0013-7227/91/1293-1257103.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 3 Printed in U.S.A.
Purification and Characterization of Mouse Decidual Calcyclin: A Novel Stimulator of Mouse Placental Lactogen-II Secretion* GUDMUNDUR THORDARSON, JONATHAN N. SOUTHARD, AND FRANK TALAMANTESt Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064
Western blots of various mouse tissue extracts and mouse serum from different physiological stages showed that the concentration of calcyclin was highest in decidual tissue. Detectable levels were found in extracts from trophoblast, lung, and stomach, but the concentrations in these tissues were about 100 times lower than in decidua. Decidual calcyclin was not detectable in mouse serum. Cultured decidual cells released calcyclin into the medium. On average, this release was about 7.8 ng//xg DNA • 24 h. The rate of release did not change significantly during 4 days of culture. The ratio of calcyclin in cells per calcyclin released during 24 h averaged 2.3 and did not change significantly during the culture period. The purified decidual calcyclin stimulated the release of mPL-II from cultured trophoblasts in a dosedependent manner at concentrations from 0.01 to 1 iig/mX. The maximum stimulation averaged about 1.5 times above control. It is concluded that decidual calcyclin may be of physiological importance for the regulation of mPL-II secretion. {Endocrinology 129: 1257-1265, 1991)
ABSTRACT. The effects of secretagogue(s) from mouse decidual tissue on the release of mouse placental lactogen-II (mPLII) were studied. Decidual tissue was obtained from 10- and 11day-pregnant mice. The tissue was homogenized, extracted, and the tissue extract was made 50% saturated with ammonium sulfate. Both the precipitate and supernatant were tested for their ability to stimulate mPL-II release from cultured trophoblasts. The supernatant contained an activity to stimulate the release of mPL-II. This activity was further purified using column chromatography. The purification resulted in isolation of a protein with a mol wt of 20 K as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and 6 K under reducing conditions. Further characterization of this protein showed that it binds calcium and has an amino acid sequence that is highly homologous with calcyclin expressed in mouse embryonic fibroblast cells and with calcyclin from other species. This protein was designated mouse decidual calcyclin. Antiserum was raised against the purified decidual calcyclin for development of an RIA and for immunoblots.
T
HE PLACENTA is an endocrine organ that produces a number of protein and steroid hormones (1, 2). Although peptide hormones from the placenta of various species have been investigated for the last two to three decades, relatively little is known about how the synthesis and secretion of these hormones are controlled. Evidence suggests that the pituitary and the ovaries of the mother are involved in regulating the secretion of placental lactogen II (PL-II) in rats and mice. Hypophysectomy (3,4) and ovariectomy (3,5,6) have been shown to enhance the circulating concentration of PL-II in these species. However, it is not known whether pituitary and ovarian hormones act directly on the placenta to influence the secretion of PL-II or if the effects are secondary, possibly through metabolic changes. For ex-
Received March 14,1991. * Presented in part at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, June 1990. This study was supported by NIH Grant HD-14966. t To whom all correspondence and requests for reprints should be addressed.
ample, it is known that the nutritional state of the mother affects the concentration of PLs in serum in some species (7, 8). It has long been speculated that the secretion of placental hormones may be regulated by local factors in an autocrine or paracrine manner and there is some evidence to support the notion that placental hormones are regulated in this manner. Handwerger and his co-workers have reported that the secretion of decidual PRL is both stimulated (9) and inhibited (10) by substances that are found in the placenta. GHRH and GnRH are produced in the placenta of some species (1116) and these peptides may affect the secretion of placental hormones (17-19). Several years ago, we observed that a coculture of trophoblast with decidual tissue enhanced the release of mPL-II (our unpublished data). In this study, we report the structural and functional characteristics of a protein from the mouse decidua that stimulates mPL-II release from cultured trophoblast cells. Partial amino acid sequence analysis of this protein revealed high sequence homology with human calcyclin (20), rat PRL receptor-associated protein (rPRA, 21),
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and mouse calcyclin (22). Therefore, we have adopted the name mouse decidual calcyclin for this protein. Materials and Methods Materials and animals Timed-pregnant Swiss Webster mice were purchased from Simonsen Laboratories (Gilroy, CA). The day a vaginal plug was found was designated day 0 of pregnancy. Mouse PL-II was purified as described previously (23). Carrier-free sodium 125 I was from Amersham (Arlington Heights, IL). Type CLS I Collagenase was from Worthington Biochemical Co. (Malvern, PA). Sephadex G-100, Percoll, and density marker beads were from Pharmacia Fine Chemicals (Uppsala, Sweden). Goat antirabbit immunoglobulin G (IgG) antiserum and normal rabbit serum were purchased from Antibodies Inc. (Davis, CA). Goat antirabbit IgG conjugated to alkaline phosphatase, 5-bromo-4chloro-3-indolyl phosphate, nitro blue tetrazolium, and molecular weight protein markers were obtained from Bio-Rad Laboratories (Richmond, CA). Medium NCTC-109 was purchased from GIBCO (Grand Island, NY), and medium 199, calf thymus DNA, used as standards in the DNA assay, BSA, phenylmethylsulfonyl fluoride (PMSF), Phenyl-Sepharose, type I deoxyribonuclease, and gentamicin sulfate were from Sigma Chemical Co. (St. Louis, MO). Iodo-gen, bicinchoninic acid (BCA) protein assay reagents, Coomassie protein assay reagents, and Iminodiacetic acid TSK HW-65F gel were obtained from Pierce Chemical Co. (Rockford, IL), and TSK-diethylaminoethyl 650S (DEAE) was purchased from Supelco, Inc. (Bellefonte, PA). Endoproteinase Glu-C (V8) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Purification of decidual calcyclin Trophoblast cell cultures were used to monitor mPL-IIstimulating activity in the initial purification of calcyclin from mouse decidual tissue. This procedure yielded a preparation of pure protein of apparent mol wt of about 20 K by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under nonreducing conditions and approximately 6 K when run under reducing conditions (data not shown). Because of the difficulty in using the bioassay for monitoring the activity of column fractions both in terms of the time required and also because of poor quantitative assessment of the assay, the purified protein was used to raise an antiserum and an RIA for the protein was developed. The RIA was then used to monitor activity in the purification procedure described below. Placental tissue was collected on dry ice from 10- and 11day-pregnant Swiss Webster mice. The tissue was kept frozen at -80 C until used. For purification, the tissue was thawed and then homogenized, using a Polytron homogenizer, in 4 volumes of extraction buffer (100 mM NH4HCO3-NH4OH, 10 mM EDTA, 10 mM EGTA, 25 mg/liter phenylmethylsulfonyl fluoride, pH 9.3). The homogenate was extracted overnight at 4 C and the pH was adjusted to 8.3 with 50% acetic acid before centrifugation at 27,000 X g for 1 h. The supernatant was made 50% saturated with (NH4)2SO4, stirred for 1 h, and centrifuged as before. The supernatant was applied to a Phenyl-Sepharose column (2.5 x 18.5 cm) equilibrated with 25 mM sodium phos-
