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Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160
Address all correspondence and requests for reprints to: Dr. Michael Soares, Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160. E-mail: msoares{at}kumc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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DASH II Wistar-Kyoto rat genomic library was
screened with a labeled d/tPRP complementary DNA, resulting in the
isolation of two phage clones, RGLd-41 [17.7 kilobases (kb)] and
RGLd-42 (15.8 kb). RGLd-41 alone was found to contain the full-length
d/tPRP gene and was used for subsequent analyses. The d/tPRP gene
possesses a six-exon, five-intron organization. Relative to other
highly conserved members of the PRL gene family, d/tPRP contains a
single small additional exon (exon 3) situated between exons 2 and 3 of
the prototypical PRL gene. The region corresponding to exon 3 of d/tPRP
encodes for a unique amino acid region found in a subset of PRL family
members. A reverse transcription-PCR (RT-PCR) tissue survey for d/tPRP
messenger RNA revealed that d/tPRP expression was restricted to
decidual and trophoblast tissues. A single transcription start site 65
bp upstream of the initiation codon was identified in decidual tissue,
whereas multiple transcription start sites ranging from 61–66 bp
upstream of the initiation codon were detected in placental tissue.
Various tissue culture systems (primary cultures and cell lines) were
evaluated for d/tPRP expression and activation of a 3.96-kb d/tPRP
promoter-luciferase reporter construct. Decidual, spongiotrophoblast,
and trophoblast giant cell populations expressed d/tPRP and were
capable of activating the d/tPRP promoter-reporter construct, whereas
other cell types were ineffective. Limited d/tPRP promoter activation
was noted in uterine stromal cell lines. In summary, d/tPRP possesses a
unique six-exon, five-intron gene structure and exhibits cell-specific
expression that is regulated at least in part by a 3.96-kb 5'-flanking
region. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functions of the decidua and placenta are probably mediated in part by their production of hormones and cytokines. In the rat, at least nine different genes, structurally related to PRL, are expressed by uterine decidual cells and/or trophoblast cells of the chorioallantoic placenta in a highly coordinated pattern during pregnancy (4). Decidual/trophoblast PRL-related protein (d/tPRP) is an example of a subset of PRL family members that is dually expressed in both decidual and trophoblast cell types (5, 6, 7). Some members of the PRL family use the PRL receptor signaling system and mimic the actions of PRL, whereas other members of the family activate apparently novel signaling pathways and possess apparently novel biological activities (4). d/tPRP falls into this latter category. It does not activate PRL receptor signaling pathways; however, d/tPRP does interact with heparin-containing molecules, and it increases the tumorigenicity of Chinese hamster ovary cells (8). The mechanisms through which the expression patterns and actions of d/tPRP facilitate the establishment and maintenance of pregnancy are yet to be resolved.
In this report, we present information on the isolation and characterization of the d/tPRP gene. We show that d/tPRP possesses a unique six-exon, five-intron gene structure and that regulatory DNA associated with the d/tPRP gene directs decidua- and trophoblast-specific expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and tissue preparation
Holtzman rats were obtained from Harlan Sprague-Dawley
(Indianapolis, IN). The animals were housed in an environmentally
controlled facility, with lights on from 0600–2000 h, and allowed free
access to food and water. Timed pregnancies and tissue dissections were
performed as previously described (9). Day 0 of pregnancy was defined
by the presence of a copulatory plug or sperm in the vaginal smear.
Protocols for the care and use of animals were approved by the
University of Kansas animal care and use committee.
Cell cultures
A series of primary cell cultures and cell lines was evaluated
for their ability to express d/tPRP and activate d/tPRP
promoter/luciferase reporter constructs. Primary decidual cell cultures
were established from deciduomal tissue collected from rats on day 7 of
pseudopregnancy. Deciduomal tissue was minced, washed three times in
Hanks’ Balanced Salt Solution, and dispersed in dispase (2.4 U/ml)
containing deoxyribonuclease I (80 U/ml) for 1.5 h at 37 C.
