Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
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ABSTRACT |
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Erythropoietin (Epo) produced by the
kidney regulates erythropoiesis. Recent evidence suggests that Epo in
the cerebrum prevents neuron death and Epo in the uterus induces
estrogen (E2)-dependent uterine angiogenesis. To elucidate
how Epo expression is regulated in these tissues, ovariectomized mice
were given E2 and/or exposed to hypoxia, and the temporal
patterns of Epo mRNA levels were examined. Epo mRNA levels in the
kidney and cerebrum were elevated markedly within 4 h after
exposure to hypoxia. Although the elevated level of Epo mRNA in the
kidney decreased markedly within 8 h despite continuous hypoxia,
the high level in the cerebrum was sustained for 24 h, indicating
that downregulation operates in the kidney but not in the brain.
E2 transiently induced Epo mRNA in the uterus but not in
the kidney and cerebrum. Interestingly, the uterine Epo mRNA was
hypoxia inducible only in the presence of E2. Thus Epo
expression appears to be regulated in a tissue-specific manner,
endorsing the tissue-specific functions of Epo.
estrogen; hypoxia; real-time polymerase chain reaction; angiogenesis; neuron survival
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INTRODUCTION |
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OXYGEN IS THE MAJOR
FACTOR that regulates production of erythropoietin (Epo)
(reviewed in Refs. 15 and 19). The hypoxic stimulation of
Epo production is largely due to the transcriptional activation of the
Epo gene, although the prolongation of mRNA half-life may also play a
partial role. The Epo gene contains the hypoxia-responsive enhancer in
the 3' flanking region, and hypoxia-inducible factor-1 (HIF-1) binds to
this enhancer under hypoxia, thereby activating the Epo promoter. HIF-1
is a basic-helix-loop-helix-PAS heterodimeric transcription factor
consisting of HIF-1 and aryl hydrocarbon receptor nuclear
translocator (ARNT). Whereas ARNT is relatively stable, HIF-1
is
rapidly degraded via the ubiquitin-proteasome pathway under normoxia.
Under hypoxia, HIF-1
is stabilized to form an active heterodimer
with ARNT. Details of these findings can be found in recent reviews
(4, 7, 33, 34, 37, 41) and references therein.
At least four production sites of Epo have been found: kidney, liver,
brain, and uterus. The kidney is a major production site in adults, and
the kidney-derived Epo is responsible for the stimulation of
erythropoiesis (15, 19). The liver produces Epo
essentially for fetal erythropoiesis (42). In the brain, there is a paracrine Epo/Epo receptor (EpoR) system that is independent of the endocrine system in erythropoiesis (5, 21-25,
27); neurons express EpoR (5, 24, 27), and
astrocytes produce Epo (22, 23, 25). Epo protects the
primary cultured cerebrocortical and hippocampal neurons from glutamate
toxicity, which is believed to be a major cause of ischemia-induced
neuron death (27). Epo prevents the ischemia-induced death
of cerebrocortical and hippocampal neurons in vivo (2, 35,
36). The Epo production by the cultured astrocytes (22,
25) and the brain Epo mRNA level (39) are also
enhanced by low oxygen tension. In the uterus, expression of Epo is
stimulated by 17-estradiol (E2), and Epo is implicated
in the uterine angiogenesis that occurs cyclically in the estrous cycle
(43). The effect of oxygen on the production of Epo in the
uterus is not known.
The existence of multiple Epo-producing sites with tissue-specific physiological functions raises important questions. Is the production of Epo stimulated by hypoxia in the uterus? Is the production of Epo in the kidney and brain stimulated by E2? Is there an interplay between two stimuli (E2 and hypoxia) for Epo production? Are the temporal patterns of stimuli-induced changes in the Epo mRNA level relevant to the assigned functions of Epo in these tissues? To answer these questions, we measured Epo mRNA quantitatively in the kidney, cerebrum, and uterus by using real-time reverse transcription-polymerase chain reaction (RT-PCR). This method has made it possible to quantify the basal levels of Epo mRNA, as well as the enhanced levels, in individual tissues. Here we report the effects of E2 treatment and exposure to hypoxia on the Epo mRNA contents in the ovariectomized (OVX) mouse.
