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Immunohistochemical localization of parathyroid hormone-related protein (PTHrP) in human term placenta and membranes
Placenta (1994), 15, 653-660
Immunohistochemical Localization of Parathyroid Hormone-Related Protein (PTHrP) in Human Term Placenta and Membranes J. F. EMLY a, J. GREGORY b, S.J. BOWDEN a, A. AHMED c, M.J. WHITTLE d, D. I. R U S H T O N e & W. A. RATCLIFFE af "Wolfson Research Laboratories, Department of Clinical Chemistry, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH bDepartment of Pathology, Queen Elizabeth Medical Centre, Edgbaston, Birmingham CDepartment of Obstetrics and Gynaecology, aDepartment of Fetal Medicine and eDepartment of Pathology, Birmingham MaterniO, Hospital, Birmingham, UK f To whom correspondenceshould be addressed Paper accepted16.2.1994
SUMMARY Parathyroid hormone-related protein (PTHrP), the major factor responsible for hypercalcaemia of malignancy, is widely expressed in normal adult and fetal tissues. In this study, the distribution of PTHrP was examined in human term placenta and membranes by immunohistochemistry using antisera to PTHrP 1-34 and 37-67. PTHrP was detected in cuboidal epithelial cells of amnion and in O~totrophoblastic cells of chorionic laeve and adherent maternal decidua. In placenta, PTHrP 1-34 was detected in the syno~tiotrophoblast, while PTHrP 37-67 activity was mainly present in the brush border of the synoJtiotrophoblast. This study also identified PTHrP 37-67 associated with fetal vessels of placental villi. These findings may retied the cellular distribution of intact PTHrP or sub-fragments derived by posttranslational processing. Postulated actions of PTHrP in the uteroplacental unit include transport of calcium across the placenta, stretch of membranes, inhibition of uterine contractility, growth and differentiation, and vasoregulation.
INTRODUCTION Parathyroid hormone-related protein (PTHrP) initially isolated from cancer cell lines and tumours from patients with hypercalcaemia of malignancy (Moseley et al, 1987) is estab0143-4004/94/060653 + 08 $08.00/0
9 1994 W. B. Saunders Company Ltd
Placenta (1994), Vol. 15
654
lished as the major humoral factor responsible for hypercalcaemia of malignancy (Martin and Suva, 1989). PTHrP exists in three isoforms of 139, 141 and 173 amino acids which differ only at the extreme carboxyl-termini. PTHrP shares sequence homology with parathyroid hormone (PTH) at its amino-terminus, allowing PTHrP to mimic the actions of P T H by interaction with classical PTH receptors in bone and kidney (Juppner et al, 1988). Receptor subtypes unique for PTHrP have yet to be identified (Orloff, Wu and Stewart, 1989). PTHrP has been localized by immunohistochemistry in a wide range of normal adult and fetal tissues (Kramer et al, 1991; Moseley et al, 1991). In the uteroplacental unit PTHrP mRNA is expressed in amnion, chorion, decidua, myometrium and placenta (Thiede et al, 1990; Ferguson et al, 1992) and PTHrP has been extracted from human term placenta and membranes and quantitated by immunoassay (Abbas et al, 1990; Bowden et al, 1994). Specific binding sites for PTH 1-34 in basal membranes and the brush border of the syncytiotrophoblast, may reflect sites of action of PTHrP (Lafond et al, 1988). A number of actions are proposed for PTHrP in placental tissue. PTHrP 67-86 amide derived from placenta or fetal parathyroid glands may stimulate calcium transport across the placenta and maintain plasma calcium in the fetus at a higher level than in the maternal circulation (Care et al, 1990). P T H 1-34 and PTHrP 1-34 are both relaxants of vascular and non-vascular smooth muscle (Winquist, Baskin and Vlasuk, 1987; Mok et al, 1989) and PTHrP produced in association with vascular tissue may have local effects on blood flow (Thiede, Grasser and Petersen, 1992). In myometrium and amnion PTHrP 1-34 may act locally to facilitate stretch and relaxation of these tissues (Daifotis et al, 1992; Ferguson et al, 1992). Thus PTHrP may act as a precursor of peptides with differing biological actions. Using antisera specific for PTHrP 1-34, positive immunostaining was demonstrated in the syncytiotrophoblast of the human placenta at term (Ishikawa et al, 1992), while PTHrP 56-86 activity was localized mainly in the cytotrophoblast (Hellman et al, 1992). Kramer et al (1991) failed to detect PTHrP in human amnion at term, although both PTHrP mRNA (Germain et al, 1992) and immunoreactivity (Bowden et al, 1994) were identified in this tissue. The aim of this study was to localize PTHrP by immunohistochemistry in human term membranes and placenta using region-specific polyclonal antisera raised to PTHrP 1-34 and 37-67. Information on the cellular distribution of PTHrP may reflect its role in the uteroplacental unit.
