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J Biol Chem. 2008 July 4; 283(27): 18957–18968. doi: 10.1074/jbc.M801826200. | PMCID: PMC2441551 |
Copyright © 2008, The American Society for Biochemistry and
Molecular Biology, Inc. Decidual Cells Produce a Heparin-binding Prolactin Family Cytokine with
Putative Intrauterine Regulatory
Actions *S. M. Khorshed Alam, Toshihiro Konno, Namita Sahgal, Lu Lu, and Michael J. Soares1 Institute of Maternal-Fetal Biology and the Division of Cancer and Developmental Biology, Departments of Pathology and Laboratory Medicine and Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas 66160 Received March 6, 2008; Revised May 7, 2008. Pregnancy in mice and rats is associated with the production of a large
family of hormones/cytokines related to prolactin (PRL). The
hormones/cytokines are hypothesized to coordinate maternal and fetal
adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known
as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of
the uterine decidua and a regulator of postimplantation intrauterine events.
PLP-J-specific antibodies and a series of recombinant PLP-J proteins were
generated and used to investigate PLP-J expression and as ligands for
investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein
and localizes to a band of decidual cells surrounding the trophoblast cell
layer on gestation day 8.5. PLP-J ligands specifically bound in situ
to the surrounding uterine stromal cells and vasculature within the decidua of
gestation day 8.5 implantation sites. We then investigated the in
vitro actions of PLP-J on uterine stromal cells and endothelial cells.
PLP-J specifically interacted with both cell populations. PLP-J promoted
uterine stromal cell proliferation and inhibited endothelial cell
proliferation. We determined that PLP-J does not interact with PRL receptors.
Instead, PLP-J interacts with heparin-containing molecules, including
syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as
in uterine stromal cells and endothelial cells. The restricted expression of
PLP-J and its specific interactions with uterine stromal cells and endothelial
cells suggests that it acts locally and regulates decidual cell development
and the endometrial vasculature. Successful pregnancy requires specialized maternal adaptations.
Decidualization is a key uterine adaptation associated with the establishment
of pregnancy and is characterized by the differentiation of uterine stromal
cells
( 1– 4).
Decidual cell differentiation is dependent upon ovarian steroid hormone
production, and in rodents, it also requires signals emanating from the
preimplantation embryo ( 1,
5). Once formed, decidual cells
establish a protective environment, facilitating the development of the
placenta and embryo. They promote the redistribution of specific populations
of leukocytes and reorganize the uterine vascular network. Intercellular
signals elaborated by decidual cells are key mediators of these uterine
adaptive responses. Among the decidual cell ligands are a family of cytokines
related to prolactin
(PRL). 2PRL is an ancient hormone with its origins as a regulator of vertebrate
environmental adaptations ( 6,
7). Some species possess a
single member of the PRL family that can be expressed in an assortment of
tissues, including the anterior pituitary and uterus through the utilization
of cell-specific promoters
( 8– 10).
Other species have undergone a gene expansion within the PRL locus
( 11). PRL family gene
expansion is particularly robust in mice and rats
( 12– 14).
Gene duplication and natural selection have yielded 2 dozen related genes in
each of these species. The PRL family genes encode cytokines/hormones that are
expressed in cell-specific and temporally specific patterns and are most
relevant to pregnancy-associated tissues, especially in the uterine decidua
and the placenta. Initial observations suggest that the expanded PRL family
participates in pregnancy-dependent adaptations to physiological stressors
( 15,
16). Although a few members
are PRL mimetics (placental lactogens), activating PRL receptor signaling
cascades, most utilize distinct strategies to regulate their cellular targets
( 11,
17). The cellular targets are
intriguing and include endothelial cells, inflammatory/immune cells, and
hematopoietic precursors
( 18– 22). Decidual cells of mice and rats express four members of the PRL family: PRL
( 23,
24), PRL-like protein-B
(PLP-B; also known as PRL family 6, subfamily a, member 1 (Prl6a1))
( 25– 28),
decidual PRL-related protein (dPRP; also known as PRL family 8, subfamily a,
member 2 (Prl8a2)) ( 27,
29,
30), and PLP-J (also known as
PRL family 3, subfamily c, member 1 (Prl3c1))
( 31– 34).
PRL is postulated to be expressed in rodent decidual cells under the direction
of a unique cell-specific promoter, as has been characterized for human
decidual cells ( 10,
35), where it acts through the
PRL receptor signaling pathway to promote decidual cell survival, regulate
leukocyte function, stimulate uterine gland development, and facilitate
vascular remodeling ( 23,
36– 38).