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MATERIALS AND METHODS |
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Animals. Animals were maintained and handled in accordance with the guidelines for the care and use of laboratory animals at Kyoto University. Outbred mice of the ICR strain (Clea) overiectomized at 3 wk of age were used for experiments at 7-9 wk of age (35-40 g).
E2 administration and hypoxic exposure. E2 (Research Biochemicals International) was dissolved in olive oil. Each mouse was given 100 µl of E2 intraperitoneally. The control mice were given 100 µl of olive oil. For hypoxic stimulation, we used an air-tight cabinet into which the premixed gas was introduced. The gas flow rate was adjusted so that 7% O2 was achieved at ~30 min after the animals were placed into the cabinet. To examine the effects of E2 and hypoxia on Epo mRNA levels in the uterus, kidney, and cerebrum, OVX mice were treated with the following three conditions. The first group of animals was given E2 and was left under normoxia. The second group was exposed to normobaric hypoxia (7% O2/93% N2 ) without E2 administration. The third group was exposed to hypoxia immediately after E2 administration. At different time points after E2 administration and/or hypoxic exposure, the animals were anesthetized with ether, and blood was collected for the determination of serum Epo concentrations, and then the tissues were quickly removed and frozen in liquid nitrogen until used for RNA extraction.
Standard plasmid containing cDNA fragment of Epo or -actin.
Sequence coordinates of mouse Epo cDNA are based on the definition of
the transcription start site as +1 (38). A 451-bp fragment
encompassing 272-722 of the mouse Epo cDNA was ligated into a
vector pCR3.1-Uni using a Eukaryotic TA Cloning Kit (Invitrogen). The
resulting plasmid was used as a standard for PCR of Epo cDNA. As a
standard plasmid for
-actin cDNA, pAL41 (accession number X03765)
was used. These cDNA fragments contained the 112-bp (Epo) and 261-bp
(
-actin) nucleotide sequences, which correspond to the PCR products
amplified from the mRNA-derived cDNAs using the primers described in
the next section.
RT and real-time PCR. Total RNA was prepared from the frozen tissues according to the protocol of the RNA Isolation System kit (Promega). RT was carried out at 45°C for 60 min in 20 µl RT mixture containing 1 µg total RNA, 200 U reverse transcriptase (GIBCO-BRL), 20 U RNase inhibitor (Takara), 0.5 mM each dNTPs, and 2.5 µM random nonamer primer. One microliter of the RT mixture was used for real-time PCR.
The PCR product of Epo mRNA-derived cDNA was quantified in real time, using a double dye-labeled fluorogenic oligonucleotide probe (12) and an automated fluorescence-based system for detection of PCR products. The probe was labeled at its 5' end with a fluorogenic reporter dye, 6-carboxy-fluoresceine (FAM), and at its 3' end with a quencher dye, 6-carboxy-tetramethylrhodamine (TAMRA). The nucleotide sequence in the probe 5'-(FAM)-TGCAGAAGGTCCCAGACTGAGTGAAAATA-3'-(TAMRA) corresponds to 397-425 in the mouse Epo cDNA. This double dye-labeled probe was obtained from PE Applied Biosystems. The Epo-specific sequences used for PCR were the forward primer 371F, 5'-GAGGCAGAAAATGTCACGATG-3', and the reverse primer 482R, 5'-CTTCCACCTCCATTCTTTTCC-3'. The forward and reverse primers correspond to the nucleotides 371-391 and 462-482 in Epo cDNA, respectively. They span exon/intron boundaries (exons II and III in the 371F, and exons III and IV in the 482R); thus amplification of contaminating genomic DNA is prevented. For PCR, we used TaqMan Universal PCR Master Mix containing dUTP instead of dTTP (PE Applied Biosystems, cat. no. 4304437). This master mixture also contained uracil-N-glycosylase (UNG), which destroys any carryover PCR products. Before PCR was started, the complete PCR mixture containing reverse-transcribed cDNA was treated at 50°C for 2 min for the action of UNG and then at 95°C for 10 min to inactivate UNG and activate DNA polymerase. Then PCR, consisting of 50 cycles at 95°C for 15 s and 60°C for 1 min, was performed. When DNA polymerase engaged in extension of the primer reaches the quencher-labeled nucleotide of the probe hybridized to cDNA, the exonuclease activity of DNA polymerase excises the labeled nucleotide, resulting in the emission of fluorescence. All procedures, including data analysis, were performed on the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) using the software provided with the instrument. Messenger RNA ofCulture of the uterus from OVX mouse. Bilateral horns of the uterus from an OVX mouse were cut into two separate horns. One horn was cultured in medium containing a test substance, and the contralateral horn was cultured without the substance as a control. They were incubated for 6 h in a humid 5% CO2 atmosphere at 37°C in phenol red-free DMEM supplemented with 10% charcoal-treated FCS. Epo protein in the culture media and sera was measured with an enzyme-linked immunoassay by use of two monoclonal antibodies that bind Epo at different epitopes (31). Recombinant human Epo, produced and isolated as described previously (10, 11), was used as a standard. This assay measures Epo as low as 1 pg/ml.
