Decidual Cells Produce a Heparin-binding Prolactin Family Cytokine with Putative Intrauterine Regulatory Actions
2008; Elsevier BV; Volume: 283; Issue: 27 Linguagem: Inglês
10.1074/jbc.m801826200
ISSN1083-351X
AutoresS. M. Khorshed Alam, Toshihiro Konno, Namita Sahgal, Lu Lu, Michael J. Soares,
Tópico(s)Bone Metabolism and Diseases
ResumoPregnancy in mice and rats is associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J-specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature. Pregnancy in mice and rats is associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J-specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature. Successful pregnancy requires specialized maternal adaptations. Decidualization is a key uterine adaptation associated with the establishment of pregnancy and is characterized by the differentiation of uterine stromal cells (1DeFeo V.J. Wynn R.M. Cellular Biology of the Uterus. Appleton-Century-Crofts, New York1967: 191-220Google Scholar, 2Aplin J. Semin. Cell Dev. Biol. 2000; 11: 115-125Crossref PubMed Scopus (63) Google Scholar, 3Bell S.C. Oxf. Rev. Reprod. Biol. 1983; 5: 220-271Google Scholar, 4Brosens J.J. Gellersen B. J. Mol. Endocrinol. 2006; 36: 389-398Crossref PubMed Scopus (106) Google Scholar). Decidual cell differentiation is dependent upon ovarian steroid hormone production, and in rodents, it also requires signals emanating from the preimplantation embryo (1DeFeo V.J. Wynn R.M. Cellular Biology of the Uterus. Appleton-Century-Crofts, New York1967: 191-220Google Scholar, 5Parr M.B. Parr E.L. Wynn R.M. Jollie W.P. Biology of the Uterus. Plenum Press, New York1989: 233-278Crossref Google Scholar). Once formed, decidual cells establish a protective environment, facilitating the development of the placenta and embryo. They promote the redistribution of specific populations of leukocytes and reorganize the uterine vascular network. Intercellular signals elaborated by decidual cells are key mediators of these uterine adaptive responses. Among the decidual cell ligands are a family of cytokines related to prolactin (PRL). 2The abbreviations used are: PRL, prolactin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CHO, Chinese hamster ovary; NTA, nitrilotriacetic acid; AP, alkaline phosphatase; BSA, bovine serum albumin; FGF2, fetal growth factor 2; BrdUrd, bromodeoxyuridine. 2The abbreviations used are: PRL, prolactin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CHO, Chinese hamster ovary; NTA, nitrilotriacetic acid; AP, alkaline phosphatase; BSA, bovine serum albumin; FGF2, fetal growth factor 2; BrdUrd, bromodeoxyuridine.PRL is an ancient hormone with its origins as a regulator of vertebrate environmental adaptations (6Bern H.A. Nicoll C.S. Rec. Prog. Horm. Res. 1968; 24: 681-720PubMed Google Scholar, 7Nicoll C.S. Bern H.A. Wolstenholme G.E.W. Knight J Lactogenic Hormones. Churchill Livingstone, London1972: 299-338Google Scholar). Some species possess a single member of the PRL family that can be expressed in an assortment of tissues, including the anterior pituitary and uterus through the utilization of cell-specific promoters (8DiMattia G.E. Gellersen B. Duckworth M.L. Friesen H.G. J. Biol. Chem. 1990; 265: 16412-16421Abstract Full Text PDF PubMed Google Scholar, 9Ben-Jonathan N.B. Mershon J.L. Allen D.L. Steinmetz R.W. Endocr. Rev. 1996; 17: 639-669PubMed Google Scholar, 10Telgmann R. Gellersen B. Hum. Reprod. Update. 