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Progesterone, but not 17β-estradiol, up-regulates erythropoietin (EPO) production in human amniotic epithelial cells
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 96, No. 5, 448–453. 2003
Progesterone, but Not 17b-Estradiol, Up-Regulates Erythropoietin (EPO) Production in Human Amniotic Epithelial Cells AKIKO OGAWA,1 SATOSHI TERADA,1* NORIO SAKURAGAWA,2 SEIJI MASUDA,3 MASAYA NAGAO,3 AND MASAO MIKI1 Department of Applied Chemistry and Biotechnology, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan,1 Department of Inherited Metabolic Disorders, National Institute of Neuroscience, NCNP, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan,2 and Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-Cho, Sakyo-ku, Kyoto 606-8502, Japan3 Received 2 May 2003/Accepted 16 August 2003
Human amniotic epithelial (HAE) cells have great potential for successful use in cell therapy, since they do not cause acute rejection upon allotransplantation. However, to date, HAE cells have not well been studied. We previously reported that HAE cells produce erythropoietin (EPO), which is known to be a regulator of hematopoiesis, and that the induction mechanism of HAE cells is unknown, although EPO production from HAE cells is not increased by hypoxia which induces several cell types to produce EPO. In this study, we determined whether female sex hormones, including progesterone and 17b-estradiol, affect the EPO production of HAE cells. Bioactive measurement of EPO activity in the culture supernatants of HAE-SV40 cells, which were immortalized by transfection with a simian virus 40 large T antigen, revealed that EPO bioactivity was significantly increased by treatment with progesterone, but not 17b-estradiol. Treatment of HAE-SV40 cells with progesterone transiently increased the EPO mRNA level by fivefold, while there was no change in response to 17b-estradiol. Furthermore, the progesterone receptor (PR)-B was detected in both HAE cells and HAE-SV40 cells by Western blotting. These results suggest that EPO synthesis in HAE-SV40 cells is stimulated by progesterone, but not by 17b-estradiol, and thus it is highly likely that the EPO synthesis of HAE cells is also regulated by progesterone. [Key words: human amniotic epithelial cells, erythropoietin, progesterone, 17b-estradiol, progesterone receptor]
stimulates the differentiation, proliferation and survival of erythroid precursors via binding to the EPO receptor (EPO-R), whereby it regulates the amount of red blood cells. When the concentration of oxygen in the blood decreases, EPO transcription is elevated. Recently, it was revealed that there are EPO/EPO-R systems not only in the hematopoiesis system, but also in the brain, uterus and oviduct. In the brain, astrocytes produce EPO to protect the neuronal cells from ischemia-induced cell death and the production is increased by hypoxia (7– 11). In the uterus, the endothelial cells of the endometrium produce EPO that induces the angiogenesis and proliferation of the endometrium, and the EPO synthesis depends on 17b-estradiol and hypoxia in the presence of this hormone (12, 13). EPO production in the oviduct is increased by 17b-estradiol and/or hypoxia, but the function of EPO released from this tissue is not clearly understood (14). EPO has also been detected in the placenta (15). In many mammalian species, such as goats, sheep and monkeys, with the exception of mice, the placenta is an effective barrier against the passage of EPO from the pregnant woman to the fetus, and also in the reverse direction (16). Furthermore, fetal EPO is produced independently from the maternal EPO production (17). These reports suggest that the origin of the EPO in the amniotic fluid must be the fetal organs
The amnion is a strong translucent membrane which contains no vessels, and when it is transplanted to a recipient, it does not incur acute allograft rejection. Due to this, the amnion has been applied to surgical regions and ophthalmologic regions. For example, it has been used as a dressing in surgery (1) and dermatology, and as a substitute for the cornea to cure some diseases such as Stevens–Johnson syndrome (2). In 1982, Adinolfi et al. found that human amniotic epithelial (HAE) cells produce lysosomal enzymes including a-induronidase, b-galactosidase and sphingomyelinase, and that culture supernatants of HAE cells were capable of correcting the abnormal accumulation in cultured fibroblasts from patients with Hurler’s syndrome (3). Moreover, Sakuragawa and coworkers found that HAE cells synthesize and release acetylcholine, catecholamines and neurotrophic factors (4–6). These studies suggest that HAE cells would be a promising source for the supply of the enzymes mentioned above and would contribute to the achievement of cell therapy in patients with specific enzyme deficiencies. Erythropoietin (EPO) is synthesized and released mainly from the kidney in adults and from the liver in fetuses. It * Corresponding author. e-mail: [email protected] phone: +81-(0)776-27-8645 fax: +81-(0)776-27-8747 448
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or fetal membrane. In 2000, Matsuura et al. reported that EPO and EPO-R are expressed in HAE cells (18). In 2001, Kim et al. reported that both EPO and its receptor are expressed in ovine fetal membranes, amnion and chorion (19). However, the functions of EPO in the placenta are still unknown. Progesterone is the female sex hormone produced in the late phase of the menstrual cycle by the corpus luteum of the ovary. When implantation succeeds, the corpus luteum continues to secrete progesterone under the influence of luteinizing hormone and prolactin for the early stage of pregnancy. After eight weeks, the placenta produces progesterone in great quantities so as to maintain the pregnancy. Progesterone, as a transcriptional regulator, is mediated by two distinct forms of the progesterone receptor (PR), PR-A and PR-B, which arise from alternative splicing (20). In most cells, including the human amnion, PR-A acts as a transcriptional inhibitor of all steroid hormone receptors, while PR-B acts as a