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RESULTS |
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Measurement of Epo mRNA.
mRNA of Epo was measured by detecting its reverse-transcribed cDNA with
the real-time PCR. To draw the standard curve of Epo cDNA, twofold
serial dilutions of the standard plasmid containing the Epo cDNA
fragment were subjected to PCR. By plotting the cycle threshold values
(CT) (12) against the log of the copy number of the plasmid, we obtained a linear curve (Fig.
1). The lower limit of quantitative
detection was ~10 copies of cDNA. Figure 1 also shows the results of
quantitative measurement of Epo mRNA in the uterus, where Epo mRNA is
markedly induced upon E2 administration, as described
later. The extracts prepared from the uterus of E2-treated and E2-untreated mice were subjected to RT. Epo cDNA in
three preparations (nondiluted, 2-fold diluted, and 4-fold diluted) of
each RT sample was amplified, and the CT values were
plotted on the standard curve. The CT values increased in
proportion to the dilution both with and without E2
treatment. The copy value of Epo mRNA in the uterus from OVX mice
(n = 6) without E2 treatment was calculated
to be 3.8 ± 0.8 × 104. To check the
reproducibility of the PCR, we did an intra-assay study in which four
samples containing Epo or -actin cDNA at different concentrations
were assayed by six runs at one time. We also performed an interassay
study in which the four samples were assayed on six different days. In
both studies, the coefficients of variation were satisfactory (<15%).
The ratio of basal levels of Epo mRNA in the kidney, cerebrum, and
uterus from OVX mice without stimulation was ~370:3:1. Hereafter, the
Epo mRNA levels induced by E2 and hypoxia are expressed as
values relative to the basal levels; Epo mRNA copy number per microgram
of total RNA in each tissue from the control animals without treatment was defined as 1.
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Hypoxia induces a marked increase of Epo mRNA in the kidney and
cerebrum, and downregulation operates in the kidney but not in the
cerebrum.
To examine the effect of E2 on the Epo mRNA level in the
kidney and cerebrum, OVX mice were given E2 or olive oil,
and they were immediately exposed to hypoxia (7% O2) or
kept under normoxia. At various time points, the tissues were removed
to measure the amount of Epo mRNA. As Fig.
2A shows, hypoxia markedly
induced Epo mRNA in the kidney, which is in agreement with previous
results (reviewed in Refs. 15 and 19). However, the Epo
mRNA level was quickly lowered despite continuous hypoxia: the level at
8 h after hypoxic stimulation was reduced to 30% of the maximum induction found at 2 h. E2 showed no significant
effect on the Epo mRNA level in the kidney under normoxia and hypoxia.
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E2 induces a transient increase of Epo mRNA in the
uterus, and the uterus responds to hypoxia only in the presence of
E2.