1998; 4: 472-479Crossref PubMed Scopus (125) Google Scholar). Other species have undergone a gene expansion within the PRL locus (11Soares M.J. Konno T. Alam S.M.K. Trends Endocrinol. Metab. 2007; 18: 114-121Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). PRL family gene expansion is particularly robust in mice and rats (12Wiemers D.O. Shao L.-J. Ain R. Dai G. Soares M.J. Endocrinology. 2003; 144: 313-325Crossref PubMed Scopus (101) Google Scholar, 13Mallon A.-M. Wilming L. Weekes J. Gilbert J.G.R. Ashurst J. Peyrefitte S. Matthews L. Cadman M. McKeone R. Sellick C.A. Arkell R. Botcherby M.R.M. Strivens M.A. Campbell R.D. Gregory S. Denny P. Hancock J.M. Rogers J. Brown S.D.M. Genome Res. 2004; 14: 1888-1901Crossref PubMed Scopus (24) Google Scholar, 14Alam S.M.K. Ain R. Konno T. Ho-Chen J.K. Soares M.J. Mamm. Genome. 2006; 17: 858-877Crossref PubMed Scopus (41) Google Scholar). Gene duplication and natural selection have yielded 2 dozen related genes in each of these species. The PRL family genes encode cytokines/hormones that are expressed in cell-specific and temporally specific patterns and are most relevant to pregnancy-associated tissues, especially in the uterine decidua and the placenta. Initial observations suggest that the expanded PRL family participates in pregnancy-dependent adaptations to physiological stressors (15Ain R. Dai G. Dunmore J.H. Godwin A.R. Soares M.J. Proc. Natl. Acad. Sci. U. S. 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Endocrinol. 2001; 15: 426-432Crossref PubMed Scopus (34) Google Scholar), PRL-like protein-B (PLP-B; also known as PRL family 6, subfamily a, member 1 (Prl6a1)) (25Croze F. Kennedy T.G. Schroedter I.C. Friesen H.G. Endocrinology. 1990; 127: 2665-2672Crossref PubMed Scopus (40) Google Scholar, 26Cohick C.B. Xu L. Soares M.J. J. Endocrinol. 1997; 152: 291-302Crossref PubMed Scopus (21) Google Scholar, 27Lin J. Poole J. Linzer D.I.H. Endocrinology. 1997; 138: 5541-5549Crossref PubMed Scopus (40) Google Scholar, 28Müller H. Ishimura R. Orwig K.E. Liu B. Soares M.J. Biol. Reprod. 1998; 58: 45-51Crossref PubMed Scopus (30) Google Scholar), decidual PRL-related protein (dPRP; also known as PRL family 8, subfamily a, member 2 (Prl8a2)) (27Lin J. Poole J. Linzer D.I.H. Endocrinology. 1997; 138: 5541-5549Crossref PubMed Scopus (40) Google Scholar, 29Roby K.F. Deb S. Gibori G. Szpirer C. Levan G. Kwok S.C.M. Soares M.J. J. Biol. 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Gellersen B. Hum. Reprod. Update. 1998; 4: 472-479Crossref PubMed Scopus (125) Google Scholar, 35Shaw-Bruha C.M. Pennington K.L. Shull J.D. Biochim. Biophys. Acta. 1998; 1442: 304-313Crossref PubMed Scopus (7) Google Scholar), where it acts through the PRL receptor signaling pathway to promote decidual cell survival, regulate leukocyte function, stimulate uterine gland development, and facilitate vascular remodeling (23Prigent-Tessier A. Tessier C. Hirosawa-Takamori M. Boyer C. Ferguson-Gottschall S. Gibori G. J. Biol. Chem. 1999; 274: 37982-37989Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 36Jabbour H.N. Critchley H.O. Boddy S.C. J. Clin. Endocrinol. Metab. 1998; 83: 2545-2553Crossref PubMed Scopus (66) Google Scholar, 37Jabbour H.N. Critchley H.O.D. Reproduction. 2001; 123: 197-205Crossref Scopus (117) Google Scholar, 38Tessier C. Prigent-Tessier A. Ferguson-Gottschall S. Gu Y. Gibori G. Endocrinology. 2001; 142: 4086-4094Crossref PubMed Scopus (59) Google Scholar). PLP-B and dPRP do not utilize the canonical PRL receptor signaling pathway (22Wang D. Ishimura R. Walia D.S. Müller H. Dai G. Hunt J.S. Lee N.A. Lee J.J. Soares M.J. J. Endocrinol. 