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Laboratory of Cellular Biochemistry, Animal Resource Science/Veterinary Medical Science, University of Tokyo, 1–1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Address all correspondence and requests for reprints to: Kunio Shiota, Ph.D., D.V.M., Laboratory of Cellular Biochemistry, Animal Resource Science/Veterinary Medical Science, University of Tokyo, 1–1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan. E-mail: ashiota{at}hongo.ecc.u-tokyo.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PRL exerts its biological function through the PRL receptor; when PRL is bound to the receptor, Janus kinase-2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) are activated by tyrosine phosphorylation (14, 15). Activated JAK2 and STAT5 play important roles in cytokine-dependent cell growth and differentiation (14, 16, 17). PL subfamily members, including PL-I, PL-II, and PL-Iv, are also able to bind to the PRL receptor and to stimulate the proliferation of Nb2 cells (18, 19, 20). The PLP subfamily includes PLP-A, PLP-B, PLP-C, PLP-D, PLP-Cv, and decidual PRL-related protein (dPRP). Of these, PLP-A, PLP-B, PLP-C, and dPRP do not bind to the PRL receptor (21, 22, 23, 24) nor do they stimulate lactogenic activity, even though they are structurally similar to PRL (13, 25). On the other hand, Jackson et al. (26) reported that proliferin and proliferin-related protein, which are members of the mouse placental PRL family, have the ability to regulate angiogenesis, which indicates that PLPs have nonlactogenic activities. Within the PLP subfamily, PLP-C, PLP-Cv, PLP-D, and dPRP are highly homologous, and their gene expression patterns are similar, except for that of dPRP (7, 8, 9, 10).
N-Glycosylation is another feature of placental PRL family members, except for PL-II (2). The glycosylation of PLs and PLPs has been investigated, and most of them were found to be glycosylated (9, 18, 20, 22, 27, 28). We have investigated the role of the N-linked sugar chain of PL-I on Nb2 proliferation and discussed its importance in signal transduction (29). On the other hand, it has been reported that the N-linked sugar chain reduces porcine PRL activity, as measured by Nb2 assay or receptor binding assay (30). Another report showed little contribution by the sugar chains of PL-I to its biological activity (18). We have recently cloned another PLP-related complementary DNA (cDNA) by the differential display method and tentatively named the gene product PLP-H. In this paper we describe the molecular cloning of this newly identified molecule and the characterization of its expression in both tissues and Rcho-1 cells. To analyze its biological function, we made a recombinant protein for PLP-H together with PLP-D and examined whether the signal is transduced through the PRL receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal treatment and tissue preparation
Wistar rats were purchased from the Imamichi Institute for
Animal Reproduction (Ibaraki, Japan). Sprague-Dawley, Fisher,
and Lewis rats were purchased from Charles River (Ibaraki, Japan). Rats
were kept under a lighting schedule of 14 h of light (lights on at
0500 h) and 10 h of darkness (lights off at 1900 h) and
were allowed food and water ad libitum. Timed pregnancies
and tissue dissections were performed as previously described (8).
Placentas collected for in situ hybridization were embedded in OCT compound (Miles, Inc., Elkhart, IN), quickly frozen in ice-cold ethanol, and stored at -80 C until use.
Molecular cloning of PLP-H cDNA and sequence analysis
In a series of experiments exploring a stage-specific placental
factor by differential display (8, 31), we obtained a cDNA fragment
encoding a late pregnancy-specific messenger RNA (mRNA), and we cloned
the full-length cDNA using 5'-RACE and full-length PCR as previously
described (8). The products of full-length PCR were ligated into the
pGEM T vector. The DNA sequence was determined for both strands by the
dideoxy chain termination method using a sequencing kit (AutoRead) and
a DNA sequencer (A. L. F. sequencer, Pharmacia
LKB) (32). Sequence analysis showed that this cDNA encodes
a novel protein, which we tentatively named PLP-H.
RT-PCR analysis
First strand cDNAs for various tissues (kidney, liver, spleen,
brain, ovary, fetus, and placenta) were prepared from total RNAs using
RT (SuperScript, Life Technologies) and a
(deoxythymidine)15-adaptor primer
[5'-GAATTCTCGAGTCGACCCGGG-(T)15-3']. 5'-Primers specific
for PLP-C (5'-GCTGCTGGGACCCCCTCCG-3'; nucleotides 133–152), PLP-D
(5'-GCTGCTGGAACCCCCTTGT-3'; nucleotides 177–195), and PLP-H
(5'-ACTGCTGGGACCTCTCCA-3'; nucleotides 180–198) were constructed and
tested for the ability to specifically amplify each clone by PCR. Each
template (PLP-C, PLP-D, and PLP-H) used for PCR contains full-length
cDNA and the adaptor primer-binding site at the 3'-end. The PCR was
performed with the 5'-specific primer, adaptor primer, the template,
and TaKaRa EX Taq polymerase. The PCR conditions for
denaturation, annealing, and elongation were 95, 65, and 72 C for 30
sec, 30 sec, and 1 min, respectively. Then, the RT-PCR was performed
using an adaptor primer and a series of 5'-primers specific for each
clone with the PCR condition stated above.