PLP-B and dPRP do not utilize the canonical PRL receptor signaling pathway
( 22,
26,
39). dPRP is a heparin binding
cytokine that is essential for pregnancy-dependent adaptive responses to
hypoxia ( 16,
22,
39). Unlike wild-type animals,
mice deficient in dPRP do not effectively adapt to hypoxia and terminate their
pregnancies. dPRP modulates decidual expression of PLP-J but not PLP-B or PRL
( 16). The expression of PLP-J
is significantly decreased in dPRP null mice, suggesting that the biology of
dPRP and PLP-J may be linked. Information on the biological functions of PLP-J
is not available. In this study, we have characterized the PLP-J protein and its expression
pattern, identified targets for its action, and determined biological
responses of its cellular targets. PLP-J is a heparin-binding cytokine with
distinct actions on uterine stromal cell and endothelial cell populations. Animals and Tissue Preparation Holtzman rats were obtained from Harlan Sprague-Dawley Inc. (Indianapolis,
IN). The animals were housed in an environmentally controlled facility, with
lights on from 0600 to 2000 h and were allowed free access to food and water.
Timed pregnancies were generated, and tissue dissections were performed as
previously detailed ( 40).
Conceptuses with associated uteri were removed on specific days of gestation.
Tissues were frozen in dry ice-cooled heptane and stored at -80 °C until
used for in situ hybridization, immunohistochemistry, and in
situ ligand binding or were frozen in liquid nitrogen and stored at -80
°C for subsequent RNA and protein analyses. The presence of sperm in the
vaginal smear was designated as day 0.5 of pregnancy. New Zealand White
rabbits were obtained from Myrtle's Rabbitry (Thompsons Station, TN) and used
for antibody production. Protocols for the care and use of animals were
approved by the University of Kansas Animal Care and Use Committee. Cell Culture U1 rat uterine stromal cells were obtained from Dr. Virginia Rider
(Pittsburg State University, Pittsburg, KS) and maintained in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS),
1 m m sodium pyruvate, and penicillin (100 units/ml) and
streptomycin (100 μg/ml)
( 41,
42). These rat uterine stromal
cells are physiologically relevant in that they can be induced to
differentiate into decidual cells
( 43). Rat aortic endothelial
cells were purchased from VEC Technologies, Inc. (Rensselaer, NY) and
maintained in MCDB-131 complete culture medium. The rat Nb2 lymphoma cell line
was provided by Dr. Peter Gout (University of British Columbia, Vancouver,
Canada) and maintained in RPMI 1640 culture medium supplemented with 10% horse
serum, 10% FBS, 50 μ m 2-mercaptoethanol, 2 m m
l-glutamine, 5 m m HEPES, penicillin (50 units/ml), and
streptomycin (50 μg/ml)
( 44). Human embryonic kidney
(HEK 293) cells were obtained from ATCC (Manassas, VA) and used as a host for
the expression of the PLP-J fusion proteins. HEK 293 cells were maintained in
DMEM/F-12 medium supplemented with 10% FBS, 1 m m sodium pyruvate,
penicillin (100 units/ml), and streptomycin (100 μg/ml). CHO cells and
heparan sulfate-deficient CHO-pgsD-677 cells were obtained from ATCC and
cultured in DMEM/MCDB 302 culture medium containing 100 units/ml penicillin,
100 μg/ml streptomycin, and 10% FBS. Raji cells stably transfected with a
syndecan-1 (Sdc1) expression vector (Raji-S1) and the parent Raji cell line
were obtained from Dr. Alan C. Rapraeger (University of Wisconsin, Madison,
WI) and were maintained in RPMI 1640 culture medium supplemented with 10% FBS
and antibiotics ( 45). All cell
cultures were maintained in a humidified atmosphere of 5% CO 2, 95%
air at 37 °C. Generation of Fusion Proteins mFLAG-PLP-J—PLP-J was expressed as a fusion protein with a
FLAG-His6-FLAG tag. The full-length mature rat PLP-J cDNA was used
as a template for PCR amplification of a PLP-J fragment with EcoRI and XbaI
restriction sites at the 5′- and 3′-ends, respectively, using
sequence specific primers, 5′-cat tta aag aat tca cac cat atg acc aga
tgt-3′ and 5′-gtt ata tgt ttc tag att acc act tgt taa taa
tg-3′. After digestion with EcoRI and XbaI restriction enzymes, the
fragment was ligated into a modified pFLAG-CMV-3 vector (mFLAG; Sigma). The
accuracy of vector construction was verified by DNA sequencing. The
mFLAG-PLP-J plasmid was transfected into HEK 293 cells using Lipofectamine
Plus according to the manufacturer's instructions (Invitrogen). The initial
selection of transfected cells was accomplished in the presence of G418 at a
concentration of 500 μg/ml. Selected cells were then maintained in 100
μg/ml G418. The mFLAG-PLP-J fusion protein was purified from serum-free conditioned
medium by incubating with an Ni2+-NTA-agarose resin (Qiagen,
Valencia, CA). In brief, Ni2+-NTA-agarose was equilibrated with
Sorensen's phosphate buffer (NaH2PO4 (66 mm)
and KH2PO4 (66 mm)) and added to conditioned
medium in a buffer containing 10 mm imidazole, 50 mm
NaH2PO4, 0.15 m NaCl, pH 8.0 (1×
binding buffer), incubated overnight at 4 °C with constant shaking. The
resin was then transferred into a column and washed with 2× binding
buffer. Recombinant mFLAG-PLP-J protein was eluted with 250 mm
imidazole and 0.15 m NaCl in Sorensen's phosphate buffer, pH 6.0.