2000; 167: 15-28Crossref PubMed Scopus (26) Google Scholar, 26Cohick C.B. Xu L. Soares M.J. J. Endocrinol. 1997; 152: 291-302Crossref PubMed Scopus (21) Google Scholar, 39Rasmussen C.A. Hashizume K. Orwig K.E. Xu L. Soares M.J. Endocrinology. 1996; 137: 5558-5566Crossref PubMed Scopus (39) Google Scholar). dPRP is a heparin binding cytokine that is essential for pregnancy-dependent adaptive responses to hypoxia (16Alam S.M.K. Konno T. Dai G. Lu L. Wang D. Dunmore J.H. Godwin A.R. Soares M.J. Development. 2007; 134: 407-415Crossref PubMed Scopus (48) Google Scholar, 22Wang D. Ishimura R. Walia D.S. Müller H. Dai G. Hunt J.S. Lee N.A. Lee J.J. Soares M.J. J. Endocrinol. 2000; 167: 15-28Crossref PubMed Scopus (26) Google Scholar, 39Rasmussen C.A. Hashizume K. Orwig K.E. Xu L. Soares M.J. Endocrinology. 1996; 137: 5558-5566Crossref PubMed Scopus (39) Google Scholar). Unlike wild-type animals, mice deficient in dPRP do not effectively adapt to hypoxia and terminate their pregnancies. dPRP modulates decidual expression of PLP-J but not PLP-B or PRL (16Alam S.M.K. Konno T. Dai G. Lu L. Wang D. Dunmore J.H. Godwin A.R. Soares M.J. Development. 2007; 134: 407-415Crossref PubMed Scopus (48) Google Scholar). The expression of PLP-J is significantly decreased in dPRP null mice, suggesting that the biology of dPRP and PLP-J may be linked. Information on the biological functions of PLP-J is not available.In this study, we have characterized the PLP-J protein and its expression pattern, identified targets for its action, and determined biological responses of its cellular targets. PLP-J is a heparin-binding cytokine with distinct actions on uterine stromal cell and endothelial cell populations.EXPERIMENTAL PROCEDURESAnimals and Tissue PreparationHoltzman rats were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The animals were housed in an environmentally controlled facility, with lights on from 0600 to 2000 h and were allowed free access to food and water. Timed pregnancies were generated, and tissue dissections were performed as previously detailed (40Ain R. Konno T. Canham L.N. Soares M.J. Methods Mol. Med. 2006; 121: 295-313PubMed Google Scholar). Conceptuses with associated uteri were removed on specific days of gestation. Tissues were frozen in dry ice-cooled heptane and stored at -80 °C until used for in situ hybridization, immunohistochemistry, and in situ ligand binding or were frozen in liquid nitrogen and stored at -80 °C for subsequent RNA and protein analyses. The presence of sperm in the vaginal smear was designated as day 0.5 of pregnancy. New Zealand White rabbits were obtained from Myrtle's Rabbitry (Thompsons Station, TN) and used for antibody production. Protocols for the care and use of animals were approved by the University of Kansas Animal Care and Use Committee.Cell CultureU1 rat uterine stromal cells were obtained from Dr. Virginia Rider (Pittsburg State University, Pittsburg, KS) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mm sodium pyruvate, and penicillin (100 units/ml) and streptomycin (100 μg/ml) (41Piva P. Flieger O. Rider V. Biol. Reprod. 1996; 55: 1333-1342Crossref PubMed Scopus (40) Google Scholar, 42Rider V. Methods Mol. Med. 2006; 121: 57-67PubMed Google Scholar). These rat uterine stromal cells are physiologically relevant in that they can be induced to differentiate into decidual cells (43Rider V. Potapova T. Dai G. Soares M.J. J. Endocrinol. 