Decidual Prolactin-Related Protein: Heterologous Expression and … · 2008-09-09 · Decidual...

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0013.7227/961$03.00/O Endocrinology Copyright 0 1996 by The Endocrine Society Vol. 137, No. 12 Prmted an U S.A. Decidual Prolactin-Related Protein: Heterologous Expression and Characterization* CHRISTINE A. RASMUSSEN-f, KAZUYOSHI HASHIZUMES, KYLE E. ORWIGS, LEI XUII, AND MICHAEL J. SOARES Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160 ABSTRACT As a first step in understanding the role of decidual PRL-related protein (dPRP) during pregnancy, we have generated recombinant dPRP protein. In this report, we present data on the generation, purification, and characterization of recombinant dPRP protein. The dPRP complementary DNA was subcloned into the pMSXND vector, and the vector was transfected into Chinese hamster ovary (CHO) cells by electroporation. After appropriate selection, amplification, and induction procedures, recombinant dPRP was purified from con- ditioned medium of the CHO-dPRP cells using ultrafiltration, size- exclusion chromatography, and reverse phase HPLC. Recombinant dPRP was found to possess electrophoretic mobility, immunoreactiv- ity, and N-terminal amino acid sequence identical to those of dPRP D ECIDUAL TISSUE represents a specialized uterine compartment arising in association with implantation of the rodent embryo. During its development, decidual cells completely surround the postimplantation embryo and are situated in direct contact with trophoblast cells at the ma- ternal-embryo interface. It has long been speculated that the decidua plays a critical role in the establishment of preg- nancy, but knowledge concerning the exact mechanism(s) by which it does so is limited. Postulated functions of decidual cells include 1) providing a nutritive role in maintenance of the embryo before development of the fetal circulatory sys- tem, 2) limiting invasion of trophoblast cells into the uterus, 3) preventing immunological rejection of the fetal allograft, and 4) producing hormones that act in paracrine or endocrine modes to influence the functions of embryonic, extraembry- onic, or maternal tissues (l-3). The latter function is probably fundamental to each of the responsibilities of decidual cells. The present study focuses on a protein produced by de- cidual cells, termed decidual PRL-related protein (dPRP). dPRP was first identified in rat decidual tissue based on its homology with members of the placental PRL family (4). The Received July 16, 1996. Address all correspondence and requests for reprints to: Dr. Michael J. Soares, Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160. E-mail: [email protected]. * This work was supported by grants from the NICHHD (HD-29036 and HD-29797). t Present address: Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160. $ Present address: Laboratory of Female Reproductive Endocrinol- ogy, National Institute of Animal Reproduction, Ibaraki, Japan. § Supported by fellowships from the Lalor and Kansas Health Foundations. I/ Present address: CytoTherapeutics, Inc., Providence, Rhode Island 02906. isolated from decidual tissue. Polyclonal antibodies were generated to the recombinant dPRP and used for Western blot analysis. dPRP is capable of binding heparin, and a significant fraction of synthesized dPRP resides within the decidual extracellular matrix. Recombinant dPRP failed to bind to PRL receptors and showed no stimulatory activity in the PRL-dependent rat Nb, lymphoma cell proliferation assay. Additional studies have shown that heterologous expression of dPRP in CHO cells significantly increased the ability of CHO cells to form tumors in athymic mice. In conclusion, recombinant dPRP pos- sesses characteristics similar to those of dPRP of decidual origin and is a heparin-binding protein that may facilitate the establishment of pregnancy. (Endocrinology 137: 5558-5566, 1996) dPRP protein and complementary DNA (cDNA) have been isolated and characterized (4). dPRP is a 29-kDa glycoprotein with approximately 70% amino acid sequence homology to a member of the placental PRL family, PRL-like protein C (PLP-C) (4,5). Another member of the PRL family, PRL-like protein B, is antigenically related to dPRP and has been shown to be expressed in rat decidual cells (6). In addition, evidence exists for the presence of a rat decidual protein related to PRL possessing actions on the ovary and uterus (7, 8). Whether dPRP or PLP-B is responsible for these ovarian and uterine PRL-like effects remains to be determined. To begin to determine the biological actions of dPRP dur- ing the establishment of pregnancy, we used the dPRP cDNA to express the dPRP protein. In this report, we describe the isolation and characterization of recombinant rat dPRP, the generation of antibodies to dPRP, and the utilization of these tools to investigate the biology of dPRP. Materials and Methods Reagents FBS and donor horse serum were purchased from JRH Bioscience (Lenexa, KS). Reagents for PAGE were purchased from Bio-Rad Labo- ratories (Hercules, CA). Adjuvant for immunizations was obtained from Organon Teknika Corp. (West Chester, PA). Methotrexate was pur- chased from Calbiochem (San Diego, CA). All restriction enzymes, poly- merases, and DNA ligase were purchased from New England Biolabs (Beverly, MA). Lactoperoxidase was acquired from Boehringer Mann- heim Biochemicals (Indianapolis, IN). Equine CC was purchased from Calbiochem (La Jolla, CA). Nitrocellulose was obtained from Schleicher and Schuell (Keene, NH). Ovine PRL was obtained from Nob1 Labora- tories (Sioux Center, IA). Na[““I] was purchased from DuPont-New England Nuclear (Boston, MA). Heparin-Sepharose was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Biotinylated Ddichos biflo- YI*S was acquired from Vector Laboratories (Burlingame, CA). Antibod- ies against von Willebrand factor were obtained from Dako Corp. (Car- penteria, CA). hCG was obtained from Schein Pharmaceutical (Port 5558 on March 9, 2006 endo.endojournals.org Downloaded from

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Page 1: Decidual Prolactin-Related Protein: Heterologous Expression and … · 2008-09-09 · Decidual Prolactin-Related Protein: Heterologous Expression and Characterization* CHRISTINE A.