Aliquots of fractions were separated using SDS-PAGE and stained with Coomassie
Blue G-250 and immunoblotted with anti-FLAG M2 antibody (Sigma).
Immunopositive fractions were pooled and dialyzed against phosphate-buffered
saline, pH 7.4, and concentrated by Centricon ultrafiltration centrifugation
devices (Millipore, Billerica, MA). Purified proteins were sterilized using
Millex filters (Millipore), and concentrations were determined with the DC
protein assay (Bio-Rad). Alkaline Phosphatase (AP)-PLP-J Fusion Protein
(AP-PLP-J)—The full-length cDNA for mature rat PLP-J was ligated
downstream of heat-stable human placental AP of pCMV-SEAP and then transfected
into HEK 293 cells, as previously described
( 46,
47). The transfected cells
were selected and maintained in DMEM/F-12 culture medium supplemented with 10%
FBS and G418 as described above. Cells were transferred to serum-free culture
medium for 72 h. Conditioned medium was collected, and cellular debris was
removed by centrifugation at 2,200 × g for 30 min at 4 °C
and then stored at -20 °C. AP activity was quantified by a colorimetric
assay at 405 nm using p-nitrophenyl phosphate as an AP substrate
( 47). AP fusion proteins were
also separated using SDS-PAGE and electrophoretically transferred to
nitrocellulose, and AP activity was detected by incubation with nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Preparation of PLP-J Antibodies Antibodies directed to PLP-J were prepared by immunizing rabbits with
recombinant His 6-tagged PLP-J protein generated in a prokaryotic
expression system, pQE-30Xa (Qiagen). The mature rat PLP-J cDNA was used as a
template for PCR amplification of a PLP-J fragment with BamHI and SacI
restriction sites at the 5′- and 3′-ends, respectively, using
sequence specific primers, 5′-cat tta aag gat cca cac cat atg acc aga
tgt-3′ and 5′-gtt ata tgt cga gct ctt acc act tgt ttt taa taa
tg-3′. The amplified fragment was ligated into the pQE-30Xa plasmid and
transformed into Escherichia coli, M15[pREP4]. His 6-PLP-J
expression was induced by 2 m m isopropyl
β- d-1-thiogalactopyranoside, and His 6-PLP-J protein
was purified from bacterial exclusion bodies by Ni 2+-NTA-agarose
affinity chromatography, as described above. Purified His 6-PLP-J
was characterized by SDS-PAGE and Western blotting with anti-His tag
antibodies (Qiagen). Purified His 6-PLP-J was used to immunize New
Zealand White rabbits as previously described
( 48,
49). Western Blot Analysis of Decidual Tissues Protein lysates were prepared by homogenizing gestation day 8.5 rat
conceptuses (decidua and extraembryonic and embryonic tissues) in radioimmune
precipitation buffer (10 m m Tris-HCl, pH 7.2, 1% Triton X-100 or 1%
Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 150 m m NaCl, 5
m m EDTA, 1 m m sodium orthovanadate, 1 m m
phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin). Protein concentrations
were determined by the DC protein assay (Bio-Rad). Fifty μg of total
protein were separated by SDS-PAGE and transferred onto nitrocellulose
membranes. Immunoreactive PLP-J and dPRP were detected with antibodies to
anti-PLP-J (present study) and anti-dPRP
( 39), respectively, and
visualized by enhanced chemiluminescence according to the manufacturer's
instructions (Amersham Biosciences). Immunocytochemistry Immunocytochemical analyses were used to localize PLP-J and dPRP proteins
and the distribution of endothelial cells in gestation day 8.5 implantation
sites, as described ( 39,
40,
50). Cryosections (10 μm)
were prepared, fixed in cold 4% paraformaldehyde solution, and blocked in 10%
normal goat serum for 1 h at room temperature. The immunodetection was
performed by incubating overnight at 4 °C with anti-PLP-J (present study),
anti-dPRP antibodies ( 39), or
RECA-1 antibodies, which recognize an uncharacterized rat endothelial
cell-specific surface antigen (Serotec, Oxford, UK)
( 41).