MATERIALS AND M E T H O D S Antisera used in this study were raised in rabbits by immunization with conjugates of PTHrP 1-34 and PTHrP 37-67. The 37-67 antiserum has previously been used to localize PTHrP in tumours from normocalcaemic and hypercalcaemic patients (Bundred et al, 1991; Dunne et al, 1993). The antibodies recognize residues 9-18 and 52-61, respectively, and do not cross-react with either PTH or other subfragments of PTHrP (Ratcliffe et al, 1991). Optimum intensity of specific immunostaining was obtained at dilutions of 1:200 in both cases. Immunohistochemistry was carried out on 5 Ixm-thick sections of formalin-fixed, paraffin-embedded tissue. Sections were deparaffinized by incubation for 5 min with Histoclear, hydrated through methanol to water and endogenous peroxidase activity was
Em& et ak Immunohistochemical Localization of PTHrP
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quenched by the addition of 0.3 per cent (v/v) of hydrogen peroxide in methanol for 15 rain. Before application of PTHrP antibodies [1:200 in phosphate buffered saline (PBS) for 30 min], non-specific binding sites were blocked by the addition of non-immune sheep serum (1:4 in PBS) for 15 min. The sections were then washed in 3 • 200ml of 0.1 per cent (v/v) polyoxylene 10 oleoyl ether (Brij 96) in PBS over a period of 15min, and incubated with affinity purified horse radish peroxidase-conjugated sheep-anti-rabbit immunoglobulin (1:100) in PBS for 30 min. Following washing, diaminobenzidine (DAB, 0.5 mg/ml and hydrogen peroxide 0.3 per cent in PBS) was applied to the sections for 6 min to visualize the peroxidase activity. Following counterstaining with Mayers haematoxylin, the sections were dehydrated and mounted. Syncytiotrophoblast was identified by immunostaining with an antiserum to human placental lactogen (Dako; diluted 1:200), following treatment of sections with 1 mg/ml trypsin, 1 mg/ml calcium chloride in water at pH 7.8 for 20 min at 37~ This was followed by addition of a second antibody as described above. Endothelial cells were visualized using monoclonal antibody QBendl0 (Unipath) (1:25 in 10 per cent goat serum) followed by a biotinylated goat anti-mouse/rabbit second antibody (Duet system from Dako) and a streptavidin-biotin HRP complex (both diluted 1:100 in PBS). For dual immunodetection of PTHrP and QBendl0 on the same section, DAB was used as the peroxidase substrate for localization of PTHrP and the substrate Vector SG (Vector Labs) was used in its place to visualize endothelial cells detected by QBendl0. Controls consisted of sections where the primary antibody was replaced by the same dilution of the appropriate non-immune serum, where the secondary antibody was omitted, or where diluted antibody was pre-incubated with either 0.4 mg/ml PTHrP 1-34 or 37-67 for 18 h at 4~
Tissues Placenta, membranes and cord were from normal term pregnancies (n = 4) delivered by caesarean section. Placental tissue was from a central location lying between the basal and chorionic plates. Amnion and chorion (chorionic laeve) were from reflected membranes formerly covering the decidua parietalis of the uterus. Chorion from the chorionic plate of the placenta was also studied.
RESULTS With antisera to PTHrP 1-34 and 37-67, positive cytoplasmic staining was identified in cuboidal epithelium (A) of reflected amnion [Figure l(a)], amnion covering the chorionic plate, and amnion covering the umbilical cord (not shown). Figure 1(b) shows chorion laeve stained with antiserum to PTHrP 1-34. This tissue consists of a reticular layer composed of fibroblasts in a fibrous network, trophoblasts that extend from the pseudo-basement membrane to the decidual layer. A degenerate villus is present in the trophoblast layer. Positive staining was found in cytotrophoblastic cells and adhering maternal decidua using antisera to both PTHrP 1-34 and 37-67. In placenta, different patterns ofPTHrP positivity were obtained with antisera to PTHrP 1-34 and 37-67. With the antiserum to PTHrP 1-34, positive staining was present in the syncytiotrophoblast [Figure l(c)], while the antiserum to PTHrP 37-67 gave positive
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Figure 1. The distribution of parathyroid hormone-related protein (PTHrP) in human term placenta and membranes. (a) Positive staining for PTHrP 1-34 in epithelial cells of amnion (A), and (b) trophoblasts (T) and decidua (D) of chorion. (c) Placenta with the syncytiotrophoblast (S) positively stained with antiserum to PTHrP 1-34. (d) Placenta with positive staining for PTHrP 37-67 in brush border of the syncytiotrophoblast (S) and in endothelial cells of fetal vessels (E). Scale bar: 30 wm for (a) and (b); 20 wm for (c) and (d). PBM, pseudobasement membrane; DV, degenerate villus.
staining in the syncytiotrophoblast brush border, and in endothelial cells of fetal vessels [Figure 1(d)]. Endothelial cells were identified by staining for QBend 10. Dual staining with QBendl0 and PTHrP 1-34 antiserum identified PTHrP 1-34 mainly in the syncytiotrophoblast. PTHrP was not localized in components of neonatal or maternal blood. In control sections, no positive staining was found when primary or secondary antibodies'were omitted or replaced with non-immune serum, or when the antiserum was pre-absorbed by excess of the appropriate peptide (Figure 2).
