Placental Lactogen-I (PL-I) Target Tissues Identified with an Alkaline Phosphatase–PL-I Fusion Protein
Total Page：16
File Type：pdf, Size：1020Kb
Volume 46(6): 737–743, 1998 The Journal of Histochemistry & Cytochemistry http://www.jhc.org ARTICLE Placental Lactogen-I (PL-I) Target Tissues Identified with an Alkaline Phosphatase–PL-I Fusion Protein Heiner Müller,1 Guoli Dai, and Michael J. Soares Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas SUMMARY 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 deter- mined. 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 KEY WORDS PL-I (AP–PL-I). The AP–PL-I construct was stably expressed in 293 human fetal kidney cells, as alkaline phosphatase fusion was the unmodified AP vector that served as a control. AP activity was monitored with a protein colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and ovary, corpus luteum prolactin PRL-like biological activities of the AP–PL-I fusion protein were demonstrated by immuno- receptors blotting and the Nb2 lymphoma cell proliferation assay, respectively. AP–PL-I specifically liver, hepatic prolactin bound to tissue sections known to express the PRL receptor, including the ovary, liver, and receptors choroid plexus. Binding of AP–PL-I to tissues was specific and could be competed with ovine Nb2 lymphoma cells PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and placental lactogen-I suggest that this labeling system may also be useful for monitoring the actions of other pregnancy members of the PRL family. (J Histochem Cytochem 46:737–743, 1998) prolactin receptor The prolactin (PRL) gene family contains at least ter requires identification and characterization of the 10 members some of which are expressed in the ante- receptor system used by the ligand. Neither of these rior pituitary, placenta, and/or uterus (Soares et al. options is readily available for nonclassical members 1998). Within this gene family, information is avail- of the PRL family whose biological actions during able on target cells and biological actions for only a pregnancy are poorly understood. Flanagan and co- subset of members utilizing the PRL receptor (classical workers developed an alternative approach, involving members) and those regulating angiogenesis (Jackson the generation of alkaline phosphatase (AP)–ligand fu- et al. 1994; Volpert et al. 1996). sion proteins, that has proved particularly useful for An important first step in understanding the actions identifying components of receptor tyrosine kinase of a hormone/cytokine is to identify its targets. Typi- signaling pathways, including ligands and receptors cally, target cells for various hormones/cytokines have (Flanagan and Leder 1990; Cheng and Flanagan 1994; been studied by radiolabeled ligand autoradiography or, Cheng et al. 1995; Chiang and Flanagan 1995,1996). when reagents are available to the designated ligand In this study, we have determined the effectiveness receptor, by immunocytochemical or in situ hybridiza- of utilizing an AP tag to monitor a prototypical mem- tion procedures. The former requires the isolation of a ber of the PRL family, placental lactogen-I (PL-I). PL-I biologically active ligand to homogeneity and the lat- is a glycoprotein, as are most members of the PRL family, and is secreted by trophoblast giant cells of the developing placenta from immediately postimplanta- Correspondence to: Dr. Michael J. Soares, Dept. of Molecular tion until midgestation (Faria et al. 1990). The actions and Integrative Physiology, U. of Kansas Medical Center, Kansas of PL-I have been primarily studied via the generation City, KS 66160–7401. of recombinant PL-I in heterologous cell types (Colosi 1 Present address: Department of Obstetrics and Gynecology, et al. 1988; Robertson et al. 1994; Dai et al. 1996). University of Rostock, Rostock, Germany. Received for publication September 3, 1997; accepted January PL-I has been shown to bind PRL receptors (MacLeod 14, 1998 (7A4458). et al. 1989; Freemark et al. 1993) and possesses a © The Histochemical Society, Inc. 0022-1554/98/$3.30 737 738 Müller, Dai, Soares number of actions previously attributed to pituitary amplified (Dai et al. 1996) and ligated into the pCMV/SEAP PRL (Soares et al. 1998). vector. Ligation with the PL-I insert resulted in a CMV pro- Collectively, the studies presented here indicate that moter-driven vector containing the ligated cDNAs encoding the AP labeling system is an effective means of moni- a SEAP-PL-I fusion protein (AP–PL-I; see Figure 1). DNA se- toring interactions of PL-I with its