Cell lines
The Rcho-1 cell line was a gift from Dr. Michael J.
Soares (University of Kansas Medical Center, Kansas City, KS).
These cells were cultured and induced to differentiate as previously
described (8). The COS-7 cell line was purchased from RIKEN Gene Bank
(Ibaraki, Japan). The Nb2 cell line was provided by Dr. Peter W. Gout
(British Columbia Cancer Research Center, Vancouver, Canada)
(33).
Northern blot analysis
DIG-labeled riboprobes were synthesized using T7 polymerase as
previously described (8).
For the Northern blot analysis, total RNA dissolved in electrophoresis buffer [20 mM 3-(N-morpholino)-propanesulfonic acid (pH 7.0), 5 mM sodium acetate, and 1 mM EDTA] containing 50% formamide and 6.5% formaldehyde was heated to 65 C for 15 min, electrophoresed on a 1.0% agarose-formamide gel, and transferred to a nylon membrane (Hybond-N, Amersham, Tokyo, Japan). The membrane was hybridized with a DIG-labeled complementary RNA (cRNA) probe in 5 x SSC (standard saline citrate) containing 50% formamide, 1.5% SDS, and 1% blocking reagent (Boehringer Mannheim Yamanouchi), washed twice with 2 x SSC-0.1% SDS at 25 C for 5 min, and then washed twice with 0.1 x SSC-0.1% SDS at 68 C for 1 h. The membrane was incubated in blocking buffer (1% blocking reagent and 150 mM NaCl in 100 mM maleic acid, pH 7.5) at 25 C for 1 h, followed by the addition of anti-DIG antibody (1:10,000 dilution). The membrane was washed three times with TBS-Tween (0.3%) for 5 min each time and finally rinsed in 100 mM Tris-HCl (pH 9.5) with 100 mM NaCl. The chemiluminescent reaction was performed in the latter solution containing CDP-Star reagent (1:100 dilution; Boehringer Mannheim Yamanouchi), and the membrane was exposed to x-ray film for 30 min.
In situ hybridization
In situ hybridization for detecting PLP-H was
performed as previously described (8) using the PLP-H cRNA probes made
for Northern blot analysis. Finally, the tissue sections were colored
with nitroblue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indolyl
phosphate (BCIP), and counterstained with either hematoxylin or methyl
green.
Generation and purification of recombinant proteins
Sets of primers were generated for PLP-H, PLP-D, and PL-I
excluding the signal sequences, and PCR reactions were performed. The
PCR products were digested with EcoRI and KpnI
restriction enzymes and ligated into the pSR
-CHFX vector (a gift
from Dr. A. Miyajima), which encodes the CD8 signal peptide,
followed by both Flag and histidine epitope tags to generate
N-terminus-tagged proteins (34). These plasmid were transfected into
COS-7 cells by the diethylaminoethyl-dextran method (35). Four days
after transfection, the culture media were collected, and the proteins
were purified using Ni+-nitrilo-triacetic acid resin
(Qiagen, Chatsworth, CA) and anti-Flag M2 affinity resin (Eastman Kodak Co., New Haven, CT) according to the manufacturer’s
instructions. Purified proteins were quantitated using the BCA
microassay kit (Pierce Chemical Co., Rockford, IL). The
purity of these proteins was checked by SDS-PAGE (10%) followed by
silver staining.
Glycosidase digestion
The purified proteins were subjected to N-glycosidase
F (Boehringer Mannheim Yamanouchi) treatment to remove
N-linked sugar chains, as described previously (29).
In brief, partially purified proteins (1 µg eluents from
Ni+-nitrilo-triacetic acid resin) were boiled for 5 min in
denaturing buffer (0.5% SDS, 0.05 M EDTA, and 0.3%
ß-mercaptoethanol). Then the mixture was incubated at 37 C for
18 h in reaction buffer (0.02 U/µl N-glycosidase F,
0.17% SDS, 1.25% Nonidet P-40, and 20 mM sodium
phosphate, pH 7.5) and subjected to SDS-PAGE followed by silver
staining and Western blotting using the anti-Flag antibody.