0013.7227/961$03.00/O Endocrinology Copyright 0 1996 by The Endocrine Society

Vol. 137, No. 12 Prmted an U S.A.

Decidual Prolactin-Related Protein: Heterologous Expression and Characterization*

CHRISTINE A. RASMUSSEN-f, KAZUYOSHI HASHIZUMES, KYLE E. ORWIGS, LEI XUII, AND MICHAEL J. SOARES

Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160

ABSTRACT As a first step in understanding the role of decidual PRL-related

protein (dPRP) during pregnancy, we have generated recombinant dPRP protein. In this report, we present data on the generation, purification, and characterization of recombinant dPRP protein. The dPRP complementary DNA was subcloned into the pMSXND vector, and the vector was transfected into Chinese hamster ovary (CHO) cells by electroporation. After appropriate selection, amplification, and induction procedures, recombinant dPRP was purified from con- ditioned medium of the CHO-dPRP cells using ultrafiltration, size- exclusion chromatography, and reverse phase HPLC. Recombinant dPRP was found to possess electrophoretic mobility, immunoreactiv- ity, and N-terminal amino acid sequence identical to those of dPRP

D ECIDUAL TISSUE represents a specialized uterine compartment arising in association with implantation

of the rodent embryo. During its development, decidual cells completely surround the postimplantation embryo and are situated in direct contact with trophoblast cells at the ma- ternal-embryo interface. It has long been speculated that the decidua plays a critical role in the establishment of preg- nancy, but knowledge concerning the exact mechanism(s) by which it does so is limited. Postulated functions of decidual cells include 1) providing a nutritive role in maintenance of the embryo before development of the fetal circulatory sys- tem, 2) limiting invasion of trophoblast cells into the uterus, 3) preventing immunological rejection of the fetal allograft, and 4) producing hormones that act in paracrine or endocrine modes to influence the functions of embryonic, extraembry- onic, or maternal tissues (l-3). The latter function is probably fundamental to each of the responsibilities of decidual cells.

The present study focuses on a protein produced by de- cidual cells, termed decidual PRL-related protein (dPRP). dPRP was first identified in rat decidual tissue based on its homology with members of the placental PRL family (4). The

Received July 16, 1996. Address all correspondence and requests for reprints to: Dr. Michael

J. Soares, Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160. E-mail: [email protected].

* This work was supported by grants from the NICHHD (HD-29036 and HD-29797).

t Present address: Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160.

$ Present address: Laboratory of Female Reproductive Endocrinol- ogy, National Institute of Animal Reproduction, Ibaraki, Japan.

§ Supported by fellowships from the Lalor and Kansas Health Foundations.

I/ Present address: CytoTherapeutics, Inc., Providence, Rhode Island 02906.

isolated from decidual tissue. Polyclonal antibodies were generated to the recombinant dPRP and used for Western blot analysis. dPRP is capable of binding heparin, and a significant fraction of synthesized dPRP resides within the decidual extracellular matrix. Recombinant dPRP failed to bind to PRL receptors and showed no stimulatory activity in the PRL-dependent rat Nb, lymphoma cell proliferation assay. Additional studies have shown that heterologous expression of dPRP in CHO cells significantly increased the ability of CHO cells to form tumors in athymic mice. In conclusion, recombinant dPRP pos- sesses characteristics similar to those of dPRP of decidual origin and is a heparin-binding protein that may facilitate the establishment of pregnancy. (Endocrinology 137: 5558-5566, 1996)

dPRP protein and complementary DNA (cDNA) have been isolated and characterized (4). dPRP is a 29-kDa glycoprotein with approximately 70% amino acid sequence homology to a member of the placental PRL family, PRL-like protein C (PLP-C) (4,5). Another member of the PRL family, PRL-like protein B, is antigenically related to dPRP and has been shown to be expressed in rat decidual cells (6). In addition, evidence exists for the presence of a rat decidual protein related to PRL possessing actions on the ovary and uterus (7, 8). Whether dPRP or PLP-B is responsible for these ovarian and uterine PRL-like effects remains to be determined.

To begin to determine the biological actions of dPRP dur- ing the establishment of pregnancy, we used the dPRP cDNA to express the dPRP protein. In this report, we describe the isolation and characterization of recombinant rat dPRP, the generation of antibodies to dPRP, and the utilization of these tools to investigate the biology of dPRP.