Endo • 1991 Vol 129 • No 3
phate, 1.0 M (NH4)2SO4, pH 6.8. The column was washed with the equilibration buffer and then eluted with a 600-ml linear gradient from equilibration buffer to H2O. The column was washed with H2O and then with 5 mM glycine, 55% ethylene glycol, pH 9.0. Decidual calcyclin eluted in the H2O wash. Active fractions were pooled, concentrated from 120 to 50 ml on an Amicon YM-10 membrane, and dialyzed against 6 1 of 17 mM Tris-HCl, pH 8.9, for 48 h. The dialyzed sample was applied to a diethylaminoethyl column (1.5 X 14 cm) equilibrated with 25 mM Tris-HCl, pH 8.0. The column was washed with equilibration buffer and eluted with two linear gradients: 1) 400 ml of 0-100 mM NaCl in equilibration buffer and 2) 400 ml of 1001000 mM NaCl in the same buffer. Decidual calcyclin eluted in a broad peak from 40 mM to 100 mM NaCl. Active fractions were pooled and loaded onto a 1 X 11 cm metal chelating column (Iminodiacetic acid TSK HW-65F saturated with Cu++) equilibrated with 25 mM Tris-HCl, 0.5 M NaCl, pH 8.0. The column was eluted with a 200-ml gradient of 0-50 mM imidazole in equilibration buffer and then washed with 100 mM EDTA, pH 8.0. Most of the applied protein bound to the column and was eluted during the gradient. More than 90% of the decidual calcyclin was not bound and eluted prior to the start of the gradient. These active fractions were pooled, and (NH4)2SO4 was added to a final concentration of 0.1 M, pH 6.8, and the pool was applied to a second Phenyl-Sepharose column (1.5 X 20 cm) equilibrated with 25 mM sodium phosphate, 0.1 M (NH4)2SO4, pH 6.8. The column was eluted with a 400-ml gradient from equilibration buffer to 25 mM sodium phosphate, 10% ethylene glycol, pH 6.8, and then washed with 5 mM glycine, 50% ethylene glycol, pH 9.0. Decidual calcyclin was detected in fractions throughout most of the gradient with the majority of the activity eluting from 2-6% ethylene glycol. Active fractions were pooled, concentrated on an Amicon YM10 membrane and loaded onto a Sephadex G-50 column (2.5 X 92 cm) equilibrated with 50 mM NH4HCO3, pH 8.3. Decidual calcyclin was eluted as a single peak with an elution volume to void volume ratio 1.8. Peak fractions were pooled, concentrated, and stored at -80 C. Antiserum production Antiserum against purified decidual calcyclin was generated in a New Zealand white rabbit. The antigen, 50-250 n% at each time, was diluted in 50 mM ammonium bicarbonate buffer, pH 8.3, and mixed with Freund's complete adjuvant for the first three injections and with incomplete adjuvant in subsequent injections. The rabbit was injected sc in the neck and shoulder region at 1- to 3-week intervals. Blood samples were collected 8-10 days after the second and fourth injection to test the titer of the antiserum and then 10 days after the fifth and last injection. Electrophoresis The molecular weight of decidual calcyclin was estimated by SDS-PAGE in the presence and absence of dithiothreitol according to the method of Giulian et al. (24). The mobility of decidual calcyclin by PAGE in the presence and absence of Ca++ was assessed by a modification of the method of Kuznicki et al. (25). Briefly, samples were stacked in 5.9% acrylamide gel with 0.5 M Tris/HCl buffer without
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EFFECT OF CALCYCLIN ON mPL-II SECRETION urea, pH 6.8. The running gel consisted of 8% acrylamide, 6 M urea with 80 mM glycine, 20 mM Tris buffer, pH 8.3. Protein samples were treated either with 0.1 mM CaCl2 or 1.0 mM EGTA and electrophoresed in 25 mM Tris, 190 mM glycine, with the anode buffer containing 0.1 M sodium acetate (26). The gels were run at constant voltage (40 V/0.75 mm gel) overnight and stained with Coomassie blue. Enzymatic digestion and amino acid sequencing Purified decidual calcyclin was enzymatically digested with Endoproteinase Glu-C. This enzyme specifically disrupts peptide bonds at the C-terminus of glutamic acid. One hundred micrograms of protein were incubated with 12 ng of Endoproteinase Glu-C in 100 nl ammonium bicarbonate buffer containing 0.01% SDS, pH 7.8. The reaction was left overnight at room temperature. Peptides were separated on a reverse-phase C-4 Aquapore RP-4 column (HPLC). The column was equilibrated with 0.1% trifluoroacetic acid in water and eluted with a linear gradient between equilibration solvent and acetonitrile containing 0.1% trifluoroacetic acid. Absorbance was monitored at 220 nm. Individual peptide peaks were collected and sequenced. Three different amino acid sequencers were used: Applied Biosystems model 470A and model 477A and Porton Instruments model PI 2020/2090. Immunoblotting Various mouse tissues were homogenized in 100 mM NH4HCO3-NH4OH, 10 mM EDTA, 10 mM EGTA, 25 mg/liter PMSF, pH 9.3. The homogenate was centrifuged at 30,000 x g for 30 min. The supernatant was collected and the protein concentration determined by the bicinchoninic acid (BCA) protein assay. All samples were diluted to contain the same concentration of total protein. Tissue samples and mouse serum from various physiological stages were electrophoresed on 15% SDS-PAGE in the presence of 10% 2-mercaptoethanol (27). The protein was transferred to an Immobilon-P membrane as described by Towbin et al. (28). The membranes were immunostained as described previously (29) using a 1:1000 dilution of antiserum to mouse decidual calcyclin. RIAs The concentration of mPL-II in medium collected from cultured mouse trophoblasts was measured by RIA as described previously (30). To measure the concentration of the decidual calcyclin, a double-antibody RIA was developed. The purified decidual calcyclin was iodinated by the iodogen method (31). Radioiodinated decidual calcyclin, approximately 10,000 cpm, and samples or standards were diluted in RIA buffer (25 mM Na2HPO4, 150 mM NaCl, 10 mM EDTA, 0.1% BSA, 0.01% Thimerosal, pH 7.5). The primary antiserum was diluted to 1:6,000 in RIA buffer containing 3% normal rabbit serum. Each reagent was added to the assay tubes in 100 n\ aliquots. The assay was incubated overnight at room temperature. The next morning, 100-/il aliquots of second antibody (goat antirabbit IgG antiserum, diluted 1:16) were added. After a 1-h incubation, 100 /xl 30% polyethylene glycol (mol wt 8 K) were added, the tubes were centrifuged for 20 min at 9,400 x g and the supernatant
