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Department of Obstetrics and Gynecology, Kobe University School of Medicine, Kobe 650-0017, Japan
Address all correspondence and requests for reprints to: Takeshi Maruo, M.D., Department of Obstetrics and Gynecology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: maruo{at}kobe-u.ac.jp
Abstract
Adrenomedullin is a multifunctional peptide expressed in a variety of tissues. This study was conducted to investigate the expression of adrenomedullin and its mRNA by human trophoblasts and the possible existence of adrenomedullin receptor in those cells. Human placentas in all three trimesters were obtained from patients undergoing therapeutic abortions and deliveries. Total RNA was extracted from placental trophoblastic tissues and JAr choriocarcinoma cells, and the expression of adrenomedullin mRNA was determined by RT-PCR. Immunohistochemical analysis was performed by the avidin/biotin immunoperoxidase method using a specific antibody to adrenomedullin. The secretion of adrenomedullin by JAr cells cultured in medium containing [35S]cysteine-[35S]methionine was determined by immunoprecipitation followed by PAGE. The presence of adrenomedullin receptor in JAr cells was examined using a binding assay with [125I]rat adrenomedullin.
Adrenomedullin mRNA was expressed by human placental trophoblastic tissues in all three trimesters and by JAr cells. Immunohistochemical analysis revealed that adrenomedullin is expressed by cytotrophoblasts in placentas in all three trimesters, but not by syncytiotrophoblasts. The expression of adrenomedullin in the cytotrophoblast was most abundant in first trimester placenta and became less abundant during the course of pregnancy. JAr cells synthesized and secreted immunoreactive adrenomedullin. Binding assay with [125I]rat adrenomedullin demonstrated specific binding of adrenomedullin to JAr cells, indicating the existence of a specific receptor for adrenomedullin in trophoblastic cells.
Adrenomedullin is transcribed and secreted by cytotrophoblastic cells that possess adrenomedullin receptor. Adrenomedullin may play a potential role as an autocrine/paracrine factor in the growth of cytotrophoblasts, especially in early gestation.
ADRENOMEDULLIN (AM) IS a potent vasodilator peptide that has been identified in pheochromocytoma tissue by its ability to elevate cAMP in rat platelets (1). Human AM with 52 amino acids shows structural homology with calcitonin gene-related peptide and amylin. AM is expressed in a variety of tissues, including heart, brain, kidney, lung, and aorta (2, 3, 4). The effect of AM as a long-lasting vasorelaxation agent has been well documented (5, 6). Apart from the vasorelaxant effect, several other functions have been reported for this peptide. AM plays an important role as a regulator of renal function with natriuretic and diuretic actions (7, 8). Endocrine function of AM includes suppression of aldosterone, ACTH, and insulin release (9, 10, 11). AM also has been reported to regulate cell growth in human tumor cell lines (12) and to act as an apoptosis survival factor (13).
It has been reported that AM is present in high concentrations in maternal and umbilical plasma and in amniotic fluid during human pregnancy, and that AM mRNA is expressed in fetal membranes (14, 15). These findings suggest that AM may play an important role in maintaining pregnancy as both an endocrine and a local factor. However, the biological function of AM in pregnancy is still not elucidated. The present study was conducted to identify whether human placentas express AM mRNA. Then we investigated the immunolocalization of AM in human placentas. Furthermore, to elucidate the potential of AM to act as a local factor in an autocrine/paracrine fashion, the presence of AM receptor in trophoblastic tumor JAr cells was examined.
Materials and Methods
Human placentas
First trimester placentas (n = 6) were obtained from patients undergoing therapeutic abortions at 7–9 wk of pregnancy. Second trimester placentas (n = 3) were obtained from premature deliveries at 20–22 wk of pregnancy, and third trimester placentas (n = 3) were obtained from spontaneous deliveries at 37–39 wk of pregnancy. Informed consent for the use of the placental tissues for this study was obtained from each patient.
RT-PCR detection of AM mRNA in human placenta and JAr cells
Total RNA was extracted from first, second, and third trimester
placentas, and from JAr choriocarcinoma cells that were purchased from
American Type Culture Collection (Manassas, VA) by the
guanidine isothianate method. Primers (12) that recognize
the most conserved regions of the AM gene were used for RT-PCR (Table 1
). The expected size of the PCR
product for AM was 410 bp. RT was performed with murine leukemia virus
reverse transcriptase (Perkin-Elmer Corp., Norwalk, CT)
for 60 min at 42 C using 1 µg total RNA, and then the reaction was
heat-inactivated for 5 min at 99 C and quick-chilled for 5 m at 5
C. Thermal cycle profiles used in this study were as follows; 1)
denaturing for 60 sec at 95 C, 2) annealing primers for 60 sec at 55 C,
and 3) extending the primers for 60 sec at 72 C. The PCR was repeated
for 30 cycles. The PCR product was electrophoresed in 2% agarose gel
in 89 mM Tris-borate and 2 mM EDTA (pH 8.0),
stained with ethidium bromide, and analyzed. Previously, the specific
bands had been isolated, cloned, and sequenced to assure that the
amplified DNA represented the expected product (data not shown).