In contrast to the kidney and cerebrum, administration of
E2 increased Epo mRNA in the uterus. The
E2-derived increase was dose dependent; 5 and 15 µg
(E2/kg body wt) caused 10- and 14-fold increases of Epo
mRNA at 3 h after administration, respectively, and the
enhancement reached a plateau at 50-100 µg E2 with a
20-fold increase. Figure 4 shows the
time-dependent changes of Epo mRNA level in the uterus. To ensure
maximal induction, a dose of 500 µg/kg was used. There was a marked
increase in Epo mRNA level at 2 h after E2
administration, but the level markedly decreased at 8 h. At
24 h after E2 administration, the Epo mRNA level was similar to that in the control mice given olive oil (not shown). The
Epo mRNA level in the uterus of animals without E2
treatment was unchanged by exposure to hypoxia. Surprisingly, the Epo
mRNA level in the uterus of animals given E2 was increased
under hypoxia. Although this hypoxia-induced increase (2.5-fold) was
small compared with the dramatic increase (30-fold) of Epo mRNA in the
kidney and brain by hypoxia (see Fig. 2), the combination of
E2 and hypoxia caused a 50-fold increase of Epo mRNA in the
uterus. The Epo mRNA level in the uterus of animals exposed to hypoxia
also markedly decreased at 8 h after E2
administration, like that in animals under normoxia. Such a transient
increase of Epo mRNA in the uterus was also seen at the low dose of 5 µg E2/kg (data not shown).
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Induction of Epo mRNA in the uterus by E2 is transient.
To determine whether the rapid reduction in the level of Epo mRNA in
the uterus after E2 administration is attributable to the
metabolic depletion of E2 or to downregulation of the
cellular response to E2, the effects of readministration of
E2 on the induction were examined. Figure
5 shows the results. A high increase in the level of Epo mRNA was seen at 4 h after treatment with 50 µg
E2/kg, but at 8 h the level markedly decreased
(experiments 2 and 3). E2 was
readministered at 4 h after the first treatment, and Epo mRNA in
the uterus was measured at 4 h after readministration (experiment 4). Readministration of E2 did not
increase the Epo mRNA; the level was similar to that at 8 h after
the first administration (compare experiments 3 and
4), if we exclude the possibility that the rapid reduction
of the Epo mRNA level is caused by the deficiency of E2. At
120 h after the first treatment with E2, the level of Epo mRNA in the uterus was low, similar to that of the control mice
(experiment 5). When E2 was readministered at
120 h after the first treatment and Epo mRNA was assayed at 4 h after readministration, a high level of Epo mRNA was induced
(experiment 6). These results suggest that the response of
Epo-producing cells in the uterus to E2 is quickly
downregulated after administration of E2 and that these
cells have restored their responsiveness to E2 at 120 h.
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E2 and tamoxifen induce Epo production by the in vitro
cultured uterus.
In addition to oxygen concentration, some substances have been shown to
influence Epo production; for example, thyroid hormones enhance
hypoxia-induced Epo production by the perfused rat kidney and HepG2
cells (9), and Epo production by cultured astrocytes is
stimulated by insulin, insulin-like growth factor (IGF) I, and IGF-II
(23). To examine whether or not various nuclear receptor ligands and these growth factors modulate Epo production by the in
vitro cultured uterus, Epo was measured in the medium after culture of
the uterus with or without test substances. There was significant Epo
production in the culture with E2, but the production was
undetectable without E2 (Fig.
6). Tamoxifen, which is believed to
function as an antiestrogen in breast tissue but acts as an E2-like ligand in uterine tissue (17), induced
Epo production. E2- or tamoxifen-induced production of Epo
was inhibited by ICI-182780, a specific E2 receptor
(ER) antagonist (40), which alone showed no effect. Epo
production was not induced by culture with progesterone, testosterone, 3,3',5-triiodo-L-thyronine,
L-thyroxin, all-trans retinoic acid, vitamin
D3, insulin (all at 1 µM), IGF-I (100 nM), and IGF-II
(100 nM) (data not shown). These results suggest that E2
induces Epo production specifically in the uterus and that the
production is mediated by ER.
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DISCUSSION |
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In adults, the kidney is the major production site of Epo,
stimulating erythropoiesis (15, 19). Recently the brain
and uterus have also been shown to produce Epo. Epo in the brain
functions as a neurotrophic factor (2, 35, 36), and Epo in
the uterus is implicated in angiogenesis, which is under the control of
E2 (43). Epo production in the kidney
(15, 19) and brain (22, 25, 39) is hypoxia
inducible, whereas that in the uterus is E2 inducible
(43). Taken together with our findings of regulatory properties of Epo production in the kidney, cerebrum, and uterus, we
propose that the regulation of Epo production is tissue specific, conformable to the specific functions of Epo produced by the individual tissues (Table 1).