Avidin-peroxidase-conjugated secondary antibody was added for 30 min at room
temperature and color-developed with an AEC kit (Zymed Laboratories, San
Francisco, CA). Tissues were counterstained with Mayer's hematoxylin. Images
were captured using a Leica MZFIII steromicroscope (Leica Microsystems GmbH,
Welzlar, Germany) or a Nikon Eclipse 55i microscope (Nikon Instruments Inc.,
Melville, NY), both equipped with Leica CCD cameras (Leica). In Situ Hybridization In situ hybridization was performed to assess the distributions of
PLP-J and dPRP transcripts in gestation day 8.5 rat implantation sites
( 40,
51). Cryosections (10-μm)
were prepared and stored at -80 °C until used. Plasmids containing cDNAs
for PLP-J ( 34) and dPRP
( 29) were used as templates to
synthesize sense and antisense digoxigenin-labeled riboprobes according to the
manufacturer's instructions (Roche Applied Science). The frozen sections were
air-dried and fixed in cold 4% paraformaldehyde in phosphate-buffered saline.
Prehybridization, hybridization, and detection of alkaline
phosphatase-conjugated anti-digoxigenin were performed as previously reported
( 12,
51). Images were captured as
described above. Northern Blot Analysis Northern blot analysis was performed as previously described
( 52). Total RNA was extracted
from gestation day 8.5 rat decidual tissues, U1 uterine stromal cells, and rat
aortic endothelial cells using TRIzol reagent (Invitrogen). Total RNA (15
μg/lane) was resolved in 1% formaldehyde-agarose gels, transferred to nylon
membranes, and cross-linked. Blots were probed with 32P-labeled
cDNAs for syndecans ( Sdc1, NM_013026; Sdc2, NM_013082;
Sdc3, NM_053893; Sdc4, NM_012649).
Glyceraldehyde-3-phosphate dehydrogenase cDNA was used to evaluate the
integrity and equal loading of RNA samples. At least three different tissue
samples from three different animals were analyzed with each probe for each
time point. AP-PLP-J Binding Tissues—An in situ AP-binding assay was performed
as previously described ( 46,
47). In brief,
8–10-μm tissue sections were prepared with a cryostat and mounted
onto glass slides. The tissue sections were washed with a modified Hanks'
balanced salt solution (HBHA; containing 20 m m HEPES, 0.5 mg/ml BSA
and 0.1% NaN3) and incubated with AP, AP-PLP-J, or AP-placental lactogen-I
(PL-I; also known as PRL family 3, subfamily d, member 1 (Prl3d1)) fusion
protein ( 46) for 75 min at
room temperature. For competition, tissue sections were incubated with various
glycosaminoglycans and/or mFLAG-PLP-J protein for 45 min prior to incubation
with AP-PLP-J. Following incubation, tissue sections were washed three times
with HBHA containing 0.1% Tween 20 and fixed for 20 min with
acetone-formaldehyde fixative. The fixed sections were washed and heated at 65
°C for 30 min to inactivate endogenous AP activity in the tissues.
Localization of AP was determined by incubation with nitro blue tetrazolium
and 5-bromo-4-chloro-3-indolyl phosphate. Cells—For AP-ligand binding with cells, 5 ×
10 4 cells were incubated with different concentrations of
conditioned medium of AP or AP-PLP-J for 60 min at room temperature. Where
indicated, cells were incubated with different concentrations of heparin for
30 min before incubation with AP-PLP-J. Cells were washed with HBHA containing
0.1% Tween 20 and HBSS. Cells were then heated for 30 min at 65 °C to
inactivate endogenous AP activity. AP activity was determined by incubation
with AP substrate ( p-nitrophenyl phosphate) and measurement of
absorbance at 405 nm. AP-PLP-J binding to heparan sulfate-deficient
CHO-pgsD-677 cells and wild-type CHO cells was also assessed as previously
described ( 22). AP-PLP-J and
AP-PL-I binding with the PRL receptor were assessed in CHO-pgsD-677 cells
( 53) transiently transfected
with the PRL receptor (pECE/long; gift from Dr. Paul Kelly, INSERM, Paris,
France) ( 54) using FusGene 6
(Roche Applied Science). The AP-PL-I fusion protein was used as a positive
control. Heparin Binding Assay PLP-J and dPRP interactions with heparin were evaluated with a
heparin-binding plate assay
( 55). Multiwell plates were
coated with either heparin conjugated to bovine serum albumin (heparin-BSA;
Sigma) or BSA (Sigma) and incubated with AP-PLP-J, AP-dPRP, or AP. Wells were
washed with HBHA containing 0.1% Tween 20 and HBSS. AP activity was determined
by incubation with AP substrate ( p-nitrophenyl phosphate) and
measurement of absorbance at 405 nm. Cell Adhesion Assay Cell adhesion assays were performed as described
( 56). 96-well microtiter
plates were coated with test proteins diluted in PBS (50 μl/well) and
