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Polar Expression and Phosphorylation of Human Leptin Receptor Isoforms in Paired, Syncytial, Microvillous and Basal Membranes from Human Term Placenta
Placenta (2002), 23, 516–521 doi:10.1053/plac.2002.0836, available online at http://www.idealibrary.com on
SHORT COMMUNICATION Polar Expression and Phosphorylation of Human Leptin Receptor Isoforms in Paired, Syncytial, Microvillous and Basal Membranes from Human Term Placenta C. F. Ebenbichlera,d, S. Kasera, M. Laimera, H. J. Wolfb, J. R. Patscha, and N. P. Illsleyc a Department of Medicine, University of Innsbruck, Austria, b Department of Gynecology and Obstetrics, University of Innsbruck, Austria and c Department of Obstetrics, Gynecology and Women’s Health, UMD-New Jersey Medical School, Newark, NJ 07103-2714, USA Paper accepted 17 April 2002
The hormone leptin (OB) and its receptor (OB-R) are key homeostatic regulators of mammalian body weight. Two predominant isoforms of OB-R are expressed by alternative splicing: the long form, OB-RL, with full signalling capacity is highly expressed in the hypothalamus and the short, signalling-defective form, OB-Rs, is ubiquitously expressed. In a previous study we detected expression of OB-RL and OB-Rs in human syncytiotrophoblast cells using in situ hybridization and immunohistochemistry (Bodner et al., 1999). The aim of this study was to investigate leptin receptor isoform expression and phosphorylation in paired, syncytial, microvillous and basal membranes from human term placenta by Western blot analysis. Both the OB-RL and the OB-Rs isoforms were detected in the syncytial membrane preparations. The OB-RL isoform was observed exclusively in microvillous membranes, whereas the OB-Rs isoform was found in both microvillous and basal membrane preparations. No significant differences were observed between syncytial membranes from normal and type 1 diabetic pregnancies. To test the phosphorylation capacity of the OB-R isoforms, microvillous and basal membrane vesicles loaded with ATP were stimulated with leptin and the phosphorylation status of the OB-R at the tyrosine 985 (Y985) was determined. A single band at the molecular weight corresponding to the molecular weight of the OB-RL isoform was detected exclusively in the ATP-loaded microvillous vesicles. We conclude that the long form OB-RL is expressed exclusively in the microvillous membrane of the syncytiotrophoblast and is capable of being phosphorylated, suggesting that it has signal transduction capacity. Placenta (2002), 23, 516–521 2002 Elsevier Science Ltd. All rights reserved.
INTRODUCTION Leptin is a circulating hormone secreted predominantly by adipose tissue but also by the placenta regulating mammalian food intake and reproduction (Chehab, 1997; Zhang et al., 1994). In mice, leptin-treated prepubertal female mice reproduced at an earlier age than nontreated mice, suggesting that leptin triggers puberty in this mouse model (Chehab et al., 1997). Leptin administration restored cyclicity in the normally acyclic ob/ob mouse, elevated LH concentrations, enhanced uterine and ovarian weights and changed uterine and ovarian d
To whom correspondence should be addressed at: Universita¨tsklinik fu¨r Innere Medizin, Universita¨t Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria. E-mail: [email protected]
0143–4004/02/$-see front matter
histology, once again suggesting that leptin acts as a trigger of reproductive maturity in mice (Chehab et al., 1996). Leptin is present in human cord blood and cord blood leptin levels correlate with birth weight and placental weight (Ashworth et al., 2000; Wolf et al., 2000). The leptin receptor (OB-R) is a single membrane spanning receptor with homology to the class I cytokine receptor family (Tartaglia et al., 1995; Tartaglia, 1997). Multiple isoforms of human and mouse OB-R have been identified, including a long form (OB-RL) with a large cytoplasmic domain capable of signaltransduction, and several receptor isoforms with short cytoplasmic domains (OB-Rs) lacking signal-transducing capabilities. The extracellular domains of the short and long forms of OB-R are identical. OB-RL transcripts were reported 2002 Elsevier Science Ltd. All rights reserved.
Ebenbichler et al.: Polar Expression and Signal Transduction of Leptin Receptor Isoforms in Human Placenta
to be expressed predominantly in regions of the hypothalamus previously thought to be important in body weight regulation whereas OB-Rs transcripts have been found in multiple tissues, including the choroid plexus, lung, kidney, and primitive hematopoietic cell populations. Signal transduction through the long isoform OB-RL occurs via phosphorylation of the tyrosines at residues 985, 1077 and 1138. Phosphorylation at the tyrosine residue 985 induces a signal cascade leading to egr-1 transcription, whereas phosphorylation at the residue 1138 leads to a feedback inhibition via the STAT 3 and socs-3 transcription (Bjorbaek et al., 1997; Bjorbaek et al., 1998; Bjorbaek et al., 2000). The leptin receptors OB-Rs and OB-RL were found in the murine placenta by RT-PCR, in situ hybridization, Western blot analysis and immunohistochemistry (Hoggard et al., 1997). Using quantitative RT-PCR, leptin receptors were also detected in the human placenta and a relative decline of the mRNA levels in advanced stages of gestation was noted (Henson et al., 1998). Both OB-RL and OB-Rs were localized to syncytiotrophoblast cells using in situ hybridization and immunohistochemistry (Bodner et al., 1999). The aim of this study was to determine the expression of human leptin receptor isoforms OB-RL and OB-Rs in paired, syncytial, microvillous and basal membranes isolated from human term placenta and to determine whether functional forms of the long signal-transducing isoform of OB-R are expressed in human placenta.