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aspirated. Nonspecific binding in the RIA was determined in tubes where the primary antiserum was replaced with 3% normal rabbit serum diluted in RIA buffer. Cell cultures Placental tissue was obtained from 11-day-pregnant mice for both the trophoblast and decidual cell cultures. The trophoblast and the decidual part of the placenta were separated under a dissecting microscope (Olympus, model SZ-Tr, Olympus Optical Co., Tokyo, Japan) and each tissue treated separately. The trophoblasts were cultured as described previously by our laboratory (32). Briefly, the tissue was minced with a razor blade and dissociated in 0.1% collagenase and 0.002% deoxyribonuclease. The dissociated cells were filtered through a 150 Mm Nitex mesh and separated on a 40% Percoll density gradient. The trophoblast cells were retrieved from the gradient, washed, and plated onto 24-well plates on top of rat tail collagen. The cells were cultured for 4 days in medium NCTC-109 without supplements. Medium was changed daily. After 3-4 days in culture, the cells were treated either with different column fractions or with purified decidual calcyclin in NCTC-109 medium or F12/Dulbecco's modified Eagle's medium (1:1, vol/vol) containing 0.018 mM calcium. Control wells received vehicle in equal amount as the treated wells. Each treatment lasted 3060 min. At the end of treatment, the medium was collected and kept at -20 C until assayed for mPL-II. Cells were harvested for assessment of DNA (33) content. The decidual tissue was dissociated in an identical enzyme mixture and for a similar period of time as the trophoblast. The dissociated cells were also fractionated on a 40% Percoll gradient. The Percoll gradient yielded several bands of cells. The top band, at a density of 1.022 g/ml, contained mostly dead cells and debris. The two bands close to the bottom of the gradient, at a density of 1.095-1.134 g/ml, contained mainly red blood cells and nuclei. Two to three layers of cells banded between the top and bottom layers at a density from 1.0401.057 g/ml. All these cells were harvested, combined, and cultured. The viability of the combined population of cells was about 90% as determined by trypan blue exclusion and the yield of viable decidual cells was 2.2 x 107 cells/g tissue. The decidual cells were plated on rat tail collagen in 24-well plates in medium NCTC-109 without supplements. About 106 cells were plated in each well. Medium was collected daily for 4 days and stored at -20 C until it was assayed for mouse decidual calcyclin by RIA and total protein (34). At the end of culture, the cells were harvested for assessment of DNA (33) and decidual calcyclin content. Statistics One way analysis of variance was used to determine if total DNA, total protein, decidual calcyclin concentration, and the ratio of calcyclin in cells/calcyclin in medium differed significantly between days of culture. One way analysis of variance was also used to assess the effect of decidual calcyclin on mPLII secretion. Parallelism in the RIA for decidual calcyclin was tested by linear regression analysis.
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Results
Endo • 1991 Vol 129 • No 3
Tissue Extract {\xg protein/ml)
Purification of mouse decidual calcyclin Purified decidual calcyclin migrated as a single band on SDS-PAGE under both nonreducing and reducing conditions (Fig. 1). The apparent molecular weight of decidual calcyclin was 20 K under nonreducing conditions and 6 K after reduction of disulfide bonds. The purification procedure yielded approximately 25 ng purified decidual calcyclin per gram of wet tissue.
1
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o m
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RIA for mouse decidual calcyclin The standard curve and validation of the RIA for mouse decidual calcyclin are shown in Fig. 2. The primary antiserum (rabbit antimouse decidual calcyclin) was diluted to 1:6000. This dilution gave from 40-50% total binding and about 3-5% nonspecific binding. The sensitivity of the assay, estimated as the concentration of standard that differed from multiple determination of zero by 10%, was about 10 ng/ml. The within assay variation was 9.6%. All samples that were directly compared were measured in the same assay. To assess the specificity of the assay, extracts of various mouse tissues
Mr
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21 K 14 .4 K 6 .4 K 2 .5 K
FIG. 1. SDS-PAGE of purified mouse decidual calcyclin on a 17.5% acrylamide gel. Lane a, Decidual calcyclin (0.1 fig) in the absence of dithiothreitol; lane b, decidual calcyclin (0.1 fig) in the presence of dithiothreitol. The gel was stained with silver.
0.1
Decidual Calcyclin (ng/ml)
FIG. 2. Characterization of the RIA for mouse decidual calcyclin. Displacement of 125I-decidual calcyclin from rabbit antimouse decidual calcyclin antiserum with different dilutions of purified mouse decidual calcyclin (•), and with different dilutions of extract from mouse decidual tissue (O). Other mouse tissue extracts had either no (heart A, fetus • , kidney • ) or a small (lung A, trophoblast O, stomach D) effect in displacing 125I-decidual calcyclin from the antiserum.
and mouse serum from different physiological stages were assayed. Only the decidual extract showed displacement of the iodinated decidual calcyclin that was parallel to the purified decidual calcyclin standard. Extracts from lung, stomach, and trophoblast showed some activity but that activity was about 100 times lower than that of the decidual extract. Enzymatic digestion and amino acid sequence analysis of mouse decidual calcyclin An attempt was made to obtain an amino terminal amino acid sequence from the intact protein. No sequence was obtained, indicating a blocked amino terminus. Therefore, an enzymatic digestion was done on the purified protein that yielded several peptides after separation of the digest on reverse-phase HPLC. The following sequences were obtained from these peptides: DGDKHTLSKKE 2) L I Q K 3)LTIGSKLQDA 4)IARLMDDLD?NKDQE 5)VNFQE 6)YVAFLGALALIYNE 7) A L K. Figure 3 shows the alignment of the mouse decidual calcyclin peptides with the cDNA-deduced sequences of several members of the S-100 protein family (20-22). Based on this alignment, the partial sequence of mouse decidual calcyclin has a high degree of sequence identity with human calcyclin (95%), rat prolactin receptor-as-
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EFFECT OF CALCYCLIN ON mPL-II SECRETION
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10 h2A9
FIG. 3. The amino acid sequences of seven peptide fragments (underlined and numbered according to text) of mouse decidual calcyclin (mDCAL) isolated from decidual tissue. The decidual calcyclin peptides have been aligned to match the amino acid sequences deduced from cDNA clones for human calcyclin (h2A9), ratprolactin receptor-associated protein (rPRA), and mouse calcyclin (mSBlO). Differences between individual sequences are in brackets. Amino acids that have not been determined for mDCAL are denoted by ?.