incubated at 4 °C for 16 h. Wells were washed three times with PBS and
blocked with 1% BSA at room temperature for 1 h. To test for specificity,
anti-PLP-J serum or normal rabbit serum was added to the wells and incubated
for 1 h at 37 °C prior to plating of cells. Cells were harvested in normal
growth medium, washed with HBSS, and resuspended at 5 × 10 5
cells/ml in serum-free normal growth medium. Where indicated, cells were
incubated with heparin prior to plating for 1 h at room temperature. A
50-μl cell suspension was added to each well and incubated 30 min at 37
°C. Adherent cells were fixed in 10% formalin in saline for 30 min at room
temperature. The cells were stained with methylene blue, and adhesion was
quantified by dye extraction and measurement of absorbance at 620 nm
( 56). Nb2 Lymphoma Cell Proliferation Assay The Nb2 lymphoma cell proliferation assay was performed with some
modifications to the previously published procedure
( 44). Twenty-four h before
initiating the assays, cells were incubated with RPMI1640 supplemented with 5%
horse serum, 2 m m l-glutamine, 5 m m HEPES, and
antibiotics (Assay Medium) to establish a quiescent state. Cells were washed
with Assay Medium, counted with a hemocytometer, and distributed to wells in a
96-well plate (2.5 × 10 4 cells/well). Cells were incubated
with ovine PRL (0.1, 1, or 10 ng/ml; NOBL Laboratories, Inc., Sioux Center,
IA) or mFLAG-PLP-J (0.1, 1, or 10 μg/ml) for 72 h. Viable cells were
quantified by the CellTiter 96 AQ uous nonradioactive cell
proliferation assay (Promega, Madison, WI). Rat Uterine Stromal Cell Proliferation Assay Rat U1 uterine stromal cells were harvested by trypsin-EDTA, washed, and
resuspended in DMEM supplemented with 10% FBS, 1 mm sodium
pyruvate, and 100 units/ml penicillin and 100 μg/ml streptomycin. Cells
were distributed in 96-well plates at a density of 1 × 103
cells/well and incubated for 24 h. To initiate the assays, cells were starved
for 24 h with DMEM medium containing 1% FBS. Cells were then treated with
mFLAG-PLP-J (0.01, 0.1, 1, or 10 μg/ml) or fibroblast growth factor 2
(FGF2; 10 ng/ml; R&D Systems, Minneapolis, MN) for 24, 48, or 72 h.
Numbers of viable cells were measured by the CellTiter 96 AQuous
nonradioactive cell proliferation assay (Promega) at the indicated times. To
assess DNA synthesis, cells were grown as described above and distributed in
24-well plates. Bromodeoxyuridine (BrdUrd; Sigma) was added to the medium (10
μm) for 16 h in the presence or absence of mFLAG-PLP-J. The
BrdUrd incorporation was detected using a BrdUrd staining kit (BD
Pharmingen). Endothelial Cell Proliferation Assay Rat aortic endothelial cells were harvested by trypsin-EDTA, washed, and
resuspended in complete MCDB-131 medium. The cells were distributed in 48-well
plates at a density of 4 × 103 cells/well and incubated for
24 h. To initiate the experiments, cells were incubated with MCDB-131 basal
medium with 1% FBS for another 24 h. Cells were then treated with different
concentrations of mFLAG-PLP-J (0.01, 0.1, 1, or 10 μg/ml) or FGF2 (10
ng/ml) for 24, 48, and 72 h. The numbers of viable cells were measured using
the CellTiter 96 AQuous nonradioactive cell proliferation assay
(Promega). The effects of PLP-J on endothelial cell DNA synthesis were
determined with the BrdUrd incorporation assay as described above. Cell Proliferation Monitored by Crystal Violet Staining Proliferation indices of U1 rat uterine stromal cells and rat aortic
endothelial cells were also evaluated morphologically by crystal violet
staining, as previously described
( 57,
58). Cells were harvested with
trypsin-EDTA, washed, resuspended at 5 × 10 4 cells/ml in
growth medium, distributed in 48-well plates, and cultured for 24 h. Cells
were washed and preincubated with medium containing 1% FBS for 24 h. Test
proteins were then added to the cells. At the indicated time intervals, cells
were stained with 0.1% crystal violet for 10 min, washed with distilled water,
and dried at room temperature. Images of cells were captured with a Leica
MZFIII stereomicroscope equipped with a CCD camera (Leica). Statistical Analysis Statistical comparisons between two means were determined with Student's
t test. Comparisons among multiple means were evaluated with analysis
of variance. The source of variation from significant F-ratios was
determined with Bonferroni's multiple comparison test
( 59). Generation of PLP-J Fusion Proteins and Antibodies—PLP-J was
engineered to be expressed in recombinant form containing FLAG and
His 6 tags at its N terminus (mFLAG-PLP-J) in HEK 293 cells.
