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Expression of alkaline phosphatase in differentiated rat labyrinthine trophoblast tissue
Placenta(1991), 12, 227-237
Expression of Alkaline Phosphatase in Diserentiated Rat Labyrinthine Trophoblast Tissue
WENDY JO CAMPBELLa9C DOUGLAS LARSEN,“, SANTANU DEB” SIMON C. M. KWOKb & MICHAEL J SOARESa9d Departmentsof Physiology” and Biochetnistryb, Ralph L. Smith MentalRetardation ResearchCenter,Universityof KansacMedical Center, Kansas City, Kansas, 66103, US4 ‘Presentaddress:TheEppleyInstitute,University OfNebraska, 42nd and DeweyAvenue, Omaha, Nebraska681986805 dTo whom correspondence should be dressed. Paper accepted17.1.1991
SUMMARY In this report, we describe the generation of specific antibodies to rat alkaline phosphatase and the temporal and regional characteristicsof alkaline phosphatase expression during maturation of the rat chorioallantoicplacenta. An antipeptide antiserum was generated to the amino terminal 15 amino acids of rat alkaline phosphatase. The antiserum specificallyrecognized alkaline phosphatase.Alkaline phosphatase expression was monitored in the junctional and labyrinth zones of the chorioallantoicplacenta by Western and Northern blot analyses. Alkalinephosphataseprotein and mRNA were present in both thejunctional and labyrinth zones on day 13 ofgestation.As gestationadvanced, alkalinephosphatasemI?hU andprotein expressiona!ecreasedbelowthe limitsofdetettionin thejunctional zone, while alkaline phosphatase expression increased in the labyrinth zone. Labyrinthine alkaline phosphatase migrated predominantly as a 95-k.Da species, whereas rat kidney expressed exclusivelythe 75-kDa species.Enzymatic deglycosylationof the 75- and 95-kDa alkalinephosphatase speciesresulted in the generation of a 55kDa species. In summary, alkaline phosphatase expression is a useful indicator of trophoblast d@rentiation.
INTRODUCTION Alkaline phosphatase has been used as a marker of trophoblast cell differentiation in a number of species (Padykula, 1959; Sherman, 1972; Chou, 1982; Soares, 1987; Hunt and Soares, 1988). In the human, the formation of syncytial trophoblast cells coincides with 0143-4004/91/030227
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elevated expression of alkaline phosphatase (Robinson et al, 1988). In the rat, alkaline phosphatase expression also accompanies differentiation and is primarily restricted to a subpopulation of trophoblast cells located in the labyrinth zone of the chorioallantoic placenta (Padykula, 1959; Soares, 1987; Hunt and Soares, 1988). The specificity of alkaline phosphatase expression has been a point of concern, especially when alkaline phosphatase is monitored by measurement of hydrolysis of p-nitrophenyl phosphate at alkaline pH. Humans possess three distinct alkaline phosphatase genes: placental, intestinal, and liver/bone/kidney (for reviews see Goldstein and Harris, 1979; Fishman, 1987; Harris, 1990), and the rat possesses two genes: intestinal and liver/ bone/kidney/placental (Thiede et al, 1988). Rat liver/bone/kidney/placental alkaline phosphatase has been purified and its cDNA cloned and sequenced (Nair et al, 1987; Nair, Majeska and Rodan, 1987; Noda et al, 1987; Theide et al, 1988). The availability of amino acid sequence information for rat alkaline phosphatase permits the development of immunological probes to specific regions of the protein (for a review of the technique, see Walter, 1986). In this study, we were interested in determining whether alkaline phosphatase could be effectively used to monitor rat trophoblast cell differentiation. We describe the generation of specific antibodies to rat alkaline phosphatase and the temporal and regional characteristics of alkaline phosphatase protein and mRNA expression during maturation of the rat chorioallantoic placenta.
MATERIALS
AND METHODS
Reagents
A complementary DNA to rat alkaline phosphatase (Noda et al, 1987) was a generous gift from Drs Masaki Noda and Gideon Rodan of Merck Sharp and Dohme (West Point, PA). Restriction enzymes and polymerases were purchased from New England BioLabs (Beverly, MA). Oligonucleotide random priming DNA labelling kits and molecular weight standards for nucleic acid agarose gels were obtained from Bethesda Research Laboratories (Gaithersburg, MD). Reagents for sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis were purchased from Bio-Rad Chemical Division (Richmond, CA). Radiolabelled nucleotides were obtained from DuPont NEN (Boston, MA). N-Glycanase was obtained from Genzyme (Boston, MA). Unless otherwise noted, all other chemical and reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and tissue isolation
Timed pregnant Holtzman rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). The presence of a copulatory plug or sperm in the vaginal smear was designated as day 0 of pregnancy. Chorioallantoic placentae were removed from uteri on various days during the second half of pregnancy (days 13-19), dissected into junctional and labyrinth zones (Soares, 1987), immediately frozen in liquid nitrogen and stored at -70°C until used for mRNA or immunoblotting analyses. The junctional zone was identified by its pale appearance, due to the absence of fetal blood, and separated from the labyrinth zone, a richly vascularized tissue, with fine forceps and 23-gauge needles (for further information concerning the placental zones, see Soares, 1987; Campbell et al, 1989). In some experiments, kidneys were obtained
Campbd et al: Placental Expression ofAlkaline Phosphatase
from pregnant rats at the time of autopsy, dissected free of mesenteries, described above.