Dispersed cells were recovered by centrifugation and washed with
Hanks’ Balanced Salt Solution to remove residual dispase. Cells were
then resuspended in DMEM-MCDB 302 culture medium containing 10% FBS
and plated at a concentration equivalent to one uterine horn (7–9
x 105 cells)/25-cm2 flask. After 20 h,
medium and unattached cells were removed and replaced with fresh medium
containing 1% FBS. Primary spongiotrophoblast cultures were
established according to previously published procedures (10) and
maintained in DMEM culture medium supplemented with 10% FBS. The UI
uterine stromal cell line was established (11) essentially as described
by Cohen et al. (12). CUS V2 and CUS V4 uterine stromal
cells are immortalized cells derived from rat uterine stroma by
transfecting primary cultures with a temperature-sensitive mutant of
the simian virus 40 large T antigen (13). All uterine stromal cell
lines were maintained in Ham’s F-10-DMEM culture medium supplemented
with 10% FBS. The Rcho-1 trophoblast cell line was derived from a rat
choriocarcinoma and is capable of differentiating along the trophoblast
giant cell lineage (14). Rcho-1 trophoblast cells were routinely
maintained in subconfluent conditions with NCTC-135 culture medium
supplemented with 20% FBS, 50 µM 2-mercaptoethanol, and
1 mM sodium pyruvate (14). Rcho-1 cells were induced to
differentiate by growing them to near confluence in FBS-supplemented
culture medium and then replacing the FBS with 10% horse serum (15, 16). The HRP-1 trophoendodermal stem cell line, which exhibits both
trophoblast and yolk sac attributes (17, 18), was maintained in RPMI
1640 medium containing 10% FBS. GH3 pituitary tumor cells
(19) were maintained in DMEM culture medium supplemented with 10% FBS,
and L929 mouse fibroblast cells were maintained in RPMI 1640 medium
containing 10% FBS. All culture media were supplemented with 100 U/ml
penicillin and 100 µg/ml streptomycin.
Isolation and characterization of the d/tPRP gene
A genomic DNA library generated from liver tissue of 12-week-old
male Wistar-Kyoto outbred rats and packaged in the DASH II vector
was obtained from Stratagene. The library was screened with a rat
d/tPRP complementary DNA (cDNA) as previously described (5). DNA
from positive plaques was amplified and used to inoculate LE 392
Escherichia coli. Phage DNA was extracted from lysates and
characterized by restriction mapping and Southern blot hybridization
(20).
Two d/tPRP genomic clones were identified, RGLd-41 and RGLd-42.
Oligonucleotides representing either the 5'- or 3'-end of d/tPRP cDNA
were end labeled with T4 polynucleotide kinase and
[
-32P]ATP and used to identify clones containing the
full-length d/tPRP gene. Genomic DNA was excised with NotI,
and a restriction map was generated using partial BamHI
digestion (Flash Nonradioactive Gene Mapping kit). Fragments containing
d/tPRP exonic DNA (determined by Southern blot hybridization) were
subcloned into the BamHI and/or NotI sites of
pBluescript SK+, flanked by T7 and T3 promoters.
Exon-containing restriction fragments in pBluescript SK+ were sequenced by the dideoxy chain termination method (21) using Sequenase and [35S]deoxy-ATP. Primers corresponding to the T7 or T3 flanking sequence or internal oligonucleotide primers were used to sequence all exons and exon/intron boundaries. Reaction products were resolved in 6% polyacrylamide urea gels, dried, and exposed to Kodak X-Omat X-ray film. Comparison with the published d/tPRP cDNA sequence confirmed the identity of the d/tPRP genomic clone (5).
Identification of the d/tPRP transcription start sites
The transcription start site of d/tPRP was determined by primer
extension analysis, essentially as described by Duckworth et
al. (22). An oligonucleotide complementary to bases +31 to +10 of
the d/tPRP cDNA (from ATG) was synthesized and end labeled using T4
polynucleotide kinase and [-32P]ATP. The labeled
primer (final concentration, 10 µM) was extracted with
phenol-chloroform, precipitated with ethanol, and hybridized with 5
µg total RNA from decidua, junctional zone placenta, or spleen.
