Endocrinology Vol. 143, No. 11 4281-4286
Copyright © 2002 by The Endocrine Society
Two Placental Hormones Are Agonists in Stimulating Megakaryocyte Growth and Differentiation
Beiyan Zhou,
Hillary E. Lum,
Jiandie Lin and
Daniel I. H. Linzer
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Daniel I. H. Linzer, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 North Sheridan Road, Evanston, Illinois 60208. E-mail: dlinzer{at}northwestern.edu.
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Abstract
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Previously, we demonstrated that a placental hormone, PRL-like protein E, stimulates megakaryocyte growth and differentiation. We now find that PRL-like protein E and a second placental hormone, PRL-like protein F (PLP-F), bind the same receptor. PLP-F, which is produced later in pregnancy, might therefore act as either an agonist or antagonist of PRL-like protein E. To resolve this question, we produced recombinant PLP-F in mammalian cell cultures, purified the secreted glycoprotein hormone, and determined its activity in primary mouse bone marrow cultures. PLP-F induces megakaryocyte differentiation and megakaryocyte progenitor growth in a dose-dependent manner, with significant activity detected at a concentration as low as 50 ng/ml. PLP-F in maternal serum reaches at least 1 µg/ml on gestational d 14.5, and thus the biological activity of PLP-F is detected at physiological concentrations. These results show that PRL-like proteins E and F have the same stimulatory effects on megakaryocyte growth and differentiation, and therefore represent gestation stage-specific agonists.
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Introduction
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IN MAMMALIAN PREGNANCY, MANY processes in hematopoiesis are dramatically altered as maternal blood volume increases and fetal hematopoiesis initiates. Early in pregnancy in the mouse, the increase in blood volume and the increased consumption of platelets leads to thrombocytopenia, or low platelet levels (1, 2, 3). Cytokines of pregnancy, especially placental hormones, are excellent candidates for factors that bring about pregnancy-specific changes in hematopoiesis. Consistent with this prediction, we have shown that a mouse placental hormone, PRL-like protein E (PLP-E), induces megakaryocyte (MK) differentiation and synergizes with IL-3, IL-6, and thrombopoietin (TPO) to increase colony-forming unit-megakaryocyte (CFU-MK) growth in primary mouse bone marrow cell cultures (4). MK cells are the precursors of platelets, and injection of PLP-E into mice stimulates platelet production (5). PLP-E binds to a receptor that couples to gp130 (4), and this receptor is also present on human MKs (6). In primary cultures of human CD34+ bone marrow cells, PLP-E synergizes with TPO or a combination of TPO, stem cell factor, and flt-3 ligand to induce growth of these cells as well as the general myeloid progenitor CFU-GEMM and the lineage-specific progenitors CFU-MK and burst-forming unit-erythroid (6). An effect of PLP-E in stimulating erythrocyte differentiation in mouse and human cell lines has also been reported (7).
PLP-E is synthesized at midgestation by trophoblast giant cells, the outermost cell layer in the rodent placenta (8). In contrast, PRL-like protein F (PLP-F), a hormone that shares more than 50% amino acid sequence identity with PLP-E, is secreted later in gestation by a distinct layer of cells, the spongiotrophoblasts (8). The pattern of stage-specific synthesis of related placental hormones is a common feature of the PRL hormone family. Placental lactogen I and proliferin are produced in early to midgestation, whereas placental lactogen II and proliferin-related protein are expressed from midgestation to term (9). The two placental lactogens bind the PRL receptor, and these proteins appear to have identical biological activities (9). In contrast, proliferin is angiogenic and proliferin-related protein is antiangiogenic (10). Thus, hormone pairs in this family may be gestational stage-specific agonists, perhaps enabling distinct compartments (such as the mother and the late-gestation fetus) to be targeted. Alternatively, these hormones may be functional antagonists, thereby initiating and then terminating processes of pregnancy (such as vascular growth at the implantation site). Given the high degree of sequence identity between PLP-E and PLP-F, it seemed possible that these two hormones would have related activities. The purpose of this study was therefore to determine the biological activities of PLP-F.