Materials and Methods

Reagents

FBS and donor horse serum were purchased from JRH Bioscience (Lenexa, KS). Reagents for PAGE were purchased from Bio-Rad Labo- ratories (Hercules, CA). Adjuvant for immunizations was obtained from Organon Teknika Corp. (West Chester, PA). Methotrexate was pur- chased from Calbiochem (San Diego, CA). All restriction enzymes, poly- merases, and DNA ligase were purchased from New England Biolabs (Beverly, MA). Lactoperoxidase was acquired from Boehringer Mann- heim Biochemicals (Indianapolis, IN). Equine CC was purchased from Calbiochem (La Jolla, CA). Nitrocellulose was obtained from Schleicher and Schuell (Keene, NH). Ovine PRL was obtained from Nob1 Labora- tories (Sioux Center, IA). Na[““I] was purchased from DuPont-New England Nuclear (Boston, MA). Heparin-Sepharose was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Biotinylated Ddichos biflo- YI*S was acquired from Vector Laboratories (Burlingame, CA). Antibod- ies against von Willebrand factor were obtained from Dako Corp. (Car- penteria, CA). hCG was obtained from Schein Pharmaceutical (Port

5558

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DECIDUAL PRL-RELATED PROTEIN

Washington, NY). The chemiluminescent detection system was pur- chased from Amersham Life Science (Arlington Heights, IL). Unless otherwise noted, all other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and tissue preparation

Holtzman rats were obtained from Harlan Sprague-Dawley (India- napolis, IN). Animals were housed in an environmentally controlled facility, with lights on from 0600-2000 h, and allowed free access to food and water. Timed pregnancies and pseudopregnancies and tissue dis- sections were performed as previously described (4,9). The presence of a copulatory plug or sperm in the vaginal smear was designated day 0 of pregnancy. Pseudopregnancy was induced by vagino-cervical stim- ulation on the evening of proestrous with a mechanical vibrator (10,ll). The first day of pseudopregnancy was defined as the first day of leu- kocytic vaginal smears after stimulation. Decidual responses were in- duced in pseudopregnant rats by the injection of 50-100 ~1 sesame oil/uterine horn on day 4 of pseudopregnancy (12). Decidual and pla- cental cytosol were prepared as previously described (13). Protein con- centrations of cytosol preparations were estimated by the method of Bradford (14).

Membrane preparations enriched for PRL receptors were isolated from hepatic and ovarian tissues of prepubertal female rats hormonally treated according to the procedure of Jayatilak et al. (15). Briefly, on day 25 postconception, rats were treated with 50 IU equine CC followed by 25 IU hCG approximately 55 h later. One week after CG treatment, animals were killed, and ovaries and livers were dissected, frozen in liquid nitrogen, and stored at -80 C until used.

BALB/cathymic mice were obtained from Charles River Laboratories (Wilmington, MA), housed in microisolator cages, and used to evaluate tumor development.

Protocols for the care and use of animals were approved by the University of Kansas institutional animal care and use committee.

Generation of recombinant dPRP

Construction of the dPRP expression vector. dPRP cDNA was subcloned into an expression vector termed pMSXND, previously constructed by Drs. S.-J. Lee and Daniel Nathans of Johns Hopkins University (16). The pMSXND vector contains a metallothionein promoter, simian virus 40 splicing and polyadenylation signals, a neomycin resistance gene, and a dihydrofolate reductase gene. The protocol we used has been previ- ously described by our laboratory for the generation of recombinant PLP-A (17) and placental lactogen I (PL-I) variant (18).

A 911-bp EcoRI fragment containing the entire coding sequence of dPRP was isolated from a dPRP-Bluescript plasmid after electrophoretic separation in a 5% polyacrylamide gel. Terminals of the isolated frag- ment were filled with Klenow fragment of DNA polymerase I. XhoI linkers were added to the ends, enabling the dPRP cDNA to be inserted into the XhoI site of the pMSXND vector. Subcloning into the XhoI site situates the dPRP insert downstream of the mouse metallothionein I promoter and upstream of the simian virus 40 splicing and polyade- nylation signals. Recombinant plasmid DNA was purified from Esche- richia coli JMlO5 cells by lysozyme and alkaline-SDS lysis of bacterial cultures, followed by two bandings on CsCl-ethidium bromide density gradients (19). The orientation of the dPRP cDNA in the expression vector was confirmed by BumHI digestion. The plasmid containing the dPRP insert in the correct orientation was designated pRdPRP.

Transfection, selection, and urnplification. Chinese hamster ovary (CHO) cells obtained from the American Type Culture Collection (ATCC CCL 61, CHO-Kl, Rockville, MD) were used as the host for the dPRP ex- pression vector. CHO cells were routinely maintained in DMEM-MCDB- 302 culture medium containing 1 mM proline, 100 U/ml penicillin, 100 wg/ml streptomycin, and 10% FBS. Transfection (via electroelution), selection (with geneticin, G418), and amplification (with methotrexate) were performed as previously described (17, 18). The final genetically modified cell line was referred to as CHO-dPRP. Expression of recom- binant dPRP protein was monitored by Western blot analysis.

Generation of conditioned medium from CHO cells transfected zuifh the dPRP expression vector. After selection and amplification, the transfected CHO

cells were seeded into roller bottles and grown to confluence in DMEM- MCDB-302 medium containing 10% FBS, antibiotics, and 25 rnM HEPES. Once confluent, the cells were washed with medium without serum and cultured in the same serum-free medium supplemented with 100 nM cadmium chloride. The conditioned medium was collected at 24-h in- tervals for 8 days. The first 24-h collection was discarded because of the presence of residual serum. The remainder of the conditioned medium was pooled and stored frozen at -25 C until further analysis.

Purification of recombinant dPRP. Approximately 3 liters conditioned medium were processed at one time. The medium was thawed, then centrifuged at 500 X g for 25 min to remove debris. The medium was concentrated using an Amicon ultrafiltration apparatus (mol wt cut-off, 10,000). The concentrated medium was chromatographed on a Sephacryl S-200 column (2.5 X 90 cm) equilibrated with 20 mM sodium phosphate buffer, pH 7.4, containing 300 mM NaCl. Fractions collected from the gel filtration column were monitored by absorbance at 280 nm and by immunoreactivity with antibodies to amino acids 140-159 of PLP-B (4) or later with antibodies to recombinant dPRP. Immunopositive fractions were pooled, concentrated by ultrafiltration, and further purified to homogeneity by reverse phase HPLC using a 15-90% acetonitrile-0.1% trifluoroacetic acid gradient over 35 min at a flow rate of 1 ml/min.