Immunoprecipitation and immunoblotting
Nb2 cells were starved in serum-free medium (Fischer’s medium)
for 5 h, then treated for 10 min with recombinant PL-I (100
ng/ml), PLP-H (1000 ng/ml), or native oPRL (100 ng/ml) at 25 C. Then
400 µg whole cell extracts made in lysis buffer [20 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonylfluoride, 50
mM sodium fluoride, and 1% Triton X-100] were incubated
with antibodies to JAK2 for 1 h at 4 C, followed by
incubation with protein A-Sepharose beads for 1 h at 4 C. The
immunoprecipitates and supernatants were separated by centrifugation,
and the supernatants were used for precipitating STAT5 by the same
procedure, except that the antibody to STAT5 was used. Then the
immunoprecipitates were washed three times with wash buffer (0.1
mM sodium orthovanadate and 0.5% Triton X-100 in PBS) and
subjected to SDS-PAGE (10%) under reducing conditions. Proteins were
transferred to a polyvinylidene difluoride membrane and blotted with
antiphosphotyrosine monoclonal antibody (4G10). The levels of
immunoprecipitated JAK2 and STAT5 were examined by reprobing the
membranes with specific antisera.
Proliferation assays
The Nb2 proliferation assay was performed as previously
described (36). Briefly, Nb2 cells in growth medium (Fischer’s medium
containing 10% FBS and 10% horse serum) were transferred to 1% FBS
and 10% horse serum in Fischer’s medium, incubated for 24 h,
collected, and washed three times in serum-free Fischer’s medium. Then
cells were divided into 96-well plates with 15,000 cells/well. Various
concentrations of histidine-Flag-tagged recombinant PLP-H, PLP-D, PL-I,
or native oPRL (NIDDK oPRL-20) were added. After 72 h at 37 C, the
number of cells in each well was determined using a colorimetric cell
counting kit that can measure the mitochondrial dehydrogenase activity
(Dojindo Laboratories, Kumamoto, Japan) (37). In brief, a mixture of
4-[6-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
sulfonate (Wst-1; 5 mM) and 1-methoxy-5-methylphenazinium
methosulfate (0.2 mM) was added to each well (10
µl/well), and the cells were incubated at 37 C for 4 h. Then,
the absorbance at 450 nm was determined using an enyzme-linked
immunosorbent assay plate reader. Values are presented as the
mean ± SEM of triplicate samples, and similar results
were obtained from three individual experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The termination codon was TAA
at nucleotides 776–778. A polyadenylation signal (38) at nucleotides
894–900 and a polyadenylase tail were present in the 3'-noncoding
region. There were two potential N-glycosylation sites,
Asn-Leu-Ser and Asn-Asp-Thr, at amino acid positions 180–183 and
188–191, respectively. This newly identified clone showed considerable
homology with previously identified members of the rat placental PRL
family, especially PLP-C and PLP-D (78% and 67% amino acid sequence
homologies, respectively; Fig. 1B).
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). DNA fragments corresponding to
PLP-C, PLP-D, and PLP-H were specifically amplified using the set of
primers described in Materials and Methods. Other than the
target molecules, no amplification of other clones was detected. RT-PCR
was then performed using these primers and first strand cDNAs from
various tissues of Wistar rats and placentas from various rat strains
(Fig. 2B). PLP-H expression was seen only in the placenta, but it was
present in every strain of rat examined. Two other related genes, PLP-C
and PLP-D, were also expressed in each strain of rat examined (Fig. 2C). This RT-PCR expression assay was also able to discriminate PLP-H
from PLP-Cv (data not shown).
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). PLP-H
mRNA was not detected on day 12, but was first detected on day 14, and
its expression level increased from then until day 20, which is just
before term.
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). Total RNA obtained from
Rcho-1 cells at each proliferation and differentiation stage
was individually subjected to Northern blot analysis. The signal was
detected 8 days after differentiation (Fig. 3B).
Glyceraldehyde-3-phosphate dehydrogenase was used as an internal
control.
Cellular localization of PLP-H mRNA
In situ hybridization analysis of PLP-H expression
showed that in day 20 placenta, PLP-H mRNA was specifically and
predominantly localized to spongiotrophoblast and trophoblast giant
cells (TGC) of the junctional zone (Fig. 4, A and B). No expression of PLP-H mRNA
was detected in day 12 placenta (data not shown). Incubation with sense
RNA probes failed to show any specific hybridization to adjacent
sections of day 20 placenta (Fig. 4C). The results of in
situ hybridization were consistent with those of Northern blot
analysis as described above.