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frozen and stored as
Generation of antisera to rat alkaline phosphatase Antibodies were generated to an amino-terminal peptide derived from the amino acid sequence of rat alkaline phosphatase (Hsu et al, 1987; Misumi et al, 1988; Nair et al, 1987; Theide et al, 1988). The selected sequence consisted of the 15 amino-terminal amino acids of alkaline phosphatase: Phe-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-Ser-Tyr-Trp-Arg-GlnGln. The peptide was synthesized as a carboxyterminal amide using a Biosearch SAM TWO solid phase peptide synthesizer (Biosearch, San Rafael, CA) with standard t-butoxycarbonyl methodology (Marglin and Merrifield, 1970). A cysteine residue was added to the amino terminus of the peptide to facilitate coupling to the carrier protein. The synthesized peptide was purified by reverse phase high performance liquid chromatography, and the amino acid composition of the purified peptide was determined. These procedures have been previously described by our laboratory (Deb et al, 1989a,b). The amino terminal alkaline phosphatase peptide was coupled to keyhole limpet hemocyanin (KLH, Calbiochem, San Diego, CA) through the amino-terminal cysteine residue by using m-maleimidobenzoyl-Nhydroxysuccinimide ester (Calbiochem) as described previously (Lerner et al, 1981; Deb et al, 1989a,b). Three adult New Zealand White rabbits obtained from White Hare Rabbitry (Stark City, MO) were immunized with the peptide-KLH preparation. The immunization and bleeding protocols were similar to that previously described for the generation of antipeptide antisera to rat placental lactogen-II peptides (Deb et al, 1989b). Antipeptide antisera were characterized by enzyme-linked immunoassay and Western blot analyses. These techniques have been previously described (Soares et al, 1988; Deb et al, 1989a,b). Enzymatic deglycosylation N-linked sugars were enzymatically removed from protein preparations via treatment with N-Glycanase (consists of a mixture of N-glycosidase F and aspartylglycosylamine aminohydrolase). Samples were exposed to N-Glycanase (final concentration: lO,&ml) in a reaction mixture containing a final concentration of 0.17 per cent SDS (Bio-Rad), 0.2 >* sodium Phosphate, pH 8.6, and 1.25 per cent Nonidet P-40 in a total volume of 3Opl at 37°C for 1620 h. The samples were then subjected to Western blot analysis (Soares et al, 1988; Deb et al, 1989b). Expression of alkaline phosphatase: estimation of protein and mRNA Relative concentrations of alkaline phosphatase protein and mRNA in the junctional and labyrinth zones of the chorioallantoic placenta during the second half of gestation were estimated by Western blot and Northern blot analyses, respectively. These techniques have been previously described (Soares et al, 1987, 1988- Campbell et al, 1989; Deb et al, 1989a,b). Relative changes in the expression of alkaline phosphatase protein and mRNA were estimated by reflectance and transmission densitometry, respectively (model 620 Video Densitometer, Bio-Rad). Alkaline phosphatase protein was extracted according to the procedure of Nair et al (1987a,b). Briefly, tissues were homogenized in Buffer No. l(10 mM tris-HCl, pH 7.2, containing 0.5 rnkt MgClz and 0.5 mu phenyl methyl sulfonyl fluoride, 0.02 per cent sodium azide; 5 ml/g tissue) for 15-30 set at 4°C. Homogenates were centrifuged at 30 000 g for 1 h. Pellets were resuspended in Buffer No. 1 (5 ml/g) and n-butanol(40% v/v) was added to the suspensions and gently stirred in the cold for 4-6 h. Butanol extracts were then centrifuged at
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10 000 g for 20 min and the aqueous layers were recovered. Cold acetone was added to the aqueous solution (60 per cent v/v), the mixture stirred in the cold for 4-6 h, and precipitates containing alkaline phosphatase retrieved by centrifugation (30 000 g for 20 min). Precipitates were solubilized in Buffer No. II (10 mu tris-HCl, pH 8.0,O.S mu MgClz, 0.2 per cent Triton X- 100) and protein concentrations determined by the method of Bradford (1976). A rat cDNA clone to alkaline phosphatase was used for hybridization (Noda et al, 1987). Alkaline phosphatase cDNA inserts were utilized as templates for the synthesis of 32Plabelled cDNA probes with an oligonucleotide random primer extension labelling kit.
RESULTS Characterization of rat alkaline phosphatase antipeptide antisera Significant antibody titers were detected to the alkaline phosphatase synthetic peptide within five weeks of the initial immunization in each of the three immunized rabbits (Figure 1). Antibody titers to the peptide showed minimal changes following subsequent booster injections. Western blot analyses of extracts from day 16 placental labyrinth zone indicated that serum collected from rabbit No. 28 showed the best reactivity with placental alkaline phosphatase. All subsequent experiments utilized antiserum collected from rabbit No. 28. Figure 2 shows the specificity of the alkaline phosphatase antipeptide antiserum with rat placental alkaline phosphatase. Substitution of preimmune serum of alkaline phosphatase antipeptide antiserum saturated with alkaline phosphatase amino-terminal peptide (10 ,ug/ml) completely eliminated the reactivity. Gestational pattern of placental alkaline phosphatase protein and mRNA expression Immunoreactive alkaline phosphatase expression was restricted to the labyrinth zone of the chorioallantoic placenta with an apparent molecular weight of 95 kDa when separated in 10 per cent polyacrylamide gels (Figure 3). Please note that the 95kDa species can be resolved
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Figure 2. Examination of the specificity of the Western blot analysis for rat placental alkaline phosphatase. Enriched preparations ofrat alkaline phosphatase extracted from the labyrinth zone ofthe day 16 chorioallantoic placenta were separated by sodium dodecyl sulfate (SDS) gel electrophoresis in 7.5 per cent gels under reducing conditions and electrophoretically transferred to nitrocellulose. Nitrocellulose membranes were probed with preimmune serum (lane A), rat alkaline phosphatase antipeptide antiserum (rabbit No. 28; lane B), or with rat alkaline phosphatase antipeptide antiserum saturated with the amino terminal rat alkaline phosphatase peptide (10 &ml; lane C). The immune and preimmune sera were used at a dilution of 1500. The migration of molecular weight standards (x1O-3) is shown.