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E2 had no significant effect on the Epo mRNA level in the kidney (Fig. 2A) or on the Epo level in the serum (data not shown). Contradictory results of the effects of E2 on serum Epo have been reported; one laboratory reported that E2 repressed (26), and another reported that E2 potentiated the hypoxia-induced elevation of serum Epo (1). In these studies, mice were exposed to hypoxia before assay to elevate the serum Epo to an assayable level. Thus the previous results cannot be directly compared with our results.
Downregulation in the hypoxic induction of the Epo mRNA in the kidney and the Epo level in the serum, despite continued hypoxia, has been well documented (8, 20, 39). The direct feedback inhibition (Epo represses its production) has been excluded as a mechanism of downregulation (8). Although the mechanism remains to be studied, the different temporal patterns of the induction of Epo mRNA by hypoxia in the kidney and cerebrum provide physiologically important implications. In the brain, Epo supports neuronal survival under hypoxia, and therefore Epo expression needs to be sustained at a high level as long as hypoxia continues. In contrast, continuous activation of Epo gene expression in the kidney overproduces erythrocytes, causing various disorders.
E2-induced accumulation of the Epo mRNA in the uterus does not require de novo protein synthesis, and the accumulation is inhibited by actinomycin D, suggesting that this accumulation is due to transcriptional activation of the Epo gene by E2 (43). The palindromic consensus sequence of the E2 response element (AGGTCAXXXTGACCT) is not present, but its half-site exists in the 5' flanking region of both mouse and human Epo genes (3). Imperfect E2 response elements, including the half-site, have been shown to confer E2 responsiveness to target genes (16, 28). Expression of the reporter gene flanked by a 5' flanking region of Epo gene was activated by E2, and this activation required the ER (unpublished observations), indicating that the E2-induced increase of Epo mRNA in the uterus is at least partly attributable to transcriptional activation of the Epo gene.
Blood vessel formation and the concomitant remodeling of uterine endometrium take place every 3-5 days in the murine estrus cycle. The downregulation of the Epo mRNA level in the uterus that occurred shortly after administration of E2 is due to the loss of the cellular response to E2. At 5 days after treatment with E2, however, the Epo-producing cells regained their response to E2. Thus the downregulation of the responsiveness to E2 of the Epo-producing cells in the uterus may be very important for preventing uterine angiogenesis in an estrous cycle stage where it should not occur. The cellular degradation of ER is stimulated by E2 (13). The half-life of ER is ~5 days in the absence of E2, but in the presence of E2 it is degraded to a half-life of 3-4 h (30, 32). The ubiquitin-proteasome pathway has been shown to mediate E2-induced degradation of ER (29, 30). It is possible that the ligand-induced ER degradation serves to downregulate the expression of E2-responsive genes, including the Epo gene.
Although the magnitude (2.5-fold) of hypoxic induction of Epo mRNA in the uterus in the presence of E2 was much smaller than that (30-fold) in the kidney and brain, this hypoxic induction in the uterus may be physiologically significant, because the uterine endometrium would be hypoxic in the proestrus stage, when the ovary produces E2 actively and the endometrium grows rapidly. Interestingly, little hypoxic induction was found in the absence of E2. A multiprotein complex including HIF-1, hepatocyte nuclear factor-4, and p300/cAMP-responsive element binding protein is involved in the hypoxic induction of the Epo gene expression (6). The hypoxic inducibility may depend on the cellular availability of these components.
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ACKNOWLEDGEMENTS |
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This work was supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan, from the "Research for the Future" program in The Japan Society for the Promotion of Science, and from the Japan Program for Promotion of Basic Research Activities for Innovative Biosciences.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Sasaki, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto Univ., Kyoto 606-8502, Japan (E-mail: rsasaki{at}kais.kyoto-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 April 2000; accepted in final form 10 July 2000.
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