MATERIALS AND METHODS Tissue samples Placental tissue from three control and three diabetic pregnancies was obtained with written, informed consent, under procedures approved by the New Jersey Medical School Institutional Review Board. Criteria for inclusion as diabetic were (i) diagnosis of White Class B diabetes, (ii) maternal age between 18 and 38, (iii) absence of medical or obstetric complications other than diabetes, (iv) term delivery (37+ weeks gestation) and (v) singleton pregnancy. Exclusion criteria included the existence of nephropathy, retinopathy, hypertension (essential or pregnancy-induced) and conditions which might indicate altered uteroplacental blood flow or substrate delivery. Normal, age-matched control placental tissue was obtained and processed in a manner identical to the diabetic tissue.
Simultaneous preparation of paired, syncytial, microvillous and basal membranes Membranes were prepared as described previously at New Jersey Medical School, Newark, NJ using magnesium precipitation and differential centrifugation (Illsley et al., 1990). All procedures were performed at 4C unless otherwise stated.
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After removal of the cord, amniochorion, chorionic plate and a decidual layer, the tissue was cut into 11 cm pieces and washed with 0.9 per cent NaCl to remove blood. The homogenate was centrifuged at 10 000g for 15 min and the supernatant was removed and retained. The pellet was resuspended and rehomogenized. The second homogenate was centrifuged at 10 000g for 15 min and the supernatants from both centrifugations were combined. The combined supernatants were centrifuged at 47 500g for 60 min and the resulting pellet was resuspended. Mg2+ -aggregated basal membranes were separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes are obtained from the low speed supernatant, while basal membranes contained in the Mg2(+)-aggregated material were resuspended and further purified on a sucrose step gradient. Purified membranes were stored at 80C prior to use. Protein concentrations were determined using the method of Bradford (Bradford, 1976).
Electrophoresis and Western blot analysis Purified paired, syncytial, microvillous and basal membrane protein (10 g protein/per lane) were subjected to SDSPAGE (4–15 per cent gradient gels, Biorad, Vienna) under reducing conditions, and were electroblotted onto PVDFmembranes (Amersham Pharmacia Biotech, Vienna). To serve as a positive control and to allow a semiquantitative analysis of the OB-R isoforms, dilutions of recombinant human leptin receptor/Fc chimera (R&D Systems, Wiesbaden, Germany) were subjected to electrophoresis on the same Western blots. Non-specific binding was saturated using 3 per cent non-fat milk powder. Blots were incubated for 1 h at room temperature with the anti-human leptin (OB) receptor antibody (R&D Systems, Wiesbaden) at a concentration of 1 g/ml. After washing with TBS containing 0.1 per cent Tween 20 for 35 min, blots were incubated at room temperature with an affinity-isolated anti-mouse Ig antibody linked to horseradishperoxidase (DAKO, Denmark) at a dilution of 1:1000 for 1 h at RT. After washing three times with TBS/Tween 20 for 5 min and TBS for 10 min, blots were developed using the ECL system (Enhanced Chemiluminescence, Amersham, Vienna).
Phosphorylation of leptin receptors Microvillous and basal membrane vesicles were loaded with ATP using a method developed previously in one of our laboratories (Illsley et al., 1988). Briefly, vesicles were loaded with 1 KNO3, 10 m Hepes (adjusted to pH 7.0 with Tris) by overnight incubation at 4C (0.2 mg membrane protein/ ml). Vesicles were pelleted by centrifugation (75 000g, 30 min) and resuspended in the same buffer at a concentration of approximately 10 mg/ml. Vesicles were mixed rapidly
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with 50 volumes of lysis buffer (10 m Hepes/Tris, pH 7.0) containing 1 m NaF, 0.1 m EGTA and 1 m ATP then incubated at room temperature for 20 min. Following incubation, vesicles were washed 3 by pelleting and resuspension in lysis buffer containing 1 m NaF and 0.1 m EGTA. Vesicles were placed on ice and used immediately after loading with ATP. ATP-loaded vesicles were incubated with 10 ionomycin and 5 m Mg Cl2, with or without 10 n leptin (Chemicon International Inc., Temecula, CA) for 30 min at room temperature then extracted into Western sample buffer (2.6 urea, 1.7 per cent SDS, 16.5 m Tris/HCl, pH 6.8) containing 150 m DTT. The extracted vesicle samples were heated to 95C for 5 min and then subjected to SDS-PAGE (Laemmli, 1970) on 10 per cent gels (20 g membrane protein/lane) and blotted on to nitrocellulose membranes (Hybond ECL, Amersham, Arlington, IL) by electrophoretic transfer. After blocking with 3 per cent bovine serum albumin in Tris-buffered saline (TBS), membranes were probed with a polyclonal antibody to phospholeptin receptor (Y985) for 18 h at 4C (2 g/ml; Upstate Biotechnology, Lake Placid, NY). Following washing in TBS containing 0.05 per cent Tween-20 (115 min, 25 min), membranes were blotted with antirabbit HRP-conjugated IgG (1:5000; Sigma Chemical Company, St Louis, MO) and washed again. Bands were visualized by chemiluminescence (Super Signal; Pierce Endogen, Rockford, IL).
