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Correspondence to: Michael J. Soares, Dept. of Molecular and Integrative Physiology, U. of Kansas Medical Center, Kansas City, KS 66160-7401.
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Summary |
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The rat placenta expresses a family of genes related to prolactin (PRL). Target tissues and physiological roles for many members of the PRL family have yet to be determined. In this investigation we evaluated the use of an alkaline phosphatase (AP) tag for monitoring the behavior of a prototypical member of the PRL family, placental lactogen-I (PL-I). A probe was generated consisting of a fusion protein of human placental AP and rat PL-I (APPL-I). The APPL-I construct was stably expressed in 293 human fetal kidney cells, as was the unmodified AP vector that served as a control. AP activity was monitored with a colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and PRL-like biological activities of the APPL-I fusion protein were demonstrated by immunoblotting and the Nb2 lymphoma cell proliferation assay, respectively. APPL-I specifically bound to tissue sections known to express the PRL receptor, including the ovary, liver, and choroid plexus. Binding of APPL-I to tissues was specific and could be competed with ovine PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and suggest that this labeling system may also be useful for monitoring the actions of other members of the PRL family. (J Histochem Cytochem 46:737743, 1998)
Key Words: alkaline phosphatase fusion, protein, ovary, corpus luteum prolactin, receptors, liver, hepatic prolactin, receptors, Nb2 lymphoma cells, placental lactogen-I, pregnancy, prolactin receptor
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Introduction |
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The prolactin (PRL) GENE FAMILY contains at least 10 members some of which are expressed in the anterior pituitary, placenta, and/or uterus (
An important first step in understanding the actions of a hormone/cytokine is to identify its targets. Typically, target cells for various hormones/cytokines have been studied by radiolabeled ligand autoradiography or, when reagents are available to the designated ligand receptor, by immunocytochemical or in situ hybridization procedures. The former requires the isolation of a biologically active ligand to homogeneity and the latter requires identification and characterization of the receptor system used by the ligand. Neither of these options is readily available for nonclassical members of the PRL family whose biological actions during pregnancy are poorly understood. Flanagan and co-workers developed an alternative approach, involving the generation of alkaline phosphatase (AP)ligand fusion proteins, that has proved particularly useful for identifying components of receptor tyrosine kinase signaling pathways, including ligands and receptors (
In this study, we have determined the effectiveness of utilizing an AP tag to monitor a prototypical member of the PRL family, placental lactogen-I (PL-I). PL-I is a glycoprotein, as are most members of the PRL family, and is secreted by trophoblast giant cells of the developing placenta from immediately postimplantation until midgestation (
Collectively, the studies presented here indicate that the AP labeling system is an effective means of monitoring interactions of PL-I with its target cells and suggest the potential use of this labeling system for determining target cells for other PRL family members.
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Materials and Methods |
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Reagents
Fetal bovine serum (FBS) and donor horse serum (HS) were purchased from Sigma (St Louis, MO). Reagents for polyacrylamide gel electrophoresis were purchased from Bio-Rad (Hercules, CA). All restriction enzymes, polymerases, and DNA ligase were purchased from New England Biolabs (Beverly, MA). The 293 cell line of human fetal kidney origin was obtained from American Type Culture Collection (Rockville, MD). Transformation competent Sure bacterial cells and random primer labeling kits were acquired from Stratagene (La Jolla, CA). DNA extraction kits were purchased from Qiagen (Chatsworth, CA). Nitrocellulose was obtained from Schleicher & Schuell (Keene, NH). Ovine PRL was purchased from Nobl Laboratories (Sioux City, IA). T7 DNA sequencing kits were acquired from United States Biochemical (Cleveland, OH). The pCMV/SEAP vector was acquired from Tropix (Bedford, MA). Radiolabeled nucleotides were purchased from DuPontNEN (Boston, MA). Reagents for detection of immune complexes by enhanced chemiluminescence were acquired from Amersham (Arlington Heights, IL). Unless otherwise noted, all other chemicals and reagents were purchased from Sigma.
Animals and Tissue Preparation
Holtzman rats were obtained from Harlan SpragueDawley (Indianapolis, IN). The animals were housed in an environmentally controlled facility, with lights on from 0600 to 2000 hr, and were allowed free access to food and water. Timed pregnancies and tissue dissections were performed as previously described (
Generation of the APPL-I Fusion Protein
Construction of the APPL-I Vector.