into at least two different species when separated in 7.5 per cent polyacrylamide gels (Figure 2). Northern blot analysis essentially resembled the Western blot analysis with one important difference (Figure 4). Alkaline phosphatase mRNA was detectable in both junctional and labyrinth zones on day 13 of gestation. In the junctional zone, alkaline phosphatase mRNA concentrations decreased below the limit of detection on days 16 and 19 of gestation, while in the labyrinth zone the relative concentration of alkaline phosphatase increased from days 1316 of gestation (Figure 4). Relative alkaline phosphatase mRNA concentrations decreased in the labyrinth zone from days 16-l 9 of gestation (Figure 4). Densitometric measurements of relative alkaline phosphatase protein and mRNA expression are shown in Figure 5. The expression pattern observed for alkaline phosphatase mRNA was not associated with compromised integrity of the labyrinth and junctional zone RNA samples. These same RNA samples have been analysed for the expression of two other placental mRNAs, prolactin-like protein-A (PLP-A) and placental lactogen-II (PL-II: Campbell et al, 1989). Unlike alkaline phosphatase, PLP-A mRNA is only expressed in the junctional zone, whereas, PL-II shows a temporally specific pattern of expression in both zones (Campbell et al, 1989). The presence of alkaline phosphatase mRNA in the junctional zone on day 13 of gestation prompted further analysis of alkaline phosphatase protein expression in the junctional zone
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Placenta (1991), Vol. I2 Alk-Phos
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Figure 3. Representative Western blot probed with antipeptide antiserum specific to rat alkalime phosphatase. Extracts were isolated from the junctional and labyrinth zones of the chorioallantoic placenta on days 13, 16 and 19 of pregnancy. Equivalent amounts of protein were separated by SDS electrophoresis in 10 per cent polyacrylamide gels under reducing conditions and transferred to nitrocellulose. Nitrocellulose membranes were probed with antipeptide antiserum to rat alkaline phosphatase (Rabbit No. 28) at a dilution of 1500. Note that immunoreactive alkaline phosphatase was localized predominantly to the labyrinth zone and had an apparent molecular weight of 95 kDa. Lane A, day 13 junctional zone 0); lane B, day 13 labyrinth zone (L); lane C, day 16 J; Lane D, day 16 L; lane E, day 19 J; lane F, day 19 L. Molecular weight standards (X 10m3) are shown.
on day 13. Alkaline phosphatase expression was examined from junctional zone isolated on day 13 of gestation, labyrinth zone from day 16, and kidney tissue under control conditions and following enzymatic deglycosylation (Figure 6). In these experiments, the alkaline phosphatase antipeptide antiserum recognized alkaline phosphatase species from day 13 placental junctional zone and kidney (Figure 6). Under control conditions immunoreactive alkaline phosphatase from the placental labyrinth zone migrated predominantly as a 95-kDa species, while alkaline phosphatase from the kidney migrated as a 7.5 kDa species. Alkaline phosphatase from the day 13 junctional zone was difficult to detect under control conditions but consisted of a mixture of both 75 and 9.5 kDa species. Treannent with N-Glycanase (Genzyme) decreased the apparent molecular sizes of all immunoreactive species from each of the tissues to 55 kDa (Figure 6). Detection of day 13 junctional zone immunoreactive alkaline phosphatase was improved following enzymatic deglycosylation (Figure 6).
DISCUSSION In this report, we have described the generation of an anitpeptide antiserum specific to rat alkaline phosphatase, the temporal and regional pattern of alkaline phosphatase protein and
233
Campbell et al: Placental Expression ofAlkaline Phosphatase Alk-Phor
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Northern blot hybridized with an alkaline phosphatase cDNA probe. Total RNA was isolated from the junctional and labyrinth zones of the chorioallantoic placenta on days 13,16 and 19 of pregnancy. The RNA (10 /&lane) was fractionated by formaldehyde agarose gel electrophoresis and transferred to nitrocellulose prior to hybridization. Single mRNA species of alkaline phosphatase were detected in the appropriate size range of 2.5 kb. Note that day 13 junctional zone contains detectable alkaline phosphatase mRNA, whereas, alkaline phosphatase mRNA levels are below the liiits of detection in the junctional zone on days 16 and 19 of gestation. Lane A, day 13 junctional zone (J); lane B, day 13 labyrinth zone (L); lane C, day 16 J; lane D, day 16 L; lane E, day 19 J; lane F, day 19 L. Molecular weight standards are shown.
mRNA expression in the developing rat chorioallantoic placenta, and some biochemical characteristics of alkaline phosphatase isolated from the chorioallantoic placenta. During the second half of gestation, alkaline phosphatase protein and mRNA are predominantly expressed in labyrinthine trophoblast cells of the chorioallantoic placenta (present study). This observation is consistent with previous reports on the distribution of alkaline phosphatase enzymatic activity within the rat placenta (Padykula, 1959; Soares, 1987) and immunohistochemical analysis of alkaline phosphatase within the rat placenta (Hunt and Soares, 1988). Alkaline phosphatase activity has recently been localized by electron microscopy to the maternal facing membrane of syncytiotrophoblast layer II of the labyrinth zone (Glazier, Jones and Sibley, 1990). Labyrinthine alkaline phosphatase has a higher apparent molecular weight than alkaline phosphatases isolated from other tissues (95 vs. 75 kDa). Mid-gestation junctional zone trophoblast tissue expresses low levels of alkaline phosphatase mRNA and low levels of the 75- and 95-kDa alkaline phosphatase forms. These two forms of alkaline phosphatase arise from differential glycosylation patterns in labyrinthine trophoblast tissue versus other tissues (present study). Removal of N-linked carbohydrates from alkaline phosphatases of kidney and labyrinthine tissue yield immunoreactive proteins of similar size (non-glycosylated precursors). Labyrinthine and junctional zone trophoblast cells diffentiate from a common precursor cell population (Jollie, 1964; Davies and Glasser, 1968). Trophoblast cell differentiation in
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the rat chorioallantoic placenta is a complex process and is associated with three specific phenomena related to alkaline phosphatase expression: (1) a termination of alkaline phosphatase mRNA and protein expression in the junctional zone; (2) an increase in the expression of alkaline phosphatase in the labyrinth zone; and (3) a change in posttranslational processing of labyrinthine alkaline phosphatase protein, shifting its apparent molecular weight to 95 kDa. Expression of the 95-kDa alkaline phosphatase appears to be linked to the differentiation of the labyrinth zone of the chorioallantoic placenta and thus, may be related to the structural and functional organization of the labyrinth zone. Unlike junctional zone trophoblast tissue, labyrinthine trophoblast cells exhibit polarity; they rest on a basement membrane overlying fetal mesenchyme (Davies and Glasser, 1968). Labyrinth trophoblast cells are involved in transport of nutrients and wastes between maternal and fetal environments (Miller, Koszalka and Brent, 1976). Alkaline phosphatases are plasma membrane associated proteins and are hypothesized to be involved in the active transport of metabolites between maternal and fetal environments (Harris, 1990). The effect of the glycosylation pattern of labyrinthine trophoblast alkaline phosphatase on its activities is not known but may represent important
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Day of pregnancy Figure 5. Relative changes in alkaline phosphatase protein concentrations (a) and mRNA concenaations (b) in junctional (0) and labyrinth (m) zones of the chorioallantoic placenta on days 13,16 and 19 of pregnancy. Values represent densitometric readings from four replicate Western and three replicate Northern blots and are expressed as a percentage of the highest value (100 per cent).