RESULTS Figure 1A shows the results of the Western blot experiments using paired, syncytial, microvillous and basal membranes from three normal pregnancies. Two bands, one at the molecular weight of approximately 110 kD, and a second at molecular weight of 70 kD were detected. The higher molecular weight band was present exclusively in the microvillous membranes, whereas the lower molecular weight band was present in both microvillous and basal membranes, but was predominant in the basal membranes. As a positive control dilutions of the recombinant human leptin receptor/Fc chimera were also subjected to electrophoresis and the Western blot procedure. The band appears at a higher molecular weight of approximately 190 kD due to fusion of the leptin receptor extracellular domain with the Fc region of human IgG1. Approximately 5 to 10 ng of the leptin receptor isoforms were present in each preparation. Figure 1B shows the results of the Western blot experiments of paired sets of microvillous and basal membranes from the pregnancies of three type 1 diabetics. Similar to results obtained from the placental membrane preparations of normal pregnancies two bands at the same molecular weight and distribution as seen in normal placental membrane preparations were detected. The quantities of the leptin isoforms present in these preparations was similar to that of the normal, control pregnancies.
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As a negative control a Western blot was prepared by omitting the primary antibody. Under these conditions no bands could be visualized (data not shown). Experiments were performed to test the potential phosphorylation capacity of leptin receptors in microvillous and basal membranes. Vesicles were loaded with 1 m ATP and 0.1 m EGTA then incubated for 30 min at room temperature with ionomycin, a magnesium-permeable ionophore, and MgCl2 in the presence or absence of 10 n leptin. Incubation with EGTA in the absence of Mg2+ prior to the incubation prevented loss of ATP. The results of Western blotting of vesicle samples using an antibody against the phosphorylated leptin receptor are shown in Figure 2. A band at a molecular weight of 110 kDa was observed in the microvillous membrane samples but no immunoreactive signal was present in the basal samples. No bands were observed following incubation in the absence of exogenous Mg2+ (data not shown), however bands were observed following incubation with Mg2+ in the presence or absence of leptin. It is clear from these results that there is phosphorylation of the leptin receptor on the microvillous membranes, while no phospholeptin receptor was observed on the basal membranes.
DISCUSSION The adipocyte-derived hormone leptin exerts its effects through the signal transduction-competent, long-form leptin receptor, OB-RL, as demonstrated in the db/db mouse (Chen et al., 1996; Lee et al., 1996). OB-RL is highly expressed in the hypothalamus and in certain peripheral tissues, including lung, pancreatic beta cells, kidney and keratinocytes. Thus, leptin mediates extrahypothalamic actions. In vitro, leptin acts as an mitogen for a growing number of cell types, including a factor-dependent hematopoetic cell line (Ghilardi and Skoda, 1997), keratinocytes (Frank et al., 2000), endothelial cells (Bouloumie et al., 1998), lung epithelial cells (Tsuchiya et al., 1999), monocytes (Santos-Alvarez et al., 1999), and pancreatic beta cells (Islam et al., 1997; Kieffer et al., 1997). In addition, leptin stimulates angiogenesis by promoting new vessel formation (Bouloumie et al., 1998). The placental syncytiotrophoblast expresses the leptin receptor (Hoggard et al., 1997; Henson et al., 1998; Hoggard et al., 2000) and, as determined by immunohistochemistry and in situ hybridization, both OB-RL and OB-Rs isoforms are present in the syncytiotrophoblast (Bodner et al., 1999). In this study we determined the distribution of the leptin receptor isoforms in paired syncytial, microvillous and basal membranes from human term placenta by the Western blot technique. The results revealed similar expression of OB-Rs in both the microvillous and basal membranes, whereas the signaltransducing isoform, OB-RL, displayed a differential expression pattern; OB-RL was exclusively expressed in syncytial microvillous membranes, but not in the syncytial basal membranes. To our knowledge this is the first description of a
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Figure 1. Western blot analysis of the OB-R isoforms in paired, syncytial, microvillous and basal membranes from human term placenta. Microvillous and basal membranes from A normal pregnancies, B type 1 diabetic pregnancies, MW denotes molecular weight, kD kilodaltons, M microvillous membranes, B basal membranes, ObRL long isoform of the leptin receptor, ObRs short isoform of the leptin receptor.
differential expression pattern of OB-R in an epithelial cell type. Although placentae from insulin-treated diabetic women have shown to have markedly elevated leptin mRNA and protein (Lepercq et al., 1998), we did not found differences in
OB-RL or OB-Rs occurrence between syncytial membranes from normal and type 1 diabetic pregnancies. Experiments were performed to test the functional significance of OB-RL expression, by investigating the potential for receptor phosphorylation. Incubation of ATP-loaded
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Figure 2. Western blot analysis of the OB-R isoforms in microvillous and basal membrane vesicles loaded with ATP from human term placenta using a polyclonal antibody to the phospholeptin receptor (Y985). kD kilodaltons, M microvillous membranes, B basal membranes.