20
rPRA
A C P L D Q A I G L L V A I F H K Y S G K E G D K H T L S K
m5B10
A C P L D Q A I G L L V A I F H K Y S G K E G D K H T L S K
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K E L K E L I Q K E L T I G S K L Q D A E I A R L M[E]D L D
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K E L K E L I Q K E L T I G [ A ] K L Q D A E I A R L M D D L D D[R]E I A R L M D D L D
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K E L K E L I Q K E L T I G S K L Q
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K E ? ? ? L I O K ? L T I G S K L O D A 2
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3
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80 V[T]F L G A L A L I Y N E A L K [ G ]
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R N K D Q E V N F Q E Y V A F L G A L A L I Y N E A L K[G]
m5B10
R N K D Q E V N F Q E Y V A F L G A L A L I Y N E A L K
mDCAL
? N K D O E V N F Q E
Y V A F L G A L A L I Y N E A L K
5
sociated protein (97%), and mouse calcyclin expressed in embryonic fibroblasts (98%). Urea gel electrophoresis
When purified decidual calcyclin was analyzed by PAGE in the presence of 6 M urea, there was a shift in mobility of the protein depending on whether Ca++ was present or absent (Fig. 4). The absence of Ca++ increased the mobility of decidual calcyclin under these conditions. These results are consistent with those of Kuznicki and
a
II
Ca++
30
A C P L D Q A I G L L V A I F H K Y S G [ R ] E G D K H T L S K
EGTA
FIG. 4. Electrophoresis of mouse decidual calcyclin on a 8% acrylamide gel in the presence of 6 M urea. The sample was incubated with either 0.1 mM CaCl2 {lane a) or 1.0 mM EGTA (lane b) before electrophoresis. The amount of protein per lane was 10 jig.
6
7
Filipek (35) who found that Ca++-binding protein isolated from Ehrlich-ascites tumor cells showed a similar shift in electrophoretic mobility. It was subsequently shown that this Ca++-binding protein is probably mouse calcyclin (25). These results further support the notion that this purified decidual Ca++-binding protein is calcyclin. Western blots The immunoblots showed that mouse decidual extract contained a protein in high concentration that corresponded in molecular weight to purified calcyclin (Fig. 5). When the total protein load was increased from 5-40 Hg per lane, a weak immunostaining that corresponded in molecular weight to purified decidual calcyclin was observed in extracts of mouse trophoblast, lung, and stomach (data not shown). No immunostaining was found in serum samples obtained from male mice and nonpregnant female mice or in serum from female mice at different stages of pregnancy. Extracts of mouse skeletal muscle, brain, pituitary, and liver were also negative by immunostaining (Fig. 5). Mouse extracts of whole fetuses, kidney, heart, and spleen and amniotic fluid were also without any detectable levels of decidual calcyclin when estimated by Western blots (data not shown). Stimulation of mPL-II release by mouse decidual calcyclin As shown in Fig. 6, purified decidual calcyclin caused a dose-dependent increase in the release of mPL-II from cultured trophoblast cells. The stimulation was significant at 0.01 /xg decidual calcyclin/ml and was highest at
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EFFECT OF CALCYCLIN ON mPL-II SECRETION
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a b c d e
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Endo'1991 Vol 129 • No 3
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FIG. 5. Western blot of various mouse tissue extracts and serum. The extracts and serum (5 ng total protein/lane) were electrophoresed on 15% SDS-PAGE, transferred to a Immobilon-P membrane and immunostained for decidual calcyclin. a) Decidual extract, 6) skeletal muscle extract, c) brain extract, d) pituitary extract, e) liver extract, /) male serum, g) serum from 17-days-pregnant female, h) serum from 10-days-pregnant female, i) serum from 6-days-pregnant female, ;") purified mouse decidual calcyclin (1 ng).
1 /itg decidual calcyclin/ml. The maximum stimulation of mPL-II release averaged about 1.5 times higher than the control, ranging from 1.4-1.7 times that of control in each culture. These effects are comparable to the maximum stimulation obtained for decidual extract after 50% ammonium sulfate precipitation. Similar stimulation of mPL-II release was obtained by decidual calcyclin whether the culture medium contained low (0.018 mM) or normal (1.8 mM) calcium. The increase in mPL-II release was most prominent after 30-60 min incubation with decidual calcyclin. Prolonged incubation beyond 1 h diminished the effect of decidual calcyclin. Decidual culture The amount of decidual calcyclin that was released into the culture medium did not change significantly during the culture period. The release of total protein was also unchanged during the 4 days of culture, but the DNA content decreased significantly after day 1 (Fig. 7). The amount of decidual calcyclin in the cells was about twice the amount of decidual calcyclin that was released into the medium during 24 h. This ratio, i.e. decidual calcyclin in tissue/decidual calcyclin released during 24 h, was unchanged during the culture period (Table 1).
0 10 100 1000 Decidual Calcyclin (ng/ml) FIG. 6. The effect of purified mouse decidual calcyclin on the secretion of mPL-II from cultured mouse trophoblasts. The cells were cultured for 4 days in medium NCTC-109 before they were treated with different concentrations of mouse decidual calcyclin for 30 min. A significant stimulation of mPL-II release was seen at 0.01 Mg/ml and maximum stimulation was obtained at 1 fig/ml. Each point represents the mean ± SEM of 18 replicates obtained from four different cultures. Asterisks denote significant difference from control (P < 0.05).
Discussion We have purified a protein from mouse decidual tissue that stimulates the release of mPL-II from cultured trophoblast cells. Partial amino acid sequence analysis of this protein showed that it is highly homologous to human calcyclin (20), rat PRA (21), and calcyclin expressed by mouse fibroblasts (22). There was one amino acid difference between the sequence that we obtained for the protein designated mouse decidual calcyclin and the cDNA-deduced sequence of mouse fibroblast calcyclin. This discrepancy may be caused by a misread amino acid, alanine for arginine, in the amino acid analysis. However, our determination of this amino acid is identical to corresponding amino acid residues of human calcyclin (20) and rat PRA (21). Alternatively, there may be more than one form of calcyclin in the mouse. Guo et al. (22) showed that there are probably five copies of the calcyclin gene per haploid mouse genome. Therefore, it is possible that these different calcyclin genes code for slightly different proteins. When the purified protein was run on SDS-PAGE in the presence and absence of reducing agents, there was a shift in the mol wt from
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EFFECT OF CALCYCLIN ON mPL-II SECRETION
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Vol. 129, No. 3 Printed in U.S.A.