2005; 184: 119-127Crossref PubMed Scopus (4) Google Scholar). Rat aortic endothelial cells were purchased from VEC Technologies, Inc. (Rensselaer, NY) and maintained in MCDB-131 complete culture medium. The rat Nb2 lymphoma cell line was provided by Dr. Peter Gout (University of British Columbia, Vancouver, Canada) and maintained in RPMI 1640 culture medium supplemented with 10% horse serum, 10% FBS, 50 μm 2-mercaptoethanol, 2 mm l-glutamine, 5 mm HEPES, penicillin (50 units/ml), and streptomycin (50 μg/ml) (44Tanaka T. Shiu R.P. Gout P.W. Beer C.T. Noble R.L. Friesen H.G. J. Clin. Endocrinol. Metab. 1980; 51: 1058-1063Crossref PubMed Scopus (565) Google Scholar). Human embryonic kidney (HEK 293) cells were obtained from ATCC (Manassas, VA) and used as a host for the expression of the PLP-J fusion proteins. HEK 293 cells were maintained in DMEM/F-12 medium supplemented with 10% FBS, 1 mm sodium pyruvate, penicillin (100 units/ml), and streptomycin (100 μg/ml). CHO cells and heparan sulfate-deficient CHO-pgsD-677 cells were obtained from ATCC and cultured in DMEM/MCDB 302 culture medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FBS. Raji cells stably transfected with a syndecan-1 (Sdc1) expression vector (Raji-S1) and the parent Raji cell line were obtained from Dr. Alan C. Rapraeger (University of Wisconsin, Madison, WI) and were maintained in RPMI 1640 culture medium supplemented with 10% FBS and antibiotics (45Lebakken C.S. Rapraeger A.C. J. Cell Biol. 1996; 132: 1209-1221Crossref PubMed Scopus (66) Google Scholar). All cell cultures were maintained in a humidified atmosphere of 5% CO2, 95% air at 37 °C.Generation of Fusion ProteinsmFLAG-PLP-J—PLP-J was expressed as a fusion protein with a FLAG-His6-FLAG tag. The full-length mature rat PLP-J cDNA was used as a template for PCR amplification of a PLP-J fragment with EcoRI and XbaI restriction sites at the 5′- and 3′-ends, respectively, using sequence specific primers, 5′-cat tta aag aat tca cac cat atg acc aga tgt-3′ and 5′-gtt ata tgt ttc tag att acc act tgt taa taa tg-3′. After digestion with EcoRI and XbaI restriction enzymes, the fragment was ligated into a modified pFLAG-CMV-3 vector (mFLAG; Sigma). The accuracy of vector construction was verified by DNA sequencing. The mFLAG-PLP-J plasmid was transfected into HEK 293 cells using Lipofectamine Plus according to the manufacturer's instructions (Invitrogen). The initial selection of transfected cells was accomplished in the presence of G418 at a concentration of 500 μg/ml. Selected cells were then maintained in 100 μg/ml G418.The mFLAG-PLP-J fusion protein was purified from serum-free conditioned medium by incubating with an Ni2+-NTA-agarose resin (Qiagen, Valencia, CA). In brief, Ni2+-NTA-agarose was equilibrated with Sorensen's phosphate buffer (NaH2PO4 (66 mm) and KH2PO4 (66 mm)) and added to conditioned medium in a buffer containing 10 mm imidazole, 50 mm NaH2PO4, 0.15 m NaCl, pH 8.0 (1× binding buffer), incubated overnight at 4 °C with constant shaking. The resin was then transferred into a column and washed with 2× binding buffer. Recombinant mFLAG-PLP-J protein was eluted with 250 mm imidazole and 0.15 m NaCl in Sorensen's phosphate buffer, pH 6.0. Aliquots of fractions were separated using SDS-PAGE and stained with Coomassie Blue G-250 and immunoblotted with anti-FLAG M2 antibody (Sigma). Immunopositive fractions were pooled and dialyzed against