target cells and sug- quencing of the insert was performed to verify the accuracy of the PCR amplification. gest the potential use of this labeling system for deter- mining target cells for other PRL family members. Transfection, Selection, and Cloning. After linearization with Bgl II, the AP–PL-I construct was electroporated into 293 cells. After a 2-week selection with 500 mg/ml G418, single Materials and Methods clones were isolated by limiting dilution and screened for AP expression. An unmodified pCMV-SEAP vector (AP) was Reagents similarly transfected, and selected, and served as a negative Fetal bovine serum (FBS) and donor horse serum (HS) were control. purchased from Sigma (St Louis, MO). Reagents for poly- acrylamide gel electrophoresis were purchased from Bio-Rad Preparation and Characterization of Medium Condi- (Hercules, CA). All restriction enzymes, polymerases, and tioned by the AP and AP–PL-I-transfected 293 Cells. DNA ligase were purchased from New England Biolabs Transfected 293 cells were cultured in MEM medium sup- (Beverly, MA). The 293 cell line of human fetal kidney ori- plemented with 20 mM HEPES, 100 U/ml penicillin, 100 mg/ gin was obtained from American Type Culture Collection ml streptomycin, and 10% FBS in an atmosphere of 5% (Rockville, MD). Transformation competent Sure bacterial CO2/95% air at 37C in a humidified incubator. After the cells and random primer labeling kits were acquired from cells reached confluence, the medium was changed to serum- Stratagene (La Jolla, CA). DNA extraction kits were pur- free MEM 1 HEPES, further conditioned for 72 hr, col- chased from Qiagen (Chatsworth, CA). Nitrocellulose was lected and clarified by centrifugation, sterile-filtered (0.22 obtained from Schleicher & Schuell (Keene, NH). Ovine mm), and stored at 220C until used. AP activity was mea- PRL was purchased from Nobl Laboratories (Sioux City, sured from conditioned medium via a colorimetric assay. IA). T7 DNA sequencing kits were acquired from United Initially, samples were heated for 30 min in a 65C waterbath States Biochemical (Cleveland, OH). The pCMV/SEAP vec- to inactivate endogenous heat-labile APs. Samples were then tor was acquired from Tropix (Bedford, MA). Radiolabeled incubated at room temperature (RT) in a glycine buffer (50 nucleotides were purchased from DuPont–NEN (Boston, MA). Reagents for detection of immune complexes by en- hanced 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 Sprague–Dawley (Indianapolis, IN). The animals were housed in an environ- mentally 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 (Soares et al. 1985; Faria et al. 1990). The presence of a copulatory plug or sperm in the vaginal smear was designated Day 0 of pregnancy. Protocols for the care and use of animals were approved by the University of Kansas Animal Care and Use Committee. Generation of the AP–PL-I Fusion Protein Construction of the AP–PL-I Vector. A fusion protein con- sisting 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-associ- Figure 1 Construction of the AP–PL-I expression vector. A fusion protein consisting of a modified human placental AP (PLAP) and ated AP. The carboxy terminus of PLAP mediates membrane rat PL-I was generated and used to monitor PL-I target cell interac- binding. Using oligodeoxynucleotide-directed mutagenesis, tions. PLAP in its native form is a heat-stable, membrane-associated Berger and co-workers (1988) engineered a carboxy-termi- AP. The carboxy terminus of PLAP was truncated, resulting in a se- nal truncation that resulted in a secreted PLAP referred to as creted PLAP referred to as SEAP and situated downstream of the SEAP. The coding sequence for SEAP was subsequently lo- CMV promoter in a vector containing ampicillin and neomycin re- calized downstream of the CMV promoter in a vector con- sistance genes (pCMV/SEAP). A nucleotide region representing the carboxy-terminal 197 amino acids of the mature rat PL-I was ampli- taining ampicillin and neomycin resistance genes (pCMV/ fied and ligated into the pCMV/SEAP vector. Ligation with the PL-I SEAP; Tropix). A nucleotide region representing the carboxy insert resulted in a CMV promoter-driven vector containing the li- terminal 197 amino acids of the mature rat PL-I protein was gated cDNAs encoding a SEAP-PL-I fusion protein (AP–PL-I). PL-I Target Cells 739 mM glycine, pH 10.4, 0.5 mM MgCl2, 0.5 mM ZnCl2) con- slips mounted in aqueous mounting medium. The AP and AP taining nitrophenylphosphate (0.5 mg/ml) in a total reaction fusion protein were used at a concentration of 450 mU/ml.