Purification and Characterization of Mouse Decidual Calcyclin: A Novel Stimulator of Mouse Placental Lactogen-II Secretion* GUDMUNDUR THORDARSON, JONATHAN N. SOUTHARD, AND FRANK TALAMANTESt Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064
Western blots of various mouse tissue extracts and mouse serum from different physiological stages showed that the concentration of calcyclin was highest in decidual tissue. Detectable levels were found in extracts from trophoblast, lung, and stomach, but the concentrations in these tissues were about 100 times lower than in decidua. Decidual calcyclin was not detectable in mouse serum. Cultured decidual cells released calcyclin into the medium. On average, this release was about 7.8 ng//xg DNA • 24 h. The rate of release did not change significantly during 4 days of culture. The ratio of calcyclin in cells per calcyclin released during 24 h averaged 2.3 and did not change significantly during the culture period. The purified decidual calcyclin stimulated the release of mPL-II from cultured trophoblasts in a dosedependent manner at concentrations from 0.01 to 1 iig/mX. The maximum stimulation averaged about 1.5 times above control. It is concluded that decidual calcyclin may be of physiological importance for the regulation of mPL-II secretion. {Endocrinology 129: 1257-1265, 1991)
ABSTRACT. The effects of secretagogue(s) from mouse decidual tissue on the release of mouse placental lactogen-II (mPLII) were studied. Decidual tissue was obtained from 10- and 11day-pregnant mice. The tissue was homogenized, extracted, and the tissue extract was made 50% saturated with ammonium sulfate. Both the precipitate and supernatant were tested for their ability to stimulate mPL-II release from cultured trophoblasts. The supernatant contained an activity to stimulate the release of mPL-II. This activity was further purified using column chromatography. The purification resulted in isolation of a protein with a mol wt of 20 K as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and 6 K under reducing conditions. Further characterization of this protein showed that it binds calcium and has an amino acid sequence that is highly homologous with calcyclin expressed in mouse embryonic fibroblast cells and with calcyclin from other species. This protein was designated mouse decidual calcyclin. Antiserum was raised against the purified decidual calcyclin for development of an RIA and for immunoblots.
T
HE PLACENTA is an endocrine organ that produces a number of protein and steroid hormones (1, 2). Although peptide hormones from the placenta of various species have been investigated for the last two to three decades, relatively little is known about how the synthesis and secretion of these hormones are controlled. Evidence suggests that the pituitary and the ovaries of the mother are involved in regulating the secretion of placental lactogen II (PL-II) in rats and mice. Hypophysectomy (3,4) and ovariectomy (3,5,6) have been shown to enhance the circulating concentration of PL-II in these species. However, it is not known whether pituitary and ovarian hormones act directly on the placenta to influence the secretion of PL-II or if the effects are secondary, possibly through metabolic changes. For ex-
Received March 14,1991. * Presented in part at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, June 1990. This study was supported by NIH Grant HD-14966. t To whom all correspondence and requests for reprints should be addressed.
ample, it is known that the nutritional state of the mother affects the concentration of PLs in serum in some species (7, 8). It has long been speculated that the secretion of placental hormones may be regulated by local factors in an autocrine or paracrine manner and there is some evidence to support the notion that placental hormones are regulated in this manner. Handwerger and his co-workers have reported that the secretion of decidual PRL is both stimulated (9) and inhibited (10) by substances that are found in the placenta. GHRH and GnRH are produced in the placenta of some species (1116) and these peptides may affect the secretion of placental hormones (17-19). Several years ago, we observed that a coculture of trophoblast with decidual tissue enhanced the release of mPL-II (our unpublished data). In this study, we report the structural and functional characteristics of a protein from the mouse decidua that stimulates mPL-II release from cultured trophoblast cells. Partial amino acid sequence analysis of this protein revealed high sequence homology with human calcyclin (20), rat PRL receptor-associated protein (rPRA, 21),
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and mouse calcyclin (22). Therefore, we have adopted the name mouse decidual calcyclin for this protein. Materials and Methods Materials and animals Timed-pregnant Swiss Webster mice were purchased from Simonsen Laboratories (Gilroy, CA). The day a vaginal plug was found was designated day 0 of pregnancy. Mouse PL-II was purified as described previously (23). Carrier-free sodium 125 I was from Amersham (Arlington Heights, IL). Type CLS I Collagenase was from Worthington Biochemical Co. (Malvern, PA). Sephadex G-100, Percoll, and density marker beads were from Pharmacia Fine Chemicals (Uppsala, Sweden). Goat antirabbit immunoglobulin G (IgG) antiserum and normal rabbit serum were purchased from Antibodies Inc. (Davis, CA). Goat antirabbit IgG conjugated to alkaline phosphatase, 5-bromo-4chloro-3-indolyl phosphate, nitro blue tetrazolium, and molecular weight protein markers were obtained from Bio-Rad Laboratories (Richmond, CA). Medium NCTC-109 was purchased from GIBCO (Grand Island, NY), and medium 199, calf thymus DNA, used as standards in the DNA assay, BSA, phenylmethylsulfonyl fluoride (PMSF), Phenyl-Sepharose, type I deoxyribonuclease, and gentamicin sulfate were from Sigma Chemical Co. (St. Louis, MO). Iodo-gen, bicinchoninic acid (BCA) protein assay reagents, Coomassie protein assay reagents, and Iminodiacetic acid TSK HW-65F gel were obtained from Pierce Chemical Co. (Rockford, IL), and TSK-diethylaminoethyl 650S (DEAE) was purchased from Supelco, Inc. (Bellefonte, PA). Endoproteinase Glu-C (V8) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Purification of decidual calcyclin Trophoblast cell cultures were used to monitor mPL-IIstimulating activity in the initial purification of calcyclin from mouse decidual tissue. This procedure yielded a preparation of pure protein of apparent mol wt of about 20 K by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under nonreducing conditions and approximately 6 K when run under reducing conditions (data not shown). Because of the difficulty in using the bioassay for monitoring the activity of column fractions both in terms of the time required and also because of poor quantitative assessment of the assay, the purified protein was used to raise an antiserum and an RIA for the protein was developed. The RIA was then used to monitor activity in the purification procedure described below. Placental tissue was collected on dry ice from 10- and 11day-pregnant Swiss Webster mice. The tissue was kept frozen at -80 C until used. For purification, the tissue was thawed and then homogenized, using a Polytron homogenizer, in 4 volumes of extraction buffer (100 mM NH4HCO3-NH4OH, 10 mM EDTA, 10 mM EGTA, 25 mg/liter phenylmethylsulfonyl fluoride, pH 9.3). The homogenate was extracted overnight at 4 C and the pH was adjusted to 8.3 with 50% acetic acid before centrifugation at 27,000 X g for 1 h. The supernatant was made 50% saturated with (NH4)2SO4, stirred for 1 h, and centrifuged as before. The supernatant was applied to a Phenyl-Sepharose column (2.5 x 18.5 cm) equilibrated with 25 mM sodium phos-