transcriptional activator of progesterone-responsive genes (21, 22). We previously reported that EPO production in HAE cells was not stimulated by hypoxia (23). We hypothesized that HAE cells would have a similar regulatory system for EPO synthesis to the uterus or oviduct where EPO synthesis is regulated by 17b-estradiol, but different from the kidney/ bone marrow system. In the present study, we determined whether female sex hormones stimulate EPO synthesis in HAE cells. MATERIALS AND METHODS Cell lines Human placentas were obtained from uncomplicated elective cesarean sections and informed consent was obtained in accordance with the requirements of several hospitals in Kodaira, Japan. HAE cells were prepared according to the method described previously (24). Because the supply of HAE cells was insufficient, immortalized HAE cells, HAE-SV40, were also used. HAE-SV40 cells were generated by transfection with an origindefective simian virus (SV) 40 large T antigen (25). The MCF-7 cell line was obtained from the Health Science Research Resources Bank (Osaka). F-36E, an EPO-dependent cell line, was obtained from the RIKEN Cell Bank (Tsukuba). Bioassay of HAE-SV40 culture supernatants HAE-SV40 cells were cultured in SF-O2 serum-free medium (Sanko, Tokyo) containing either 1 mM 17b-estradiol (Sigma, St. Louis, MO, USA), 3 mM progesterone (Sigma), or no additive in a 5% CO2 atmosphere at 37°C. The culture supernatants of HAE-SV40 cells were collected and condensed by centrifugation (Ultrafree-MC Centrifugal Filter Units; Millipore, Billerica, MA, USA). Each supernatant was then re-diluted with RPMI 1640 medium (Nissui, Tokyo) containing 10% fetal bovine serum (FBS) to the same volume as before the condensation. F-36E cells were cultured in these media containing 10% FBS supplemented with a concentrated conditioned medium for the EPO bioassay. Purified recombinant EPO reagent (Kirin, Tokyo) was used as a positive control. An anti-EPO antibody was used for neutralizing the effect of EPO. The viabilities of the F-36E cells were determined by counting in a hemacytometer under a phase contrast microscope using the trypan blue exclusion assay. Proliferation assay of HAE-SV40 cells treated with progesterone HAE-SV40 cells were seeded into 24-well culture plates and cultured for 9 d in RPMI 1640 medium supplemented with 10% FBS with or without progesterone. The viability was deter-
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mined by counting in a hemacytometer under a phase contrast microscope using the trypan blue exclusion assay. RT-PCR To determine the effect of 17b-estradiol or progesterone, HAE-SV40 cells were cultured with 17b-estradiol (240 mM) or progesterone (3.2 mM) for 0, 1, 4, and 24 h in phenol red-free RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 15% charcoal-treated FBS. Total RNA was extracted from the cells using the acid guanidinium/phenol extraction method. Total RNA (5 mg) was heated to 70°C for 10 min before reverse transcription (RT). First strand cDNA synthesis was performed using an oligo(dT)15 primer and SuperScript™ II RT (Invitrogen, Carlsbad, CA, USA). The reaction was carried out at 42°C for 50 min in an iCycler thermal cycler (Bio-Rad, Hercules, CA, USA). To amplify the G3PDH target cDNA fragment, 50 ml of the PCR reaction mixture contained 1 ml of RT reaction product, 1.5 mM MgCl2, 0.2 mM dNTP mixture, 1.25 U of Taq DNA polymerase (Toyobo, Osaka) and 0.2 mM primers. The primers were 5¢-ACCACAGTCC ATGCCATCAC-3¢ (sense) and 5¢-TCCACCACCCTGTTCCTGTA3¢ (antisense). We performed 19 amplification cycles with 1 min of initial denaturation at 94°C, followed by cycles of 30 s at 94°C for denaturation, 30 s at 64°C for annealing and 1 min at 72°C for elongation. The terminal polymerase reaction time was 10 min at 72°C. To amplify the EPO target cDNA fragment, 50 ml of the PCR reaction mixture contained 1 ml of RT reaction product, 1.5 mM MgCl2, 0.2 mM dNTP mixture, 1.25 U of Taq DNA polymerase and 0.5 mM primers. The primers were 5¢-GCCAGAGGAACTGT CCAGAG-3¢ (sense) and 5¢-ATGGTAGGTGCGAAAACAGG-3¢ (antisense). We performed 28 amplification cycles with 1 min of initial denaturation at 94°C, followed by cycles of 30 s at 94°C for denaturation, 30 s at 61.7°C for annealing and 2 min at 72°C for elongation. The terminal polymerase reaction time was 10 min at 72°C. To detect the progesterone receptor cDNA fragment, 50 ml of the PCR reaction mixture contained 1 ml of RT reaction product, 1.5 mM MgCl2, 0.2 mM dNTP mixture, 1.25 U of HotStarTaq™ DNA polymerase (Qiagen, Valencia, CA, USA) and 0.4 mM primers. The primers were 5¢-GTCAGTGGGCAGATGCTGTA-3¢ (sense) and 5¢-AGCCCTTCCAAAGGAATTGT-3¢ (antisense). The reaction reagent was heated at 95°C for 15 min according to the protocol of the HotStarTaq™ DNA polymerase system. We then performed 40 amplification cycles with 1 min of initial denaturation at 94°C, followed by cycles of 30 s at 94°C for denaturation, 1 min at 53°C for annealing and 1 min at 72°C for elongation. The terminal polymerase reaction time was 10 min at 72°C. The expected band lengths were 452 bp for G3PDH, 207 bp for EPO, and 194 bp for the progesterone receptor. The PCR products were visualized in 2% agarose gels stained with ethidium bromide. Quantitative analysis of EPO mRNA EPO mRNA was reverse-transcribed and amplified by the RT-PCR method as described above. The products were quantified using a microchip electrophoretic analysis system (Hitachi, Tokyo). Western blotting MCF-7, HAE and HAE-SV40 cells were suspended in M-PER® Mammalian Protein Extraction Reagent (Pierce, Milwaukee, WI, USA) with protease inhibitors (Nacalai Tesque, Kyoto). An aliquot of 20 ml of each culture supernatant of these cells was separated by 8% SDS–PAGE and probed with a rabbit polyclonal antibody against human progesterone receptors A and B (Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated secondary antibodies (Chemicon International, Temecula, CA, USA) were used and immunoreactive bands were visualized using the Enhanced Chemiluminescence System (ECL plus; Amersham Bioscience, Piscataway, NJ, USA) on Polaroid film in an A-cassette apparatus (Fuji Film, Tokyo).