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Materials and Methods
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Protein expression and purification
Three distinct PLP-F expression constructs were generated. First, the coding region from the PLP-F cDNA for only the mature protein was inserted downstream of the glutathione S-transferase (GST) coding sequence to produce GST-PLP-F fusion protein in bacteria. Second, the full-length PLP-F cDNA, including the coding region for the secretion signal sequence, was cloned into the pMT2 mammalian expression vector (11) and stably transfected into Chinese hamster ovary (CHO) cells. Third, the PLP-F coding region was positioned downstream of sequences encoding a secreted alkaline phosphatase (AP) in pCMV-SEAP to generate AP-PLP-F, as described previously for PLP-E (4); this construct was also stably transfected into CHO cells. CHO cells were selected for growth in G418, and individual clones were assayed by immunoblotting for secretion of PLP-F or AP-PLP-F. GST-PLP-F and the previously described GST-PLP-E (4) were purified from bacterial cell lysates by glutathione-Sepharose affinity chromatography. PLP-F secreted from CHO cells was precipitated from conditioned medium with 100 mM ZnSO4. After solubilization in 0.5 M EDTA (pH 8.0), 1 mM ß-mercaptoethanol, the protein preparation was applied to a fast protein liquid chromatography S-200 sizing column (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated with 50 mM Na 3-[N-morpholino]propanesulfonic acid (pH 8.0), 1 mM ß-mercaptoethanol. Hormone-containing fractions were identified by immunoblot analysis. Those fractions were pooled, concentrated with a Centricon-30 mini column (Amicon, Beverly, MA) and applied to a fast protein liquid chromatography ion exchange TSK DEAE column (Supleco, Bellefonte, PA) in 50 mM Na 3-[N-morpholino]propanesulfonic acid (pH 8.0), 1 mM ßmercaptoethanol. Bound proteins were eluted with a gradient of sodium acetate, and positive fractions were again identified by immunoblot analysis. PLP-F elutes at 230–280 mM sodium acetate. Fractions were pooled and applied to a lentil lectin Sepharose matrix (Amersham Pharmacia Biotech). PLP-F protein was eluted with 0.2-M 
-D-methylmannoside, and protein purity was determined by silver staining after polyacrylamide gel electrophoresis.
Hormone-binding assay
Mouse spleen sections were incubated with or without competitor protein for 30 min at room temperature before adding AP-PLP-F or AP-PLP-E for 45 min. Slides were then rinsed three times in Hanks’ balanced salt solution and fixed in 20 mM HEPES (pH 7.4), 60% acetone, and 3% formaldehyde. After washing and fixation, the slides were heated for 30 min at 65 C to inactivate endogenous AP, and bound fusion protein was detected by addition of the chromogenic substrate acetylthiocholiniodide (Sigma, St. Louis, MO).
Colony formation assay
Femurs from CD1 female mice were flushed with 5 ml Iscove’s modification of Dulbecco’s medium, 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD). Marrow cells were passed through gauge 21 and 25 needles sequentially and cultured for 45 min at 37 C to remove stromal cells by attachment to the culture dish surface. Nonadherent cells were collected, washed with Iscove’s modification of Dulbecco’s medium, 1% Nutridoma (Roche Molecular Biochemicals, Indianapolis, IN), and plated in MegaCult medium (Stem Cell Technology, Vancouver, Canada) following instructions provided by the manufacturer. About 5 x 105 nucleated bone marrow cells were cultured for 7–10 d in each well on two-well chamber slides. Colonies were dried and stained for acetylcholinesterase (AchE) activity before processing for microscopy and colony scoring. Colonies of MK cells were defined as clusters of at least three cells staining positive for AchE.