Reverse transcription was performed using a Superscript
Preamplification kit. The reaction was stopped by the addition of
sample running buffer (98% formamide, 2.5 mM EDTA, 0.1%
bromophenol blue, and 0.1% xylene cyanol) and electrophoretically
separated on a 6% polyacrylamide-7 M urea sequencing gel.
A known DNA sequence was separated on the same gel to indicate the size
of the primer-extended product.
Analysis of d/tPRP expression
Western blot analyses for d/tPRP were performed as previously
described (8). Samples were separated by 12.5% PAGE under reducing
conditions and transferred to nitrocellulose membranes. Immunoreactive
bands were visualized using a chemiluminescent detection system
(Arlington Heights, IL).
Tissue and cellular localization of d/tPRP was confirmed by immunocytochemistry using a streptavidin-biotin immunoperoxidase kit for rabbit IgG and the chromagen, diaminobenzidine (7). The immunostained sections were counterstained with hematoxylin. The specificity of the immunoreactions was demonstrated using preimmune serum and preadsorbed antibodies.
Northern blots were performed as previously described (5, 23). Total RNA was extracted from tissues and cells essentially as described by Chomczynski and Sacchi (24), using TRIzol. Blots were probed with 32P-labeled d/tPRP cDNA (5).
RT was performed using 0.5 µg oligo(deoxythymidine) primers and 5 µg total RNA. The resulting cDNAs were amplified by PCR for 35 cycles with a denaturing temperature of 94 C (1 min), an annealing temperature of 60 C (2 min), and an extension temperature of 72 C (2 min), using a Perkin-Elmer Thermocycler (model 480, Norwalk, CT). Oligonucleotide primers specifically amplified a 342-bp region of the d/tPRP cDNA: upstream primer, 5'-CATGGACCTGAACATGAAAACATCAAA-3' (sense, 325–354; located on exon 4); and downstream primer, 3'-GTGACGGATGCACAACTATATAAGATG-5' (antisense, 637–666; located on exon 6). The PCR reaction mixture also contained primers that amplified a 244-bp region of rat ß-actin (25). ß-Actin bands were readily detectable on ethidium bromide-stained gels and used to demonstrate equal loading and integrity of the messenger RNA (mRNA) template. Reaction products were fractionated in agarose gels and transferred to nitrocellulose. Southern blots were performed using 32P-labeled d/tPRP cDNA and visualized on Kodak X-Omat X-ray film (Eastman Kodak, Rochester, NY).
d/tPRP promoter analysis
Cell-specific d/tPRP promoter activation was evaluated in
various primary cell cultures and cell lines. PCR was used to amplify
3960 bp of d/tPRP 5'-flanking DNA from the 6.9-kilobase (kb)
restriction fragment of the d/tPRP genomic clone. The 3960-bp amplified
fragment extended to the cytosine residue located 38 bp downstream of
the transcription start site and 28 bp upstream of the translation
start site (ATG; see Fig. 2
). The Advantage Genomic PCR kit was used to
amplify a promoter fragment using the high fidelity Tth DNA
polymerase. PCR primers were designed so the amplified fragment
contained a KpnI restriction site at the 5'-end and an
XhoI site at the 3'-end. This allowed directional cloning of
the d/tPRP 5'-flanking DNA into the KpnI and XhoI
sites of the pGL-2 basic, luciferase reporter vector. We will refer to
this construct as d/tPRP-luc.
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Rcho-1 trophoblast cells were also cotransfected with the d/tPRP-luc construct (3 µg/ml; 10-fold excess) and pSV2 neo (0.3 µg/ml; a plasmid providing neomycin resistance). Cells were selected for 2 weeks in the presence of geneticin (G418; 250 µg/ml) as we previously described (28). Stably transfected clones were isolated by limiting dilution. Cellular lysates were collected from stably transfected clones at various times during the transition from proliferation to differentiation states and evaluated for luciferase activity as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DASH II rat genomic library with labeled
d/tPRP cDNA resulted in the isolation of two phage clones, RGLd-41
(17.7 kb) and RGLd-42 (15.8 kb). Dot blot analysis revealed that
RGLd-41 alone contained the full-length d/tPRP gene. A restriction map
was generated based on a partial digestion with BamHI (Fig. 1). Complete NotI/BamHI digestion
resulted in the generation of five fragments, ranging from 2.1–6.9 kb
in size (Fig. 1). Southern blots were performed on the resulting
digestion products using random primer-labeled d/tPRP cDNA as a probe.