microvillous vesicles revealed a band, identified by a specific anti-phospholeptin antibody, running at a molecular weight similar to that observed for the long-form leptin receptor. No bands were observed in the basal membranes or in the absence of Mg2+ . It is interesting to note however that bands were observed following incubation in the presence or absence of leptin, suggesting that at this concentration of MgATP, the accessory proteins are capable of phosphorylating the receptor in the absence of ligand. The presence of a phospholeptin band on the microvillous membrane but not on the basal membrane not only confirms the presence of OB-RL on the microvillous membrane, but also shows that this receptor is capable of being phosphorylated and may serve as a viable means of signal transduction. What mechanism could explain the selective transport of OB-RL to the syncytial microvillous membrane? The Janus kinase 2 (JAK 2) is required for ligand-induced receptor activation and the subsequent activation of JAK 2 (Ghilardi and Skoda, 1997). JAK 2 phosphorylates a number of substrates, including members of the signal transducer and activator (STAT) family. Both OB-RL and OB-Rs preferentially form homodimers, whereas OB-RL selectively
interacts with JAK 2, and concomitantly establishes its signal capacity (White et al., 1997; White and Tartaglia, 1999). This interaction may represent an important functional characteristic separating the OB-R isoforms and may result in cellular sorting of two distinct pools of OB-R. The homodimerization occurs at the level of the endoplasmatic reticulum or by a post-folding step, and once sorted into distinct pools, OB-RL may be transported selectively to the syncytial microvillous membrane. In summary this is, to our knowledge, the first report of the polar expression of OB-RL. In addition OB-RL expressed in the microvillous membrane exhibits phosphorylation in the residue Y985 of the intracellular domain. The substantial production and secretion of leptin by the placenta into the maternal circulation means that the maternal-facing, microvillous OB-RL isoform may play an important role in coordinating placental function with maternal metabolic homeostasis. This polar and functional expression of OB-RL may be the basis for further work on biological consequences of the maternal and/or foetal leptin/OB-RL interaction on placental and foetal growth.
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ACKNOWLEDGEMENTS This study was supported by grant Nr. 9078 of the Jubila¨umsfonds of the O } sterreichische Nationalbank and by NIH grant R01 DK55369 (NPI). The authors would like to thank the nursing staff and physicians of the Labor and Delivery Unit of University Hospital, Newark, for their assistance in helping to obtain the placental tissue used in this study.
REFERENCES Ashworth C, Hoggard N, Thomas L, Mercer J, Wallace J & Lea R (2000) Placental leptin. Rev Reprod, 5, 18–24. Bjorbaek C, Elmquist JK, Frantz JD, Shoelson SE & Flier JS (1998) Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol Cell, 1, 619–625. Bjorbaek C, Lavery HJ, Bates SH, Olson RK, Davis SM, Flier JS & Myers MG (2000) SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985. J Biol Chem. Bjorbaek C, Uotani S, da Silva B & Flier JS (1997) Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem, 272, 32686–32695. Bodner J, Ebenbichler C, Wolf H, Mu¨ller-Holzner E, Stanzl R, Huter O & Patsch J (1999) Leptin receptor in human term placenta: in situ hybridization and immunohistochemical localization. Placenta, 20, 677–682. Bouloumie A, Drexler H, Lafontan M & Busse R (1998) Leptin, the product of the Ob gene, promotes angiogenesis. Circ Res, 83, 1059–1066. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248–254. Chehab FF (1997) The reproductive side of leptin. Nature Medicine, 3, 952–953. Chehab FF, Lim ME & Lu R (1996) Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nature Genetics, 12, 318–320. Chehab FF, Mounzih K, Lu R & Lim ME (1997) Early onset of reproductive function in normal female mice treated with leptin. Science, 275, 88–90. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI & Morgenstern JP (1996) Evidence that the diabetes gene encodes the leptin receptor: Identification of a mutation in the leptin receptor gene in db/db mice. Cell, 84, 491–495. Frank S, Stallmeyer B, Kampfer H, Kolb N & Pfeilschifter J (2000) Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair. J Clin Invest, 106, 501–509. Ghilardi N & Skoda R (1997) The leptin receptor activates janus kinase 2 and signals for proliferation in a factor-dependent cell line. Mol Endokrinol, 11, 393–399. Henson M, Swan K & O’Neil J (1998) Expression of placental leptin and leptin receptor transcripts in early pregnancy and at term. Obstet Gynecol, 92, 1020–1028. Hoggard N, Hunter L, Duncan J, Williams L, Trayhurn P & Mercer J (1997) Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta. Proc Natl Acad Sci USA, 94, 11073–11078. Hoggard N, Hunter L, Lea R, Trayhurn P & Mercer J (2000) Ontogeny of the expression of leptin and its receptor in the murine fetus and placenta. Br J Nutr, 83, 317–326.