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RESULTS AND DISCUSSION Effect of female sex hormones on the EPO production of HAE-SV40 cells The concentration of female sex hormones, such as progesterone and 17b-estradiol, in the blood plasma of pregnant women increases markedly as pregnancy progresses, and most of the female sex hormones are produced in the placenta. Therefore, we hypothesized that such female sex hormones have important roles in the amniotic metabolism system and that they are involved in the EPO production by HAE cells. To test this hypothesis, we determined whether these hormones increased the EPO production of HAE-SV40 cells. HAE-SV40 cells were cultured in serum-free medium in the absence or presence of progesterone (3 mM) or 17b-estradiol (1 mM). The total EPO protein quantities in the culture supernatants of HAE-SV40 cells were measured by the EPO bioactivity. F-36E cells, known as EPO-dependent cells, were cultured in medium including the culture supernatant of HAE-SV40 cells and at the same time, they were cultured with or without the EPO reagent for several days. Then we assessed the EPO activities in the culture supernatants by measuring the viability of the F-36E cells and the result is shown in Fig. 1. The viability of the F-36E cells treated with the culture supernatant of HAE-SV40 cells was higher than that of the negative control. Treatment with the culture supernatant of HAE-SV40 cells cultured in the presence of progesterone increased the viability of F-36E cells significantly more than treatment with that of HAE-SV40 cells cultured in the absence of progesterone. The anti-EPO neutralizing antibody completely neutralized the effect of 0.1 U/ml of purified EPO on the survival of F-36E cells. In contrast, the anti-EPO antibody also neu-
FIG. 1. Effect of progesterone on the EPO production of HAESV40 cells. HAE-SV40 cells were cultured in SF-O2 medium with or without progesterone (3 mM) for 3 d. On reaching confluence, the culture supernatants were collected, concentrated once and then rediluted with the medium used for F-36E culture. F-36E cells were seeded at 20,000 cells/ml and cultured with (closed bars) or without (open bars) an anti-EPO antibody (2 mg/ml). After a 5-d culture, the viability of the F-36E cells was measured under a microscope using a hemocytometer and the trypan blue exclusion assay. The data indicate the mean viabilities of the F-36E cells. 1, Negative control; 2, addition of the culture supernatant of HAE-SV40 cells; 3, addition of the culture supernatant of HAE-SV40 cells treated with progesterone; 4, addition of EPO solution (0.1 U/ml). Ab represents the anti-EPO antibody. Error bars: SD (n = 3).
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TABLE 1. The effect of 17b-estradiol on the EPO production of HAE-SV40 cells Culture condition Viability of F-36E (%) SD (%) 1 26.1 1.4 2 32.0 2.9 3 33.2 1.9 4 88.7 0.4 HAE-SV40 cells were cultured in SF-O2 medium with or without 17b-estradiol (1 mM) for 6 d. On reaching confluence, the culture supernatants were collected, concentrated and then rediluted with the medium used for F-36E culture. F-36E cells were seeded at 18,400 cells/ml and cultured in medium including the culture supernatant of HAE-SV40 cells treated with or without 17b-estradiol, or EPO solution (0.25 U/ml), or neither. After a 3-d culture, the viability of F-36E cells was measured under a microscope using a hemocytometer and trypan blue exclusion assay. The data represent the mean viabilities of F-36E cells. 1, Negative control; 2, addition of the culture supernatant of HAE-SV40 cells; 3, addition of the culture supernatant of HAESV40 cells treated with 17b-estradiol; 4, addition of EPO solution.
tralized, but not completely, the effect in the culture supernatant of HAE-SV40 cells cultured with progesterone on the survival of F-36E cells. Moreover, the viabilities of F-36E cells treated with the supernatant of HAE-SV40 cells cultured both in the presence or absence of progesterone were higher than that of the negative control when F-36E cells were cultured with the anti-EPO antibody. These results suggest that the EPO activity in the culture supernatant of HAE-SV40 cells was increased by progesterone, and that HAE-SV40 cells secreted some factor or factors which functioned as a growth factor(s) for F-36E cells independent of progesterone. However, there was no significant effect of 17b-estradiol on the EPO activity of the culture supernatant of HAESV40 cells (Table 1), which suggests that progesterone, but not 17b-estradiol, increased the EPO production of HAESV40 cells. Effect of progesterone on the proliferation of HAESV40 cells There is another possible explanation for how progesterone increased the EPO production of HAE-SV40 cells. This possible mechanism was that progesterone stimulated the proliferation of HAE-SV40 cells with a concomitant increase in the amount of EPO in the culture. To exclude this possibility, we determined whether progesterone increases the cell population of HAE-SV40 cells. HAE-SV40 cells were seeded in the medium with or without progesterone at 14,400 cells/well, a much lower density than used in other experiments in the present study to determine conclusively the effect of progesterone on cell proliferation. When the cells were seeded at this lower density, the culture period was prolonged so that any mitogenic effect was more easily detected. The HAE-SV40 cell line proliferates very slowly and divides approximately every 3 d. Thus the culture period in this experiment was as long as 9 d. After a 9-d culture period, we determined the viable cell numbers. There were no significant differences in the viable cell numbers among the various cultures (Fig. 2), which indicated that progesterone did not stimulate the proliferation of HAE-SV40 cells at this concentration range. These results suggest that progesterone directly stimulates the EPO production pathway of HAE-SV40 cells, with no effect on
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FIG. 2. Effect of progesterone on the proliferation of HAE-SV40 cells. HAE-SV40 cells were seeded at 14,400 cells/well and cultured with or without progesterone (300 nM, 3000 nM) for 9 d. Error bars: SD (n = 3).