Antiserum against PLP-F, immunodepletion, and immunoblot analysis
A polyclonal antiserum against PLP-F was produced by immunization of rabbits with GST-PLP-F. To remove PLP-F from protein preparations, immunoglobulins in the antiserum or in normal rabbit serum were bound to protein G-Sepharose beads (Amersham Pharmacia Biotech), the beads were washed extensively with PBS, and either crude or purified protein samples were added. After centrifugation, unbound proteins were recovered and analyzed for the extent of removal of PLP-F. Samples were separated on 10% SDS polyarylamide gels and transferred to nitrocellulose membranes. The membranes were treated with blocking buffer (20 mM Tris, pH 7.6; 150 mM NaCl; 0.5% Triton X-100; and 5% nonfat milk), incubated with antiserum (1:1000 dilution), exposed to donkey-antirabbit immunoglobulins coupled to horseradish peroxidase, and then incubated with SuperSignal WestPico chemiluminescent substrate (Pierce Chemical Co., Rockford, IL).
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Results
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With the expectation that PLP-F, like PLP-E, would bind to a receptor on MK cells, AP-PLP-F was prepared and incubated with mouse spleen tissue sections. AP-PLP-F does bind to MK cells in these sections (Fig. 1A
). These cells are identified as MK based on size and morphology and expression of the MK marker, AchE, as we have shown previously for PLP-E binding (4). AP-PLP-F also binds to subcellular-size material in the spleen, which may correspond to platelets, as was also seen with PLP-E binding to spleen (4). Addition of excess PLP-E in the form of a fusion protein with GST (GST-PLP-E) abolishes AP-PLP-F binding, whereas the related hormone PLP-B (again as a GST fusion protein) is unable to block AP-PLP-F interaction with its receptor (Fig. 1, B and C). In the reverse experiment, binding of AP-PLP-E to MK cells (Fig. 1D) is blocked by PLP-F (Fig. 1E) but not be PLP-B (Fig. 1F). For these competitions, PLP-F and PLP-B were added as conditioned medium from COS cells transiently transfected with the corresponding expression constructs. (These preparations proved sufficient to analyze the identity of PLP-F target cells before undertaking large-scale production and purification of this protein.) Also, binding of AP-PLP-E is not competed by other cytokines known to target MK cells or by PRL or GH (5). Thus, PLP-E and PLP-F share a specific receptor on MK cells.
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Figure 1. Binding of AP-PLP-F to mouse MK cells. Mouse spleen sections were incubated with AP-PLP-F either without competitor protein (A) or after addition of excess GST-PLP-E (B) or GST-PLP-B (C). Additional sections were incubated with AP-PLP-E without competitor (D) or with PLP-F (E) or PLP-B (F) as competitor. Binding was scored by staining for AP activity, which is seen as dark staining of MK cells. Bar, 100 µm.
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To determine whether PLP-E and PLP-F are agonists, with both hormones capable of stimulating MK growth and differentiation, or whether PLP-F is an antagonist and prevents PLP-E action, purified PLP-F was generated. Because GST-PLP-E is biologically active (4), we initially synthesized and purified PLP-F as a bacterial GST-PLP-F fusion protein. Unlike GST-PLP-E, GST-PLP-F shows no receptor binding and is therefore not suitable for functional assays (data not shown). Nevertheless, the bacterial fusion protein was of value in producing a rabbit polyclonal antiserum against PLP-F. This antiserum is specific for PLP-F because it does not recognize the closely related hormone PLP-E (Fig. 2A).