Three exon-containing fragments were identified (open boxes
in Fig. 1) and subcloned into pBluescript SK+ for
sequencing.
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and 2). Relative to the other members
of the PRL family, d/tPRP contains one additional exon, exon 3 (Fig. 3). A homologous exon 3 is also present in PRL-like
protein-C variant (PLP-Cv) (29). Exon 3 encodes a region that is also
homologous with unique regions of mouse proliferin-related protein
(mPRP) (30), PLP-C (31), and PLP-D (32). However, the genomic
structures of these PRL family members have not been characterized.
This exon is part of a larger 14-amino acid region that has been termed
the aromatic domain because it contains three phenylalanines and one
tyrosine (see Fig. 2).
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). A weaker d/tPRP signal was also observed in the
labyrinth zone, but probably resulted from junctional zone
contamination. In support of this contention, d/tPRP expression was not
detectable in labyrinthine trophoblast by in situ or
immunocytochemical analyses (7).
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, upper panel). Immunoreactive
d/tPRP increased from days 1–3 of culture, as demonstrated by Western
blot (Fig. 5, lower panel) and immunocytochemical analyses
(Fig. 6). In vivo, d/tPRP immunoreactivity
was localized predominantly to the antimesometrial region of the
decidua (Fig. 6d).
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). In addition to the primary decidual
cell culture, d/tPRP expression was monitored in primary
spongiotrophoblast culture, the Rcho-1 trophoblast cell line, several
uterine stromal cell lines (CUS V2, CUS V4, and UI), L929 mouse
fibroblasts, HRP-1 trophoendodermal cells, and GH3
pituitary tumor cells. Only the primary decidual cells, primary
spongiotrophoblast cells, and differentiated (day 9) Rcho-1 trophoblast
cells were capable of expressing detectable levels of d/tPRP mRNA.
Collectively, these results indicate that d/tPRP is expressed in both a
cell-specific and differentiation-dependent manner.
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). In contrast, the placenta had several apparent
transcription start sites between 61–66 bases upstream of the
translation start site (Fig. 8). Spleen RNA served as a negative
control and had no primer-extended products. The sequence of the
proximal promoter revealed a consensus TATA box (TATATAA) at positions
-28 to -22 relative to the transcription start site (Fig. 2, underlined and shaded).
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demonstrate that 3960 bp of d/tPRP
5'-flanking DNA promoted a 60-fold induction of the luciferase reporter
relative to the empty pGL-2 vector in primary decidual cells. Lower
levels of d/tPRP promoter activity were also detected in CUS V4 and UI
uterine stromal cell lines (Fig. 9, upper panel). Similarly,
the 3960-bp d/tPRP promoter was able to drive reporter expression in
Rcho-1 trophoblast cells and the spongiotrophoblast primary culture
(Fig. 9, lower panel; 700- and 400-fold induction over
pGL-2, respectively). No significant reporter activity was observed in
L-929, CUS V2, GH3, or HRP-1 cells. The positive control,
RSV-luc construct, was functional in all cell lines tested.