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Figure 2. (a) Human term amnion, chorion and decidua and (b) placenta incubated with non-immune serum in place of primary antibody. Scale bar: 120 Ixm.
DISCUSSION This study has confirmed the presence of PTHrP in cells of different histological types within the human uteroplacental unit at term, suggesting that PTHrP may have diverse physiological roles in these tissues. Strong positivity was found exclusively in cuboidal epithelium of amnion from all sites examined. Recent studies have reported high PTHrP mRNA levels in human amnion, and demonstrated secretion of PTHrP by human amnion in vitro (Germain et al, 1992), and the PTHrP concentration in amnion has been quantitated by immunoassay (Bowden et al, 1994). Vasoactive peptides such as PTHrP and endothelin (Casey, Word and MacDonald, 1991) produced by amnion may act locally to regulate blood flow in fetal vessels of the chorionic plate, while PTHrP from reflected amnion and chorion may act locally to facilitate stretch of the membranes and inhibit contractility of the pregnant uterus (Shew et al, 1991; Paspaliaris et al, 1992). PTHrP mRNA concentrations in amnion were lower after labour than after elective caesarean section (Germain et al, 1992) and in labouring women, tissue PTHrP concentrations were inversely correlated with the time between rupture of the membranes and delivery (Bowden et al, 1994), suggesting that PTHrP gene expression in amnion is regulated by mechanical stretch or intraluminal pressure as in the uterus and bladder (Thiede et al, 1990; Yamamoto et al, 1992). The failure of earlier studies to detect PTHrP in amnion may reflect the duration of labour (Kramer et al, 1991). Amnion is a likely source of the high PTHrP concentrations found in amniotic fluid at term (Ferguson et al, 1992; Bowden et al, 1994). PTHrP is widely expressed in human fetal tissues and changes in expression with gestation suggest that it may have roles in growth and differentiation (Moseley et al, 1991). PTHrP in amniotic fluid may also have effects in the fetus either by intestinal absorption or by direct effects on skin, or the intestinal and respiratory tracts. In contrast PTHrP 1-86 levels are only modestly increased in maternal and cord venous plasma at term, suggesting that PTHrP acts predominantly by a local mechanism in placenta.(Bowden et al, 1994). The increase in epidermal growth factor (EGF) receptors in cultured trophoblasts seen in response to P T H (Alsat et al, 1991) may
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reflect interactive roles for EGF and PTHrP in regulating growth and endocrine function of these cells. Positive immunostaining was also localized in extravillous trophoblasts and maternal decidua associated with chorionic laeve. These cells are a likely source of the high PTHrP levels measured in chorion and membranes by immunoassay (Abbas et al, 1990; Bowden et al, 1994). PTHrP produced at these sites may have important actions as an inhibitor of uterine contractility during pregnancy (Paspaliaris et al, 1992). PTHrP mRNA has previously been identified in endometrial stroma cells (Casey et al, 1993) which are progenitors of decidua. Proteolytic processing of native PTHrP occurs between residues 37 and 38 in normal and malignant cells in vitro, yielding a major secretory form with residue 38 at its aminoterminus (Soifer et al, 1992). The same study also showed differing intracellular locations of amino-terminal and mid-region PTHrP. The epitopes of the antisera to PTHrP 1-34 and 37-67 used in the present study span the 37-38 cleavage point and would be expected to localize both intact PTHrP and subfragments derived by proteolytic processing. Using these antisera, PTHrP 1-34 was localized to the cytoplasm of the syncytium, and PTHrP 37-67 to its brush border and also endothelial cells of fetal vessels. It is unclear whether these differences reflect the specificities of the antibodies, or processing of PTHrP to subfragments with differing intracellular locations. The higher levels of 1-34 and 37-67 immunoreactivities relative to 1-86 immunoreactivity in human term placenta, and the molecular heterogeneity seen on chromatography (Bowden et al, 1994), are supporting evidence that proteolytic processing occurs between the epitopes of the 1-34 and 37-67 antibodies. In contrast an earlier study co-localized PTHrP 1-34 and 56-86 mainly in cytotrophoblasts (Hellman et al, 1992), while PTHrP 1-34 was identified in the syncytium and Hofbauer cells of human term placenta (Ishikawa et al, 1992). Production of PTHrP by vascular tissue and specific binding of PTH and PTHrP to membranes of vascular smooth muscle cells provide support for its role in the local regulation of vascular tone (Mok et al, 1989). We postulate that PTHrP produced by endothelial cells may have a similar action in modulating blood flow in fetal vessels, possibly by interaction with the extravascular contractile system of the placenta (Grafet al, 1993). In the isolated perfused cotyledon from human term placenta, PTHrP gave a concentrationdependent vasodflator effect and reduced perfusion pressure by approximately 50 per cent (Mandsager, Brewer and Myatt, 1993). Specific regulatory factors are increasingly implicated in the regulation of placental growth and function and in the interaction of decidua and trophoblasts. The wide tissue distribution of PTHrP in the uteroplacental unit suggests that it may have a range of potential physiological actions, including growth and differentiation, inhibition of smooth muscle contractility, vasoregulation and calcium transport. It remains to be established whether it has a role in calcium homeostasis in the fetus or in initiation of labour. ACKNOWLEDGEMENTS We thank the Departmentof Health and the EndowmentFund of the UnitedBirminghamHospitalsfor financial support. REFERENCES Abbas, S. K., Pickard, D. W., Illingworth, D., Storer,J., Purdie, D. W., Moniz, C., Dixit, M., Caple, I. W., Ebeling, P. R., Rodda, C. P., Martin, T. J. & Care, A. D. (1990) Measurementof parathyroid hormone-
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related protein in extracts of fetal parathyroid glands and placental membranes. Journal of Endocrinology, 124, 319-325. Alsat, E., Mirlesse, V., Fondacei, C., Dodeur, M. & Evain-Brion, D. (1991) Parathyroid hormone increases epidermal growth factor receptors in cultured human trophoblastic cells from early and term placenta.Journal of Clinical Endocrinologyand Metabolism, 73,288-295. Bowden, S.J., Emly, J. F., Hughes, S. V., Powell, G., Ahmed, A., Whittle, M.J., Ratcliffe,J. G. & Ratcliffe, W. A. (1994) Parathyroid hormone-related protein (PTHrP) in human term placenta and membranes. Journal of Endocrinology (in press). Bundred, N. J., Ratcliffe, W. A., Walker, R. A., Coley, S., Morrison, J. M. & Ratcliffe, J. G. (1991) Parathyroid hormone related protein and hypercalcaemia in breast cancer. British MedicalJournal, 303, 15061509. Care, A. D., Abbas, S. K., Pickard, D. W., Barri, M., Drinkhill, M., Findlay, J. B. C., White, I. R. & Caple, I. W. (1990) Stimulation of ovine placental transport of calcium and magnesium by mid-molecule fragments of human parathyroid hormone-related protein. Journal of ExperimentalPhysiology, 75, 605-608. Casey, M. L., Word, R. A. & MacDonald, P. C. (1991) Endothelin-1 gene expression and regulation of endothelin mRNA and protein biosynthesis in avascular human amnion: potential source of amniotic fluid endothelin. Journal of Biological Chemistry, 266, 5762-5768. Casey, M. L., Mibe, M. & MacDonald, P. C. (1993) Regulation of parathyroid hormone-related protein gene expression in human endomelxial stroma cells in culture. Journal of Clinical Endocrinology and Metabolism, 77, 188-194. Daifotis, A. G., Weir, E. C., Dreyer, B. E. & Broadus, A. E. (1992) Parathyroid hormone-related peptide gene expression in the rat uterus.Journal of Biological Chemistry, 267, 23 455-23 458. Dunne, F. D., Lee, S., Ratcliffe, W. A., Hutehesson, A. C., Bundred, N. J. & Heath, D. A. (1993) Parathyroid hormone-related protein (PTHrP) gene expression in solid tumours associated with normocalcaemia and hypercalcaemia. Journal of Pathology, 171, 215-221. Ferguson, J. E. II, Gorman, J. V., Bruns, D. E., Weir, E. C., Burtis, W. J., Martin, T. J. & Bruns, N. E. (1992) Abundant expression of parathyroid hormone-related protein in human amnion and its association with labor. Proceedingof the NationalAcademy of Sdences USA, 89, 8384-8388. Germain, A. M., Attaroglu, H., MacDonald, P. C. & Casey, M. L. (1992) Parathyroid hormone-related protein mRNA in avascular human amnion. Journal of ClinicalEndocrinologyand Metabolism, 75, 1173-1175. Graf, R., Langer, J.-U., Sehonfelder, G., HarteI-Schenk, S. & Reutter, W. (1993) The extravascular contractile system (EVCS) of the human placenta. Placenta, 14, A25. Hellman, P., Ridefelt, P., Juhlin, C., Akestrom, G., Rastad, J. & Gylfe, E. (1992) Parathyroid-like regulation of parathyroid-hormone-related protein release and cytoplasmic calcium in cytotrophoblast cells of the human placenta. Archives of Biochemistry and Biophysics, 293, 174-180. Ishikawa, E., Katakami, H., Hidaka, H., Ushiroda, Y., Ikeda, T., Ikenoue, T. & Matsukura, S. (1992) Characterization of parathyroid hormone-related protein in the human term placenta. EndocrinoligicaJaponica, 39, 555-561. Juppner, H., Abou-Samra, A.