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Figure 2. Purification of PLP-F. A, PLP-F was initially produced as a GST fusion protein in bacteria, and GST-PLP-F was used as immunogen to generate a rabbit-anti-PLP-F polyclonal antiserum. This antiserum recognizes PLP-F secreted from COS cells transiently transfected with a PLP-F cDNA expression construct but not similarly produced PLP-E. In contrast, the polyclonal antiserum against PLP-E reacts with PLP-E but not PLP-F. B, Recombinant PLP-F was purified from the conditioned medium of CHO cells (Crude) stably transfected with a PLP-F expression construct. Purity was assessed by SDS-PAGE and silver staining after S-200 gel filtration, DEAE ion exchange, and lectin affinity chromatography. Total protein in each lane is 1.2 µg (Crude), 500 ng (S-200), 430 ng (DEAE), and 18 ng (Lectin). After the final purification step, a single stained band with an apparent size of approximately 60 kDa is detected, consistent with extensive N-linked glycosylation of the PLP-F polypeptide. C, Purified protein was mock immunoprecipitated without antiserum (-) or immunoprecipitated with normal rabbit serum or rabbit-anti-PLP-F serum. Soluble protein remaining after immunoprecipitation was analyzed by gel electrophoresis and immunoblotting.
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In addition to generating a bacterial expression protein, we therefore turned to producing functional hormone by stable transfection of mammalian cells with a PLP-F expression construct. CHO cells transfected with this construct secrete glycosylated PLP-F protein, which was purified by size exclusion, ion exchange, and lectin affinity chromatography. The result is a protein that is detected as a single, silver-stained band after PAGE (Fig. 2B
). The polyclonal antiserum against PLP-F recognizes the native protein as evidenced by the ability of the antiserum, but not normal rabbit serum, to remove purified PLP-F protein from solution (Fig. 2C). This antiserum also interacts with PLP-F after denaturation and transfer to a membrane (Fig. 2C).
Purified PLP-F protein was applied to primary mouse bone marrow cell cultures at a concentration of 125 ng/ml, and MK cells were identified by staining for AchE. This concentration of hormone is well below the level of PLP-F in maternal serum, which is in excess of 1 µg/ml on gestational d 14.5 as judged by a semiquantitative comparison of pregnant mouse serum to known amounts of GST-PLP-F protein (Fig. 3). At d 14.5, peak levels of PLP-F mRNA are detected in the placenta (8). In the presence of IL-3, MK cells proliferate but remain small (Fig. 4A); addition of either PLP-E (Fig. 4B) or PLP-F (Fig. 4C) along with IL-3 promotes MK differentiation as seen by increased size of AchE-positive cells. The combination of PLP-E and PLP-F similarly induces MK differentiation (Fig. 4D).
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Figure 3. PLP-F levels in maternal serum. GST-PLP-F was used as a standard for comparison with unknown amounts of PLP-F present in maternal mouse serum on d 14.5 of gestation. The protein samples were fractionated by SDS-PAGE, transferred to a filter, and probed with anti-PLP-F antiserum. The antiserum was generated against GST-PLP-F, so antibodies that recognize both GST and PLP-F are present. About 60% of the GST-PLP-F fusion protein is GST, and we therefore estimate that a corresponding 60% of the signal in the standards is due to antibody binding to GST. Comparison of the 3-ng standard (about 1.2 ng of PLP-F) to the 1-µl serum sample or the 6-ng standard (about 2.4 ng of PLP-F) to the 2- and 3-µl serum samples suggests a concentration of more than 1 µg/ml.
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Figure 4. PLP-F and differentiation of MK colonies. Primary mouse bone marrow cultures were grown in the presence of 10 ng/ml IL-3 to induce proliferation. Cultures were also treated with (A) no other cytokines, 4 µg/ml GST-PLP-E (B), 125 ng/ml PLP-F (C), GST-PLP-E plus PLP-F (D), GST-PLP-E plus PLP-F after immunodepletion with anti-PLP-F antiserum (E), or GST-PLP-E plus PLP-F after immunodepletion with normal rabbit serum (F). After 1 wk, the cultures were stained for AchE activity. Large MK cells with intense AchE staining are seen in cultures treated with GST-PLP-E or PLP-F. Scale bar, 100 µm. G, Quantitative analysis of the various treatments. Results are mean ± SD, n = 220. Statistical significance was determined by a one-way ANOVA and a post hoc Tukey’s test, a vs. b, P < 0.001.