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Campbell et al: Placental Expression ofAlkaline Phosphatase
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products following enzymatic deglycosylation. Tissue Figwe 6. Analysis of alkaline phosphatase immunoreactive extracts were treated with N-glycanase overnight at 37X, separated by SDS electrophoresis in 10 per cent polyacrylamide gels under reducing conditions, transferred to nitrocellulose, and subjected to Western blot analysis. Lanes A and D, day 13 junctional zone (J); lanes B and E, day 16 labyrinth zone (L); lanes C and F, kidney. Lanes A-C contain control preparations and lanes D-F contain the same preparations treated with N-glycanase. Note that the apparent molecular weight of each of the N-glycanase treated samples was similar. Rat alkaline phosphatase antipeptide antiserum (rabbit No. 28) was used at a dilution of 1:.500. Molecular weight standards (X 10m3) are shown.
modifications necessary for the specialized actions of placental alkaline phosphatase in transport between maternal and fetal compartments. In conclusion, regionally and temporally specific inhibitory and stimulatory events are involved in the regulation of alkaline phosphatase expression during trophoblast differentiation. Labyrinthine trophoblast tissue specifically expresses a 95-kDa glycosylated alkaline phosphatase isoform. The specific factors responsible for the regulation of trophoblast cell alkaline phosphatase expression, post-translational processing of labyrinthine alkaline phosphatase, or the specific biological roles served by this protein during development remain to be determined. ACKNOWLEDGEMENTS We thank Drs Masaki Noda and Gideon Rodan for generously supplying the rat alkaline phosphatase cDNA and Dr Allen Rawitch and Mr Richard Grabbe for assistance in the synthesis of the amino-terminal rat alkaline phosphatase peptide. We also acknowledge Mr Michael McMaster for assistance in early stages ofthii project and Linda Carr for help in the preparation of the manuscript. We also express appreciation to Dr Glen .i\ndrews, Teresa Faria, and Carla Green for advice and assistance with the mRNA analysis. This study was supported by grants from the National Institute of Child Health and Human Development (HD 22208, HD 20676). W.J.C. and S.D. are recipients of a Wesley Foundation postdoctoral fellowship.
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Placenta (1991), Vol. 12 REFERENCES
Bradford, M. (1976) A rapid and sensitive method for quantitation principle of dye binding. Analytical Biochemistry,72, 248-254.
of microgram
quantities
of protein utilizing the
Campbell, W. J., Deb, S., Kwok, S. C. M., JosIin, J. A. & Soares, M. J. (1989) Differential expression ofplacental lactogen-II and prolactin-lie protein-A in the rat chorioallantoic placenta. Endocrinology,125, 1565-1574. Chou, J. Y. (1982) Effects of retinoic acid on differentiation of choriocarcinoma cells in vitro. Journal of Clinical Endocrinologyand Metabolism, 54, 1174-l 180. Davies, J. & Glasser, S. R. (1968) Histological and fine structural observation on the placenta of the rat. .4ctu Anatomica, 69, 542-608. Deb, S., Youngblood, T., Rawitch, A. B. & Soares, M. J. (1989a) Placental prolactin-like protein-A. Identification and characterization of two major glycoprotein species with antipeptide antibodies. 3oournal of BiologicalChemisty, 264,14348-14353. Deb, S., Hashizume, K., Boone, K., Southard, J. N., Talamantes, F., Rawitch, A. & Soares, M. L. (1989b) Antipeptide antibodies reveal structural and functional characteristics of rat placental lactogen-II. Molecular and Celhtlar Endoctinologv,63, 45-56. Fishman, W. H. (1987) Oncotrophoblast gene expression: placental alkaline phosphatase. Advances in Cancer Research, 48, l-35. Glazier, J. D., Jones, C. J. P. & Sibley, C. P. (1990) Preparation ofplasma membrane vesicles from the rat placenta at term and measurement of Na+ uptake. Placenta, 11,45 l-463. Goldstein, D. J. & Harris, H. (1979) Human placental alkaline phosphatase differs from that of other species. Nature, 280, 602-605. Harris, H. (1990) The human alkaline phosphatases: what we know and what we don’t know. Clinica ChimicaALta, 186,133-150. Hsu, H. H. T., Rouse, J., Hamilton, J. &Anderson, H. C. (1987) Purification and partial amino acid sequencing of rat bone tumour (UMR 106) alkaline phosphatase. BiochemiraBiophysicsActu, 913,329-334. Hunt, J. S. & Soares, M. J. (1988) Expression of histocompatibility antigens, transferrin receptors, intermediate filaments and alkaline phosphatase by in vitro cultured rat placental cells and rat placental cells in situ. Plucpntu,9, 159-171. Jollie, W. P. (1964) Radiographic observations on variations in desoxyribonucleic acid synthesis in rat placenta with increasing gestational age. Americun~oumal ofAnatomy, 114, 161-171. Lemer, R. A., Green, N., Alexander, H., Liu, F.-T., Sutcliffe, J. G. & Shinnick, T. M. (1981) Chemically synthesized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope protein of Dane particles. Proceedingsof the NationalAcademyof Sciences of the United States ofAmerica, 78,3403-3407. Marglin, A. & Merrifield, R. B.‘(1970) Ch emical synthesis ofpeptides and proteins. Annual Raiew ofBiochemistry, 39 841-866. Miller, R. K., Koszalka, T. R. & Brent, R. L. (1976) The transport of molecules across placental membranes. In The Cell Surface in Animal Embyogenesis and Development (Eds) Poste, G. & Nicolson, G.L. pp. 145-223. New York: Elsevier/North Holland Biomedical Press. Misumi, Y., Tashiro, K., Hattori, M., Sal&i, Y. & Ikehara, Y. (1988) Primary structure of rat liver alkaline phosphatase deduced from its cDNA. BiochemicalJwmal, 249,661-668. Nair, B. C., Majeska, R. J. & Rodan, G. A. (1987) Rat alkaline phosphatase. I. Purification and characterization of the enzyme from osteosarcoma: generation of monoclonal and polyclonal antibodies. Archives ofBiochemisty and Biophysics,254, 18-27. Nair, B. C., Johnson, D. E., Majeska, R. J., Rodkey, J. A., Bennett, C. D. & Rodan, G. A. (1987) Rat alkaline phosphatase. II. Structural similarities between the osteosarcoma, bone, kidney, and placenta isoenzymes. .4rchives ofBiochemisty and Biophysics,254, 28-34. Noda, M., Yoon, K., Thiede, M., Buenaga, R., Weiss, M., Henthorn, P., Harris, H. & Rodan, G. A. (1987) cDNA cloning of alkaline phosphatase from rat osteosarcoma (ROS 17/2.8) cells. 