-B., Uneno, S., Gu, W.-X., Potts, J. T. & Segre, G. V. (1988) The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 ceils. TheJournal of Biological Chemistry,263, 85578560. Kramer, S., Reynolds, F. H., Casdllo, M., Valenzuela, D. M., Thorikay, M. & Sorvillo, J. M. (1991) Immunological identification and distribution of parathyroid hormone-like protein polypeptides in normal and malignant tissues. Endocrinology, 128, 1927-1937. Lafond, J., Auger, D., Fortler, J. & Brunette, M. G. (1988) Hormone receptor in human placental syncytiotrophobiast brush border and basal plasma membranes. Endocrinology, 123, 2834-2840. Mandsager, N. T., Brewer A. & Myatt L. (1993) Actions of parathyroid hormone, parathyroid hormone-related peptide and calcitonin gene-related peptide in the fetal-placental circulation. Placenta, 14, A48. Martin, 1".J. & Suva, L.J. (1989) Parathyroid hormone-related protein in hypercalcaemia of malignancy. Clinical Endocrinology, 31, 631-647. Mok, L. L. S., Nichols, G. A., Thompson, J. C. & Cooper, C. W. (1989) Parathyroid hormone as a smooth muscle relaxant. EndocrineReviews, 10, 420-436. Moseley, J. M., Kubota, M., Diefenbaeh-Jagger, H. D., Wettenhall, R. E. H., Kemp, B. E., Suva, L. J., Rodda, C. P., Ebeling, P. R., Hudson, P. J., Zajac, J. D. & Martin, T. J. (1987) Parathyroid hormonerelated protein purified from a human lung cancer cell line. Proceedingsof the NationalAcademy of Sciences USA, 84, 5048-5052. Moseley, J. M., Hayman, J. A., Danks, J. A., Alcorn, D., Grill, V., Southby, J. & Horton, M. A. (1991) Immunohistochemical detection of parathyroid hormone-related protein in human fetal epithelia. Journal of Clinical Endocrinologyand Metabolism, 73,478-484. Orloff, J. J., Wu, T. L. & Stewart, A. F. (1989) Parathyroid hormone-like proteins: biochemical responses and receptor interactions. EndocrineReviews, 10, 476-495. Paspaliaris, V., Vargas, S.J., Gillespie, M. T., Williams, E. D., Danks, J. A., Moseley, J. M., Story, M. E., Pennefather, J. N., Leaver, D. D. & Martin, T. J. (1992) Oestrogen enhancement of the myometrial
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response to exogenous parathyroid hormone-related protein (PTHrP) and tissue localization of endogenous PTHrP and its mRNA in the virgin rat uterus.Journal of Endocrinolo~, 134, 415-425. Rateliffe, W. A., Norbury, S., Stott, R. A., Heath, D. A. & Rateliffe, J. G. (1991) Immunoreactivity of plasma parathyrin-related peptide: three region specific radioimmunoassays and a two-site immunoradiometric assay compared. Clinical Chemistry, 37, 1781-1787. Shew, R. L., Yee, J. A., Kliewer, D. B., Keflemariam, Y. J. & MeNeill, D. L. (1991) Parathyroid hormonerelated protein inhibits stimulated uterine contraction in vitro. Journal of Bone and Mineral Research, 6, 955-959. Soifer, N. E., Dee, K. E., Insogna, K. L., Burtis, W.J., Matoveik, L. M., Wu, T., Milstone, L. M., Broadus, A. E., Philbriek, W. M. & Stewart, A. F. (1992) Parathyroid hormone-related protein. Evidence for secretion of a novel mid-region fragment by three different cell types.Journal of Biological Chemistry, 267, 18 236-18 243. Thiede, M. A., Grasser, W. A. & Petersen, D. N. (1992) Regulated expression of parathyroid hormone-related protein in mammary blood supply supports a role in mammary blood flow. In Calcium Regulating Hormones and Bone Metabolism: Basic and ClinicalAspects (Eds) Cohn, D. V., Gennari, C. & Tashjian, A. H. Vol. II, pp. 62-68. Amsterdam: Excerpta Medica. Thiede, M. A., Daifotis, A. G., Weir, E., Brines, M. L., Burtis, W.J., Ikeda, K., Dreyer, B. E., Garfield, R. E. & Broadus, A. E. (1990) Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in pre-term rat myometrium. Proceedingsof the NationalAcademy of Sciences USA, 87, 6969-6973. Winquist, R. J., Baskin, E. P. & Vlasuk, G. P. (1987) Synthetic tumor derived human hypercalcemic factor exhibits parathyroid hormone-like vasorelaxation in renal arteries. Biochemical and Biophysical Research Communications, 149, 227-232. Yamamoto, M., Harm, S. C., Grasser, W. A. & Thiede, M. A. (1992) Parathyroid hormone-related protein in the rat urinary bladder: A smooth muscle relaxant produced locally in response to mechanical stretch. Proceedings of the National Academy of Sciences USA, 89, 5326-5330.