Characterization of recombinant dPRP

Amino-terminal sequence analysis. Amino acid sequences were determined by the Edman degradation method, using a gas phase protein sequencer at the University of California-Los Angeles Protein Microsequencing Facility. Approximately 100 pmol protein were sequenced. Cysteine residues were not protected and thus could not be determined from the analysis.

Generntion ofantibodies to recombinant dPRP. Purified recombinant dPRP (-150 kg/rabbit) was used to immunize two New Zealand White rab- bits (Myrtle Rabbitry, Thompson Station, TN). Booster injections (75 pg each) were given at 2-week intervals. The remainder of the immuniza- tion protocol and serum collection were performed as previously de- scribed (13).

Western blot unulysis. Western blot analyses were performed as previ- ously described (13,20). Samples were separated by SDS-PAGE in 12.5% gels under reducing conditions. Proteins from the gels were electro- phoretically transferred to nitrocellulose. Antibodies generated to a syn- thetic peptide corresponding to amino acids 140-159 of PLP-B (4), re- combinant PLP-A (17), and recombinant dPRP were used as probes. The nitrocellulose filters were then developed by exposure to either an al- kaline phosphatase-labeled antirabbit IgG with subsequent histochem- ical staining for alkaline phosphatase or a peroxidase-labeled antirabbit IgG followed by a chemiluminescent detection system. In some exper- iments, preimmune serum or antibodies saturated with the respective antigens were used as controls.

Characterization of dPRP-heparin interactions

Hepurin-Sephurose chromatography. Minicolumns containing either hep- arinSepharose CL-6B or unconjugated Sepharose 68 were prepared and equilibrated with equilibration buffer (10 mM PBS, pH 7.4, containing 0.15 M NaCl) (21). Samples were diluted to 1 bed vol with equilibration buffer, applied directly to the minicolumns, and placed on a platform rocker for 30 min at 4 C. Unbound proteins were collected by centrif- ugation and precipitated with cold acetone. Minicolumns were washed with 5 bed vol equilibration buffer, which was also collected and pre- cipitated with cold acetone. Bound proteins were eluted from the resin by boiling in a buffer containing 20% glycerol, 125 mM Tris-HCl, 1.4 M

2-mercaptoethanol, and 4% SDS, pH 6.8. The processed samples in- cluded decidual cytosol (native dPRP), recombinant dPRP, and heat- denatured recombinant dPRP. The existence of a possible heparin-bind- ing domain located between amino acids 83-100 of dPRP was also evaluated (22). This region was synthesized (Bio-Synthesis, Lewisville, TX), and heparin-Sepharose minicolumns were prepared as before and pretreated with either 800 pg synthetic peptide or equilibration buffer. After washing, 8 pg dPRP were loaded onto the minicolumns, and fractions were separated by SDS-PAGE and immunoblotted with anti- bodies to dPRP as described above.

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5560 DECIDUAL PRL-RELATED PROTEIN Endo . 1996 Vol 137. No 12

Heparimsc nssuy. CHO cells transfected with pRdPRl? were grown to confluence in 75-cm’ flasks, washed with Hanks’ Buffered Salt Solution, and subsequently incubated in 4 ml DMEM-MCDB-302 culture medium with or without 2 U of the glycosaminoglycanase, heparinase III, for 90 min at 37 C (23). Culture medium was collected, dialyzed for 24 h against water, and precipitated with 100 mM ZnSO,. Samples were separated by SDS-PAGE and immunoblotted with anti-dPRP antibodies.

Drc-ic/tlnl &mcclltrlnr tffatri,~ e?tirnctio!f. Extracellular matrix (ECM) from day 10 decidual tissue was isolated according to the procedure of Led- better et nl. (24). Day 10 decidual tissue was homogenized in 3.4 M

NaCI-0.05 M Tris-HCI, pH 7.4, containing 4 mM EDTA and 0.5 mM phenylmethylsulfonylfluoride at 4 C. Insoluble material was collected by centrifugation at 47,000 X g for 20 min. The extraction and centrif- ugation steps were repeated three times. The ECM protein fraction was solubilized from the cumulative pellet by extraction overnight in 2 vol 0.5 M NaCl-0.05 M Tris-HCl, pH 7.4, containing 4 mM EDTA and 0.5 mM phenylmethylsulfonylfluoride and recovered by centrifugation as de- scribed above. The ECM protein fraction was then dialyzed overnight in 2 M urea-O.05 M Tris-HCl, pH 7.4, and centrifuged at 10,000 X ,g for 30 min. The resulting supernatant containing the ECM protein fraction was collected; dialyzed against 0.15 M NaCl-0.05 M Tris-HCl, pH 7.4, overnight; and frozen at -20 C until analyzed for the presence of dPRP by immunoblotting. Laminin, a structural protein present in the ECM, was used as a positive control and was monitored by immunoblotting with antibodies to rat laminin (25).

Characterization of PRL-like activities

PRL RRA. The procedures used for radioiodination and membrane preparation, and the assay conditions were based on the protocols of Haro and Talamantes (26) and have been described in detail previously m

PRL irl ~itrr> bionssny. PRL-like bioactivity was assessed by evaluating the proliferation of Nb2 rat lymphoma cells as previously described (18,27, 28).