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Although production of GST-PLP-E in bacteria enabled the activity of that hormone to be tested without the possibility of contaminating mammalian cytokines, PLP-F was purified from mammalian cell cultures. To determine whether the observed activity is actually caused by PLP-F and not to a contaminant, purified PLP-F was removed by immunodepletion with the antiserum against PLP-F (Fig. 2B). As a control, a mock immunodepletion was carried out with normal rabbit serum. These samples were then applied to primary bone marrow cell cultures. Removal of PLP-F completely eliminates the activity of the purified hormone preparation (Fig. 4E), whereas the mock depletion is without effect (Fig. 4F), demonstrating that the MK differentiative activity corresponds to PLP-F. A quantitative analysis of MK cell size under these different conditions is shown (Fig. 4G).
In the absence of IL-3, MK cells in primary bone marrow cultures do not proliferate, but they are still able to differentiate in response to PLP-E (Fig. 5B), PLP-F (Fig. 5C), or the combination of these two hormones (Fig. 5D). The quantitative results from these treatments are shown (Fig. 5E). Thus, PLP-F can induce MK differentiation by itself, consistent with previous results with PLP-E (4).
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Figure 5. PLP-F and differentiation of nonproliferating MK cells. Primary bone marrow cultures were grown in the absence of IL-3. Cultures were untreated (A), or treated with 4 µg/ml GST-PLP-E (B), 125 ng/ml PLP-F (C), or GST-PLP-E plus PLP-F (D). After a week, the cultures were stained for AchE activity. Large MK cells with intense AchE staining are seen in cultures treated with GST-PLP-E or with PLP-F. Scale bar, 100 µm. E, Quantitative analysis of the various treatments. Results are mean ± SD, n = 150. Statistical significance was determined by a one-way ANOVA and a post hoc Tukey’s test, a vs. b, P < 0.001.
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PLP-E and PLP-F are also indistinguishable in their abilities to enhance the growth of the MK progenitor, CFU-MK. Neither hormone alone is able to stimulate CFU-MK growth above background levels, but each induces a significant increase in CFU-MK numbers when combined in the culture medium with IL-3 (Fig. 6). Immunodepletion of the PLP-F preparation with antibodies against PLP-F removes the CFU-MK growth stimulatory activity, whereas antibodies present in normal rabbit serum have no effect (Fig. 6). A concentration of PLP-F as low as 50 ng/ml in combination with IL-3 has a significant effect on CFU-MK growth, and the number of colonies further increases in response to higher PLP-F doses (Fig. 7).
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Figure 6. CFU-MK growth. Primary bone marrow cultures were treated with combinations of 10 ng/ml IL-3, 4 µg/ml GST-PLP-E, and 125 ng/ml PLP-F (purified hormone or that preparation after immunodepletion with anti-PLP-F antiserum or normal rabbit serum) and grown in semisolid media. After a week, clusters of at least three AchE-positive cells were scored as colonies. Data shown are the mean ± SEM and were evaluated by a one-way ANOVA followed by a Tukey’s test; a vs. b, P = 0.001; a vs. c, P < 0.001; b vs. c, P < 0.01; n = 4.
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Figure 7. PLP-F dose response. Concentrations of PLP-F from 50 to 250 ng/ml along with 10 ng/ml IL-3 were applied to primary bone marrow cultures. After a week, clusters of at least three AchE-positive cells were scored as colonies. Data shown are the mean ± SEM (n = 4) and were evaluated by a one-way ANOVA followed by a Tukey’s test; concentrations of PLP-F at 50 ng/ml (P < 0.01), 100 ng (P < 0.001), 150 ng (P = 0.001), and 250 ng (P < 0.001) gave statistically significant increases, compared with the control.