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. To measure the purity of these
proteins, they were subjected to SDS-PAGE followed by silver staining
(Fig. 5D). One major (lower) band and one minor (upper) band were
detected for each protein. After treatment with
N-glycosidase F, the minor band disappeared (Fig. 5, B and
C), suggesting that the minor portions of the recombinant proteins were
glycosylated forms.
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).
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, no
PRL-like proliferative activity was seen for PLP-H and PLP-D, whereas
both PL-I and oPRL stimulated proliferation dose dependently. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PLP-H expression was first detected on day 14, and the level of its expression increased during pregnancy, similar to the expression patterns of PLP-C, PLP-Cv, and PLP-D (7, 10, 8). The cell types that expressed PLP-H were the spongiotrophoblast cells and the secondary TGCs, both of which express PLP-C, PLP-Cv, and PLP-D. These cell types are located at the maternal interface, which is called the junctional zone of the placenta. Rcho-1, a rat choriocarcinoma cell line, the lineage of which is thought to be TGC, expresses various placental PRL family proteins depending on the cell’s stage of differentiation (39, 40). Our data showed that PLP-H was expressed by Rcho-1 cells only after they had differentiated, confirming that TGC express PLP-H.
The placental PRL family members have been searched for both proteins and genes. Ogilvie et al. (41) sequenced four GH-like proteins secreted from placental explants that we now consider the glycosylated and nonglycosylated forms of PLP-C (7, 42), PLP-D (8), and an uncharacterized protein. The amino acid sequence we deduced from the PLP-H cDNA exactly matches the 37 N-terminal amino acids of the uncharacterized protein they reported (41). Therefore, PLP-H is probably a secretory protein, and it may exist in the maternal and fetal circulatory system or in the extraplacental space.
To characterize the functions of PLP-H and PLP-D, we prepared a recombinant protein for each of them using the COS-7 transfection system. Purified PLP-H and PLP-D expressed in COS-7 cells both had two populations analyzed by SDS-PAGE and Western blotting. The upper minor bands (29 and 31 kDa, respectively) were probably the N-glycosylated forms of PLP-H and PLP-D, respectively, as these bands disappeared after N-glycosidase F treatment. The low frequency of glycosylation of PLP-H and PLP-D seems to be protein specific, as PL-I expressed in COS-7 cells is heavily glycosylated (29). There are some indications of the role of N-linked sugar chains in the placental PRL family. Recently, Manzella et al. (43) reported that the N-linked sugar chains of these PRL family proteins are developmentally regulated. We examined the role of the N-linked sugar chain of PL-I by site-directed mutagenesis, and they demonstrated that it is involved in signal transduction through the PRL receptor, using the Nb2 proliferation assay and RRA (29).
As PLP-H and PLP-D are structurally similar to PRL and are glycosylated, we examined their lactogenic activity using the PRL-dependent Nb2 cell line. However, we did not detect signal transduction through the PRL receptor (JAK2 and STAT5 phosphorylation), and we did not observe Nb2 cell proliferation. PLP-H is most similar to PLP-C, PLP-Cv, PLP-D, and dPRP within the PLP subfamilies. The N- and C-terminal regions that are reported to be important for rat PRL biological function (44, 45) are not conserved in PLP subfamilies. These data show that PLP-H does not have lactogenic activities like the other PLP proteins (21, 22, 23, 24).
The functions of PLP-H and the other PLP subfamily members remain to be elucidated; however, the site- and stage-specific expression patterns and the variations between the related molecules suggest that they may have unique biological actions. Therefore, it is likely that the PLP family has an unknown role and that the molecules in this family function through receptors specific for them. Further studies are now underway using the recombinant PLP-H (and PLP-D) made in this study to elucidate the roles of these molecules.
In conclusion, we have identified PLP-H in rat placenta, which produces at least 10 members of the PRL family. The molecular structure, expression pattern, and effect on PRL-dependent cells of PLP-H suggest that it may share common features with PLP-C and PLP-D.
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Acknowledgments |
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-CHFX vector and Dr. Peter W. Gout for providing Nb2
cells. |
Footnotes |
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Received April 20, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid
dehydrogenase (20-HDS) in the rat. Endocr J [Suppl]
41:S43–S56
2,6GalNAcß1,4GlcNAc, on select
members of the rat placental prolactin family. J Biol Chem 272:4775–4782This article has been cited by other articles:
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