the proliferation. Effect of female sex hormones on the EPO production of HAE-SV40 cells at the EPO mRNA transcription level To determine whether progesterone or 17b-estradiol increases the EPO transcription in HAE-SV40 cells, we studied the time course of EPO mRNA transcript production in HAE-SV40 cells treated with progesterone (3 mM) or 17b-estradiol (240 mM). Total RNA transcripts derived from HAESV40 cells were extracted at specific intervals after the addition of the hormone. The EPO and G3PDH mRNAs were measured semiquantitatively by setting the PCR cycles to be approximately proportional to the band intensity of the amplified cDNA. We used G3PDH as a control of the total RNA amount because it is one of the housekeeping genes and expressed constitutively. Figure 3a shows the EPO mRNA- and G3PDH mRNA-derived products from HAESV40 cells. There was a distinct increase in the EPO mRNA at 4 h after the progesterone treatment, but not after the 17b-estradiol treatment. The products were quantified using a microchip electrophoretic analysis system (Hitachi). Figure 3b shows the quantities of EPO mRNA at 0 h, 1 h, 4 h and 24 h after the addition of a female sex hormone to the HAE-SV40 culture. When HAE-SV40 cells were treated with progesterone, the amount of EPO mRNA was increased after 1 h and reached a maximum of 5-fold the level of the control at 4 h. Twenty-four hours later, the amount of EPO mRNA was reduced almost to the level in the cells not treated with progesterone. A small increase in EPO mRNA was observed when HAE-SV40 cells were treated with 17b-estradiol. These results indicate that progesterone upregulates EPO transcription in HAE-SV40 cells, while 17b-estradiol does not. Detection of the progesterone receptor on HAE and HAE-SV40 cells It is commonly thought that steroid hormones bind to a specific receptor on a target cell and then function as transcription regulators. Thus, HAE-SV40 cells should express the PR for progesterone to be able to regulate their EPO synthesis. To determine whether HAE-SV40 cells and the wild-type HAE cells present PR, we used an
FIG. 3. Time course of the EPO transcription activity of HAESV40 cells treated with progesterone or 17b-estradiol. At intervals after the addition of progesterone (3 mM) or 17b-estradiol (240 mM) to the HAE-SV40 culture, EPO mRNA and G3PDH mRNA were amplified by RT-PCR, as described in Materials and Methods. These products were measured quantitatively using a microchip electrophoretic analysis system. (a) Result of agarose gel electrophoresis. Lane C indicates the control. The upper panel shows EPO mRNA-derived RT-PCR products (207 bp) and lower panel shows G3PDH mRNA-derived RTPCR products (452 bp). (b) Result of quantitative measurement of the EPO mRNA product. Open bars indicate the amount of EPO mRNA product derived from HAE-SV40 cells treated with 17b-estradiol. Closed bars indicate the amount of EPO mRNA product derived from HAE-SV40 cells treated with progesterone.
RT-PCR method and a Western blotting method. Figure 4a shows that PR mRNA- and G3PDH mRNA-derived products were observed in both HAE cells and HAESV40 cells, and in the MCF-7 cell line as a positive control which is known to present PR on its surface. HAE cells and HAE-SV40 cells were cultured in phenol red-free RPMI 1640 medium supplemented with 15% charcoal-treated FBS to remove estrogenic activity. Phenol red has estrogenic activity and charcoal treatment removes estrogen from the serum. PR mRNA-derived bands were observed for all of the cell types, but the intensities of the bands were different. The band intensity for HAE cells was more than threefold that for HAE-SV40 cells. HAE-SV40 cells expressed less PR than HAE cells and this may be due to immortalization which often decreases the functions of cells. Therefore, HAE-SV40 cells might exhibit decreased PR synthesis during immortalization, but this is unclear. Figure 4b shows the result of the Western blotting analysis. HAE and HAE-SV40 cells were cultured in RPMI 1640 medium including 10% FBS. For HAE-SV40 cells, the PR-B band was detected but not the PR-A band. For HAE cells, the PR-B band was distinctly detected, and the PR-A band
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and that progesterone might regulate EPO production in the human amnion in vivo. ACKNOWLEDGMENTS We thank Dr. H. Yoshimoto of Kirin Brewery Co. Ltd. for the generous gift of the EPO. We also thank Dr. I. Kano, Department of Toxicology, The Tokyo Metropolitan Research Laboratory of Public Health, for informative technical advice regarding SDS– PAGE. This work was partly supported by a Sasagawa Scientific Research Grant to Ms. A. Ogawa.
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FIG. 4. Detection of the PR on HAE cells and HAE-SV40 cells. (a) Detection of the PR mRNA product by RT-PCR. The process is described in Materials and Methods. The upper panel shows PR mRNA-derived RT-PCR products (194 bp) and the lower panel shows G3PDH mRNA-derived RT-PCR products (452 bp). (b) Detection of PR-A and -B by Western blotting analysis. PR-A indicates progesterone receptor-A, and its Mw is approximately 82 kDa. PR-B indicates progesterone receptor-B, and its Mw is approximately 120 kDa. MCF-7 is a mammary carcinoma cell line known to express PR-A and -B.
