling | 4.37×10-2 | PERP, HIF1A |
| Canonical pathways | P value | Molecules |
|---|---|---|
| Glycolysis | 3.16×10-12 | PGK1, ENO1, GPI, TPI1, PKM2, ALDOA, PFKP, ALDOC |
| Sucrose degradation | 4.47×10-6 | TPI1, ALDOA, ALDOC |
| Systemic lupus erythematosus signaling | 6.17×10-4 | RNU1-3, RNU4-2, RNU6-1 |
| Estrogen biosynthesis | 3.31 ×10-3 | CYP1A1, CYP2J2 |
| Vitamin C transport | 7.76×10-3 | SLC2A1, SLC2A3 |
| TR/RXR activation | 2.09×10-2 | ENO1, SLC2A1, PFKP |
| HIF1 a signaling | 2.95×10-2 | SLC2A1, LDHA, SLC2A3 |
TR/RXR, thyroid hormone receptor/retinoid X receptor.
BioSpherix (Lacona, NY). Constant oxygen levels were maintained in the hypoxia chamber throughout the experimental procedure and incubations. All plastic ware, pipette aids, tissue culture media, and buffers were equilibrated in the hypoxic conditions before use.
Incubations
Under an institutional review board-approved human experimenta- tion protocol, fetal adrenal tissues was transported in a full tube of
| Name | Sequence | Size of product (bp) |
|---|---|---|
| CYP11A1 forward | 5'- TCC AGA AGT ATG GCC CGATT -3' | 75 |
| CYP11A1 reverse | 5'- CAT CTT CAG GGT CGATGA CAT AAA -3' | |
| CYP17A1 forward | 5'- TCT CTG GGC GGC CTC AA -3' | 63 |
| CYP17A1 reverse | 5'- AGG CGATAC CCT TAC GGTTGT -3' | |
| HSD3B2 forward | 5'- GGA AGA GAA GGA ACT GAA GGA G -3' | 194 |
| HSD3B2 reverse | 5'- AGA CAT CAATGA TAC AGG CGG -3' | |
| StAR forward | 5'- CCA CCC CTA GCA CGT GGAT -3' | 88 |
| StAR reverse | 5'-TCCTGGTCA CTG TAG AGA GTCTCTTC-3' | |
| GAPDH forward | 5'- CGG GGCTCT CCA GAA CAT CAT CC -3' | 199 |
| GAPDH reverse | 5'- CGA CGC CTG CTT CAC CAC CTT CTT -3' | |
| ACTIN forward | 5'- AACTCCATCATGAAGTGTGACG -3' | 234 |
| ACTIN reverse | 5'- GATCCACATCTGCTGGAAGG -3' |
qRT-PCR, quantitative reverse transcription PCR.
phosphate-buffered saline that had been degassed so as to minimize exposure to oxygen before arriving in the laboratory. All manipula- tions were done under hypoxic conditions. Fetal adrenals were de- encapsulated, the two adrenals were combined, minced into small pieces, rinsed twice with Ca/Mg-free Hank’s balanced salt solution, digested with 44 mg dispase (Life Technologies, Carlsbad, CA), 20 mg collagenase type I (Worthington Biochemical, Lakewood, NJ) at 37℃ for 40 min, filtered through 100 um nylon mesh, layered onto 5ml ficoll-paque plus (GE Healthcare, Piscataway, NJ), and centrifuged at 600g for 30 min at room temperature on an IEC centraGP8R cen- trifuge (Thermo Fisher Scientific, Waltham, MA). The cells from the resulting interphase was collected and washed, resuspended in RPMI medium, and incubated in the hypoxic environment. Cells from each fetus were incubated separately in duplicate cultures grown in hypoxic or normoxic conditions; subsequent RNA preparations and analyses were done separately.
NCI-H295A cells were incubated under hypoxic conditions for 15 d and were split twice before the start of the experiment. After 15 d, six 10-cm plates of cells were moved from hypoxic conditions to an incubator with room air (normoxia). RNA was isolated from three plates after 1 d in normoxia and from the other three plates after 2 d in normoxia. RNA was also isolated from three control plates main- tained in hypoxia.
RNA Analysis
Total RNA was isolated using TRIzol (Life Technologies) according to the manufacturer’s recommended protocol. For cells kept in hypoxia, homogenization with TRIzol was done under hypoxic conditions. RNA was quantitated using an ND-1000 NanoDrop spectrophotom- eter (Thermo Scientific).
For real-time quantitative reverse transcription-PCR, 1 µg total RNA was reverse transcribed using Superscript II reverse transcrip- tase (Life Technologies), and PCR was performed at 94 °℃ for 5 min, followed by 40 cycles of 94 ℃ for 0.5 min, 55 ℃ for 0.5 min, and 72 ℃ for 1 min using primers and probes for the cholesterol side- chain cleavage enzyme (P450scc, encoded by CYP11A1), P450c17, 3ßHSD2, StAR, glyceraldehyde-3-phosphate dehydrogenase, and actin (Table 6). Reactions were performed in a total volume 25 ul con- taining 2 ul complementary DNA and 12.5 ul FastStart SYBR Green Master (Roche, Mannheim, Germany) on an iCycler iQ Real Time Detection System (Bio-Rad, Hercules, CA).
For microarray experiments, 300 ng total RNA was used to pro- duce biotin-labeled complementary RNA using Illumina TotalPrep RNA amplification kit (Life Technologies) according to the manu- facturer’s recommended protocol. The biotinylated complementary
Oxygen-induced perinatal adrenal changes
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RNA was eluted in nuclease-free water and was quantitated by NanoDrop spectrophotometer. Hybridization to the HumanHT-12 v4 BeadChip array (Illumina, San Diego, CA) was done at a com- plementary RNA concentration of 150 ng/ul in the UCSF Genomic Core Facility. The chips were scanned, and data were analyzed using Genome Studio Gene Expression Module (Illumina). Data were normalized using the “quantile” method of normalization in the software. Normalized data containing the signal levels and detection P values were exported into Microsoft Excel for gene expression analysis. The fold changes in gene expression levels were calculated as the gene signal levels under normoxic conditions divided by the signal levels under hypoxic conditions for control cells. The data are expressed as mean fold change in normoxia over control in hypoxia ± SD. Genes with signal detection of P > 0.05 in both normoxia and control groups were excluded from further analysis. An arbitrary fold change cutoff of >1.5-fold or <0.67-fold over control was chosen.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at http:// www.nature.com/pr
ACKNOWLEDGMENTS
We thank Emin Maltepe for productive discussions and for the use of the hypoxia chamber, Marcus Schoneman for helpful advice, and the staff and faculty at San Francisco General Hospital Women’s Options Center for assistance in the collection of human fetal tissues. V.A. is currently Assistant Professor, Centre for Microbial Biotechnology, Panjab University, Chandigarh, India.
STATEMENT OF FINANCIAL SUPPORT
This work was supported by the University of California-San Francisco Molecular Endocrinology Fund; J.Q. was supported by the School of Medi- cine, Shanghai Jiao Tong University, Shanghai, China.
Disclosure: The authors have nothing to disclose. The authors report no con- flict of interest.
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