phosphate-buffered saline, pH 7.4, and concentrated by Centricon ultrafiltration centrifugation devices (Millipore, Billerica, MA). Purified proteins were sterilized using Millex filters (Millipore), and concentrations were determined with the DC protein assay (Bio-Rad).Alkaline Phosphatase (AP)-PLP-J Fusion Protein (AP-PLP-J)—The full-length cDNA for mature rat PLP-J was ligated downstream of heat-stable human placental AP of pCMV-SEAP and then transfected into HEK 293 cells, as previously described (46Müller H. Dai G. Soares M.J. J. Histochem. Cytochem. 1998; 46: 737-743Crossref PubMed Scopus (13) Google Scholar, 47Müller H. Soares M.J. Methods Mol. Med. 2006; 122: 331-340PubMed Google Scholar). The transfected cells were selected and maintained in DMEM/F-12 culture medium supplemented with 10% FBS and G418 as described above. Cells were transferred to serum-free culture medium for 72 h. Conditioned medium was collected, and cellular debris was removed by centrifugation at 2,200 × g for 30 min at 4 °C and then stored at -20 °C. AP activity was quantified by a colorimetric assay at 405 nm using p-nitrophenyl phosphate as an AP substrate (47Müller H. Soares M.J. Methods Mol. Med. 2006; 122: 331-340PubMed Google Scholar). AP fusion proteins were also separated using SDS-PAGE and electrophoretically transferred to nitrocellulose, and AP activity was detected by incubation with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.Preparation of PLP-J AntibodiesAntibodies directed to PLP-J were prepared by immunizing rabbits with recombinant His6-tagged PLP-J protein generated in a prokaryotic expression system, pQE-30Xa (Qiagen). The mature rat PLP-J cDNA was used as a template for PCR amplification of a PLP-J fragment with BamHI and SacI restriction sites at the 5′- and 3′-ends, respectively, using sequence specific primers, 5′-cat tta aag gat cca cac cat atg acc aga tgt-3′ and 5′-gtt ata tgt cga gct ctt acc act tgt ttt taa taa tg-3′. The amplified fragment was ligated into the pQE-30Xa plasmid and transformed into Escherichia coli, M15[pREP4]. His6-PLP-J expression was induced by 2 mm isopropyl β-d-1-thiogalactopyranoside, and His6-PLP-J protein was purified from bacterial exclusion bodies by Ni2+-NTA-agarose affinity chromatography, as described above. Purified His6-PLP-J was characterized by SDS-PAGE and Western blotting with anti-His tag antibodies (Qiagen). Purified His6-PLP-J was used to immunize New Zealand White rabbits as previously described (48Deb S. Hashizume K. Boone K. Southard J.N. Talamantes F. Rawitch A. Soares M.J. Mol. Cell. Endocrinol. 1989; 63: 45-56Crossref PubMed Scopus (44) Google Scholar, 49Deb S. Youngblood T. Rawitch A. Soares M.J. J. Biol. Chem. 1989; 264: 14348-14353Abstract Full Text PDF PubMed Google Scholar).Western Blot Analysis of Decidual TissuesProtein lysates were prepared by homogenizing gestation day 8.5 rat conceptuses (decidua and extraembryonic and embryonic tissues) in radioimmune precipitation buffer (10 mm Tris-HCl, pH 7.2, 1% Triton X-100 or 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 5 mm EDTA, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin). Protein concentrations were determined by the DC protein assay (Bio-Rad). Fifty μg of total protein were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Immunoreactive PLP-J and dPRP were detected with antibodies to anti-PLP-J (present study) and anti-dPRP (39Rasmussen C.A. Hashizume K. Orwig K.E. Xu L. Soares M.J. Endocrinology. 