Illsley N, Wang Z, Gray A, Sellers M & Jacobs M (1990) Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. Biochim Biophys Acta, 1029, 218–226. Illsley NP, Goodman JG, Verkman AS & Davis B (1988) Macromolecule entrapment in human placental microvillous membrane vesicles. Anal Biochem, 175, 106–113. Islam M, Morton N, Hansson A & Emilsson V (1997) Rat insulinomaderived pancreatic beta-cells express a functional leptin receptor that mediates a proliferative response. Biochem Biophys Res Commun, 238, 851–855. Kieffer TJ, Heller RS, Leech CA, Holz GG & Habener JF (1997) Leptin suppression of insulin secretion by the activation of ATP-sensitive K+ channels in pancreatic beta-cells. Diabetes, 46, 1087–1093. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI & Friedman JM (1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature, 379, 632–635. Lepercq J, Cauzac M, Lahlou N, Timsit J, Girard J, Auwerx J & Hauguel-de Mouzon S (1998) Overexpression of placental leptin in diabetic pregnancy: a critical role for insulin. Diabetes, 47, 847–850. Santos-Alvarez J, Goberna R & Sanchez-Margalet V (1999) Human leptin stimulates proliferation and activation of human circulating monocytes. Cell Immunol, 194, 6–11. Tartaglia LA (1997) The leptin receptor. Journal of Biological Chemistry, 272, 6093–6096. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moriarty A, Moore KJ, Smutko JS, Mays GG, Woolf EA, Monroe CA & Tepper RI (1995) Identification and expression cloning of a leptin receptor, OB-R. Cell, 83, 1263–1271. Tsuchiya T, Shimizu H, Horie T & Mori M (1999) Expression of leptin receptor in lung: leptin as a growth factor. Eur J Pharmacol, 365, 273–279. White D, Kuropatwinski K, Devos R, Baumann H & Tartaglia L (1997) Leptin receptor (OB-R) signaling. Cytoplasmic domain mutational analysis and evidence for receptor homo-oligomerization. J Biol Chem, 272, 4065–4071. White D & Tartaglia L (1999) Evidence for ligand-independent homooligomerization of leptin receptor (OB-R) isoforms: a proposed mechanism permitting productive long-form signaling in the presence of excess short-form expression. J Cell Biochem, 73, 278–288. Wolf H, Ebenbichler C, Huter O, Bodner J, Lechleitner M, Foger B, Patsch J & Desoye G (2000) Fetal leptin and insulin levels only correlate in large-for-gestational age infants. Eur J Endocrinol, 142, 623–629. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L & Friedman JM (1994) Positional cloning of the mouse obese gene and its human homologue. Nature, 372, 425–432.
SHORT COMMUNICATION Polar Expression and Phosphorylation of Human Leptin Receptor Isoforms in Paired, Syncytial, Microvillous and Basal Membranes from Human Term Placenta C. F. Ebenbichlera,d, S. Kasera, M. Laimera, H. J. Wolfb, J. R. Patscha, and N. P. Illsleyc a Department of Medicine, University of Innsbruck, Austria, b Department of Gynecology and Obstetrics, University of Innsbruck, Austria and c Department of Obstetrics, Gynecology and Women’s Health, UMD-New Jersey Medical School, Newark, NJ 07103-2714, USA Paper accepted 17 April 2002
The hormone leptin (OB) and its receptor (OB-R) are key homeostatic regulators of mammalian body weight. Two predominant isoforms of OB-R are expressed by alternative splicing: the long form, OB-RL, with full signalling capacity is highly expressed in the hypothalamus and the short, signalling-defective form, OB-Rs, is ubiquitously expressed. In a previous study we detected expression of OB-RL and OB-Rs in human syncytiotrophoblast cells using in situ hybridization and immunohistochemistry (Bodner et al., 1999). The aim of this study was to investigate leptin receptor isoform expression and phosphorylation in paired, syncytial, microvillous and basal membranes from human term placenta by Western blot analysis. Both the OB-RL and the OB-Rs isoforms were detected in the syncytial membrane preparations. The OB-RL isoform was observed exclusively in microvillous membranes, whereas the OB-Rs isoform was found in both microvillous and basal membrane preparations. No significant differences were observed between syncytial membranes from normal and type 1 diabetic pregnancies. To test the phosphorylation capacity of the OB-R isoforms, microvillous and basal membrane vesicles loaded with ATP were stimulated with leptin and the phosphorylation status of the OB-R at the tyrosine 985 (Y985) was determined. A single band at the molecular weight corresponding to the molecular weight of the OB-RL isoform was detected exclusively in the ATP-loaded microvillous vesicles. We conclude that the long form OB-RL is expressed exclusively in the microvillous membrane of the syncytiotrophoblast and is capable of being phosphorylated, suggesting that it has signal transduction capacity. Placenta (2002), 23, 516–521 2002 Elsevier Science Ltd. All rights reserved.