3oumal of Bone and Mineral Research, 2, 161-164. Padykula, H.A. (1959) A histochemical and quantitative study of enzymes of the rat’s placenta._?obumalofAnatotny, 92,118-129. Robinson, B. G., Emanuel, R. L., Frim, D. M. & Majzoub, J. A. (1988) Gl ucocorticoid stimulates corticotrophin releasing hormone gene expression in human placenta. Proceedingsofthe NationalAcademy ofSciences of the United States ofAmerica, 85, 52445248. Sherman, M. I. (1972) The biochemisay of differentiation of mouse trophoblast: alkaline phosphatase. Dmelopmental Biology, 27,337-350. Soares, M. J. (1987) Developmental changes in the intraplacental distribution of placental lactogen and alkaline phosphatase in the rat.Joumal OfReproductionand Fertili@, 79,93-98. Soares, M. J., De, S. K., Foster, B. A., Julian, J. A. & Glasser, S. R. (1988) Identification of multiple low molecular weight placental prolactin-like proteins produced by rat trophoblast cells._?ooumal ofEndotinology, 116, 101-106.
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Soares, M. J., Schaberg, K. D., Pinal, C. S., De, S. K., Bhatia, P. & Andrews, G. K. (1987) Establishment of a rat cell line expressing characteristics of extraembryonic membranes. Developmental Biology, 124, 134-144. Thiede, M. A., Yoon, K., Golub, E., Noda, M. & Rodan, G. A. (1988) Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene. Proceedings of the Nutional ~4cademy of Sciences of the United States ofAmerica, 85,3 19-323. Walter, G. (1986) Production and use of antibodies against synthetic peptides.Jounal ofImmunologiculMethods, 88, 148-l 61.
Expression of Alkaline Phosphatase in Diserentiated Rat Labyrinthine Trophoblast Tissue
WENDY JO CAMPBELLa9C DOUGLAS LARSEN,“, SANTANU DEB” SIMON C. M. KWOKb & MICHAEL J SOARESa9d Departmentsof Physiology” and Biochetnistryb, Ralph L. Smith MentalRetardation ResearchCenter,Universityof KansacMedical Center, Kansas City, Kansas, 66103, US4 ‘Presentaddress:TheEppleyInstitute,University OfNebraska, 42nd and DeweyAvenue, Omaha, Nebraska681986805 dTo whom correspondence should be dressed. Paper accepted17.1.1991
SUMMARY In this report, we describe the generation of specific antibodies to rat alkaline phosphatase and the temporal and regional characteristicsof alkaline phosphatase expression during maturation of the rat chorioallantoicplacenta. An antipeptide antiserum was generated to the amino terminal 15 amino acids of rat alkaline phosphatase. The antiserum specificallyrecognized alkaline phosphatase.Alkaline phosphatase expression was monitored in the junctional and labyrinth zones of the chorioallantoicplacenta by Western and Northern blot analyses. Alkalinephosphataseprotein and mRNA were present in both thejunctional and labyrinth zones on day 13 ofgestation.As gestationadvanced, alkalinephosphatasemI?hU andprotein expressiona!ecreasedbelowthe limitsofdetettionin thejunctional zone, while alkaline phosphatase expression increased in the labyrinth zone. Labyrinthine alkaline phosphatase migrated predominantly as a 95-k.Da species, whereas rat kidney expressed exclusivelythe 75-kDa species.Enzymatic deglycosylationof the 75- and 95-kDa alkalinephosphatase speciesresulted in the generation of a 55kDa species. In summary, alkaline phosphatase expression is a useful indicator of trophoblast d@rentiation.
INTRODUCTION Alkaline phosphatase has been used as a marker of trophoblast cell differentiation in a number of species (Padykula, 1959; Sherman, 1972; Chou, 1982; Soares, 1987; Hunt and Soares, 1988). In the human, the formation of syncytial trophoblast cells coincides with 0143-4004/91/030227
+ 11 $05.00/O
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Placenta(1991), Vol. 12
elevated expression of alkaline phosphatase (Robinson et al, 1988). In the rat, alkaline phosphatase expression also accompanies differentiation and is primarily restricted to a subpopulation of trophoblast cells located in the labyrinth zone of the chorioallantoic placenta (Padykula, 1959; Soares, 1987; Hunt and Soares, 1988). The specificity of alkaline phosphatase expression has been a point of concern, especially when alkaline phosphatase is monitored by measurement of hydrolysis of p-nitrophenyl phosphate at alkaline pH. Humans possess three distinct alkaline phosphatase genes: placental, intestinal, and liver/bone/kidney (for reviews see Goldstein and Harris, 1979; Fishman, 1987; Harris, 1990), and the rat possesses two genes: intestinal and liver/ bone/kidney/placental (Thiede et al, 1988). Rat liver/bone/kidney/placental alkaline phosphatase has been purified and its cDNA cloned and sequenced (Nair et al, 1987; Nair, Majeska and Rodan, 1987; Noda et al, 1987; Theide et al, 1988). The availability of amino acid sequence information for rat alkaline phosphatase permits the development of immunological probes to specific regions of the protein (for a review of the technique, see Walter, 1986). In this study, we were interested in determining whether alkaline phosphatase could be effectively used to monitor rat trophoblast cell differentiation. We describe the generation of specific antibodies to rat alkaline phosphatase and the temporal and regional characteristics of alkaline phosphatase protein and mRNA expression during maturation of the rat chorioallantoic placenta.