Effects of dPRP on angiogenesis

Eurioilrrlinl cc/l ~~rol$rntio?~ assny. Bovine aortic endothelial cells (29) or mouse heart small vessel endothelial cells (30) were grown in DMEM plus 10% FBS and 10% calf serum or DMEM plus 10% FBS, respectively. To assess proliferation, cells were plated at a density of 1 X 10” cells/well in 24-well plates containing 1 ml medium. Cells were allowed to attach for 24 h, then were incubated with varying concentrations of dPRP and serum in medium (100 pg to 1.0 pg dPRP with and without heparin; O-10% FBS or 10% calf or horse serum). Cells were incubated for 5 days, with a change in treatments on alternate days. At the end of the 5-day incubation period, cultures were washed with 10 mM PBS, pH 7.3, and stained with crystal violet (300 pl/ well; 5% formalin, 50% ethanol, 150 mM NaCI, and 0.5% crystal violet) for 5 min. Plates were extensively washed with water and eluted with 1 ml ethylene glycol. Cell density was estimated by absorbance of the eluate at 600 nm (31, 32).

If1 z>ii~ transplur~fntion ofgenetically e~~girmwri C)-iO cc//s. CHO cells trans- fected with expression vectors for dPRP (present study), PLP-A (17), or control CHO cells were grown in 75-cm- flasks in DMEM-MCDB-302 plus 10% FBS. Parental CHO cells (nontransfected) were grown as a control. Cells (1 X 107) were SC injected into the upper dorsal areas of female athymic mice (33). To stimulate the expression of dPRP through the metallothionein promoter, ZnSO, was added to the drinking water (6 rnM final concentration). Tumor growth and volume were monitored over a 3-week period. Representative samples of tumor tissue were either frozen in liquid nitrogen for analysis of transgene expression or immersion fixed in Bouin’s fluid for histological, immunocytochemical, or histochemical analysis. Cytosol was prepared from frozen tumors as previously described (13). Cytosolic proteins were separated by SDS- PAGE, transferred to nitrocellulose, and immunoblotted with dPRP antibodies. Fixed tissues were processed for paraffin embedding, sec- tioned at 6 wrn, and stained with hematoxylin-eosin for routine histo- logical analysis, with antibodies to dPRP for the immunocytochemical analysis of dPRP, or with biotinylated Doiichos b$orus agglutinin for histochemical detection of tumor vascularization. Briefly, sections were

deparaffinized, rehydrated, and endogenous peroxldase-blocked with 3% hydrogen peroxide. Slides were then incubated with blotmylated Dol~clzos blflorus agglutinin (15 Fg/ml) for 30 min and subsequently with a streptavidin-peroxldase conjugate and diaminobenzidine substrate chromogen. Sections were counterstained with hematoxylin.

III mtro pdlfernt~a~~ ofparentnl nr~d trmsfected CHO cc//s. For determination ot CHO cell proliferation l?j mtro, stable CHO cell transfectants and parental CHO cells were plated at 1 x 10’ cells/well in 24-well plates containing DMEM-MCDB-302 medium plus 10% FBS and allowed to grow in culture for 72 h. At the end of the culture period, cultures were washed, stained with crystal violet, eluted with ethylene glycol, and monitored at 600 nm, as described above, to determine endothehal cell proliferation (see above).

Results

Heterologous dPRP expression and purification

CHO cells transfected with the dPRP expression vector produced a 29-kDa protein with immunochemical, electro- phoretic, and biochemical characteristics similar to those of native dPRP (Figs. l-3). dPRP production was initially ex- amined with antipeptide antibodies directed to amino acids 140-159 of PLP-B. These antibodies are known to recognize dPRP (4) and specifically cross-reacted with a 29-kDa protein

A 0 c D

\

43-

31 -

21 -

anti-PLP-B

43.

31

21 .

Excess peptide

anti-PLP-A

FIG. 1. Immunochemical analysis of dPRP produced by engineered CHO cells. All samples were separated in 12.5% polyacrylamide gels, transferred to nitrocellulose, and subjected to immunoblotting. The final dilution of all antisera was 1:2000. M, standards (X10-‘) are shown. Top panel, Conditioned media from CHO-dPRP cells (lanes A and C) or CHO-PLP-A cells (lanes B and D) were probed with anti- peptide antibodies to amino acids 140-159 of PLP-B (lanes A and B) or antibodies to PLP-A (lanes C and D). Bottom panel, Examination of the specificity of the reaction of dPRP produced by CHO cells (lanes A and C) or deciduoma (lanes B and D) with antipeptide antibodies to PLP-B. Lanes A and B were incubated with antipeptide antibodies to amino acids 140-159 ofPLP-B. Lanes C and D were incubated with the same antipeptide antibodies saturated with the PLP-B peptide antigen.