INTRODUCTION Leptin is a circulating hormone secreted predominantly by adipose tissue but also by the placenta regulating mammalian food intake and reproduction (Chehab, 1997; Zhang et al., 1994). In mice, leptin-treated prepubertal female mice reproduced at an earlier age than nontreated mice, suggesting that leptin triggers puberty in this mouse model (Chehab et al., 1997). Leptin administration restored cyclicity in the normally acyclic ob/ob mouse, elevated LH concentrations, enhanced uterine and ovarian weights and changed uterine and ovarian d
To whom correspondence should be addressed at: Universita¨tsklinik fu¨r Innere Medizin, Universita¨t Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria. E-mail: [email protected]
0143–4004/02/$-see front matter
histology, once again suggesting that leptin acts as a trigger of reproductive maturity in mice (Chehab et al., 1996). Leptin is present in human cord blood and cord blood leptin levels correlate with birth weight and placental weight (Ashworth et al., 2000; Wolf et al., 2000). The leptin receptor (OB-R) is a single membrane spanning receptor with homology to the class I cytokine receptor family (Tartaglia et al., 1995; Tartaglia, 1997). Multiple isoforms of human and mouse OB-R have been identified, including a long form (OB-RL) with a large cytoplasmic domain capable of signaltransduction, and several receptor isoforms with short cytoplasmic domains (OB-Rs) lacking signal-transducing capabilities. The extracellular domains of the short and long forms of OB-R are identical. OB-RL transcripts were reported 2002 Elsevier Science Ltd. All rights reserved.
Ebenbichler et al.: Polar Expression and Signal Transduction of Leptin Receptor Isoforms in Human Placenta
to be expressed predominantly in regions of the hypothalamus previously thought to be important in body weight regulation whereas OB-Rs transcripts have been found in multiple tissues, including the choroid plexus, lung, kidney, and primitive hematopoietic cell populations. Signal transduction through the long isoform OB-RL occurs via phosphorylation of the tyrosines at residues 985, 1077 and 1138. Phosphorylation at the tyrosine residue 985 induces a signal cascade leading to egr-1 transcription, whereas phosphorylation at the residue 1138 leads to a feedback inhibition via the STAT 3 and socs-3 transcription (Bjorbaek et al., 1997; Bjorbaek et al., 1998; Bjorbaek et al., 2000). The leptin receptors OB-Rs and OB-RL were found in the murine placenta by RT-PCR, in situ hybridization, Western blot analysis and immunohistochemistry (Hoggard et al., 1997). Using quantitative RT-PCR, leptin receptors were also detected in the human placenta and a relative decline of the mRNA levels in advanced stages of gestation was noted (Henson et al., 1998). Both OB-RL and OB-Rs were localized to syncytiotrophoblast cells using in situ hybridization and immunohistochemistry (Bodner et al., 1999). The aim of this study was to determine the expression of human leptin receptor isoforms OB-RL and OB-Rs in paired, syncytial, microvillous and basal membranes isolated from human term placenta and to determine whether functional forms of the long signal-transducing isoform of OB-R are expressed in human placenta.
MATERIALS AND METHODS Tissue samples Placental tissue from three control and three diabetic pregnancies was obtained with written, informed consent, under procedures approved by the New Jersey Medical School Institutional Review Board. Criteria for inclusion as diabetic were (i) diagnosis of White Class B diabetes, (ii) maternal age between 18 and 38, (iii) absence of medical or obstetric complications other than diabetes, (iv) term delivery (37+ weeks gestation) and (v) singleton pregnancy. Exclusion criteria included the existence of nephropathy, retinopathy, hypertension (essential or pregnancy-induced) and conditions which might indicate altered uteroplacental blood flow or substrate delivery. Normal, age-matched control placental tissue was obtained and processed in a manner identical to the diabetic tissue.
Simultaneous preparation of paired, syncytial, microvillous and basal membranes Membranes were prepared as described previously at New Jersey Medical School, Newark, NJ using magnesium precipitation and differential centrifugation (Illsley et al., 1990). All procedures were performed at 4C unless otherwise stated.
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After removal of the cord, amniochorion, chorionic plate and a decidual layer, the tissue was cut into 11 cm pieces and washed with 0.9 per cent NaCl to remove blood. The homogenate was centrifuged at 10 000g for 15 min and the supernatant was removed and retained. The pellet was resuspended and rehomogenized. The second homogenate was centrifuged at 10 000g for 15 min and the supernatants from both centrifugations were combined. The combined supernatants were centrifuged at 47 500g for 60 min and the resulting pellet was resuspended. Mg2+ -aggregated basal membranes were separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes are obtained from the low speed supernatant, while basal membranes contained in the Mg2(+)-aggregated material were resuspended and further purified on a sucrose step gradient. Purified membranes were stored at 80C prior to use. Protein concentrations were determined using the method of Bradford (Bradford, 1976).