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Fresh trophoblastic tissues obtained from first, second, and third trimester placentas were fixed in 4% buffered neutral formaldehyde, dehydrated in graded alcohols, and embedded in paraffin. Sections of 4 µm were prepared for immunohistochemical staining. Immunohistochemical staining was performed by the avidin/biotin immunoperoxidase method using a polyvalent immunoperoxidase kit (Omnitag, Lipshaw, MI). The primary antibody was a rabbit polyclonal antibody against human AM (Peninsula Laboratories, Inc., Belmont, CA). Avidin-horseradish peroxidase was used in the second incubation. Chromogenic reaction was developed with a freshly prepared solution of tetrahydrochloride diaminobenzidine and hydrogen peroxidase. The sections were counterstained with Harris hematoxylin.
The following control procedures were undertaken to assure the specificity of the immunological reactions. The control section was subjected to the same immunoperoxidase method, except that the primary antibody to AM was replaced by nonimmune rabbit serum at the same dilution as the specific primary antibody. In the control, no positive staining was observed.
Immunoprecipitation of AM produced and secreted by JAr cells
JAr cells were cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) with 10% FBS (Sigma, St. Louis, MO) containing [35S]cysteine-[35S]methionine (Amersham Pharmacia Biotech, Little Chalfont, UK) for 12 h at 37 C under a 5% CO2-95% air atmosphere. The medium was collected and extracted with Sep-Pak C14 columns. The extract was incubated with antihuman AM antibody at 4 C for 12 h. The antigen-antibody complex was absorbed with Pansorbin (Calbiochem, La Jolla, CA) and loaded on an SDS-PAGE. After electrophoresis, the gels were dried and autoradiographed.
Binding assay of AM to JAr cells
Confluent JAr cells (106 cells/well) were washed with PBS and incubated with [125I]rat AM (5.0 x 10-15 mol) for 12 h at 4 C in 0.5 ml PBS in the absence or presence of unlabeled rat AM (Peptide Laboratories, Tokyo, Japan). After completion, cells were extensively washed with PBS, and the cell-bound radioactivity was determined. Specific binding was obtained by subtracting nonspecific binding in the presence of excess (10-6 M) unlabeled rat AM from total binding.
Results
AM mRNA expression in human placenta and JAr cells
When RT-PCR with specific primers for AM was performed using RNA
extracted from human placental trophoblastic tissues obtained in the
first, second, and third trimesters of pregnancy and from JAr cells, a
band of 410 bp was observed in each sample (Fig. 1). Sequencing of this band revealed that
it was consistent with the AM mRNA nucleotide sequence previously
published (16). When reverse transcriptase was omitted, no
band was apparent. As the condition of PCR was saturating, no
difference in the intensity of the bands was observed.
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Cytotrophoblasts in first trimester placental tissues showed
prominent staining with the anti-AM antibody, whereas
syncytiotrophoblasts and fetal stroma cells did not show any
appreciable staining. The intensity of the staining for AM in the
tissue sections of placenta varied during the course of pregnancy.
Cytotrophoblasts in first trimester placenta displayed the most
pronounced staining for AM. Cytotrophoblasts in second trimester
placenta stained less than those in first trimester placenta, and
cytotrophoblasts in third trimester placenta stained only faintly (Fig. 2).
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JAr cells were metabolically labeled with
[35S]cysteine-[35S]methionine,
and the protein secreted into the medium was immunoprecipitated with a
specific antibody. A radioactive band of 6 kDa, which corresponds to AM
peptide, was demonstrated, suggesting the production and secretion of
AM peptide by JAr cells (Fig. 3).
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Competitive binding of [125I]rat AM to JAr
cells by unlabeled rat AM is shown in Fig. 4. The inhibition of the binding of
[125I]rat AM was proportional to the amount of
unlabeled AM added. The specific binding of
[125I]rat AM was 48.5% of the total binding.
This result suggests the existence of specific receptors for AM in JAr
cells. As estimated from Scatchard analysis of the binding study, the
dissociation constant (Kd) was 1.35 x
10-7 M, and the binding capacity was
6.0 x 105 sites/cell.