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).
d/tPRP-Rcho-3 luciferase activity increased 95-fold between days 2–13
of culture.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inclusion in the PRL gene family is largely based on structural relationships among the members. Sequence analysis of the original d/tPRP cDNA placed it in the PRL family (5). Among the many striking similarities of members of the PRL gene family is their exon/intron organization. The PRL gene has retained a characteristic five-exon/four-intron configuration across all species where a structure has been reported (33). Data for other members of the PRL family, including placental lactogens (PLs), PRL-like proteins, and PRL-related proteins, are less abundant. Nonetheless, bovine PL, mouse PL-II, and mouse proliferin contain a similar five-exon/four-intron organization (34, 35, 36). d/tPRP exhibits a distinctive variation from this common alignment and represents an exonic structure likely to be characteristic of a subset of PRL family members referred to as the PLP-C subfamily. The PLP-C subfamily includes PLP-C, PLP-Cv, PLP-D, and d/tPRP (5, 29, 31, 32). Each constituent of this subfamily shows considerable overall homology and contains a unique segment of amino acids that are rich in aromatic residues, which is not present in other members of the PRL family. The genomic structures of two members of the subfamily (PLP-Cv and d/tPRP) have now been reported (Ref. 29 and the present findings). Both possess a structure homologous to those of other PRL family genes, except for an additional small exon located between exons 2 and 3 of the prototypical PRL gene structure. This unique exon 3 encodes a region rich in aromatic amino acids. We hypothesize that PLP-C and PLP-D also exhibit a six-exon/five-intron organization similar to those of PLP-Cv and d/tPRP. A member of the mouse PRL family, mPRP, although showing limited overall homology to the PLP-C subfamily, does contain the aromatic-rich domain characteristic of the PLP-C subfamily (30). Although the mPRP gene has not been cloned, we postulate that its aromatic-rich region may also be encoded by an additional exon. Previously, this similarity led us to speculate that members of the PLP-C subfamily may possess biological activities similar to those of proliferin-related protein, a potent antiangiogenic factor (8, 37). Initial experimentation has not supported a role of d/tPRP in the modulation of angiogenesis in the uteroplacental environment (8). Thus, inclusion of the aromatic domain in a protein structure does not appear to be solely responsible for instilling endothelial cell-targeted actions. The availability of the d/tPRP gene provides us with a necessary prerequisite for the implementation of additional molecular and transgenic strategies directed toward understanding the role of d/tPRP in the physiology of pregnancy in the rat.
The d/tPRP gene is specifically expressed in maternal and extraembryonic cells (Ref. 7 and the present study). Immediately, postimplantation d/tPRP expression is initiated in decidual cells located predominantly in the antimesometrial compartment (7). The antimesometrial decidua regresses around midgestation, and d/tPRP expression arises in trophoblast giant cells and in newly forming spongiotrophoblast cells of the junctional zone in the chorioallantoic placenta (7). Thus, there are three recognizable differentiated cell types capable of expressing d/tPRP: 1) antimesometrial decidual cells, 2) trophoblast giant cells, and 3) spongiotrophoblast cells. The decidua-trophoblast shift in d/tPRP expression resembles that reported for PLP-B (38, 39, 40). However, in contrast to d/tPRP, PLP-B is expressed at relatively low levels in decidua, and within the chorioallantoic placenta its expression is restricted to spongiotrophoblast cells (38, 39, 40).
Analysis of regulatory mechanisms controlling cell-specific gene expression is facilitated by the availability of in vitro strategies. In the present report, we demonstrated the usefulness of primary culture systems for decidual and spongiotrophoblast cells and the value of the Rcho-1 trophoblast cell line. The latter paradigm represents a valuable method of dissecting regulatory mechanisms controlling trophoblast giant cell-specific gene expression (41). The three cell culture models exhibit characteristics that reflect the three cell types capable of d/tPRP expression in situ. Each of these cell populations was shown in vitro to express d/tPRP and to specifically activate a 3960-bp d/tPRP promoter. Modest d/tPRP promoter activation was also seen in two uterine stromal cell lines (UI and CUS V4). Although these cell lines show minimal endogenous d/tPRP expression, they may represent a stem cell population with some potential to differentiate into decidual cells. Initial attempts to induce an antimesometrial decidual cell phenotype after progesterone and/or PGE2 treatment of the uterine stromal cell lines have proven unsuccessful (Orwig, K. E., and M. J. Soares, unpublished observations). Rcho-1 trophoblast cells, which exhibit controlled and robust trophoblast giant cell differentiation, activated the 3960-bp d/tPRP promoter in a differentiation-dependent manner. Using a similar experimental paradigm, the PL-I and cholesterol side-chain cleavage cytochrome P450 promoters were previously shown to be transcriptionally activated during trophoblast giant cell differentiation (28, 42, 43).