Electrophoresis and Western blot analysis Purified paired, syncytial, microvillous and basal membrane protein (10 g protein/per lane) were subjected to SDSPAGE (4–15 per cent gradient gels, Biorad, Vienna) under reducing conditions, and were electroblotted onto PVDFmembranes (Amersham Pharmacia Biotech, Vienna). To serve as a positive control and to allow a semiquantitative analysis of the OB-R isoforms, dilutions of recombinant human leptin receptor/Fc chimera (R&D Systems, Wiesbaden, Germany) were subjected to electrophoresis on the same Western blots. Non-specific binding was saturated using 3 per cent non-fat milk powder. Blots were incubated for 1 h at room temperature with the anti-human leptin (OB) receptor antibody (R&D Systems, Wiesbaden) at a concentration of 1 g/ml. After washing with TBS containing 0.1 per cent Tween 20 for 35 min, blots were incubated at room temperature with an affinity-isolated anti-mouse Ig antibody linked to horseradishperoxidase (DAKO, Denmark) at a dilution of 1:1000 for 1 h at RT. After washing three times with TBS/Tween 20 for 5 min and TBS for 10 min, blots were developed using the ECL system (Enhanced Chemiluminescence, Amersham, Vienna).
Phosphorylation of leptin receptors Microvillous and basal membrane vesicles were loaded with ATP using a method developed previously in one of our laboratories (Illsley et al., 1988). Briefly, vesicles were loaded with 1 KNO3, 10 m Hepes (adjusted to pH 7.0 with Tris) by overnight incubation at 4C (0.2 mg membrane protein/ ml). Vesicles were pelleted by centrifugation (75 000g, 30 min) and resuspended in the same buffer at a concentration of approximately 10 mg/ml. Vesicles were mixed rapidly
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with 50 volumes of lysis buffer (10 m Hepes/Tris, pH 7.0) containing 1 m NaF, 0.1 m EGTA and 1 m ATP then incubated at room temperature for 20 min. Following incubation, vesicles were washed 3 by pelleting and resuspension in lysis buffer containing 1 m NaF and 0.1 m EGTA. Vesicles were placed on ice and used immediately after loading with ATP. ATP-loaded vesicles were incubated with 10 ionomycin and 5 m Mg Cl2, with or without 10 n leptin (Chemicon International Inc., Temecula, CA) for 30 min at room temperature then extracted into Western sample buffer (2.6 urea, 1.7 per cent SDS, 16.5 m Tris/HCl, pH 6.8) containing 150 m DTT. The extracted vesicle samples were heated to 95C for 5 min and then subjected to SDS-PAGE (Laemmli, 1970) on 10 per cent gels (20 g membrane protein/lane) and blotted on to nitrocellulose membranes (Hybond ECL, Amersham, Arlington, IL) by electrophoretic transfer. After blocking with 3 per cent bovine serum albumin in Tris-buffered saline (TBS), membranes were probed with a polyclonal antibody to phospholeptin receptor (Y985) for 18 h at 4C (2 g/ml; Upstate Biotechnology, Lake Placid, NY). Following washing in TBS containing 0.05 per cent Tween-20 (115 min, 25 min), membranes were blotted with antirabbit HRP-conjugated IgG (1:5000; Sigma Chemical Company, St Louis, MO) and washed again. Bands were visualized by chemiluminescence (Super Signal; Pierce Endogen, Rockford, IL).
RESULTS Figure 1A shows the results of the Western blot experiments using paired, syncytial, microvillous and basal membranes from three normal pregnancies. Two bands, one at the molecular weight of approximately 110 kD, and a second at molecular weight of 70 kD were detected. The higher molecular weight band was present exclusively in the microvillous membranes, whereas the lower molecular weight band was present in both microvillous and basal membranes, but was predominant in the basal membranes. As a positive control dilutions of the recombinant human leptin receptor/Fc chimera were also subjected to electrophoresis and the Western blot procedure. The band appears at a higher molecular weight of approximately 190 kD due to fusion of the leptin receptor extracellular domain with the Fc region of human IgG1. Approximately 5 to 10 ng of the leptin receptor isoforms were present in each preparation. Figure 1B shows the results of the Western blot experiments of paired sets of microvillous and basal membranes from the pregnancies of three type 1 diabetics. Similar to results obtained from the placental membrane preparations of normal pregnancies two bands at the same molecular weight and distribution as seen in normal placental membrane preparations were detected. The quantities of the leptin isoforms present in these preparations was similar to that of the normal, control pregnancies.
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As a negative control a Western blot was prepared by omitting the primary antibody. Under these conditions no bands could be visualized (data not shown). Experiments were performed to test the potential phosphorylation capacity of leptin receptors in microvillous and basal membranes. Vesicles were loaded with 1 m ATP and 0.1 m EGTA then incubated for 30 min at room temperature with ionomycin, a magnesium-permeable ionophore, and MgCl2 in the presence or absence of 10 n leptin. Incubation with EGTA in the absence of Mg2+ prior to the incubation prevented loss of ATP. The results of Western blotting of vesicle samples using an antibody against the phosphorylated leptin receptor are shown in Figure 2. A band at a molecular weight of 110 kDa was observed in the microvillous membrane samples but no immunoreactive signal was present in the basal samples. No bands were observed following incubation in the absence of exogenous Mg2+ (data not shown), however bands were observed following incubation with Mg2+ in the presence or absence of leptin. It is clear from these results that there is phosphorylation of the leptin receptor on the microvillous membranes, while no phospholeptin receptor was observed on the basal membranes.