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To determine whether human placenta produces AM mRNA, RT-PCR with specific primers for AM using RNA obtained from placentas in the first, second, and third trimesters was performed. As human placenta is composed of heterogeneous components, including endothelial cells and vascular smooth muscle cells, which are known to produce AM (17, 18), RNA obtained from the choriocarcinoma cell line JAr was also subjected to RT-PCR. These experiments revealed the presence of AM mRNA in human placentas in all three trimesters as well as in JAr cells. This demonstrates that AM mRNA is transcribed by the trophoblast. Furthermore, immunoprecipitation of the culture medium metabolically labeled with [35S]cysteine-[35S]methionine by JAr cells with a specific antibody to AM followed by electrophoresis on SDS-PAGE demonstrated a band corresponding to 35S-labeled AM. This indicates that AM is not only transcribed, but is also synthesized and secreted by the trophoblast. Immunohistochemical staining for AM in human placentas revealed that localization of AM is prominent in cytotrophoblasts, but not in syncytiotrophoblasts and fetal stroma cells. Moreover, it was of interest to note that AM was most abundant in cytotrophoblasts of first trimester placenta, less abundant in those of second trimester placenta, and least abundant in those of third trimester placenta. These findings imply that placental AM is likely to exert its function particularly in the early stage of gestation. Interestingly, no immunostaining for AM was observed in the endothelial cells of fetal stroma, suggesting that extensive vasculature does not exist during the stage of placental development or that AM is differentially regulated in fetal stroma. In this connection, specific binding of AM to JAr cells suggests that trophoblastic cells possess the specific AM receptors. Due to the technical difficulty of isolating pure population of primary cytotrophoblastic cells, JAr cells, which are mainly composed of cytotrophoblast-like cells, were used to assess the specific binding of AM in the present study. These data obtained with JAr cells must be interpreted with caution for understanding the presence of AM receptors in placental cytotrophoblasts.
It has been reported that AM is present in second trimester human amniotic fluid, and AM protein is synthesized by amniotic membrane (14), and that AM production by amniotic membrane increases during gestation (19). It also has been noted that maternal plasma AM concentrations are lower in pregnant women with preeclampsia than in normotensive pregnant women (20). On the other hand, AM levels are significantly increased in the fetal membranes and umbilical arteries of pregnant women with preeclampsia compared with those of normotensive pregnant woman (21). Furthermore, AM levels in amniotic fluid are reported to be elevated in cases of premature rupture of membranes and preterm labor (22). These findings suggest that AM expressed by the feto-placental unit may play a role in the physiology of pregnancy.
On the other hand, it has been reported that AM and its receptor are expressed in numerous human cancer cell lines of various origins and act as a modulator of cell growth in those cells (12). Kato et al. (13) reported that AM acts as an apoptosis survival factor for endothelial cells in an autocrine/paracrine manner. These reports support that AM may be vital in the proliferation of cells in which AM is expressed.
Marinoni et al. (23) reported that AM is expressed by syncytiotrophoblasts, but not by cytotrophoblasts, in the placental tissue obtained at term. This report is the opposite of our current findings. Although the reasons for the difference between our results and those of Marinoni et al. are unknown, they may be due to a difference in the polyvalent immunoperoxidase kit used in their study and ours. When the embryo implants, it starts to invade the endometrium, with trophoblasts aggressively proliferating and forming placenta. Trophoblast is composed of two layers of cytotrophoblast and syncytiotrophoblast, but it is cytotrophoblast that is responsible for proliferation (24). Our current finding that AM is expressed by cytotrophoblast, but not by syncytiotrophoblast, may be attributed to the difference in the proliferative potentials of those two cells. As AM receptor was present in JAr cells, that is cytotrophoblast-like cells, it could be postulated that AM expressed by cytotrophoblast may act as an autocrine/paracrine factor in the regulation of trophoblast proliferation. We previously demonstrated the expression and action of epidermal growth factor and IGF-I and their receptors in the placenta and reported their involvement in the growth and differentiation of the trophoblast (25, 26, 27). Further studies of the interaction of AM with other growth factors are needed to elucidate the biological action of AM on human trophoblast.
Footnotes
This work was supported in part by Grant-in-Aid for Scientific Research (10470346) from the Japanese Ministry of Education, Science and Culture and by the Japan Association of Obstetricians and Gynecologists Ogyaa-Donation Foundation (JODF).
Abbreviations: AM, Adrenomedullin.
Received October 30, 2000.
Accepted April 21, 2001.
References
. Biochem Biophys Res
Commun 203:719–726[CrossRef][Medline]
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