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Discussion
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Analysis of purified PLP-F demonstrated that this hormone shares the same receptor as PLP-E, although the relative affinity of these two hormones for receptor is not yet established. Quantitative analysis of hormone-receptor interaction, and characterization of the receptor protein, will likely depend on identifying homogenous cell populations (cell lines) that bind PLP-E and PLP-F. Not only do these two hormones bind the same receptor, but they also both induce the same responses in the MK lineage. PLP-F induces MK cell differentiation and in conjunction with IL-3 promotes CFU-MK growth. These activities, coupled with the timing of PLP-F expression in the placenta, are consistent with this hormone extending the midgestational stimulatory effect of PLP-E on platelet production into late gestation.
The selective pressure leading to the synthesis of two distinct placental hormones with indistinguishable activities can only be speculated on at this time. The synthesis of two agonists for the PRL receptor, placental lactogen I and placental lactogen II, provides a potentially useful model for thinking about PLP-E and PLP-F. Among its many functions, placental lactogen I has been implicated in binding PRL receptor in the hypothalamus to initiate a signal that blocks pituitary PRL secretion (12). Placental lactogen II has been detected in the fetal compartment and is the major ligand for the PRL receptor during late fetal development (13, 14). Thus, these two hormones may have evolved to reach generally inaccessible sites, with placental lactogen I able to transit the blood-brain barrier and placental lactogen II able to cross extraembryonic membranes. PLP-E and PLP-F may also have evolved to optimize access to two distinct populations of hematopoietic cells, possibly enabling these hormones to make different contributions to hematopoiesis in the maternal bone marrow, extraembryonic blood islands, and fetal liver.
Our previous studies of PLP-E activity relied on purified bacterial fusion protein. Higher concentrations of GST-PLP-E from bacteria (1–4 µg/ml) were required to stimulate cell responses (4) than is typical for many cytokines, but this is anticipated because the bacterial protein is nonglycosylated, because GST constitutes more than half of the fusion protein, and because at least some of the fusion protein recovered from bacteria may be inactive. In contrast, PLP-F purified from mammalian cell cultures has significant activity at a concentration of 50 ng/ml. All of the rodent placental hormones in the PRL family have been found to accumulate in maternal serum at high concentrations. For example, circulating placental lactogen I levels can reach 8 µg/ml (13), and proliferin can reach a concentration in the maternal serum of more than 5 µg/ml (15). Thus, our estimate of more than 1 µg/ml for the serum PLP-F concentration on gestational d 14.5 is consistent with measurements of related hormones and indicates that the hormone concentrations of 50–250 ng/ml that were used in these experiments are well within the physiological range.
Initially, we thought to compare GST-PLP-E and GST-PLP-F in terms of bioactivity, but the latter has no detectable ability to bind receptor. The N-terminal GST portion of the fusion protein might prevent proper folding or block a receptor binding site, but the results with GST-PLP-E argue against these explanations. Also, in contrast to the bacterial fusion protein, a mammalian PLP-F fusion protein with AP appended at the N terminus is able to bind receptor, suggesting that an extension at the N terminus does not prevent receptor interaction. Thus, the PLP-F polypeptide itself may be less competent than PLP-E to adopt its correct three-dimensional structure in bacteria. Both of these hormones are glycoproteins, and the mobility on polyacrylamide gels of PLP-F protein from maternal serum or secreted from mammalian cell cultures suggests that glycosylation is extensive; instead of the predicted size of 25 kDa based on the primary amino acid sequence, the PLP-F glycoprotein migrates with an apparent size of more than 50 kDa. It may be that the lack of glycosylation of GST-PLP-F is an important factor in the inactivity of this bacterial expressed protein.
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Acknowledgments
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We thank Diane Christiansen and Weimin Song for expert technical assistance.
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Footnotes
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This work was supported by NIH Grant R-01-HD-24518.
Abbreviations: AchE, Acetylcholinesterase; AP, alkaline phosphatase; CFU-MK, colony-forming unit-megakaryocyte; CHO, Chinese hamster ovary; GST, glutathione S-transferase; MK, megakaryocyte; PLP-E, PRL-like protein E; PLP-F, PRL-like protein F; TPO, thrombopoietin.
Received April 25, 2002.
Accepted for publication July 26, 2002.
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Abstract
Introduction