Endo • 1991 Vol 129 • No 3
phate, 1.0 M (NH4)2SO4, pH 6.8. The column was washed with the equilibration buffer and then eluted with a 600-ml linear gradient from equilibration buffer to H2O. The column was washed with H2O and then with 5 mM glycine, 55% ethylene glycol, pH 9.0. Decidual calcyclin eluted in the H2O wash. Active fractions were pooled, concentrated from 120 to 50 ml on an Amicon YM-10 membrane, and dialyzed against 6 1 of 17 mM Tris-HCl, pH 8.9, for 48 h. The dialyzed sample was applied to a diethylaminoethyl column (1.5 X 14 cm) equilibrated with 25 mM Tris-HCl, pH 8.0. The column was washed with equilibration buffer and eluted with two linear gradients: 1) 400 ml of 0-100 mM NaCl in equilibration buffer and 2) 400 ml of 1001000 mM NaCl in the same buffer. Decidual calcyclin eluted in a broad peak from 40 mM to 100 mM NaCl. Active fractions were pooled and loaded onto a 1 X 11 cm metal chelating column (Iminodiacetic acid TSK HW-65F saturated with Cu++) equilibrated with 25 mM Tris-HCl, 0.5 M NaCl, pH 8.0. The column was eluted with a 200-ml gradient of 0-50 mM imidazole in equilibration buffer and then washed with 100 mM EDTA, pH 8.0. Most of the applied protein bound to the column and was eluted during the gradient. More than 90% of the decidual calcyclin was not bound and eluted prior to the start of the gradient. These active fractions were pooled, and (NH4)2SO4 was added to a final concentration of 0.1 M, pH 6.8, and the pool was applied to a second Phenyl-Sepharose column (1.5 X 20 cm) equilibrated with 25 mM sodium phosphate, 0.1 M (NH4)2SO4, pH 6.8. The column was eluted with a 400-ml gradient from equilibration buffer to 25 mM sodium phosphate, 10% ethylene glycol, pH 6.8, and then washed with 5 mM glycine, 50% ethylene glycol, pH 9.0. Decidual calcyclin was detected in fractions throughout most of the gradient with the majority of the activity eluting from 2-6% ethylene glycol. Active fractions were pooled, concentrated on an Amicon YM10 membrane and loaded onto a Sephadex G-50 column (2.5 X 92 cm) equilibrated with 50 mM NH4HCO3, pH 8.3. Decidual calcyclin was eluted as a single peak with an elution volume to void volume ratio 1.8. Peak fractions were pooled, concentrated, and stored at -80 C. Antiserum production Antiserum against purified decidual calcyclin was generated in a New Zealand white rabbit. The antigen, 50-250 n% at each time, was diluted in 50 mM ammonium bicarbonate buffer, pH 8.3, and mixed with Freund's complete adjuvant for the first three injections and with incomplete adjuvant in subsequent injections. The rabbit was injected sc in the neck and shoulder region at 1- to 3-week intervals. Blood samples were collected 8-10 days after the second and fourth injection to test the titer of the antiserum and then 10 days after the fifth and last injection. Electrophoresis The molecular weight of decidual calcyclin was estimated by SDS-PAGE in the presence and absence of dithiothreitol according to the method of Giulian et al. (24). The mobility of decidual calcyclin by PAGE in the presence and absence of Ca++ was assessed by a modification of the method of Kuznicki et al. (25). Briefly, samples were stacked in 5.9% acrylamide gel with 0.5 M Tris/HCl buffer without
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EFFECT OF CALCYCLIN ON mPL-II SECRETION urea, pH 6.8. The running gel consisted of 8% acrylamide, 6 M urea with 80 mM glycine, 20 mM Tris buffer, pH 8.3. Protein samples were treated either with 0.1 mM CaCl2 or 1.0 mM EGTA and electrophoresed in 25 mM Tris, 190 mM glycine, with the anode buffer containing 0.1 M sodium acetate (26). The gels were run at constant voltage (40 V/0.75 mm gel) overnight and stained with Coomassie blue. Enzymatic digestion and amino acid sequencing Purified decidual calcyclin was enzymatically digested with Endoproteinase Glu-C. This enzyme specifically disrupts peptide bonds at the C-terminus of glutamic acid. One hundred micrograms of protein were incubated with 12 ng of Endoproteinase Glu-C in 100 nl ammonium bicarbonate buffer containing 0.01% SDS, pH 7.8. The reaction was left overnight at room temperature. Peptides were separated on a reverse-phase C-4 Aquapore RP-4 column (HPLC). The column was equilibrated with 0.1% trifluoroacetic acid in water and eluted with a linear gradient between equilibration solvent and acetonitrile containing 0.1% trifluoroacetic acid. Absorbance was monitored at 220 nm. Individual peptide peaks were collected and sequenced. Three different amino acid sequencers were used: Applied Biosystems model 470A and model 477A and Porton Instruments model PI 2020/2090. Immunoblotting Various mouse tissues were homogenized in 100 mM NH4HCO3-NH4OH, 10 mM EDTA, 10 mM EGTA, 25 mg/liter PMSF, pH 9.3. The homogenate was centrifuged at 30,000 x g for 30 min. The supernatant was collected and the protein concentration determined by the bicinchoninic acid (BCA) protein assay. All samples were diluted to contain the same concentration of total protein. Tissue samples and mouse serum from various physiological stages were electrophoresed on 15% SDS-PAGE in the presence of 10% 2-mercaptoethanol (27). The protein was transferred to an Immobilon-P membrane as described by Towbin et al. (28). The membranes were immunostained as described previously (29) using a 1:1000 dilution of antiserum to mouse decidual calcyclin. RIAs The concentration of mPL-II in medium collected from cultured mouse trophoblasts was measured by RIA as described previously (30). To measure the concentration of the decidual calcyclin, a double-antibody RIA was developed. The purified decidual calcyclin was iodinated by the iodogen method (31). Radioiodinated decidual calcyclin, approximately 10,000 cpm, and samples or standards were diluted in RIA buffer (25 mM Na2HPO4, 150 mM NaCl, 10 mM EDTA, 0.1% BSA, 0.01% Thimerosal, pH 7.5). The primary antiserum was diluted to 1:6,000 in RIA buffer containing 3% normal rabbit serum. Each reagent was added to the assay tubes in 100 n\ aliquots. The assay was incubated overnight at room temperature. The next morning, 100-/il aliquots of second antibody (goat antirabbit IgG antiserum, diluted 1:16) were added. After a 1-h incubation, 100 /xl 30% polyethylene glycol (mol wt 8 K) were added, the tubes were centrifuged for 20 min at 9,400 x g and the supernatant