A fusion protein consisting of a modified human placental AP (PLAP) and rat PL-I was generated and used to monitor PL-I target cell interactions. PLAP, in its native form, is a heat-stable, membrane-associated AP. The carboxy terminus of PLAP mediates membrane binding. Using oligodeoxynucleotide-directed mutagenesis, Berger and co-workers (1988) engineered a carboxy-terminal truncation that resulted in a secreted PLAP referred to as SEAP. The coding sequence for SEAP was subsequently localized downstream of the CMV promoter in a vector containing ampicillin and neomycin resistance genes (pCMV/SEAP; Tropix). A nucleotide region representing the carboxy terminal 197 amino acids of the mature rat PL-I protein was amplified (
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Transfection, Selection, and Cloning. After linearization with Bgl II, the APPL-I construct was electroporated into 293 cells. After a 2-week selection with 500 µg/ml G418, single clones were isolated by limiting dilution and screened for AP expression. An unmodified pCMV-SEAP vector (AP) was similarly transfected, and selected, and served as a negative control.
Preparation and Characterization of Medium Conditioned by the AP and APPL-I-transfected 293 Cells. Transfected 293 cells were cultured in MEM medium supplemented with 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS in an atmosphere of 5% CO2/95% air at 37C in a humidified incubator. After the cells reached confluence, the medium was changed to serum-free MEM + HEPES, further conditioned for 72 hr, collected and clarified by centrifugation, sterile-filtered (0.22 µm), and stored at -20C until used. AP activity was measured from conditioned medium via a colorimetric assay. Initially, samples were heated for 30 min in a 65C waterbath to inactivate endogenous heat-labile APs. Samples were then incubated at room temperature (RT) in a glycine buffer (50 mM glycine, pH 10.4, 0.5 mM MgCl2, 0.5 mM ZnCl2) containing nitrophenylphosphate (0.5 mg/ml) in a total reaction volume of 200 µl. After a 5-min incubation absorbance was measured at 405 nm. One unit of AP is defined as the amount of enzyme that hydrolyzes 1 µmole of p-nitrophenylphosphate to p-nitrophenol in 1 min at 37C in a volume of 1 ml.
Western Blot Analysis of PL-I
Western blot analysis was performed as previously described (
Biological Characterization of APPL-I
PRL-like biological activities were assessed by use of the rat Nb2 lymphoma cell proliferation assay (
In Situ Analysis of APPL-I Binding to Rat Tissues
Tissues were frozen in liquid nitrogen and stored at -70C until tissue sections were prepared with the aid of a cryostat. Sections were mounted onto glass slides, washed in a modified Hank's balanced salt solution (HBHA; containing 20 mM HEPES, 0.5 mg/ml BSA, and 0.1% NaN3), and incubated with AP, APPL-I, or APPL-I + excess PRL for 75 min at RT. After incubation, the sections were washed with HBHA supplemented with 0.1% Tween 20 and fixed for 2 min in a 20 mM HEPES buffer containing acetone (60%) and formaldehyde (3%). The fixed sections were washed, heated at 65C for 30 min to inactivate endogenous tissue AP activity, and then processed for detection of the heat-stable AP activity associated with the fusion proteins, and coverslips mounted in aqueous mounting medium. The AP and AP fusion protein were used at a concentration of 450 mU/ml. The specificity of binding was assessed by the addition of ovine PRL (5 µg/ml) to some of the tissue section incubations.
Statistical Analysis
Data were analyzed by one-way ANOVA. The source of variation from significant F ratios was determined with Student's two-tailed t-test (
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Results |
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As an important step towards demonstrating the suitability of the AP labeling system for monitoring activities of members of the PRL family, we generated and characterized an APPL-I fusion protein. The construction of the APPL-I vector was achieved by in-frame insertion of the cDNA sequence of mature rat PL-I downstream of the SEAP coding sequence within the pCMV-SEAP vector (Figure 1).
Generation and Characterization of the APPL-I Fusion Protein
The AP-PL-I construct and an unmodified AP control vector were transfected via electroporation into 293 cells. The presence of AP activity in conditioned medium from transfected cells and nontransfected cells was evaluated using a colorimetric assay. Several conditions for the production of recombinant protein were evaluated (Figure 2). Heat-stable AP activity secreted by AP- and APPL-I-transfected 293 cells was enhanced by the presence of FBS in the culture medium. The elevated AP activity in serum-containing cultures likely reflected stronger CMV promoter activity or possibly decreased protein degradation. Therefore, the generation of the APPL-I fusion protein did not interfere with AP enzymatic activity.
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Anti-PL-I antibodies specifically recognized the APPL-I fusion protein. AP and the APPL-I fusion protein were initially enriched by immunoprecipitation with a monoclonal antibody to human PLAP conjugated to agarose. As shown by Western blot analysis, PL-I antibodies recognized an APPL-I protein species approximating 110 kD and native PL-I protein species synthesized by differentiated Rcho-1 trophoblast cells with sizes ranging from 36 to 45 kD (Figure 3). The 110-kD Mr of the APPL-I fusion protein was consistent with the predicted Mr of its AP and PL-I components. The AP control preparation was not recognized by the PL-I antibodies (data not shown).