DISCUSSION The adipocyte-derived hormone leptin exerts its effects through the signal transduction-competent, long-form leptin receptor, OB-RL, as demonstrated in the db/db mouse (Chen et al., 1996; Lee et al., 1996). OB-RL is highly expressed in the hypothalamus and in certain peripheral tissues, including lung, pancreatic beta cells, kidney and keratinocytes. Thus, leptin mediates extrahypothalamic actions. In vitro, leptin acts as an mitogen for a growing number of cell types, including a factor-dependent hematopoetic cell line (Ghilardi and Skoda, 1997), keratinocytes (Frank et al., 2000), endothelial cells (Bouloumie et al., 1998), lung epithelial cells (Tsuchiya et al., 1999), monocytes (Santos-Alvarez et al., 1999), and pancreatic beta cells (Islam et al., 1997; Kieffer et al., 1997). In addition, leptin stimulates angiogenesis by promoting new vessel formation (Bouloumie et al., 1998). The placental syncytiotrophoblast expresses the leptin receptor (Hoggard et al., 1997; Henson et al., 1998; Hoggard et al., 2000) and, as determined by immunohistochemistry and in situ hybridization, both OB-RL and OB-Rs isoforms are present in the syncytiotrophoblast (Bodner et al., 1999). In this study we determined the distribution of the leptin receptor isoforms in paired syncytial, microvillous and basal membranes from human term placenta by the Western blot technique. The results revealed similar expression of OB-Rs in both the microvillous and basal membranes, whereas the signaltransducing isoform, OB-RL, displayed a differential expression pattern; OB-RL was exclusively expressed in syncytial microvillous membranes, but not in the syncytial basal membranes. To our knowledge this is the first description of a
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Figure 1. Western blot analysis of the OB-R isoforms in paired, syncytial, microvillous and basal membranes from human term placenta. Microvillous and basal membranes from A normal pregnancies, B type 1 diabetic pregnancies, MW denotes molecular weight, kD kilodaltons, M microvillous membranes, B basal membranes, ObRL long isoform of the leptin receptor, ObRs short isoform of the leptin receptor.
differential expression pattern of OB-R in an epithelial cell type. Although placentae from insulin-treated diabetic women have shown to have markedly elevated leptin mRNA and protein (Lepercq et al., 1998), we did not found differences in
OB-RL or OB-Rs occurrence between syncytial membranes from normal and type 1 diabetic pregnancies. Experiments were performed to test the functional significance of OB-RL expression, by investigating the potential for receptor phosphorylation. Incubation of ATP-loaded
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Figure 2. Western blot analysis of the OB-R isoforms in microvillous and basal membrane vesicles loaded with ATP from human term placenta using a polyclonal antibody to the phospholeptin receptor (Y985). kD kilodaltons, M microvillous membranes, B basal membranes.
microvillous vesicles revealed a band, identified by a specific anti-phospholeptin antibody, running at a molecular weight similar to that observed for the long-form leptin receptor. No bands were observed in the basal membranes or in the absence of Mg2+ . It is interesting to note however that bands were observed following incubation in the presence or absence of leptin, suggesting that at this concentration of MgATP, the accessory proteins are capable of phosphorylating the receptor in the absence of ligand. The presence of a phospholeptin band on the microvillous membrane but not on the basal membrane not only confirms the presence of OB-RL on the microvillous membrane, but also shows that this receptor is capable of being phosphorylated and may serve as a viable means of signal transduction. What mechanism could explain the selective transport of OB-RL to the syncytial microvillous membrane? The Janus kinase 2 (JAK 2) is required for ligand-induced receptor activation and the subsequent activation of JAK 2 (Ghilardi and Skoda, 1997). JAK 2 phosphorylates a number of substrates, including members of the signal transducer and activator (STAT) family. Both OB-RL and OB-Rs preferentially form homodimers, whereas OB-RL selectively
interacts with JAK 2, and concomitantly establishes its signal capacity (White et al., 1997; White and Tartaglia, 1999). This interaction may represent an important functional characteristic separating the OB-R isoforms and may result in cellular sorting of two distinct pools of OB-R. The homodimerization occurs at the level of the endoplasmatic reticulum or by a post-folding step, and once sorted into distinct pools, OB-RL may be transported selectively to the syncytial microvillous membrane. In summary this is, to our knowledge, the first report of the polar expression of OB-RL. In addition OB-RL expressed in the microvillous membrane exhibits phosphorylation in the residue Y985 of the intracellular domain. The substantial production and secretion of leptin by the placenta into the maternal circulation means that the maternal-facing, microvillous OB-RL isoform may play an important role in coordinating placental function with maternal metabolic homeostasis. This polar and functional expression of OB-RL may be the basis for further work on biological consequences of the maternal and/or foetal leptin/OB-RL interaction on placental and foetal growth.
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ACKNOWLEDGEMENTS This study was supported by grant Nr. 9078 of the Jubila¨umsfonds of the O } sterreichische Nationalbank and by NIH grant R01 DK55369 (NPI). The authors would like to thank the nursing staff and physicians of the Labor and Delivery Unit of University Hospital, Newark, for their assistance in helping to obtain the placental tissue used in this study.
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