1996; 137: 5558-5566Crossref PubMed Scopus (39) Google Scholar), respectively, and visualized by enhanced chemiluminescence according to the manufacturer's instructions (Amersham Biosciences).ImmunocytochemistryImmunocytochemical analyses were used to localize PLP-J and dPRP proteins and the distribution of endothelial cells in gestation day 8.5 implantation sites, as described (39Rasmussen C.A. Hashizume K. Orwig K.E. Xu L. Soares M.J. Endocrinology. 1996; 137: 5558-5566Crossref PubMed Scopus (39) Google Scholar, 40Ain R. Konno T. Canham L.N. Soares M.J. Methods Mol. Med. 2006; 121: 295-313PubMed Google Scholar, 50Konno T. Rempel L.A. Arroyo J.A. Soares M.J. Biol. Reprod. 2007; 76: 709-718Crossref PubMed Scopus (42) Google Scholar). Cryosections (10 μm) were prepared, fixed in cold 4% paraformaldehyde solution, and blocked in 10% normal goat serum for 1 h at room temperature. The immunodetection was performed by incubating overnight at 4 °C with anti-PLP-J (present study), anti-dPRP antibodies (39Rasmussen C.A. Hashizume K. Orwig K.E. Xu L. Soares M.J. Endocrinology. 1996; 137: 5558-5566Crossref PubMed Scopus (39) Google Scholar), or RECA-1 antibodies, which recognize an uncharacterized rat endothelial cell-specific surface antigen (Serotec, Oxford, UK) (41Piva P. Flieger O. Rider V. Biol. Reprod. 1996; 55: 1333-1342Crossref PubMed Scopus (40) Google Scholar). Avidin-peroxidase-conjugated secondary antibody was added for 30 min at room temperature and color-developed with an AEC kit (Zymed Laboratories, San Francisco, CA). Tissues were counterstained with Mayer's hematoxylin. Images were captured using a Leica MZFIII steromicroscope (Leica Microsystems GmbH, Welzlar, Germany) or a Nikon Eclipse 55i microscope (Nikon Instruments Inc., Melville, NY), both equipped with Leica CCD cameras (Leica).In Situ HybridizationIn situ hybridization was performed to assess the distributions of PLP-J and dPRP transcripts in gestation day 8.5 rat implantation sites (40Ain R. Konno T. Canham L.N. Soares M.J. Methods Mol. Med. 2006; 121: 295-313PubMed Google Scholar, 51Ain R. Canham L.N. Soares M.J. Dev. Biol. 2003; 260: 176-190Crossref PubMed Scopus (186) Google Scholar). Cryosections (10-μm) were prepared and stored at -80 °C until used. Plasmids containing cDNAs for PLP-J (34Dai G. Wang D. Liu B. Kasik J.W. Müller H. White R.A. Hummel G.S. Soares M.J. J. Endocrinol. 2000; 166: 63-75Crossref PubMed Scopus (26) Google Scholar) and dPRP (29Roby K.F. Deb S. Gibori G. Szpirer C. Levan G. Kwok S.C.M. Soares M.J. J. Biol. Chem. 1993; 268: 3136-3142Abstract Full Text PDF PubMed Google Scholar) were used as templates to synthesize sense and antisense digoxigenin-labeled riboprobes according to the manufacturer's instructions (Roche Applied Science). The frozen sections were air-dried and fixed in cold 4% paraformaldehyde in phosphate-buffered saline. Prehybridization, hybridization, and detection of alkaline phosphatase-conjugated anti-digoxigenin were performed as previously reported (12Wiemers D.O. Shao L.-J. Ain R. Dai G. Soares M.J. Endocrinology. 