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Biological Characterization of APPL-I
For the APPL-I fusion protein to be a useful probe for monitoring PL-I interactions with its target tissues, the APPL-I should be able to mimic PRL biological actions. This issue was addressed by examining the actions of the APPL-I fusion protein on rat Nb2 lymphoma cells. The APPL-I fusion protein was capable of significantly stimulating the proliferation of rat Nb2 lymphoma cells in a concentration-dependent manner (Figure 4). Rat Nb2 lymphoma cell proliferation is dependent on activation of the PRL receptor signaling pathway, which can be achieved by PRL or other ligands capable of interacting with the PRL receptor, such as PL-I (
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Because the APPL-I fusion protein retained the ability to bind and activate PRL receptor signaling systems in vitro, we next evaluated its utility as an in situ probe for identifying PL-I target cells within tissue sections. AP, APPL-I, or APPL-I + excess ovine PRL were incubated with sections from several tissues, including ovaries from midgestation rats, liver tissues from midgestation rats, and brains from normal female rats (Figure 5). AP failed to demonstrate significant binding to any structures in the tissues investigated. APPL-I showed cell type-specific patterns of binding to the tissue sections that could be competed with excess ovine PRL (Figure 5). In the ovary, APPL-I was specifically localized to the corpus luteum, and binding within the brain was most prominent in the choroid plexus. APPL-I binding to liver sections was relatively homogeneous. On the basis of present evidence, the APPL-I fusion protein appears to be a useful probe for monitoring PL-I interactions with its target tissues and the localization of PRL receptors.
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Discussion |
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In this report we have described the generation and characterization of an APPL-I fusion protein. The fusion protein was generated by stably expressing a fusion gene containing the heat-stable human placental AP cDNA upstream of the rat PL-I cDNA in the 293 human fetal kidney cell line. The APPL-I fusion protein retained AP enzymatic activity, PL-I immunological and biological activities, and represents an important advance in the generation of a probe for monitoring PL-I interactions with its target cells. Therefore, in addition to investigating growth factor receptortyrosine kinase interactions (
PL-I is a PRL receptor agonist expressed from implantation until midgestation by trophoblast giant cells situated at the maternalplacental interface (
PL-I is a member of a larger family of hormones/cytokines that comprise the PRL family. PL-I is typical of most members of the PRL family in that it is a glycoprotein synthesized by trophoblast cells of the developing placenta (
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Footnotes |
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1 Present address: Department of Obstetrics and Gynecology, University of Rostock, Rostock, Germany.
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Acknowledgments |
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Supported by grants from the National Institute of Child Health and Human Development (HD 20676, HD 29797, HD 33994). HM was supported by a fellowship from the Deutsche Forschungsgemeinschaft of Germany (Mu 1183/1-1).
We thank Donna Millard, Bing Liu, and Belinda Chapman for advice and assistance in the preparation of the tissue sections used in this study and Christopher Cohick for assistance in preparing the figures. We also would like to thank Youngsoo Lee and Dr James Voogt for providing the rat brain tissue sections.
Received for publication September 3, 1997; accepted January 14, 1998.
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Literature Cited |
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Berger J, Hauber J, Hauber R, Geiger R, Cullen BR (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66:1-10[Medline]
Cheng HJ, Flanagan JG (1994) Identification and cloning of ELF-1, a developmentally expressed ligand for the Mek4 and Sek receptor tyrosine kinases. Cell 79:157-168[Medline]
Cheng HJ, Nakamoto M, Bergemann AD, Flanagan JG (1995) Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell 82:371-381[Medline]
Chiang MK, Flanagan JG (1995) Interactions between the Flk-1 receptor, vascular endothelial growth factor, and cell surface proteoglycan identified with a soluble receptor reagent. Growth Factors 12:1-10[Medline]
Chiang MK, Flanagan JG (1996) PIP NP, a new member of the receptor protein tyrosine phosphastase family, implicated in development of nervous system and pancreatic endocrine cells. Development 122:2239-2250