MATERIALS
AND METHODS
Reagents
A complementary DNA to rat alkaline phosphatase (Noda et al, 1987) was a generous gift from Drs Masaki Noda and Gideon Rodan of Merck Sharp and Dohme (West Point, PA). Restriction enzymes and polymerases were purchased from New England BioLabs (Beverly, MA). Oligonucleotide random priming DNA labelling kits and molecular weight standards for nucleic acid agarose gels were obtained from Bethesda Research Laboratories (Gaithersburg, MD). Reagents for sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis were purchased from Bio-Rad Chemical Division (Richmond, CA). Radiolabelled nucleotides were obtained from DuPont NEN (Boston, MA). N-Glycanase was obtained from Genzyme (Boston, MA). Unless otherwise noted, all other chemical and reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and tissue isolation
Timed pregnant Holtzman rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). The presence of a copulatory plug or sperm in the vaginal smear was designated as day 0 of pregnancy. Chorioallantoic placentae were removed from uteri on various days during the second half of pregnancy (days 13-19), dissected into junctional and labyrinth zones (Soares, 1987), immediately frozen in liquid nitrogen and stored at -70°C until used for mRNA or immunoblotting analyses. The junctional zone was identified by its pale appearance, due to the absence of fetal blood, and separated from the labyrinth zone, a richly vascularized tissue, with fine forceps and 23-gauge needles (for further information concerning the placental zones, see Soares, 1987; Campbell et al, 1989). In some experiments, kidneys were obtained
Campbd et al: Placental Expression ofAlkaline Phosphatase
from pregnant rats at the time of autopsy, dissected free of mesenteries, described above.
229
frozen and stored as
Generation of antisera to rat alkaline phosphatase Antibodies were generated to an amino-terminal peptide derived from the amino acid sequence of rat alkaline phosphatase (Hsu et al, 1987; Misumi et al, 1988; Nair et al, 1987; Theide et al, 1988). The selected sequence consisted of the 15 amino-terminal amino acids of alkaline phosphatase: Phe-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-Ser-Tyr-Trp-Arg-GlnGln. The peptide was synthesized as a carboxyterminal amide using a Biosearch SAM TWO solid phase peptide synthesizer (Biosearch, San Rafael, CA) with standard t-butoxycarbonyl methodology (Marglin and Merrifield, 1970). A cysteine residue was added to the amino terminus of the peptide to facilitate coupling to the carrier protein. The synthesized peptide was purified by reverse phase high performance liquid chromatography, and the amino acid composition of the purified peptide was determined. These procedures have been previously described by our laboratory (Deb et al, 1989a,b). The amino terminal alkaline phosphatase peptide was coupled to keyhole limpet hemocyanin (KLH, Calbiochem, San Diego, CA) through the amino-terminal cysteine residue by using m-maleimidobenzoyl-Nhydroxysuccinimide ester (Calbiochem) as described previously (Lerner et al, 1981; Deb et al, 1989a,b). Three adult New Zealand White rabbits obtained from White Hare Rabbitry (Stark City, MO) were immunized with the peptide-KLH preparation. The immunization and bleeding protocols were similar to that previously described for the generation of antipeptide antisera to rat placental lactogen-II peptides (Deb et al, 1989b). Antipeptide antisera were characterized by enzyme-linked immunoassay and Western blot analyses. These techniques have been previously described (Soares et al, 1988; Deb et al, 1989a,b). Enzymatic deglycosylation N-linked sugars were enzymatically removed from protein preparations via treatment with N-Glycanase (consists of a mixture of N-glycosidase F and aspartylglycosylamine aminohydrolase). Samples were exposed to N-Glycanase (final concentration: lO,&ml) in a reaction mixture containing a final concentration of 0.17 per cent SDS (Bio-Rad), 0.2 >* sodium Phosphate, pH 8.6, and 1.25 per cent Nonidet P-40 in a total volume of 3Opl at 37°C for 1620 h. The samples were then subjected to Western blot analysis (Soares et al, 1988; Deb et al, 1989b). Expression of alkaline phosphatase: estimation of protein and mRNA Relative concentrations of alkaline phosphatase protein and mRNA in the junctional and labyrinth zones of the chorioallantoic placenta during the second half of gestation were estimated by Western blot and Northern blot analyses, respectively. These techniques have been previously described (Soares et al, 1987, 1988- Campbell et al, 1989; Deb et al, 1989a,b). Relative changes in the expression of alkaline phosphatase protein and mRNA were estimated by reflectance and transmission densitometry, respectively (model 620 Video Densitometer, Bio-Rad). Alkaline phosphatase protein was extracted according to the procedure of Nair et al (1987a,b). Briefly, tissues were homogenized in Buffer No. l(10 mM tris-HCl, pH 7.2, containing 0.5 rnkt MgClz and 0.5 mu phenyl methyl sulfonyl fluoride, 0.02 per cent sodium azide; 5 ml/g tissue) for 15-30 set at 4°C. Homogenates were centrifuged at 30 000 g for 1 h. Pellets were resuspended in Buffer No. 1 (5 ml/g) and n-butanol(40% v/v) was added to the suspensions and gently stirred in the cold for 4-6 h. Butanol extracts were then centrifuged at
Plamzta (1991), Vol. I2
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10 000 g for 20 min and the aqueous layers were recovered. Cold acetone was added to the aqueous solution (60 per cent v/v), the mixture stirred in the cold for 4-6 h, and precipitates containing alkaline phosphatase retrieved by centrifugation (30 000 g for 20 min). Precipitates were solubilized in Buffer No. II (10 mu tris-HCl, pH 8.0,O.S mu MgClz, 0.2 per cent Triton X- 100) and protein concentrations determined by the method of Bradford (1976). A rat cDNA clone to alkaline phosphatase was used for hybridization (Noda et al, 1987). Alkaline phosphatase cDNA inserts were utilized as templates for the synthesis of 32Plabelled cDNA probes with an oligonucleotide random primer extension labelling kit.