was also detected but the intensity was much weaker than that of the PR-B band. Moreover, the PR-B band intensity for HAE cells was about twofold than that for HAE-SV40 cells, which indicated that HAE cells expressed PR-B more strongly than HAE-SV40 cells. This result corresponded with the result of the RT-PCR. When HAE cells and HAE-SV40 cells were cultured in phenol red-free RPMI 1640 medium supplemented with 15% charcoal-treated FBS, the PR expression pattern was the same (data not shown). According to the report by Rossmanith et al. (26), the developing human placenta expresses the progesterone receptor but not the estrogen receptor. We failed to detect estrogen receptor mRNA by RT-PCR in either HAE or HAESV40 cells, although it was detected in HeLa cells as a positive control. Therefore, the estrogen receptor is not involved in the EPO production of HAE cells. We investigated the effect of progesterone and 17b-estradiol on EPO production in HAE cells. Treatment with progesterone increased EPO bioactivity in the culture supernatant and the EPO mRNA level, but 17b-estradiol did not. The results of this research showed that progesterone, but not 17b-estradiol, enhanced EPO synthesis in HAE cells
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Progesterone, but Not 17b-Estradiol, Up-Regulates Erythropoietin (EPO) Production in Human Amniotic Epithelial Cells AKIKO OGAWA,1 SATOSHI TERADA,1* NORIO SAKURAGAWA,2 SEIJI MASUDA,3 MASAYA NAGAO,3 AND MASAO MIKI1 Department of Applied Chemistry and Biotechnology, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan,1 Department of Inherited Metabolic Disorders, National Institute of Neuroscience, NCNP, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan,2 and Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-Cho, Sakyo-ku, Kyoto 606-8502, Japan3 Received 2 May 2003/Accepted 16 August 2003
Human amniotic epithelial (HAE) cells have great potential for successful use in cell therapy, since they do not cause acute rejection upon allotransplantation. However, to date, HAE cells have not well been studied. We previously reported that HAE cells produce erythropoietin (EPO), which is known to be a regulator of hematopoiesis, and that the induction mechanism of HAE cells is unknown, although EPO production from HAE cells is not increased by hypoxia which induces several cell types to produce EPO. In this study, we determined whether female sex hormones, including progesterone and 17b-estradiol, affect the EPO production of HAE cells. Bioactive measurement of EPO activity in the culture supernatants of HAE-SV40 cells, which were immortalized by transfection with a simian virus 40 large T antigen, revealed that EPO bioactivity was significantly increased by treatment with progesterone, but not 17b-estradiol. Treatment of HAE-SV40 cells with progesterone transiently increased the EPO mRNA level by fivefold, while there was no change in response to 17b-estradiol. Furthermore, the progesterone receptor (PR)-B was detected in both HAE cells and HAE-SV40 cells by Western blotting. These results suggest that EPO synthesis in HAE-SV40 cells is stimulated by progesterone, but not by 17b-estradiol, and thus it is highly likely that the EPO synthesis of HAE cells is also regulated by progesterone. [Key words: human amniotic epithelial cells, erythropoietin, progesterone, 17b-estradiol, progesterone receptor]
stimulates the differentiation, proliferation and survival of erythroid precursors via binding to the EPO receptor (EPO-R), whereby it regulates the amount of red blood cells. When the concentration of oxygen in the blood decreases, EPO transcription is elevated. Recently, it was revealed that there are EPO/EPO-R systems not only in the hematopoiesis system, but also in the brain, uterus and oviduct. In the brain, astrocytes produce EPO to protect the neuronal cells from ischemia-induced cell death and the production is increased by hypoxia (7– 11). In the uterus, the endothelial cells of the endometrium produce EPO that induces the angiogenesis and proliferation of the endometrium, and the EPO synthesis depends on 17b-estradiol and hypoxia in the presence of this hormone (12, 13). EPO production in the oviduct is increased by 17b-estradiol and/or hypoxia, but the function of EPO released from this tissue is not clearly understood (14). EPO has also been detected in the placenta (15). In many mammalian species, such as goats, sheep and monkeys, with the exception of mice, the placenta is an effective barrier against the passage of EPO from the pregnant woman to the fetus, and also in the reverse direction (16). Furthermore, fetal EPO is produced independently from the maternal EPO production (17). These reports suggest that the origin of the EPO in the amniotic fluid must be the fetal organs
The amnion is a strong translucent membrane which contains no vessels, and when it is transplanted to a recipient, it does not incur acute allograft rejection. Due to this, the amnion has been applied to surgical regions and ophthalmologic regions. For example, it has been used as a dressing in surgery (1) and dermatology, and as a substitute for the cornea to cure some diseases such as Stevens–Johnson syndrome (2). In 1982, Adinolfi et al. found that human amniotic epithelial (HAE) cells produce lysosomal enzymes including a-induronidase, b-galactosidase and sphingomyelinase, and that culture supernatants of HAE cells were capable of correcting the abnormal accumulation in cultured fibroblasts from patients with Hurler’s syndrome (3). Moreover, Sakuragawa and coworkers found that HAE cells synthesize and release acetylcholine, catecholamines and neurotrophic factors (4–6). These studies suggest that HAE cells would be a promising source for the supply of the enzymes mentioned above and would contribute to the achievement of cell therapy in patients with specific enzyme deficiencies. Erythropoietin (EPO) is synthesized and released mainly from the kidney in adults and from the liver in fetuses. It * Corresponding author. e-mail: [email protected] phone: +81-(0)776-27-8645 fax: +81-(0)776-27-8747 448