Purified mFLAG-PLP-J consisted of three species with molecular masses ranging
from ~31 to 37 kDa (). PLP-J possesses two putative N-linked
glycosylation sites
( 31– 34);
thus, differential glycosylation may account for the mFLAG-PLP-J size
variants. mFLAG-PLP-J was used as a ligand to evaluate PLP-J cellular actions
(see below). PLP-J was also expressed as a His 6-tagged fusion
protein in a bacterial expression system. The His 6-tagged PLP-J
fusion protein migrated predominantly as a 26-kDa protein
() and was used
as an immunogen in rabbits. PLP-J antibodies reacted with both the mFLAG-PLP-J
and His 6-tagged PLP-J fusion proteins
(). AP and AP-PLP-J fusion proteins were generated in HEK
293 cells ()
and used as probes to assess PLP-J interactions with putative cellular and
molecular targets. Intrauterine PLP-J Expression and Binding—PLP-J expression
patterns and PLP-J targets were investigated. The distributions of PLP-J and
dPRP mRNAs within the uterus of pregnant rats were assessed by in
situ hybridization and were consistent with earlier reports
( 29,
30,
32,
34). PLP-J and dPRP mRNAs were
predominantly observed in the uterine decidua, especially within the
antimesometrial decidua (). PLP-J and dPRP protein immunolocalizations
mirrored the distribution of PLP-J and dPRP mRNAs
(). Decidual PLP-J migrated as a single 29-kDa protein
and thus differed from the multiple molecular species of recombinant HEK 293
cell-engineered mFLAG-PLP-J (). The specificity of the PLP-J immunoreactivity was
demonstrated by competition with purified mFLAG-PLP-J
(, lanes 2 and 4). PLP-J interactions
with structures within gestation day 8.5 implantation sites were also
investigated (). AP-PLP-J
binding was detected throughout the uterus and was particularly evident in
association with the uterine vasculature
() and the undifferentiated uterine stromal cell
compartment ().
Intrauterine distributions of AP-PLP-J binding and RECA-1 immunoreactivity (a
rat endothelial cell-specific antibody) overlapped. Intrauterine AP-PLP-J
binding was specific () and could be competed by mFLAG-PLP-J
(), heparin
(), heparan
sulfate (), and
dermatan sulfate () but not chondroitin sulfate A or C
(). In summary, PLP-J protein is synthesized by uterine
decidual cells situated proximal to the developing embryo and specifically
interacts with intrauterine structures, especially the vasculature and
undifferentiated uterine stromal cells, in a heparin/heparan sulfate- and/or
dermatan sulfate-dependent manner. PLP-J Interacts with Heparin—Based on the heparin-dependence
of the AP-PLP-J-uterine tissue binding, we further investigated PLP-J-heparin
interactions. dPRP, another decidual cell cytokine, also specifically
interacts with heparin ( 22,
39) and was used as a positive
control in some of the analyses. AP-PLP-J and AP-dPRP specifically bound
heparin-BSA-coated plates but not BSA-coated plates, whereas AP did not bind
to either plate (). AP-PLP-J also exhibited binding to CHO cells that
was dependent upon the presence of heparan sulfate
(). Additional PLP-J-cellular interactions that are
potentially physiologically relevant (uterine stromal cells and endothelial
cells; see ) were
assessed. AP-PLP-J specifically interacted with both uterine stromal cells
() and
endothelial cells (). Heparin was an effective competitor of AP-PLP-J
binding to uterine stromal cells () but was less effective in competing with AP-PLP-J
binding to endothelial cells (). Collectively, the results indicate that PLP-J
associates with heparin-containing molecules, potentially located in
extracellular matrices and on cell surfaces. PLP-J Interacts with Syndecans—Syndecans are a family of
four transmembrane proteins possessing heparan sulfate sugar chains associated
with their extracellular domains
( 60,
61). They have been implicated
in cell adhesion and growth factor signal transduction and were considered
molecular candidates for PLP-J interaction with its target cells. Initially,
uterine decidua, uterine stromal cells, and endothelial cells were assessed
for their expression of syndecan family members by Northern blotting
(). Gestation
day 8.5 uterine decidua expressed Sdc1 and Sdc4 mRNAs,
whereas uterine stromal cells and endothelial cells expressed only
Sdc1 mRNA. Sdc2 and Sdc3 were not detected in
decidua and the two cell populations. Raji cells are devoid of heparan
containing molecules on their surfaces and represent a useful cell model for
testing the biology of specific heparan sulfate proteoglycans
( 45). The ability of PLP-J to
bind syndecans was evaluated in Raji cells genetically engineered to express
Sdc1 ( 45). AP-PLP-J
interactions with Raji cells were shown to be dependent upon the surface
expression of Sdc1 (). Thus, syndecans are expressed in the uterine decidua
and are potential mediators of PLP-J-cellular interactions. PLP-J Does Not Activate the Canonical PRL Receptor Signaling
Pathway—A subset of members of the PRL family influence target cell
function through activation of the PRL receptor signaling pathway
( 11). This subset includes
PRL, PL-I, and PL-II (also known as PRL family 3, subfamily b, member 1
(Prl3b1)). Interestingly, PLP-J is also a member of the PL-I and PL-II
subfamily. Its interactions with the PRL receptor signaling pathway were
evaluated in the next series of experiments. Initially, binding experiments
were performed with heparan sulfate-deficient CHO-pgsD-677 cells transfected
with an expression vector containing the long form of the rat PRL receptor.