Immunohistochemical Localization of Parathyroid Hormone-Related Protein (PTHrP) in Human Term Placenta and Membranes J. F. EMLY a, J. GREGORY b, S.J. BOWDEN a, A. AHMED c, M.J. WHITTLE d, D. I. R U S H T O N e & W. A. RATCLIFFE af "Wolfson Research Laboratories, Department of Clinical Chemistry, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH bDepartment of Pathology, Queen Elizabeth Medical Centre, Edgbaston, Birmingham CDepartment of Obstetrics and Gynaecology, aDepartment of Fetal Medicine and eDepartment of Pathology, Birmingham MaterniO, Hospital, Birmingham, UK f To whom correspondenceshould be addressed Paper accepted16.2.1994
SUMMARY Parathyroid hormone-related protein (PTHrP), the major factor responsible for hypercalcaemia of malignancy, is widely expressed in normal adult and fetal tissues. In this study, the distribution of PTHrP was examined in human term placenta and membranes by immunohistochemistry using antisera to PTHrP 1-34 and 37-67. PTHrP was detected in cuboidal epithelial cells of amnion and in O~totrophoblastic cells of chorionic laeve and adherent maternal decidua. In placenta, PTHrP 1-34 was detected in the syno~tiotrophoblast, while PTHrP 37-67 activity was mainly present in the brush border of the synoJtiotrophoblast. This study also identified PTHrP 37-67 associated with fetal vessels of placental villi. These findings may retied the cellular distribution of intact PTHrP or sub-fragments derived by posttranslational processing. Postulated actions of PTHrP in the uteroplacental unit include transport of calcium across the placenta, stretch of membranes, inhibition of uterine contractility, growth and differentiation, and vasoregulation.
INTRODUCTION Parathyroid hormone-related protein (PTHrP) initially isolated from cancer cell lines and tumours from patients with hypercalcaemia of malignancy (Moseley et al, 1987) is estab0143-4004/94/060653 + 08 $08.00/0
9 1994 W. B. Saunders Company Ltd
Placenta (1994), Vol. 15
654
lished as the major humoral factor responsible for hypercalcaemia of malignancy (Martin and Suva, 1989). PTHrP exists in three isoforms of 139, 141 and 173 amino acids which differ only at the extreme carboxyl-termini. PTHrP shares sequence homology with parathyroid hormone (PTH) at its amino-terminus, allowing PTHrP to mimic the actions of P T H by interaction with classical PTH receptors in bone and kidney (Juppner et al, 1988). Receptor subtypes unique for PTHrP have yet to be identified (Orloff, Wu and Stewart, 1989). PTHrP has been localized by immunohistochemistry in a wide range of normal adult and fetal tissues (Kramer et al, 1991; Moseley et al, 1991). In the uteroplacental unit PTHrP mRNA is expressed in amnion, chorion, decidua, myometrium and placenta (Thiede et al, 1990; Ferguson et al, 1992) and PTHrP has been extracted from human term placenta and membranes and quantitated by immunoassay (Abbas et al, 1990; Bowden et al, 1994). Specific binding sites for PTH 1-34 in basal membranes and the brush border of the syncytiotrophoblast, may reflect sites of action of PTHrP (Lafond et al, 1988). A number of actions are proposed for PTHrP in placental tissue. PTHrP 67-86 amide derived from placenta or fetal parathyroid glands may stimulate calcium transport across the placenta and maintain plasma calcium in the fetus at a higher level than in the maternal circulation (Care et al, 1990). P T H 1-34 and PTHrP 1-34 are both relaxants of vascular and non-vascular smooth muscle (Winquist, Baskin and Vlasuk, 1987; Mok et al, 1989) and PTHrP produced in association with vascular tissue may have local effects on blood flow (Thiede, Grasser and Petersen, 1992). In myometrium and amnion PTHrP 1-34 may act locally to facilitate stretch and relaxation of these tissues (Daifotis et al, 1992; Ferguson et al, 1992). Thus PTHrP may act as a precursor of peptides with differing biological actions. Using antisera specific for PTHrP 1-34, positive immunostaining was demonstrated in the syncytiotrophoblast of the human placenta at term (Ishikawa et al, 1992), while PTHrP 56-86 activity was localized mainly in the cytotrophoblast (Hellman et al, 1992). Kramer et al (1991) failed to detect PTHrP in human amnion at term, although both PTHrP mRNA (Germain et al, 1992) and immunoreactivity (Bowden et al, 1994) were identified in this tissue. The aim of this study was to localize PTHrP by immunohistochemistry in human term membranes and placenta using region-specific polyclonal antisera raised to PTHrP 1-34 and 37-67. Information on the cellular distribution of PTHrP may reflect its role in the uteroplacental unit.