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DECIDUAL PRL-RELATED PROTEIN 5561

ABC D E F G A 6 C D E F

67-” 43 -rr)

31-- 21 -u 14-11

Coomassie blue

a-dPRP

FIG. 2. Electrophoretic separation of protein samples from various steps in the purification of dPRP from CHO cell-conditioned medium. Samples were separated in 12.5% polyacrylamide gels and stained with Coomassie blue (lanes B-D) or transferred to nitrocellulose and immunoblotted with antibodies to recombinant dPRP (lanes E-G). Lanes B and E, Concentrated conditioned medium from CHO cells transfected with the pRdPRP expression vector; lanes C and F, pooled peak fractions containing dPRP coilected from the Sephacryl S-200 gel chromatography column; lanes D and G, peak collected from re- verse phase HPLC. Lane A, M, standards (~10-~).

present in CHO-dPRP cell-conditioned medium (Fig. 1). An- tibodies to recombinant PLP-A did not recognize any pro- teins in the CHO-dPRP cell-conditioned medium (Fig. 1). The 29-kDa recombinant dPRP was purified to homogeneity from conditioned medium of CHO cells stably transfected with the pRdPRP expression vector using gel filtration and reverse phase HPLC (Fig. 2). N-Terminal sequence analysis of the 29-kDa protein isolated from conditioned medium (Val’-Pro*-Ala3-XxX4-His5) confirmed its identity with the N-terminal sequence of native dPRP and the amino acid sequence predicted from the dPRP cDNA (4). Recombinant dPRP was used to generate polyclonal antibodies. Immuno- blot analysis of the reactivity of the antisera indicated that antibodies directed to recombinant dPRP specifically recog- nized a protein with a mol wt of 29 kDa; this was evident in immunoblots made from decidual cytosol preparations and conditioned medium from decidual and CHO-dPRP cells (Fig. 3).

dPRP-heparin interactions

dPRP was not detectable in serum from pseudopregnant decidualized rats, as measured by Western blot analysis (data not shown). In contrast, other members of the PRL family are abundantly present in maternal serum (34). Thus, we hypothesized that dPRP may have a more restricted distribution and possibly associate with the decidual ECM. Heparan sulfate proteoglycan is known to bind an array of cytokines and growth factors and was evaluated as a possible reservoir for dPRP (35). Heparin-Sepharose affinity chroma- tography has been used to characterize and purify a number of heparin-binding molecules, such as basic fibroblast growth factor (23), lipoprotein lipase (21), and heparin-bind- ing epidermal growth factor (36). In the present experiments, dPRP was shown to bind strongly to heparin, requiring treat- ment with SDS for dissociation. Both native and recombinant dPRP bound to heparin in uitvo (Fig. 4). dPRP did not bind to unconjugated Sepharose, and heat denaturation of dPRP

ExcessdPRP - - - -t + +

FIG. 3. Characterization of antibodies generated to recombinant dPRP. Lanes A and D, Medium conditioned by decidual cell cultures; lanes B and E, deciduoma cytosolic preparations; lanes C and F, conditioned medium from CHO-dPRP cells. Samples were separated by SDS-PAGE in 12.5% gels, electrophoretically transferred to nitro- cellulose, and examined by Western blot analysis using an antiserum to recombinant dPRP at a final dilution of 1:20,000. Antibodies used in lanes D, E, and F were saturated with recombinant dPRP. M, standards (X 10m3) are shown.

abrogated its ability to bind heparin, providing evidence for the specificity of the dPRP-heparin interaction (data not shown). Studies with other heparin-binding proteins have demonstrated a consensus heparin-binding domain. Two motifs, -B-B-X-B- and -B-B-B-X-X-B-, where B represents a basic residue and X represents a nonbasic residue, have been shown to be heparin-binding domains (22). A region of dPRP spanning amino acids 84-98 resembles a heparin-binding domain. The putative heparin-binding domain was synthe- sized and tested for its ability to bind heparin. In a variety of experiments, we failed to show any significant affinity of the putative dPRP heparin-binding domain for heparin. To further characterize dPRP’s ability to bind to heparin in viuo, CHO-dPRP cells were incubated with the glycosaminogly- can-degrading enzyme heparinase. As shown in Fig. 5, he- parinase-treated cultures released dPRP from the cellular monolayer. In contrast, there was little release of dPRP from vehicle-treated cultures. Collectively, these results suggest that dPRP may bind to a heparan sulfate proteoglycan. To further establish an in viva relationship between dPRP and decidual ECM, we extracted ECM from decidual tissue and tested for the presence of laminin and dPRP. Western blot analysis with antibodies to rat laminin revealed the presence of laminin in the decidual ECM preparation (data not shown). Our results also show the presence of dPRP in the ECM of decidual tissue (Fig. 5).

Characterization of PRL-like activities

dPRP did not displace ovine PRL from ovarian or hepatic PRL receptors (Fig. 6) and showed negligible stimulating activity in the rat Nb, lymphoma cell proliferation assay (Table 1). These results indicate that dPRP does not use the PRL signaling pathway.

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5562 DECIDUAL PRL-RELATED PROTEIN Endo. 1996 Vol 137 . No 12

a b c a b

43.

31%

21,

43,

31,

21,

a b c

FIG. 4. Analysis of dPRP binding to heparin-Sepharose. Samples were subjected to heparin-Sepharose chromatography, and fractions were collected and analyzed by SDS-PAGE and Western blotting using anti-dPRP antibodies. Pooled flow-through (lane A), wash (lane B), and eluate (lane C) fractions were assessed. The samples pro- cessed included native dPRP (top panel) and recombinant dPRP (hot- tom panel). M, standards (X 10e3) are indicated at the left of each blot.