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aspirated. Nonspecific binding in the RIA was determined in tubes where the primary antiserum was replaced with 3% normal rabbit serum diluted in RIA buffer. Cell cultures Placental tissue was obtained from 11-day-pregnant mice for both the trophoblast and decidual cell cultures. The trophoblast and the decidual part of the placenta were separated under a dissecting microscope (Olympus, model SZ-Tr, Olympus Optical Co., Tokyo, Japan) and each tissue treated separately. The trophoblasts were cultured as described previously by our laboratory (32). Briefly, the tissue was minced with a razor blade and dissociated in 0.1% collagenase and 0.002% deoxyribonuclease. The dissociated cells were filtered through a 150 Mm Nitex mesh and separated on a 40% Percoll density gradient. The trophoblast cells were retrieved from the gradient, washed, and plated onto 24-well plates on top of rat tail collagen. The cells were cultured for 4 days in medium NCTC-109 without supplements. Medium was changed daily. After 3-4 days in culture, the cells were treated either with different column fractions or with purified decidual calcyclin in NCTC-109 medium or F12/Dulbecco's modified Eagle's medium (1:1, vol/vol) containing 0.018 mM calcium. Control wells received vehicle in equal amount as the treated wells. Each treatment lasted 3060 min. At the end of treatment, the medium was collected and kept at -20 C until assayed for mPL-II. Cells were harvested for assessment of DNA (33) content. The decidual tissue was dissociated in an identical enzyme mixture and for a similar period of time as the trophoblast. The dissociated cells were also fractionated on a 40% Percoll gradient. The Percoll gradient yielded several bands of cells. The top band, at a density of 1.022 g/ml, contained mostly dead cells and debris. The two bands close to the bottom of the gradient, at a density of 1.095-1.134 g/ml, contained mainly red blood cells and nuclei. Two to three layers of cells banded between the top and bottom layers at a density from 1.0401.057 g/ml. All these cells were harvested, combined, and cultured. The viability of the combined population of cells was about 90% as determined by trypan blue exclusion and the yield of viable decidual cells was 2.2 x 107 cells/g tissue. The decidual cells were plated on rat tail collagen in 24-well plates in medium NCTC-109 without supplements. About 106 cells were plated in each well. Medium was collected daily for 4 days and stored at -20 C until it was assayed for mouse decidual calcyclin by RIA and total protein (34). At the end of culture, the cells were harvested for assessment of DNA (33) and decidual calcyclin content. Statistics One way analysis of variance was used to determine if total DNA, total protein, decidual calcyclin concentration, and the ratio of calcyclin in cells/calcyclin in medium differed significantly between days of culture. One way analysis of variance was also used to assess the effect of decidual calcyclin on mPLII secretion. Parallelism in the RIA for decidual calcyclin was tested by linear regression analysis.
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Results
Endo • 1991 Vol 129 • No 3
Tissue Extract {\xg protein/ml)
Purification of mouse decidual calcyclin Purified decidual calcyclin migrated as a single band on SDS-PAGE under both nonreducing and reducing conditions (Fig. 1). The apparent molecular weight of decidual calcyclin was 20 K under nonreducing conditions and 6 K after reduction of disulfide bonds. The purification procedure yielded approximately 25 ng purified decidual calcyclin per gram of wet tissue.
1
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RIA for mouse decidual calcyclin The standard curve and validation of the RIA for mouse decidual calcyclin are shown in Fig. 2. The primary antiserum (rabbit antimouse decidual calcyclin) was diluted to 1:6000. This dilution gave from 40-50% total binding and about 3-5% nonspecific binding. The sensitivity of the assay, estimated as the concentration of standard that differed from multiple determination of zero by 10%, was about 10 ng/ml. The within assay variation was 9.6%. All samples that were directly compared were measured in the same assay. To assess the specificity of the assay, extracts of various mouse tissues
Mr
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—
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—
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21 K 14 .4 K 6 .4 K 2 .5 K
FIG. 1. SDS-PAGE of purified mouse decidual calcyclin on a 17.5% acrylamide gel. Lane a, Decidual calcyclin (0.1 fig) in the absence of dithiothreitol; lane b, decidual calcyclin (0.1 fig) in the presence of dithiothreitol. The gel was stained with silver.
0.1
Decidual Calcyclin (ng/ml)
FIG. 2. Characterization of the RIA for mouse decidual calcyclin. Displacement of 125I-decidual calcyclin from rabbit antimouse decidual calcyclin antiserum with different dilutions of purified mouse decidual calcyclin (•), and with different dilutions of extract from mouse decidual tissue (O). Other mouse tissue extracts had either no (heart A, fetus • , kidney • ) or a small (lung A, trophoblast O, stomach D) effect in displacing 125I-decidual calcyclin from the antiserum.
and mouse serum from different physiological stages were assayed. Only the decidual extract showed displacement of the iodinated decidual calcyclin that was parallel to the purified decidual calcyclin standard. Extracts from lung, stomach, and trophoblast showed some activity but that activity was about 100 times lower than that of the decidual extract. Enzymatic digestion and amino acid sequence analysis of mouse decidual calcyclin An attempt was made to obtain an amino terminal amino acid sequence from the intact protein. No sequence was obtained, indicating a blocked amino terminus. Therefore, an enzymatic digestion was done on the purified protein that yielded several peptides after separation of the digest on reverse-phase HPLC. The following sequences were obtained from these peptides: DGDKHTLSKKE 2) L I Q K 3)LTIGSKLQDA 4)IARLMDDLD?NKDQE 5)VNFQE 6)YVAFLGALALIYNE 7) A L K. Figure 3 shows the alignment of the mouse decidual calcyclin peptides with the cDNA-deduced sequences of several members of the S-100 protein family (20-22). Based on this alignment, the partial sequence of mouse decidual calcyclin has a high degree of sequence identity with human calcyclin (95%), rat prolactin receptor-as-
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EFFECT OF CALCYCLIN ON mPL-II SECRETION
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10 h2A9
FIG. 3. The amino acid sequences of seven peptide fragments (underlined and numbered according to text) of mouse decidual calcyclin (mDCAL) isolated from decidual tissue. The decidual calcyclin peptides have been aligned to match the amino acid sequences deduced from cDNA clones for human calcyclin (h2A9), ratprolactin receptor-associated protein (rPRA), and mouse calcyclin (mSBlO). Differences between individual sequences are in brackets. Amino acids that have not been determined for mDCAL are denoted by ?.