2003; 144: 313-325Crossref PubMed Scopus (101) Google Scholar, 51Ain R. Canham L.N. Soares M.J. Dev. Biol. 2003; 260: 176-190Crossref PubMed Scopus (186) Google Scholar). Images were captured as described above.Northern Blot AnalysisNorthern blot analysis was performed as previously described (52Faria T.N. Deb S. Kwok S.C.M. Talamantes F. Soares M.J. Dev. Biol. 1990; 141: 279-291Crossref PubMed Scopus (113) Google Scholar). Total RNA was extracted from gestation day 8.5 rat decidual tissues, U1 uterine stromal cells, and rat aortic endothelial cells using TRIzol reagent (Invitrogen). Total RNA (15 μg/lane) was resolved in 1% formaldehyde-agarose gels, transferred to nylon membranes, and cross-linked. Blots were probed with 32P-labeled cDNAs for syndecans (Sdc1, NM_013026; Sdc2, NM_013082; Sdc3, NM_053893; Sdc4, NM_012649). Glyceraldehyde-3-phosphate dehydrogenase cDNA was used to evaluate the integrity and equal loading of RNA samples. At least three different tissue samples from three different animals were analyzed with each probe for each time point.AP-PLP-J BindingTissues—An in situ AP-binding assay was performed as previously described (46Müller H. Dai G. Soares M.J. J. Histochem. Cytochem. 1998; 46: 737-743Crossref PubMed Scopus (13) Google Scholar, 47Müller H. Soares M.J. Methods Mol. Med. 2006; 122: 331-340PubMed Google Scholar). In brief, 8–10-μm tissue sections were prepared with a cryostat and mounted onto glass slides. The tissue sections were washed with a modified Hanks' balanced salt solution (HBHA; containing 20 mm HEPES, 0.5 mg/ml BSA and 0.1% NaN3) and incubated with AP, AP-PLP-J, or AP-placental lactogen-I (PL-I; also known as PRL family 3, subfamily d, member 1 (Prl3d1)) fusion protein (46Müller H. Dai G. Soares M.J. J. Histochem. Cytochem. 1998; 46: 737-743Crossref PubMed Scopus (13) Google Scholar) for 75 min at room temperature. For competition, tissue sections were incubated with various glycosaminoglycans and/or mFLAG-PLP-J protein for 45 min prior to incubation with AP-PLP-J. Following incubation, tissue sections were washed three times with HBHA containing 0.1% Tween 20 and fixed for 20 min with acetone-formaldehyde fixative. The fixed sections were washed and heated at 65 °C for 30 min to inactivate endogenous AP activity in the tissues. Localization of AP was determined by incubation with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.Cells—For AP-ligand binding with cells, 5 × 104 cells were incubated with different concentrations of conditioned medium of AP or AP-PLP-J for 60 min at room temperature. Where indicated, cells were incubated with different concentrations of heparin for 30 min before incubation with AP-PLP-J. Cells were washed with HBHA containing 0.1% Tween 20 and HBSS. Cells were then heated for 30 min at 65 °C to inactivate endogenous AP activity. AP activity was determined by incubation with AP substrate (p-nitrophenyl phosphate) and measurement of absorbance at 405 nm. AP-PLP-J binding to heparan sulfate-deficient CHO-pgsD-677 cells and wild-type CHO cells was also assessed as previously described (22Wang D. Ishimura R. Walia D.S. Müller H. Dai G. Hunt J.S. Lee N.A. Lee J.J. Soares M.J. J. Endocrinol. 2000; 167: 15-28Crossref PubMed Scopus (26) Google Scholar). AP-PLP-J and AP-PL-I binding with the PRL receptor were assessed in CHO-pgsD-677 cells (53Lidholt K. Weinke J.L. Kiser C.S. Lugenwa F.N. Bame K.J. Cheifetz S. Massague J. Lindhahl U. Esko J.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2267-2271Crossref PubMed Scopus (234) Google Scholar) transiently transfected with the PRL receptor (pECE/long; gift from Dr. Paul Kelly, INSERM, Paris, France) (54Ali S. Edery M. Pellegrin I. Lesueur L. Paly J. Djiane J. Kelly P.A. Mol. Endocrinol. 1992; 6: 1242-1248Crossref PubMed Scopus (0) Google Scholar) usi
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