Effects of dPRP on angiogenesis

Members of the PRL family have been shown to have effects on angiogenesis (37,38). This information in addition to the localization of dPRP to an area of the pregnant uterus with limited vacularization (39) led us to hypothesize that dPRP may be involved in the control of uterine blood vessel development. To examine the effects of dPRP on endothelial cells in vitro, we tested its ability to affect endothelial cell proliferation. Our experiments showed that dPRP had lim- ited effects on endothelial cell proliferation (data not shown). A number of factors can stimulate angiogenesis in vivo with- out stimulatory effects on endothelial cells in vitro (40). To determine whether dPRP modulates angiogenesis in vivo, we tested its ability to affect tumor development in athymic

2

a b

FIG. 5. dPRP-ECM interactions. The association of dPRP with the ECM was further investigated via its enzymatic release and extract- ability from ECM. Top panel, Effect of heparinase on the release of cell- and/or ECM-associated dPRP. Transfected CHO cell cultures were treated with (lane A) or without (lane B) heparinase. Samples were separated by SDS-PAGE and immunoblotted with antibodies to dPRP. M, standards (X10A3) are indicated at the left of the panel. Bottom panel, Association of dPRP with decidual ECM. Decidual cytosolic (lane A) or ECM (lane B) fractions were separated by SDS- PAGE and analyzed by Western blotting with antibodies to dPRP. M, standards (~10~~) are indicated at the left of the panel.

mice, similar to the procedure described by Ueki et al. (33). Control CHO cells or CHO cells expressing dPRP or PLP-A generated solid tumors after SC transplantation in athymic mice. Histological examination of tumor tissue revealed no significant differences in the architecture or morphology of dPRP-producing tumors compared to that of control tumors (Fig. 7). Lectin histochemical detection of endothelial cells detailed prominent neovascularization in both types of tu- mors (Fig. 7). Immunoreactive dPRP was identified in the

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DECIDUAL PRL-RELATED PROTEIN 5563

FIG. 6. Interaction of dPRP with PRL receptors. Displacement curves for [‘25110vine PRL binding to liver (upper panel) and ovarian (lower panel) mem-

g 80-

$ E

p (So-

G

kg 40-

d 0

20 -

.Ol .I 1 IO 100 1000 10000 100000

HORMONE (ng)

brane preparations were generated with ovine PRL and dPRP. Membranes were incubated with [125110vine PRL alone or with increasing concentrations of ovine PRL or dPRP.

0 oPRL

I ’ ’ ’ ““‘I ’ ’ ’ ““‘I ’ ’ ’ ““‘I ’ * ’ *e-l - ’ - .Ol .I

CHO-dPRP tumors by Western blot analysis (Fig. 8). Al- though dPRP expression did not affect tumor structure, a striking effect was noted in the incidence of the tumors (Table 2). There was a significantly greater percentage of mice in- jected with CHO cells expressing dPRP-generated tumors than of mice injected with control CHO cells or CHO cells expressing PLP-A (Table 2). The difference in tumor inci- dence was not correlated with in vitro growth rates of the cell populations. Control CHO cells grew significantly faster in vitvo (1.1 + 0.4 absorbance units) than either CHO-dPRP cells (0.6 2 0.1 absorbance units) or CHO-PLP-A cells (0.7 ? 0.03 absorbance units).

Discussion

Control dPRP (rig/ml)

0.1 1.0 10 100 1000

Ovine PRL (rig/ml) 0.1 1.0 10 100 1000

1.0 t 0.08 1.1 + 0.08 1.0 + 0.08 0.9 2 0.07 1.0 t 0.02

2.9 + 0.08 7.0 + 0.34" 8.8 2 0.61" 8.8 k 0.11” 7.4 lr 0.42"

In the present report, we expressed dPRP in CHO cells, Values are the mean t SEM. characterized recombinant dPRP protein, generated poly- n Significantly different than control cultures, P < 0.01.

1 IO 100 1000 10000 100000

HORMONE (ng)

TABLE 1. Effects of dPRP and ovine PRL on the proliferation of rat Nb2 lymphoma cells

Treatment Cell no. (X10”)

1.0 2 0.02

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5564 DECIDUAL PRL-RELATED PROTEIN Endo. 1996 Vol 137 . No 12

FIG. 7. Morphology and vessel formation in CHO cell tumors. Tissue sections from control CHO cell tumors (A and C) and CHO-dPRP-expressing tumors (B and D) were stained with hematoxylin and eosin (A and B; magnification, X100) or Dolichos biflorus agglutinin (C and D; magnification, X40). Both types of tumors show spindle-shaped cells with prominent nuclei and are well vascularized.

2 1 2 s TUMORS

FIG. 8. Western blot analysis of dPRP expression in CHO-dPRP tu- mors. CHO-dPRP tumor cytosolic preparations (designated 1 and 2 and obtained from tumors from different mice) were electrophoreti- tally separated on a 12.5% polyacrylamide gel, transferred to nitro- cellulose, and examined for the presence of dPRP by Western blot analysis using antiserum to dPRP. Recombinant dPRP was used as a control. M, standards (x10-“) are shown.

clonal antibodies to recombinant dPRP, and used these re- agents to dissect possible roles of dPRP in the uterus during pregnancy. Our strategy involved subcloning a dPRP cDNA into the pMSXND expression vector and using CHO cells as a host for the production of recombinant dPRP protein. This approach has been used for the generation of biologically active proteins representing other members of the PRL fam- ily, including proliferin (16), PL-I (41, 42), PL-II (43), and PLP-A (17). Polyclonal antibodies were generated to recom- binant dPRP and greatly facilitated the characterization of native dPRP protein. Our findings indicate that recombinant dPRP is immunochemically and biochemically similar to native dPRP and possesses biologically relevant activities.