RESULTS Characterization of rat alkaline phosphatase antipeptide antisera Significant antibody titers were detected to the alkaline phosphatase synthetic peptide within five weeks of the initial immunization in each of the three immunized rabbits (Figure 1). Antibody titers to the peptide showed minimal changes following subsequent booster injections. Western blot analyses of extracts from day 16 placental labyrinth zone indicated that serum collected from rabbit No. 28 showed the best reactivity with placental alkaline phosphatase. All subsequent experiments utilized antiserum collected from rabbit No. 28. Figure 2 shows the specificity of the alkaline phosphatase antipeptide antiserum with rat placental alkaline phosphatase. Substitution of preimmune serum of alkaline phosphatase antipeptide antiserum saturated with alkaline phosphatase amino-terminal peptide (10 ,ug/ml) completely eliminated the reactivity. Gestational pattern of placental alkaline phosphatase protein and mRNA expression Immunoreactive alkaline phosphatase expression was restricted to the labyrinth zone of the chorioallantoic placenta with an apparent molecular weight of 95 kDa when separated in 10 per cent polyacrylamide gels (Figure 3). Please note that the 95kDa species can be resolved
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Campbrli et al: Placental Eqression
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Figure 2. Examination of the specificity of the Western blot analysis for rat placental alkaline phosphatase. Enriched preparations ofrat alkaline phosphatase extracted from the labyrinth zone ofthe day 16 chorioallantoic placenta were separated by sodium dodecyl sulfate (SDS) gel electrophoresis in 7.5 per cent gels under reducing conditions and electrophoretically transferred to nitrocellulose. Nitrocellulose membranes were probed with preimmune serum (lane A), rat alkaline phosphatase antipeptide antiserum (rabbit No. 28; lane B), or with rat alkaline phosphatase antipeptide antiserum saturated with the amino terminal rat alkaline phosphatase peptide (10 &ml; lane C). The immune and preimmune sera were used at a dilution of 1500. The migration of molecular weight standards (x1O-3) is shown.
into at least two different species when separated in 7.5 per cent polyacrylamide gels (Figure 2). Northern blot analysis essentially resembled the Western blot analysis with one important difference (Figure 4). Alkaline phosphatase mRNA was detectable in both junctional and labyrinth zones on day 13 of gestation. In the junctional zone, alkaline phosphatase mRNA concentrations decreased below the limit of detection on days 16 and 19 of gestation, while in the labyrinth zone the relative concentration of alkaline phosphatase increased from days 1316 of gestation (Figure 4). Relative alkaline phosphatase mRNA concentrations decreased in the labyrinth zone from days 16-l 9 of gestation (Figure 4). Densitometric measurements of relative alkaline phosphatase protein and mRNA expression are shown in Figure 5. The expression pattern observed for alkaline phosphatase mRNA was not associated with compromised integrity of the labyrinth and junctional zone RNA samples. These same RNA samples have been analysed for the expression of two other placental mRNAs, prolactin-like protein-A (PLP-A) and placental lactogen-II (PL-II: Campbell et al, 1989). Unlike alkaline phosphatase, PLP-A mRNA is only expressed in the junctional zone, whereas, PL-II shows a temporally specific pattern of expression in both zones (Campbell et al, 1989). The presence of alkaline phosphatase mRNA in the junctional zone on day 13 of gestation prompted further analysis of alkaline phosphatase protein expression in the junctional zone
232
Placenta (1991), Vol. I2 Alk-Phos
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Figure 3. Representative Western blot probed with antipeptide antiserum specific to rat alkalime phosphatase. Extracts were isolated from the junctional and labyrinth zones of the chorioallantoic placenta on days 13, 16 and 19 of pregnancy. Equivalent amounts of protein were separated by SDS electrophoresis in 10 per cent polyacrylamide gels under reducing conditions and transferred to nitrocellulose. Nitrocellulose membranes were probed with antipeptide antiserum to rat alkaline phosphatase (Rabbit No. 28) at a dilution of 1500. Note that immunoreactive alkaline phosphatase was localized predominantly to the labyrinth zone and had an apparent molecular weight of 95 kDa. Lane A, day 13 junctional zone 0); lane B, day 13 labyrinth zone (L); lane C, day 16 J; Lane D, day 16 L; lane E, day 19 J; lane F, day 19 L. Molecular weight standards (X 10m3) are shown.
on day 13. Alkaline phosphatase expression was examined from junctional zone isolated on day 13 of gestation, labyrinth zone from day 16, and kidney tissue under control conditions and following enzymatic deglycosylation (Figure 6). In these experiments, the alkaline phosphatase antipeptide antiserum recognized alkaline phosphatase species from day 13 placental junctional zone and kidney (Figure 6). Under control conditions immunoreactive alkaline phosphatase from the placental labyrinth zone migrated predominantly as a 95-kDa species, while alkaline phosphatase from the kidney migrated as a 7.5 kDa species. Alkaline phosphatase from the day 13 junctional zone was difficult to detect under control conditions but consisted of a mixture of both 75 and 9.5 kDa species. Treannent with N-Glycanase (Genzyme) decreased the apparent molecular sizes of all immunoreactive species from each of the tissues to 55 kDa (Figure 6). Detection of day 13 junctional zone immunoreactive alkaline phosphatase was improved following enzymatic deglycosylation (Figure 6).