20
rPRA
A C P L D Q A I G L L V A I F H K Y S G K E G D K H T L S K
m5B10
A C P L D Q A I G L L V A I F H K Y S G K E G D K H T L S K
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? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? G D K H T L S K 1
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K E L K E L I Q K E L T I G [ A ] K L Q D A E I A R L M D D L D D[R]E I A R L M D D L D
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K E L K E L I Q K E L T I G S K L Q
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K E ? ? ? L I O K ? L T I G S K L O D A 2
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3
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80 V[T]F L G A L A L I Y N E A L K [ G ]
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R N K D Q E V N F Q E Y V A F L G A L A L I Y N E A L K[G]
m5B10
R N K D Q E V N F Q E Y V A F L G A L A L I Y N E A L K
mDCAL
? N K D O E V N F Q E
Y V A F L G A L A L I Y N E A L K
5
sociated protein (97%), and mouse calcyclin expressed in embryonic fibroblasts (98%). Urea gel electrophoresis
When purified decidual calcyclin was analyzed by PAGE in the presence of 6 M urea, there was a shift in mobility of the protein depending on whether Ca++ was present or absent (Fig. 4). The absence of Ca++ increased the mobility of decidual calcyclin under these conditions. These results are consistent with those of Kuznicki and
a
II
Ca++
30
A C P L D Q A I G L L V A I F H K Y S G [ R ] E G D K H T L S K
EGTA
FIG. 4. Electrophoresis of mouse decidual calcyclin on a 8% acrylamide gel in the presence of 6 M urea. The sample was incubated with either 0.1 mM CaCl2 {lane a) or 1.0 mM EGTA (lane b) before electrophoresis. The amount of protein per lane was 10 jig.
6
7
Filipek (35) who found that Ca++-binding protein isolated from Ehrlich-ascites tumor cells showed a similar shift in electrophoretic mobility. It was subsequently shown that this Ca++-binding protein is probably mouse calcyclin (25). These results further support the notion that this purified decidual Ca++-binding protein is calcyclin. Western blots The immunoblots showed that mouse decidual extract contained a protein in high concentration that corresponded in molecular weight to purified calcyclin (Fig. 5). When the total protein load was increased from 5-40 Hg per lane, a weak immunostaining that corresponded in molecular weight to purified decidual calcyclin was observed in extracts of mouse trophoblast, lung, and stomach (data not shown). No immunostaining was found in serum samples obtained from male mice and nonpregnant female mice or in serum from female mice at different stages of pregnancy. Extracts of mouse skeletal muscle, brain, pituitary, and liver were also negative by immunostaining (Fig. 5). Mouse extracts of whole fetuses, kidney, heart, and spleen and amniotic fluid were also without any detectable levels of decidual calcyclin when estimated by Western blots (data not shown). Stimulation of mPL-II release by mouse decidual calcyclin As shown in Fig. 6, purified decidual calcyclin caused a dose-dependent increase in the release of mPL-II from cultured trophoblast cells. The stimulation was significant at 0.01 /xg decidual calcyclin/ml and was highest at
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EFFECT OF CALCYCLIN ON mPL-II SECRETION
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a b c d e
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Endo'1991 Vol 129 • No 3
j 150
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FIG. 5. Western blot of various mouse tissue extracts and serum. The extracts and serum (5 ng total protein/lane) were electrophoresed on 15% SDS-PAGE, transferred to a Immobilon-P membrane and immunostained for decidual calcyclin. a) Decidual extract, 6) skeletal muscle extract, c) brain extract, d) pituitary extract, e) liver extract, /) male serum, g) serum from 17-days-pregnant female, h) serum from 10-days-pregnant female, i) serum from 6-days-pregnant female, ;") purified mouse decidual calcyclin (1 ng).
1 /itg decidual calcyclin/ml. The maximum stimulation of mPL-II release averaged about 1.5 times higher than the control, ranging from 1.4-1.7 times that of control in each culture. These effects are comparable to the maximum stimulation obtained for decidual extract after 50% ammonium sulfate precipitation. Similar stimulation of mPL-II release was obtained by decidual calcyclin whether the culture medium contained low (0.018 mM) or normal (1.8 mM) calcium. The increase in mPL-II release was most prominent after 30-60 min incubation with decidual calcyclin. Prolonged incubation beyond 1 h diminished the effect of decidual calcyclin. Decidual culture The amount of decidual calcyclin that was released into the culture medium did not change significantly during the culture period. The release of total protein was also unchanged during the 4 days of culture, but the DNA content decreased significantly after day 1 (Fig. 7). The amount of decidual calcyclin in the cells was about twice the amount of decidual calcyclin that was released into the medium during 24 h. This ratio, i.e. decidual calcyclin in tissue/decidual calcyclin released during 24 h, was unchanged during the culture period (Table 1).
0 10 100 1000 Decidual Calcyclin (ng/ml) FIG. 6. The effect of purified mouse decidual calcyclin on the secretion of mPL-II from cultured mouse trophoblasts. The cells were cultured for 4 days in medium NCTC-109 before they were treated with different concentrations of mouse decidual calcyclin for 30 min. A significant stimulation of mPL-II release was seen at 0.01 Mg/ml and maximum stimulation was obtained at 1 fig/ml. Each point represents the mean ± SEM of 18 replicates obtained from four different cultures. Asterisks denote significant difference from control (P < 0.05).
Discussion We have purified a protein from mouse decidual tissue that stimulates the release of mPL-II from cultured trophoblast cells. Partial amino acid sequence analysis of this protein showed that it is highly homologous to human calcyclin (20), rat PRA (21), and calcyclin expressed by mouse fibroblasts (22). There was one amino acid difference between the sequence that we obtained for the protein designated mouse decidual calcyclin and the cDNA-deduced sequence of mouse fibroblast calcyclin. This discrepancy may be caused by a misread amino acid, alanine for arginine, in the amino acid analysis. However, our determination of this amino acid is identical to corresponding amino acid residues of human calcyclin (20) and rat PRA (21). Alternatively, there may be more than one form of calcyclin in the mouse. Guo et al. (22) showed that there are probably five copies of the calcyclin gene per haploid mouse genome. Therefore, it is possible that these different calcyclin genes code for slightly different proteins. When the purified protein was run on SDS-PAGE in the presence and absence of reducing agents, there was a shift in the mol wt from
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EFFECT OF CALCYCLIN ON mPL-II SECRETION
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