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or fetal membrane. In 2000, Matsuura et al. reported that EPO and EPO-R are expressed in HAE cells (18). In 2001, Kim et al. reported that both EPO and its receptor are expressed in ovine fetal membranes, amnion and chorion (19). However, the functions of EPO in the placenta are still unknown. Progesterone is the female sex hormone produced in the late phase of the menstrual cycle by the corpus luteum of the ovary. When implantation succeeds, the corpus luteum continues to secrete progesterone under the influence of luteinizing hormone and prolactin for the early stage of pregnancy. After eight weeks, the placenta produces progesterone in great quantities so as to maintain the pregnancy. Progesterone, as a transcriptional regulator, is mediated by two distinct forms of the progesterone receptor (PR), PR-A and PR-B, which arise from alternative splicing (20). In most cells, including the human amnion, PR-A acts as a transcriptional inhibitor of all steroid hormone receptors, while PR-B acts as a transcriptional activator of progesterone-responsive genes (21, 22). We previously reported that EPO production in HAE cells was not stimulated by hypoxia (23). We hypothesized that HAE cells would have a similar regulatory system for EPO synthesis to the uterus or oviduct where EPO synthesis is regulated by 17b-estradiol, but different from the kidney/ bone marrow system. In the present study, we determined whether female sex hormones stimulate EPO synthesis in HAE cells. MATERIALS AND METHODS Cell lines Human placentas were obtained from uncomplicated elective cesarean sections and informed consent was obtained in accordance with the requirements of several hospitals in Kodaira, Japan. HAE cells were prepared according to the method described previously (24). Because the supply of HAE cells was insufficient, immortalized HAE cells, HAE-SV40, were also used. HAE-SV40 cells were generated by transfection with an origindefective simian virus (SV) 40 large T antigen (25). The MCF-7 cell line was obtained from the Health Science Research Resources Bank (Osaka). F-36E, an EPO-dependent cell line, was obtained from the RIKEN Cell Bank (Tsukuba). Bioassay of HAE-SV40 culture supernatants HAE-SV40 cells were cultured in SF-O2 serum-free medium (Sanko, Tokyo) containing either 1 mM 17b-estradiol (Sigma, St. Louis, MO, USA), 3 mM progesterone (Sigma), or no additive in a 5% CO2 atmosphere at 37°C. The culture supernatants of HAE-SV40 cells were collected and condensed by centrifugation (Ultrafree-MC Centrifugal Filter Units; Millipore, Billerica, MA, USA). Each supernatant was then re-diluted with RPMI 1640 medium (Nissui, Tokyo) containing 10% fetal bovine serum (FBS) to the same volume as before the condensation. F-36E cells were cultured in these media containing 10% FBS supplemented with a concentrated conditioned medium for the EPO bioassay. Purified recombinant EPO reagent (Kirin, Tokyo) was used as a positive control. An anti-EPO antibody was used for neutralizing the effect of EPO. The viabilities of the F-36E cells were determined by counting in a hemacytometer under a phase contrast microscope using the trypan blue exclusion assay. Proliferation assay of HAE-SV40 cells treated with progesterone HAE-SV40 cells were seeded into 24-well culture plates and cultured for 9 d in RPMI 1640 medium supplemented with 10% FBS with or without progesterone. The viability was deter-
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mined by counting in a hemacytometer under a phase contrast microscope using the trypan blue exclusion assay. RT-PCR To determine the effect of 17b-estradiol or progesterone, HAE-SV40 cells were cultured with 17b-estradiol (240 mM) or progesterone (3.2 mM) for 0, 1, 4, and 24 h in phenol red-free RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 15% charcoal-treated FBS. Total RNA was extracted from the cells using the acid guanidinium/phenol extraction method. Total RNA (5 mg) was heated to 70°C for 10 min before reverse transcription (RT). First strand cDNA synthesis was performed using an oligo(dT)15 primer and SuperScript™ II RT (Invitrogen, Carlsbad, CA, USA). The reaction was carried out at 42°C for 50 min in an iCycler thermal cycler (Bio-Rad, Hercules, CA, USA). To amplify the G3PDH target cDNA fragment, 50 ml of the PCR reaction mixture contained 1 ml of RT reaction product, 1.5 mM MgCl2, 0.2 mM dNTP mixture, 1.25 U of Taq DNA polymerase (Toyobo, Osaka) and 0.2 mM primers. The primers were 5¢-ACCACAGTCC ATGCCATCAC-3¢ (sense) and 5¢-TCCACCACCCTGTTCCTGTA3¢ (antisense). We performed 19 amplification cycles with 1 min of initial denaturation at 94°C, followed by cycles of 30 s at 94°C for denaturation, 30 s at 64°C for annealing and 1 min at 72°C for elongation. The terminal polymerase reaction time was 10 min at 72°C. To amplify the EPO target cDNA fragment, 50 ml of the PCR reaction mixture contained 1 ml of RT reaction product, 1.5 mM MgCl2, 0.2 mM dNTP mixture, 1.25 U of Taq DNA polymerase and 0.5 mM primers. The primers were 5¢-GCCAGAGGAACTGT CCAGAG-3¢ (sense) and 5¢-ATGGTAGGTGCGAAAACAGG-3¢ (antisense). We performed 28 amplification cycles with 1 min of initial denaturation at 94°C, followed by cycles of 30 s at 94°C for denaturation, 30 s at 61.7°C for annealing and 2 min at 72°C for elongation. The terminal polymerase reaction time was 10 min at 72°C. To detect the progesterone receptor cDNA fragment, 50 ml of the PCR reaction mixture contained 1 ml of RT reaction product, 1.5 mM MgCl2, 0.2 mM dNTP mixture, 1.25 U of HotStarTaq™ DNA polymerase (Qiagen, Valencia, CA, USA) and 0.4 mM primers. The primers were 5¢-GTCAGTGGGCAGATGCTGTA-3¢ (sense) and 5¢-AGCCCTTCCAAAGGAATTGT-3¢ (antisense). The reaction reagent was heated at 95°C for 15 min according to the protocol of the HotStarTaq™ DNA polymerase system. We then performed 40 amplification cycles with 1 min of initial denaturation at 94°C, followed by cycles of 30 s at 94°C for denaturation, 1 min at 53°C for annealing and 1 min at 72°C for elongation. The terminal polymerase reaction time was 10 min at 72°C. The expected band lengths were 452 bp for G3PDH, 207 bp for EPO, and 194 bp for the progesterone receptor. The PCR products were visualized in 2% agarose gels stained with ethidium bromide. Quantitative analysis of EPO mRNA EPO mRNA was reverse-transcribed and amplified by the RT-PCR method as described above. The products were quantified using a microchip electrophoretic analysis system (Hitachi, Tokyo). Western blotting MCF-7, HAE and HAE-SV40 cells were suspended in M-PER® Mammalian Protein Extraction Reagent (Pierce, Milwaukee, WI, USA) with protease inhibitors (Nacalai Tesque, Kyoto). An aliquot of 20 ml of each culture supernatant of these cells was separated by 8% SDS–PAGE and probed with a rabbit polyclonal antibody against human progesterone receptors A and B (Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated secondary antibodies (Chemicon International, Temecula, CA, USA) were used and immunoreactive bands were visualized using the Enhanced Chemiluminescence System (ECL plus; Amersham Bioscience, Piscataway, NJ, USA) on Polaroid film in an A-cassette apparatus (Fuji Film, Tokyo).