AP-PL-I effectively interacted with the engineered cells, whereas AP-PLP-J was
ineffective (). AP-PLP-J was also ineffective in binding to
tissue sections of the rat corpus luteum and liver, tissues with abundant PRL
receptor expression (). Rat Nb2 lymphoma cell proliferation has been
used as a bioassay for canonical PRL receptor activation
( 44). PRL stimulated Nb2
lymphoma cell proliferation; however, PLP-J was ineffective
(). The results
indicate that PLP-J is not a PRL receptor agonist. PLP-J Promotes Uterine Stromal Cell and Endothelial Cell
Adhesion—Since PLP-J interacts with heparin-containing molecules
and heparin-related molecules are located on the cell surface of uterine
stromal cells and endothelial cells, we next assessed the effectiveness of
PLP-J in promoting uterine stromal cell and endothelial cell adhesion. In
these experiments, fibronectin was used as a positive control and PRL as a
negative control. PLP-J and fibronectin facilitated both uterine stromal cell
and endothelial cell adhesion, whereas PRL was ineffective
(). Co-incubation with heparin decreased cellular
adhesion to PLP-J. Heparin was more potent in interfering with PLP-J-mediated
endothelial cell adhesion () than with PLP-J-mediated uterine stromal cell
adhesion ().
PLP-J-dependent adhesion was also disrupted by co-incubation with PLP-J
antibodies (). The same antibodies did not interfere with
fibronectin-mediated cellular adhesion. The findings indicate that PLP-J can
act as an adhesion molecule for both uterine stromal cells and endothelial
cells. PLP-J Modulation of Uterine Stromal Cell and Endothelial Cell
Proliferation—Uterine stromal cells and endothelial cells undergo
dynamic changes during the establishment of pregnancy
( 1– 4).
The effects of PLP-J in modulating uterine stromal cell and endothelial cell
proliferation were investigated with three complementary assays (Figs.
and
). PLP-J stimulated uterine
stromal cell proliferation () and uterine stromal cell incorporation of
BrdUrd (). In
contrast, PLP-J inhibited endothelial cell proliferation and endothelial cell
incorporation of BrdUrd (). The effects of PLP-J on its cellular targets were
most impressive after 72 h of incubation (Figs.
and
). The minimal
effective PLP-J concentration was 1 μg/ml (Figs.
and ). PLP-J also inhibited FGF2-stimulated endothelial
cell proliferation (). In summary, PLP-J stimulates uterine stromal cell
proliferation and inhibits endothelial cell proliferation. Evidence is accumulating for the involvement of the PRL family of cytokines
in the regulation of maternal adaptations to pregnancy
( 11). In this report, we
provide insights into a role for PLP-J, a member of the PRL family, in
controlling intrauterine events during the establishment of pregnancy. The
PLP-J protein was characterized, and its molecular and cellular interactions
were investigated. PLP-J is expressed in uterine decidual cells and is a
heparin-binding cytokine with potential paracrine actions on stromal and
vascular cell development. PLP-J is an intrauterine cytokine. The PLP-J protein is localized to a
subpopulation of decidual cells situated proximal to the developing embryo
(present study). This observation is consistent with earlier reports on PLP-J
mRNA expression patterns
( 31– 34).
The decidual PLP-J protein has a molecular size of ~29 kDa and possesses
heparin-binding properties. The expression profile and heparin binding
features are also characteristics of another decidual PRL family cytokine,
dPRP ( 22,
27,
29,
30,
39). PLP-J expression is also
positively modulated by dPRP
( 16). This relationship may be
indirect through dPRP promotion of decidual cell survival. In humans, PRL is
the sole member of the PRL family
( 11,
62). It is expressed in both
pituitary and extrapituitary sites, including decidual cells, and also
possesses heparin binding properties
( 9,
10,
63). Rodent PRLs and most
members of the PRL family do not exhibit heparin binding characteristics. The
decidual cell expression of PLP-J, dPRP, and human PRL and their restricted
distribution through interactions with heparin-containing molecules suggest
that they act locally as cytokines within the vicinity of the embryo
implantation site. PLP-J interacts with and regulates specific cellular constituents of the
uterus. Stromal cells and endothelial cells are among the putative
intrauterine PLP-J targets. PLP-J can promote uterine stromal and endothelial
cell adhesion. These interactions are mediated, at least in part, through
heparin-containing molecules. Heparin-containing molecules are prominently
displayed on the plasma membrane of cells and within the extracellular
matrices of tissues ( 64).