Tissue distribution of a hormone or cytokine has direct bearing on its accessibility to target cells. Some extracellular signals circulate free or bound to transport proteins, whereas others act locally, often tethered to the surface of cells or the

TABLE 2. Tumor incidence in mice injected with parental CHO cells and transfected CHO cells

Cell type

CHO CHO-dPRP CHO-PLP-A

No. of mice injected

13 15

5

% Tumor incidence

39 37 40

ECM (35, 44). Western blot analysis of serum from pseudo- pregnant decidualized rats indicated that unlike other mem- bers of the PRL family, dPRP did not circulate at detectable levels. dPRP was found to associate with heparin and was localized at least in part within the decidual ECM. Heparin binding is not an attribute previously reported for the PRL family; however, it is a feature of a number of growth factors and cytokines, such as fibroblast growth factor and heparin- binding epidermal growth factor, that influence cellular be- havior principally via a paracrine mode of action (23, 36). Modulatory factors present in the decidual ECM, such as dPRP, would be well situated to influence the behavior of decidual cells and other cell types traversing the decidual ECM, such as trophoblast, endothelial, and various immune cells.

The actions of members of the extrapituitary PRL family can be distinguished based on their utilization of the PRL receptor signaling pathway. PL-I, PL-II, and PL-I variant bind to PRL receptors and appear to exhibit biological actions similar to those of pituitary PRL (classical members) (18,42, and 45-47) (Dai, G., and M. J. Soares, unpublished results). dPRP does not use the PRL receptor and does not effectively stimulate the proliferation of lactogen-dependent Nb, lym- phoma cells (present study). Gibori and colleagues have demonstrated that the decidual luteotrophin acts on the cor-

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DECIDUAL PRL-RELATED PROTEIN

pus luteum via its utilization of the PRL receptor signaling system (7). Thus, our findings do not support a relationship between dPRP and the decidual luteotrophin. This places dPRP in a subgroup of PRL structural relatives with distinc- tive biological actions (nonclassical members). Also included in the nonclassical subgroup are a few members of the rat placental PRL family, PLP-A, PLP-B, and PLP-C (17, 48) (Cohick, C. B., and M. J. Soares, unpublished results); two members of the mouse placental PRL family, proliferin and proliferin-related protein (16, 38); and a 16-kDa fragment of pituitary PRL (37).

Progress in identifying the biological activities of nonclas- sical members of the PRL family has been limited. Proliferin, proliferin-related protein, and the 16-kDa fragment of PRL have been shown to specifically participate in the control of blood vessel development (37, 38). Proliferin stimulates an- giogenesis, whereas proliferin-related protein and the 16- kDa fragment of PRL inhibit angiogenesis. This information coupled with the intrauterine expression pattern of dPRP in a region relatively devoid of vasculature (39) prompted us to hypothesize that dPRP would be antiangiogenic. dPRP did not markedly influence the development of vascular struc- tures, as evaluated in both in vitro and in vivo assays. Al- though our prediction was not supported by the experimen- tal evidence, results from the in vivo analyses implicated dPRP in alternative physiological roles within the uterus. We used a tumor development model to evaluate the role of dPRP on angiogenesis. In this model, the putative modulator is stably overexpressed in CHO cells and then transplanted into athymic mice (33). Tumor growth is directly related to the vascularization of the transplant. We did not observe any significant differences in the morphology, vascularity, or growth of control vs. dPRP-expressing tumors. Heterologous expression of dPRP in CHO cells was, however, associated with an increase in the ability of CHO cells to form tumors after transplantation into athymic mice. Thus, dPRP expres- sion was correlated with an altered relationship between the tumor cells and their host, thereby increasing the success rate for establishing a tumor. In the future, it will be necessary to directly examine the effects of dPRP on tumor cell-host relationships.

Parallels between host regulatory events in tumor trans- plantation and establishment of the genetically foreign em- bryo within the uterus are evident (49). Natural killer (NK) cells and macrophages are potential effector cells in tumor resistance (50, 51) and have also been implicated in the ma- ternal immune response to pregnancy (52, 53). Inhibition of these immune cells may account for the facilitation of tumor development in athymic mice transplanted with CHO cells overexpressing dPRP. Similarly, dPRP may be regulating maternal immune cell responses during the establishment of pregnancy. Previous studies have shown that decidual prod- ucts influence macrophage function (54,55). In addition, and most interestingly, there is a virtual absence of macrophages and NK lineage cells in regions of the uterus that express dPRP (Refs. 53 and 56-58 and the present study) (Rasmus- sen, C. A., K. E. Orwig, and M. J. Soares, unpublished results). Thus, dPRP is a candidate cytokine responsible for the re- distribution and altered function of resident intrauterine im- mune cells during the initiation of pregnancy. Information on

the effects of dPRP on host macrophage and NK cell pop- ulations in tumors growing in athymic mice may aid in the evaluation of this prediction.

In conclusion, we have generated reagents that can help expand our understanding of the biological actions of dPRP, one of the principal secretory products of the deciduum. dPRP is a heparin-binding cytokine residing at least in part within the decidual ECM that probably assists in the estab- lishment of pregnancy through paracrine actions within the uteroplacental compartment.

Acknowledgments

We thank S.-J. Lee, Daniel Nathans, and Daniel I. H. Linzer for the pMSXND vector; Drs. Lawrence Reynolds and Robert Auerbach for the endothelial cells; and Dr. Peter W. Gout for the Nb2 cells. We also acknowledge the advice and assistance of Belinda Chapman and Christopher Cohick.

5.

h.

7.

8.

9.

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11.

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17.

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21.

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