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RESULTS AND DISCUSSION Effect of female sex hormones on the EPO production of HAE-SV40 cells The concentration of female sex hormones, such as progesterone and 17b-estradiol, in the blood plasma of pregnant women increases markedly as pregnancy progresses, and most of the female sex hormones are produced in the placenta. Therefore, we hypothesized that such female sex hormones have important roles in the amniotic metabolism system and that they are involved in the EPO production by HAE cells. To test this hypothesis, we determined whether these hormones increased the EPO production of HAE-SV40 cells. HAE-SV40 cells were cultured in serum-free medium in the absence or presence of progesterone (3 mM) or 17b-estradiol (1 mM). The total EPO protein quantities in the culture supernatants of HAE-SV40 cells were measured by the EPO bioactivity. F-36E cells, known as EPO-dependent cells, were cultured in medium including the culture supernatant of HAE-SV40 cells and at the same time, they were cultured with or without the EPO reagent for several days. Then we assessed the EPO activities in the culture supernatants by measuring the viability of the F-36E cells and the result is shown in Fig. 1. The viability of the F-36E cells treated with the culture supernatant of HAE-SV40 cells was higher than that of the negative control. Treatment with the culture supernatant of HAE-SV40 cells cultured in the presence of progesterone increased the viability of F-36E cells significantly more than treatment with that of HAE-SV40 cells cultured in the absence of progesterone. The anti-EPO neutralizing antibody completely neutralized the effect of 0.1 U/ml of purified EPO on the survival of F-36E cells. In contrast, the anti-EPO antibody also neu-
FIG. 1. Effect of progesterone on the EPO production of HAESV40 cells. HAE-SV40 cells were cultured in SF-O2 medium with or without progesterone (3 mM) for 3 d. On reaching confluence, the culture supernatants were collected, concentrated once and then rediluted with the medium used for F-36E culture. F-36E cells were seeded at 20,000 cells/ml and cultured with (closed bars) or without (open bars) an anti-EPO antibody (2 mg/ml). After a 5-d culture, the viability of the F-36E cells was measured under a microscope using a hemocytometer and the trypan blue exclusion assay. The data indicate the mean viabilities of the F-36E cells. 1, Negative control; 2, addition of the culture supernatant of HAE-SV40 cells; 3, addition of the culture supernatant of HAE-SV40 cells treated with progesterone; 4, addition of EPO solution (0.1 U/ml). Ab represents the anti-EPO antibody. Error bars: SD (n = 3).
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TABLE 1. The effect of 17b-estradiol on the EPO production of HAE-SV40 cells Culture condition Viability of F-36E (%) SD (%) 1 26.1 1.4 2 32.0 2.9 3 33.2 1.9 4 88.7 0.4 HAE-SV40 cells were cultured in SF-O2 medium with or without 17b-estradiol (1 mM) for 6 d. On reaching confluence, the culture supernatants were collected, concentrated and then rediluted with the medium used for F-36E culture. F-36E cells were seeded at 18,400 cells/ml and cultured in medium including the culture supernatant of HAE-SV40 cells treated with or without 17b-estradiol, or EPO solution (0.25 U/ml), or neither. After a 3-d culture, the viability of F-36E cells was measured under a microscope using a hemocytometer and trypan blue exclusion assay. The data represent the mean viabilities of F-36E cells. 1, Negative control; 2, addition of the culture supernatant of HAE-SV40 cells; 3, addition of the culture supernatant of HAESV40 cells treated with 17b-estradiol; 4, addition of EPO solution.
tralized, but not completely, the effect in the culture supernatant of HAE-SV40 cells cultured with progesterone on the survival of F-36E cells. Moreover, the viabilities of F-36E cells treated with the supernatant of HAE-SV40 cells cultured both in the presence or absence of progesterone were higher than that of the negative control when F-36E cells were cultured with the anti-EPO antibody. These results suggest that the EPO activity in the culture supernatant of HAE-SV40 cells was increased by progesterone, and that HAE-SV40 cells secreted some factor or factors which functioned as a growth factor(s) for F-36E cells independent of progesterone. However, there was no significant effect of 17b-estradiol on the EPO activity of the culture supernatant of HAESV40 cells (Table 1), which suggests that progesterone, but not 17b-estradiol, increased the EPO production of HAESV40 cells. Effect of progesterone on the proliferation of HAESV40 cells There is another possible explanation for how progesterone increased the EPO production of HAE-SV40 cells. This possible mechanism was that progesterone stimulated the proliferation of HAE-SV40 cells with a concomitant increase in the amount of EPO in the culture. To exclude this possibility, we determined whether progesterone increases the cell population of HAE-SV40 cells. HAE-SV40 cells were seeded in the medium with or without progesterone at 14,400 cells/well, a much lower density than used in other experiments in the present study to determine conclusively the effect of progesterone on cell proliferation. When the cells were seeded at this lower density, the culture period was prolonged so that any mitogenic effect was more easily detected. The HAE-SV40 cell line proliferates very slowly and divides approximately every 3 d. Thus the culture period in this experiment was as long as 9 d. After a 9-d culture period, we determined the viable cell numbers. There were no significant differences in the viable cell numbers among the various cultures (Fig. 2), which indicated that progesterone did not stimulate the proliferation of HAE-SV40 cells at this concentration range. These results suggest that progesterone directly stimulates the EPO production pathway of HAE-SV40 cells, with no effect on
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FIG. 2. Effect of progesterone on the proliferation of HAE-SV40 cells. HAE-SV40 cells were seeded at 14,400 cells/well and cultured with or without progesterone (300 nM, 3000 nM) for 9 d. Error bars: SD (n = 3).