These molecules modulate development and are disrupted in some disease
processes. PLP-J may not discriminate and instead associate with any uterine
cell type possessing surface heparan sulfate proteoglycans in its proximity.
Stromal cells and endothelial cells are prominent components of the
preimplantation uterus, which undergo marked changes following embryo
implantation ( 3,
5). Stromal cells serve as
precursors for decidual cell differentiation, and endothelial cells line the
intrauterine vasculature. During the establishment of pregnancy in mice and
rats, the intrauterine vasculature undergoes specific regional modifications
( 3,
5,
65). Blood vessels in the
mesometrial compartment (site of the developing chorioallantoic placenta) are
significantly expanded in comparison with the anti-mesometrial uterine
vasculature. In our in vitro analysis, PLP-J differentially affected
the behavior of uterine stromal cells and endothelial cells. PLP-J stimulated
proliferation of uterine stromal cells while inhibiting the proliferation of
endothelial cells. These results indicate that PLP-J is a biologically active
cytokine with the potential to regulate the intrauterine environment. The
in vivo biological impact of decidual cell PLP-J production will be
influenced by its access to its cellular targets and its relative contribution
to the milieu of other growth factors and cytokines elaborated at the
uteroplacental interface. Relevant biologically active concentrations are difficult to determine for
PLP-J. The heparin-binding properties of PLP-J will direct PLP-J to the cell
surface or extracellular matrix, where it will have a juxtacrine mode of
action. Thus, in an in vivo setting, PLP-J is sequestered and
requires liberation from its tether to find its targets, or alternatively, the
cellular targets may have to be brought to the sequestered PLP-J. PLP-J modulates cell activities through mechanisms that are not yet
defined. Some insights about PRL family member activation of signal
transduction cascades are available. A subgroup of PRL family members (PRL,
PL-I, and PL-II) utilizes the canonical PRL receptor signaling pathway
( 11). Among all members of the
PRL family, PLP-J is most similar in amino acid sequence to the placental
lactogens
( 31– 34);
however, PLP-J does not bind to or activate the PRL receptor (present study).
Apparently, the critical amino acids dictating PRL receptor recognition are
not present in PLP-J. Other differences from its closest relatives (PL-I and
PL-II) are also evident. PLP-J possesses a domain(s) facilitating interactions
with heparin-containing molecules. This ligand-directed heparin binding may be
a key to understanding the cellular actions of PLP-J. Some heparan sulfate
proteoglycans, such as syndecans, are transmembrane proteins capable of
interacting with heparin-binding growth factors and mediating the activation
of signal transduction cascades
( 66,
67). Decidual cells, uterine
stromal cells, and endothelial cells express members of the syndecan family,
and PLP-J specifically binds to Sdc1. Thus, PLP-J could modulate cellular
action directly via engaging transmembrane heparan sulfate proteoglycans (such
as syndecans) or by interfering with the actions of other heparin-binding
growth factors ( e.g. fibroblast growth factors) that utilize
transmembrane heparan sulfate proteoglycans. Differential expression of
heparan sulfate proteoglycans could be responsible for the distinct effects of
PLP-J on uterine stromal cells versus endothelial cells. Components
of PLP-J activities were independent of interactions with heparin-containing
molecules, suggesting that there may be additional modes of PLP-J action for
some of its cellular targets. Among these may be interactions with specific
membrane-associated receptor signaling pathways or possibly other
glycosaminoglycans, including dermatan sulfate. PLP-J is capable of
interacting with dermatan sulfate, and the uterine endometrium possesses
dermatan sulfate proteoglycans
( 68). However, possible roles
for dermatan sulfate proteoglycans in modulating PLP-J signaling have yet to
be determined. The PRL gene family expanded during the evolution of rodents
( 11,
17). New genes were derived
from an ancestral template, which encode hormones and cytokines that are
linked to viviparity and reproductive adaptations. PLP-J is part of the
expanded rodent PRL family. It possesses a unique intrauterine tissue
distribution and a novel intrauterine mode of action that is mediated, at
least in part, through interactions with heparan sulfate proteoglycans. We acknowledge the technical assistance of Chunbin Li and acknowledge the
generous gifts of the U1 cell line from Dr. Virginia Rider, the Nb2 cell line
from Dr. Peter Gout, the Raji cell lines from Dr. Alan C. Rapraeger, and the
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