MATERIALS AND M E T H O D S Antisera used in this study were raised in rabbits by immunization with conjugates of PTHrP 1-34 and PTHrP 37-67. The 37-67 antiserum has previously been used to localize PTHrP in tumours from normocalcaemic and hypercalcaemic patients (Bundred et al, 1991; Dunne et al, 1993). The antibodies recognize residues 9-18 and 52-61, respectively, and do not cross-react with either PTH or other subfragments of PTHrP (Ratcliffe et al, 1991). Optimum intensity of specific immunostaining was obtained at dilutions of 1:200 in both cases. Immunohistochemistry was carried out on 5 Ixm-thick sections of formalin-fixed, paraffin-embedded tissue. Sections were deparaffinized by incubation for 5 min with Histoclear, hydrated through methanol to water and endogenous peroxidase activity was
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quenched by the addition of 0.3 per cent (v/v) of hydrogen peroxide in methanol for 15 rain. Before application of PTHrP antibodies [1:200 in phosphate buffered saline (PBS) for 30 min], non-specific binding sites were blocked by the addition of non-immune sheep serum (1:4 in PBS) for 15 min. The sections were then washed in 3 • 200ml of 0.1 per cent (v/v) polyoxylene 10 oleoyl ether (Brij 96) in PBS over a period of 15min, and incubated with affinity purified horse radish peroxidase-conjugated sheep-anti-rabbit immunoglobulin (1:100) in PBS for 30 min. Following washing, diaminobenzidine (DAB, 0.5 mg/ml and hydrogen peroxide 0.3 per cent in PBS) was applied to the sections for 6 min to visualize the peroxidase activity. Following counterstaining with Mayers haematoxylin, the sections were dehydrated and mounted. Syncytiotrophoblast was identified by immunostaining with an antiserum to human placental lactogen (Dako; diluted 1:200), following treatment of sections with 1 mg/ml trypsin, 1 mg/ml calcium chloride in water at pH 7.8 for 20 min at 37~ This was followed by addition of a second antibody as described above. Endothelial cells were visualized using monoclonal antibody QBendl0 (Unipath) (1:25 in 10 per cent goat serum) followed by a biotinylated goat anti-mouse/rabbit second antibody (Duet system from Dako) and a streptavidin-biotin HRP complex (both diluted 1:100 in PBS). For dual immunodetection of PTHrP and QBendl0 on the same section, DAB was used as the peroxidase substrate for localization of PTHrP and the substrate Vector SG (Vector Labs) was used in its place to visualize endothelial cells detected by QBendl0. Controls consisted of sections where the primary antibody was replaced by the same dilution of the appropriate non-immune serum, where the secondary antibody was omitted, or where diluted antibody was pre-incubated with either 0.4 mg/ml PTHrP 1-34 or 37-67 for 18 h at 4~
Tissues Placenta, membranes and cord were from normal term pregnancies (n = 4) delivered by caesarean section. Placental tissue was from a central location lying between the basal and chorionic plates. Amnion and chorion (chorionic laeve) were from reflected membranes formerly covering the decidua parietalis of the uterus. Chorion from the chorionic plate of the placenta was also studied.
RESULTS With antisera to PTHrP 1-34 and 37-67, positive cytoplasmic staining was identified in cuboidal epithelium (A) of reflected amnion [Figure l(a)], amnion covering the chorionic plate, and amnion covering the umbilical cord (not shown). Figure 1(b) shows chorion laeve stained with antiserum to PTHrP 1-34. This tissue consists of a reticular layer composed of fibroblasts in a fibrous network, trophoblasts that extend from the pseudo-basement membrane to the decidual layer. A degenerate villus is present in the trophoblast layer. Positive staining was found in cytotrophoblastic cells and adhering maternal decidua using antisera to both PTHrP 1-34 and 37-67. In placenta, different patterns ofPTHrP positivity were obtained with antisera to PTHrP 1-34 and 37-67. With the antiserum to PTHrP 1-34, positive staining was present in the syncytiotrophoblast [Figure l(c)], while the antiserum to PTHrP 37-67 gave positive
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Figure 1. The distribution of parathyroid hormone-related protein (PTHrP) in human term placenta and membranes. (a) Positive staining for PTHrP 1-34 in epithelial cells of amnion (A), and (b) trophoblasts (T) and decidua (D) of chorion. (c) Placenta with the syncytiotrophoblast (S) positively stained with antiserum to PTHrP 1-34. (d) Placenta with positive staining for PTHrP 37-67 in brush border of the syncytiotrophoblast (S) and in endothelial cells of fetal vessels (E). Scale bar: 30 wm for (a) and (b); 20 wm for (c) and (d). PBM, pseudobasement membrane; DV, degenerate villus.
staining in the syncytiotrophoblast brush border, and in endothelial cells of fetal vessels [Figure 1(d)]. Endothelial cells were identified by staining for QBend 10. Dual staining with QBendl0 and PTHrP 1-34 antiserum identified PTHrP 1-34 mainly in the syncytiotrophoblast. PTHrP was not localized in components of neonatal or maternal blood. In control sections, no positive staining was found when primary or secondary antibodies'were omitted or replaced with non-immune serum, or when the antiserum was pre-absorbed by excess of the appropriate peptide (Figure 2).
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Figure 2. (a) Human term amnion, chorion and decidua and (b) placenta incubated with non-immune serum in place of primary antibody. Scale bar: 120 Ixm.