Regulatory mechanisms controlling the expression of decidua- and trophoblast-specific genes are just beginning to be elucidated. In the present report, we showed that regions located within 4 kb upstream of the d/tPRP gene are capable of specifically directing reporter gene expression to decidual, spongiotrophoblast, and trophoblast giant cells. It is likely that at least some of the regulatory elements controlling decidua-, spongiotrophoblast-, or trophoblast giant cell-specific expression will be differentially located within the 3960-bp d/tPRP promoter.
Decidual cell-specific promoter analyses have been limited to studies
on human insulin-like growth factor binding protein-1, human PRL, and
rat d/tPRP genes. The insulin-like growth factor binding protein-1 gene
is regulated in part via the interaction of the Sp3 protein with
cis-elements located between -2.8 to -2.6 kb of its
5'-flanking DNA (44). Decidual expression of PRL is directed by a 3-kb
regulatory region located approximately 6 kb upstream of the pituitary
transcription start site (45, 46, 47). This decidual regulatory region is
distinct from the region controlling pituitary PRL gene expression (46, 47). Progesterone and PGE2 are activators of pathways
controlling decidualization (1, 47, 48, 49, 50, 51) and may also activate
transcription of decidual-specific genes. Distinct expression patterns
for a few DNA-binding proteins, including the basic helix-loop-helix
transcription factor (bHLH) Hed/Thing-2/d-HAND (52, 53, 54), Wilms’
tumor-1 (55), and the retinoid X receptor-
(56), suggest the
existence of additional candidate regulators controlling
decidualization and decidual cell-specific gene expression. The
involvement of any of these factors or regulatory mechanisms in the
control of decidua-specific d/tPRP gene expression is currently being
evaluated.
d/tPRP expression in the placenta is restricted to trophoblast giant cells and spongiotrophoblast cells. The Rcho-1 trophoblast cell line has proven useful in the identification of at least some regulatory mechanisms controlling trophoblast giant cell-specific gene expression. AP-1, GATA, and E-box regulatory elements and their respective activators, fos, jun, GATA-2, GATA-3, and the bHLH transcription factor, Hxt/Thing-1/eHAND, have been implicated in transcriptional control of the trophoblast giant cell PL-I gene (42, 52, 57). Hxt/Thing-1/eHAND has also been postulated to direct differentiation along the trophoblast giant cell lineage (52). Calzonetti and colleagues (58) used a transgenic strategy to identify a 340-bp regulatory region that is essential for targeting the 4311 gene to spongiotrophoblast cells. This region contains E-boxes that may interact with a bHLH transcription factor, Mash-2, which was previously shown to be essential for development of the spongiotrophoblast cell lineage (59). Whether similar regulatory mechanisms are involved in trophoblast giant cell- and spongiotrophoblast-specific d/tPRP gene expression remain to be determined.
In summary, we have demonstrated that the d/tPRP gene is organized in a novel exon/intron arrangement and that the d/tPRP promoter directs cell type-specific and differ-entiation-dependent expression. Further evaluation of the d/tPRP promoter will facilitate the identification of cis-acting elements and trans-acting factors required for de-cidual- and trophoblast-specific gene expression and the molecular mechanisms controlling decidual and trophoblast differentiation.
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Acknowledgments |
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Footnotes |
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2 Supported by fellowships from the Lalor and Kansas Health
Foundations.
3 Present address: Department of Anatomy and Cell Biology,
University of Kansas Medical Center, Kansas City, Kansas 66160.
Received November 19, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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K. E. Orwig, R. Ishimura, H. Muller, B. Liu, and M. J. Soares Identification and Characterization of a Mouse Homolog for Decidual/Trophoblast Prolactin-Related Protein Endocrinology, December 1, 1997; 138(12): 5511 - 5517. [Abstract] [Full Text] [PDF]
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