Clarke DL, Linzer DIH (1993) Changes in prolactin receptor expression during pregnancy in the mouse ovary. Endocrinology 133:224-232[Abstract]
Cohick CB, Dai G, Xu L, Deb S, Kamei T, Levan G, Szpirer C, Szpirer J, Kwok SCM, Soares MJ (1996) Placental lactogen I variant utilizes the prolactin receptor signaling pathway. Mol Cell Endocrinol 116:49-58[Medline]
Cohick CB, Xu L, Soares MJ (1997) Prolactin-like-B: heterologous expression and characterization of placental and decidual species. J Endocrinol 152:291-302[Abstract]
Colosi P, Ogren L, Southard JN, Thordarson G, Linzer DI, Talamantes F (1988) Biological, immunological, and binding properties of recombinant mouse placental lactogen-I. Endocrinology 123:2662-2667[Abstract]
Conliffe PR, Farmerie WG, Charles GD, Buhi WC, Kelly PA, Simmen RCM, Shiverick KT (1994) Expression and characterization of recombinant rat placental prolactin-like protein C. Mol Cell Endocrinol 106:121-130[Medline]
Dai G, Imagawa W, Liu B, Szpirer C, Levan G, Kwok SCM, Soares MJ (1996) Rcho-1 trophoblastcell placental lactogens: complementary deoxyribonucleic acids, heterologous expression, and biological activities. Endocrinology 137:5020-5027[Abstract]
Deb S, Hamlin GP, Roby KF, Kwok SCM, Soares MJ (1993) Heterologous expression and characterization of prolactin-like protein-A. Identification of serum binding proteins. J Biol Chem 268:3298-3305
Faria TN, Deb S, Kwok SCM, Talamantes F, Soares MJ (1990) Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta. Dev Biol 141:279-291[Medline]
Flanagan JG, Leder P (1990) The kit ligand: a cell surface molecule altered in steel mutant fibroblasts. Cell 63:185-194[Medline]
Freemark M, Kirk K, Pihoker C, Robertson MC, Shiu RPC, Driscoll P (1993) Pregnancy lactogens in the rat conceptus and fetus: circulating levels, distribution of binding, and expression of receptor messenger ribonucleic acid. Endocrinology 133:1830-1842[Abstract]
Galosy SS, Talamantes F (1995) Luteotropic actions of placental lactogens at midpregnancy in the mouse. Endocrinology 136:3993-4003[Abstract]
Hamlin GP, Lu X-J, Roby KF, Soares MJ (1994) Recapitulation of the pathway for trophoblast giant cell differentiation in vitro: stage-specific expression of members of the prolactin gene family. Endocrinology 134:2390-2396[Abstract]
Iwatsuki K, Shinozaki M, Hattori N, Hirasawa K, Itagaki S-I, Shiota K, Ogawa T (1996) Molecular cloning and characterization of a new member of the rat placental prolactin (PRL) family PRL-like protein D (PLP-D). Endocrinology 137:3849-3855[Abstract]
Jackson D, Volpert OV, Bouck N, Linzer DIH (1994) Stimulation and inhibition of angiogenesis by placental proliferin and proliferin-related protein. Science 266:1581-1584[Medline]
Keppel G (1973) Design and Analysis. Englewood Cliffs, NJ, Prentice Hall
MacLeod KR, Smith WC, Ogren L, Talamantes F (1989) Recombinant mouse placental lactogen-I binds to lactogen receptors in mouse liver and ovary: partial characterization of the ovarian receptor. Endocrinology 125:2258-2266[Abstract]
Ouhtit A, Morel G, Kelly PA (1993) Visualization of gene expression of short and long forms ofprolactin receptor in rat reproductive tissues. Biol Reprod 49:528-536[Abstract]
Pihoker C, Robertson MC, Freemark M (1993) Rat placental lactogen-I binds to the choroid plexus and hypothalamus of the pregnant rat. J Endocrinol 139:235-242[Abstract]
Rasmussen CA, Hashizume K, Orwig KE, Xu L, Soares MJ (1996) Decidual prolactin-related protein: heterologous expression and characterization. Endocrinology 137:5558-5566[Abstract]
Robertson MC, Cosby H, Fresnoza A, Cattini PA, Shiu RP, Friesen HG (1994) Expression, purification, and characterization of recombinant rat placental lactogen-I: a comparison with the native hormone. Endocrinology 134:393-400[Abstract]
Sakal E, Robertson MC, ChapnikCohen N, Tchelet A, Gertler A (1996) Interaction of recombinantrat placental lactogen-I with extracellular domains of prolactin receptors from three species. Receptors Signal Transduct 6:35-42[Medline]
Schuler LA, Kessler MA (1992) Bovine placental prolactin-related hormones. Trends Endocrinol Metab 3:334-338
Soares MJ, Julian JA, Glasser SR (1985) Trophoblast giant cell release of placental lactogens:temporal and regional characteristics. Dev Biol 107:520-526[Medline]
Soares MJ, Müller H, Orwig KE, Peters TJ, Dai G (1998) The uteroplacental prolactin family and pregnancy. Biol Reprod 58:273-284[Medline]
Tanaka T, Shiu RPC, Gout PW, Beer CT, Noble RL, Friesen HG (1980) A new sensitive and specific bioassay for lactogenic hormones: measurement of prolactin and growth hormone in human serum. J Clin Endocrinol Metab 51:1058-1063[Abstract]
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[Medline]
Volpert O, Jackson D, Bouck N, Linzer DIH (1996) The insulin-like growth factor II/mannose 6-phosphate receptor is required for proliferin-induced angiogenesis. Endocrinology 137:3871-3876[Abstract]