DISCUSSION In this report, we have described the generation of an anitpeptide antiserum specific to rat alkaline phosphatase, the temporal and regional pattern of alkaline phosphatase protein and
233
Campbell et al: Placental Expression ofAlkaline Phosphatase Alk-Phor
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Northern blot hybridized with an alkaline phosphatase cDNA probe. Total RNA was isolated from the junctional and labyrinth zones of the chorioallantoic placenta on days 13,16 and 19 of pregnancy. The RNA (10 /&lane) was fractionated by formaldehyde agarose gel electrophoresis and transferred to nitrocellulose prior to hybridization. Single mRNA species of alkaline phosphatase were detected in the appropriate size range of 2.5 kb. Note that day 13 junctional zone contains detectable alkaline phosphatase mRNA, whereas, alkaline phosphatase mRNA levels are below the liiits of detection in the junctional zone on days 16 and 19 of gestation. Lane A, day 13 junctional zone (J); lane B, day 13 labyrinth zone (L); lane C, day 16 J; lane D, day 16 L; lane E, day 19 J; lane F, day 19 L. Molecular weight standards are shown.
mRNA expression in the developing rat chorioallantoic placenta, and some biochemical characteristics of alkaline phosphatase isolated from the chorioallantoic placenta. During the second half of gestation, alkaline phosphatase protein and mRNA are predominantly expressed in labyrinthine trophoblast cells of the chorioallantoic placenta (present study). This observation is consistent with previous reports on the distribution of alkaline phosphatase enzymatic activity within the rat placenta (Padykula, 1959; Soares, 1987) and immunohistochemical analysis of alkaline phosphatase within the rat placenta (Hunt and Soares, 1988). Alkaline phosphatase activity has recently been localized by electron microscopy to the maternal facing membrane of syncytiotrophoblast layer II of the labyrinth zone (Glazier, Jones and Sibley, 1990). Labyrinthine alkaline phosphatase has a higher apparent molecular weight than alkaline phosphatases isolated from other tissues (95 vs. 75 kDa). Mid-gestation junctional zone trophoblast tissue expresses low levels of alkaline phosphatase mRNA and low levels of the 75- and 95-kDa alkaline phosphatase forms. These two forms of alkaline phosphatase arise from differential glycosylation patterns in labyrinthine trophoblast tissue versus other tissues (present study). Removal of N-linked carbohydrates from alkaline phosphatases of kidney and labyrinthine tissue yield immunoreactive proteins of similar size (non-glycosylated precursors). Labyrinthine and junctional zone trophoblast cells diffentiate from a common precursor cell population (Jollie, 1964; Davies and Glasser, 1968). Trophoblast cell differentiation in
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Placenta (1991), Vol. 12
the rat chorioallantoic placenta is a complex process and is associated with three specific phenomena related to alkaline phosphatase expression: (1) a termination of alkaline phosphatase mRNA and protein expression in the junctional zone; (2) an increase in the expression of alkaline phosphatase in the labyrinth zone; and (3) a change in posttranslational processing of labyrinthine alkaline phosphatase protein, shifting its apparent molecular weight to 95 kDa. Expression of the 95-kDa alkaline phosphatase appears to be linked to the differentiation of the labyrinth zone of the chorioallantoic placenta and thus, may be related to the structural and functional organization of the labyrinth zone. Unlike junctional zone trophoblast tissue, labyrinthine trophoblast cells exhibit polarity; they rest on a basement membrane overlying fetal mesenchyme (Davies and Glasser, 1968). Labyrinth trophoblast cells are involved in transport of nutrients and wastes between maternal and fetal environments (Miller, Koszalka and Brent, 1976). Alkaline phosphatases are plasma membrane associated proteins and are hypothesized to be involved in the active transport of metabolites between maternal and fetal environments (Harris, 1990). The effect of the glycosylation pattern of labyrinthine trophoblast alkaline phosphatase on its activities is not known but may represent important
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235
Campbell et al: Placental Expression ofAlkaline Phosphatase
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products following enzymatic deglycosylation. Tissue Figwe 6. Analysis of alkaline phosphatase immunoreactive extracts were treated with N-glycanase overnight at 37X, separated by SDS electrophoresis in 10 per cent polyacrylamide gels under reducing conditions, transferred to nitrocellulose, and subjected to Western blot analysis. Lanes A and D, day 13 junctional zone (J); lanes B and E, day 16 labyrinth zone (L); lanes C and F, kidney. Lanes A-C contain control preparations and lanes D-F contain the same preparations treated with N-glycanase. Note that the apparent molecular weight of each of the N-glycanase treated samples was similar. Rat alkaline phosphatase antipeptide antiserum (rabbit No. 28) was used at a dilution of 1:.500. Molecular weight standards (X 10m3) are shown.
modifications necessary for the specialized actions of placental alkaline phosphatase in transport between maternal and fetal compartments. In conclusion, regionally and temporally specific inhibitory and stimulatory events are involved in the regulation of alkaline phosphatase expression during trophoblast differentiation. Labyrinthine trophoblast tissue specifically expresses a 95-kDa glycosylated alkaline phosphatase isoform. The specific factors responsible for the regulation of trophoblast cell alkaline phosphatase expression, post-translational processing of labyrinthine alkaline phosphatase, or the specific biological roles served by this protein during development remain to be determined. ACKNOWLEDGEMENTS We thank Drs Masaki Noda and Gideon Rodan for generously supplying the rat alkaline phosphatase cDNA and Dr Allen Rawitch and Mr Richard Grabbe for assistance in the synthesis of the amino-terminal rat alkaline phosphatase peptide. We also acknowledge Mr Michael McMaster for assistance in early stages ofthii project and Linda Carr for help in the preparation of the manuscript. We also express appreciation to Dr Glen .i\ndrews, Teresa Faria, and Carla Green for advice and assistance with the mRNA analysis. This study was supported by grants from the National Institute of Child Health and Human Development (HD 22208, HD 20676). W.J.C. and S.D. are recipients of a Wesley Foundation postdoctoral fellowship.
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Placenta (1991), Vol. 12 REFERENCES
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of microgram
quantities
of protein utilizing the
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