the proliferation. Effect of female sex hormones on the EPO production of HAE-SV40 cells at the EPO mRNA transcription level To determine whether progesterone or 17b-estradiol increases the EPO transcription in HAE-SV40 cells, we studied the time course of EPO mRNA transcript production in HAE-SV40 cells treated with progesterone (3 mM) or 17b-estradiol (240 mM). Total RNA transcripts derived from HAESV40 cells were extracted at specific intervals after the addition of the hormone. The EPO and G3PDH mRNAs were measured semiquantitatively by setting the PCR cycles to be approximately proportional to the band intensity of the amplified cDNA. We used G3PDH as a control of the total RNA amount because it is one of the housekeeping genes and expressed constitutively. Figure 3a shows the EPO mRNA- and G3PDH mRNA-derived products from HAESV40 cells. There was a distinct increase in the EPO mRNA at 4 h after the progesterone treatment, but not after the 17b-estradiol treatment. The products were quantified using a microchip electrophoretic analysis system (Hitachi). Figure 3b shows the quantities of EPO mRNA at 0 h, 1 h, 4 h and 24 h after the addition of a female sex hormone to the HAE-SV40 culture. When HAE-SV40 cells were treated with progesterone, the amount of EPO mRNA was increased after 1 h and reached a maximum of 5-fold the level of the control at 4 h. Twenty-four hours later, the amount of EPO mRNA was reduced almost to the level in the cells not treated with progesterone. A small increase in EPO mRNA was observed when HAE-SV40 cells were treated with 17b-estradiol. These results indicate that progesterone upregulates EPO transcription in HAE-SV40 cells, while 17b-estradiol does not. Detection of the progesterone receptor on HAE and HAE-SV40 cells It is commonly thought that steroid hormones bind to a specific receptor on a target cell and then function as transcription regulators. Thus, HAE-SV40 cells should express the PR for progesterone to be able to regulate their EPO synthesis. To determine whether HAE-SV40 cells and the wild-type HAE cells present PR, we used an
FIG. 3. Time course of the EPO transcription activity of HAESV40 cells treated with progesterone or 17b-estradiol. At intervals after the addition of progesterone (3 mM) or 17b-estradiol (240 mM) to the HAE-SV40 culture, EPO mRNA and G3PDH mRNA were amplified by RT-PCR, as described in Materials and Methods. These products were measured quantitatively using a microchip electrophoretic analysis system. (a) Result of agarose gel electrophoresis. Lane C indicates the control. The upper panel shows EPO mRNA-derived RT-PCR products (207 bp) and lower panel shows G3PDH mRNA-derived RTPCR products (452 bp). (b) Result of quantitative measurement of the EPO mRNA product. Open bars indicate the amount of EPO mRNA product derived from HAE-SV40 cells treated with 17b-estradiol. Closed bars indicate the amount of EPO mRNA product derived from HAE-SV40 cells treated with progesterone.
RT-PCR method and a Western blotting method. Figure 4a shows that PR mRNA- and G3PDH mRNA-derived products were observed in both HAE cells and HAESV40 cells, and in the MCF-7 cell line as a positive control which is known to present PR on its surface. HAE cells and HAE-SV40 cells were cultured in phenol red-free RPMI 1640 medium supplemented with 15% charcoal-treated FBS to remove estrogenic activity. Phenol red has estrogenic activity and charcoal treatment removes estrogen from the serum. PR mRNA-derived bands were observed for all of the cell types, but the intensities of the bands were different. The band intensity for HAE cells was more than threefold that for HAE-SV40 cells. HAE-SV40 cells expressed less PR than HAE cells and this may be due to immortalization which often decreases the functions of cells. Therefore, HAE-SV40 cells might exhibit decreased PR synthesis during immortalization, but this is unclear. Figure 4b shows the result of the Western blotting analysis. HAE and HAE-SV40 cells were cultured in RPMI 1640 medium including 10% FBS. For HAE-SV40 cells, the PR-B band was detected but not the PR-A band. For HAE cells, the PR-B band was distinctly detected, and the PR-A band
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and that progesterone might regulate EPO production in the human amnion in vivo. ACKNOWLEDGMENTS We thank Dr. H. Yoshimoto of Kirin Brewery Co. Ltd. for the generous gift of the EPO. We also thank Dr. I. Kano, Department of Toxicology, The Tokyo Metropolitan Research Laboratory of Public Health, for informative technical advice regarding SDS– PAGE. This work was partly supported by a Sasagawa Scientific Research Grant to Ms. A. Ogawa.
REFERENCES
FIG. 4. Detection of the PR on HAE cells and HAE-SV40 cells. (a) Detection of the PR mRNA product by RT-PCR. The process is described in Materials and Methods. The upper panel shows PR mRNA-derived RT-PCR products (194 bp) and the lower panel shows G3PDH mRNA-derived RT-PCR products (452 bp). (b) Detection of PR-A and -B by Western blotting analysis. PR-A indicates progesterone receptor-A, and its Mw is approximately 82 kDa. PR-B indicates progesterone receptor-B, and its Mw is approximately 120 kDa. MCF-7 is a mammary carcinoma cell line known to express PR-A and -B.
was also detected but the intensity was much weaker than that of the PR-B band. Moreover, the PR-B band intensity for HAE cells was about twofold than that for HAE-SV40 cells, which indicated that HAE cells expressed PR-B more strongly than HAE-SV40 cells. This result corresponded with the result of the RT-PCR. When HAE cells and HAE-SV40 cells were cultured in phenol red-free RPMI 1640 medium supplemented with 15% charcoal-treated FBS, the PR expression pattern was the same (data not shown). According to the report by Rossmanith et al. (26), the developing human placenta expresses the progesterone receptor but not the estrogen receptor. We failed to detect estrogen receptor mRNA by RT-PCR in either HAE or HAESV40 cells, although it was detected in HeLa cells as a positive control. Therefore, the estrogen receptor is not involved in the EPO production of HAE cells. We investigated the effect of progesterone and 17b-estradiol on EPO production in HAE cells. Treatment with progesterone increased EPO bioactivity in the culture supernatant and the EPO mRNA level, but 17b-estradiol did not. The results of this research showed that progesterone, but not 17b-estradiol, enhanced EPO synthesis in HAE cells
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