DISCUSSION This study has confirmed the presence of PTHrP in cells of different histological types within the human uteroplacental unit at term, suggesting that PTHrP may have diverse physiological roles in these tissues. Strong positivity was found exclusively in cuboidal epithelium of amnion from all sites examined. Recent studies have reported high PTHrP mRNA levels in human amnion, and demonstrated secretion of PTHrP by human amnion in vitro (Germain et al, 1992), and the PTHrP concentration in amnion has been quantitated by immunoassay (Bowden et al, 1994). Vasoactive peptides such as PTHrP and endothelin (Casey, Word and MacDonald, 1991) produced by amnion may act locally to regulate blood flow in fetal vessels of the chorionic plate, while PTHrP from reflected amnion and chorion may act locally to facilitate stretch of the membranes and inhibit contractility of the pregnant uterus (Shew et al, 1991; Paspaliaris et al, 1992). PTHrP mRNA concentrations in amnion were lower after labour than after elective caesarean section (Germain et al, 1992) and in labouring women, tissue PTHrP concentrations were inversely correlated with the time between rupture of the membranes and delivery (Bowden et al, 1994), suggesting that PTHrP gene expression in amnion is regulated by mechanical stretch or intraluminal pressure as in the uterus and bladder (Thiede et al, 1990; Yamamoto et al, 1992). The failure of earlier studies to detect PTHrP in amnion may reflect the duration of labour (Kramer et al, 1991). Amnion is a likely source of the high PTHrP concentrations found in amniotic fluid at term (Ferguson et al, 1992; Bowden et al, 1994). PTHrP is widely expressed in human fetal tissues and changes in expression with gestation suggest that it may have roles in growth and differentiation (Moseley et al, 1991). PTHrP in amniotic fluid may also have effects in the fetus either by intestinal absorption or by direct effects on skin, or the intestinal and respiratory tracts. In contrast PTHrP 1-86 levels are only modestly increased in maternal and cord venous plasma at term, suggesting that PTHrP acts predominantly by a local mechanism in placenta.(Bowden et al, 1994). The increase in epidermal growth factor (EGF) receptors in cultured trophoblasts seen in response to P T H (Alsat et al, 1991) may
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reflect interactive roles for EGF and PTHrP in regulating growth and endocrine function of these cells. Positive immunostaining was also localized in extravillous trophoblasts and maternal decidua associated with chorionic laeve. These cells are a likely source of the high PTHrP levels measured in chorion and membranes by immunoassay (Abbas et al, 1990; Bowden et al, 1994). PTHrP produced at these sites may have important actions as an inhibitor of uterine contractility during pregnancy (Paspaliaris et al, 1992). PTHrP mRNA has previously been identified in endometrial stroma cells (Casey et al, 1993) which are progenitors of decidua. Proteolytic processing of native PTHrP occurs between residues 37 and 38 in normal and malignant cells in vitro, yielding a major secretory form with residue 38 at its aminoterminus (Soifer et al, 1992). The same study also showed differing intracellular locations of amino-terminal and mid-region PTHrP. The epitopes of the antisera to PTHrP 1-34 and 37-67 used in the present study span the 37-38 cleavage point and would be expected to localize both intact PTHrP and subfragments derived by proteolytic processing. Using these antisera, PTHrP 1-34 was localized to the cytoplasm of the syncytium, and PTHrP 37-67 to its brush border and also endothelial cells of fetal vessels. It is unclear whether these differences reflect the specificities of the antibodies, or processing of PTHrP to subfragments with differing intracellular locations. The higher levels of 1-34 and 37-67 immunoreactivities relative to 1-86 immunoreactivity in human term placenta, and the molecular heterogeneity seen on chromatography (Bowden et al, 1994), are supporting evidence that proteolytic processing occurs between the epitopes of the 1-34 and 37-67 antibodies. In contrast an earlier study co-localized PTHrP 1-34 and 56-86 mainly in cytotrophoblasts (Hellman et al, 1992), while PTHrP 1-34 was identified in the syncytium and Hofbauer cells of human term placenta (Ishikawa et al, 1992). Production of PTHrP by vascular tissue and specific binding of PTH and PTHrP to membranes of vascular smooth muscle cells provide support for its role in the local regulation of vascular tone (Mok et al, 1989). We postulate that PTHrP produced by endothelial cells may have a similar action in modulating blood flow in fetal vessels, possibly by interaction with the extravascular contractile system of the placenta (Grafet al, 1993). In the isolated perfused cotyledon from human term placenta, PTHrP gave a concentrationdependent vasodflator effect and reduced perfusion pressure by approximately 50 per cent (Mandsager, Brewer and Myatt, 1993). Specific regulatory factors are increasingly implicated in the regulation of placental growth and function and in the interaction of decidua and trophoblasts. The wide tissue distribution of PTHrP in the uteroplacental unit suggests that it may have a range of potential physiological actions, including growth and differentiation, inhibition of smooth muscle contractility, vasoregulation and calcium transport. It remains to be established whether it has a role in calcium homeostasis in the fetus or in initiation of labour. ACKNOWLEDGEMENTS We thank the Departmentof Health and the EndowmentFund of the UnitedBirminghamHospitalsfor financial support. REFERENCES Abbas, S. K., Pickard, D. W., Illingworth, D., Storer,J., Purdie, D. W., Moniz, C., Dixit, M., Caple, I. W., Ebeling, P. R., Rodda, C. P., Martin, T. J. & Care, A. D. (1990) Measurementof parathyroid hormone-
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