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Endocrinology, doi:10.1210/en.2004-0415
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Endocrinology Vol. 145, No. 11 5243-5251
Copyright © 2004 by The Endocrine Society

Expression and the Biological Activities of Insulin-Like Growth Factor-Binding Protein Related Protein 1 in Rat Uterus during the Periimplantation Period

Kazuhiro Tamura, Takahiko Hara, Masahiko Kutsukake, Ken Iwatsuki, Mayuko Yanagida, Mikihiro Yoshie and Hiroshi Kogo

Department of Endocrine Pharmacology (K.T., M.K., M.Ya., M.Yo., H.K.), Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan; and Department of Tumor Biochemistry (T.H., K.I.), The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-0032, Japan

Address all correspondence and requests for reprints to: Kazuhiro Tamura or Hiroshi Kogo, Department of Endocrine Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1, Hachioji-shi 192-0392, Japan. E-mail: hiro{at}ps.toyaku.ac.jp; or kogo{at}ps.toyaku.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF binding protein-related protein 1 (IGFBP-rP1) is highly expressed in the rat uterus around the time of implantation. In the present study, we determined the periimplantation localization of IGFBP-rP1 mRNA and assessed the effects of recombinant IGFBP-rP1 on the proliferative and prostacyclin (PGI2)-producing abilities of cultured endometrial cells early in pregnancy. IGFBP-rP1 mRNA was detected at high levels in endometrial stromal cells close to the smooth muscle of interimplantation sites around the time of implantation but absent from decidual zones surrounding the embryo. Differential uterine IGFBP-rP1 expression was also recognized in the delayed implanting pregnant model, but the level of mRNA decreased as decidual tissues formed in the decidualization model. Recombinant IGFBP-rP1 inhibited the proliferation of endometrial stromal cells in vitro and arrested them in the G1 phase of the cell cycle. Furthermore, IGFBP-rP1 significantly stimulated PGI2 synthesis and cyclooxygenase II mRNA expression in myometrial cells, both of which are essential molecules for successful implantation. These data suggest that IGFBP-rP1 is an implantation-associated protein and that it modulates the proliferation of rat uterine cells and their production of PGI2 during the periimplantation period.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IMPLANTATION IS A complex process in which the embryo makes close contact with the maternal endometrium during the establishment of pregnancy. Successful communication between the blastocyst and uterus leads to normal implantation. In rats, a nidatory surge of estrogen on the afternoon of d 5 of pregnancy is essential for initiating implantation later that evening and in the early morning of d 6, thereby allowing the blastocyst to attach to the epithelial layers of the receptive uterus (1, 2). Extensive endometrial stromal cell (ESC) proliferation and differentiation (decidualization) are also necessary for the successful establishment of pregnancy (2, 3). Decidual tissues play a role in supplying nutrition to the developing embryo, protecting it from maternal immunologic responses and regulating trophoblast invasion into the uterine stroma.

We previously determined by a library subtraction technique that IGF binding protein-related protein (IGFBP-rP) 1 mRNA is highly expressed in the rat uterus on d 6 of pregnancy (4). The IGFBP-rP1 gene, which resides on chromosome 4q12, was originally identified by its decreased level of expression in meningioma cell lines (5), and it encodes a follistatin-like protein of 281 amino acids that binds to activin (6). It also exhibits high homology with IGF binding protein (IGFBP), it interacts with both IGF and insulin, and its C-terminal region shares sequence similarity with the fibroblast growth factor receptor. IGFBP-rP1 has also been termed meningioma-associated cDNA 25 (mac25), angiomodulin, and tumor-derived adhesion factor, and more recently IGFBP-7 (7, 8, 9). The IGFBP family, a crucial component of the IGF system, consists of two groups, high affinity binding proteins (IGFBP-1 to -6) and low affinity binding proteins (IGFBP-7 to -10) (9). Low-affinity IGFBPs, including IGFBP-rP1, may have other biological properties independent of their IGF binding capacity. IGFBP-rP1 modulates the metabolism and distribution of IGF, and it influences the ability of IGF to bind the IGF receptor. Recently a potential role for IGFBP-rP1 in human endometrial receptivity has been suggested (10), based on cDNA microarray data showing that it is the second most up-regulated gene in models of endometrial and epithelial receptivity. In the present study, we investigated the ontology of IGFBP-rP1 mRNA during early pregnancy and the biological activity of recombinant IGFBP-rP1 in cultured cells derived from the preimplantation rat uterus. We show that IGFBP-rP1 expression is enhanced in ESCs during the time of implantation, and we describe possible roles for IGFBP-rP1 in the control of uterine cell proliferation and prostacyclin (PGI2) production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and protocols
The animal care and surgery protocols used in the present studies were reviewed and approved by the Institutional Animal Care Committees of Tokyo University of Pharmacy and Life Science, in compliance with institutional guidelines for experimental animal care. Eight week-old female rats of the Wistar-Imamichi strain (Imamichi Institute for Animal Reproduction, Ibaraki, Japan) were mated at proestrus with 10-wk-old males of the same strain. The presence of a vaginal plug or sperm was considered as d 1 of pregnancy. Uteri were excised each morning (0900–0930 h) between d 3 and 14 of pregnancy and fixed with 4% paraformaldehyde solution for in situ hybridization or frozen in liquid nitrogen for RNA analysis. For the preparation of uterine cells, d 4 uteri were collected and subjected to enzyme digestion. Identification of implantation sites on d 6 was conducted as previously described (4). The tissues between the blue bands (nonimplantation sites) were defined as interimplantation sites. The implantation sites of d 6 uteri were separated from interimplantation sites by careful dissection, and tissues were pooled for RNA extraction. Delayed implantation was induced by hypophysectomy (11) on d 3, and pregnancy was maintained by daily sc injection of progesterone (3 mg/rat) dissolved in sesame oil. Six days after surgery, each rat was given sc 0.5 µg of 17ß-estradiol (E2) to initiate implantation, and implantation sites were isolated 24 h later. To induce pseudopregnancy, female rats were mated with vasectomized males of the same strain. On d 5 of pseudopregnancy, when the uteri were optimally sensitized to deciduogenic stimulus, 100 µl sesame oil were infused into the lumen of one of the uterine horns to induce artificial decidualization. The contralateral uterine horn, which was not infused with oil, served as a control. At 0–96 h after the intraluminal oil infusion, the rats were killed, and the uterine horns were isolated.

Northern blot analysis of IGFBP-rP1 mRNA
Uterine poly (A)+ RNA was prepared and transferred to nylon membranes, as previously described (4). For the preparation of cRNA probes, the digested cDNA (about 300 bp) of the IGFBP-rP1 gene cloned from rat uterus was subcloned into pGEM-T Easy Vector (Promega Corp., Madison, WI). Digoxigenin (DIG)-labeled antisense riboprobe was prepared using an RNA transcription kit (TOYOBO, Tokyo, Japan). After linearization of the plasmid, the cRNA probe was synthesized by in vitro transcription with DIG-11-UTP using T7 RNA polymerase. Northern blot analyses were performed using the DIG system (4). Glyceraldehyde phosphate dehydrogenase (G3PDH) or ß-actin was used as an internal control. Bands on scientific imaging film (X-Omat XB-1, Kodak, Rochester, NY) were quantitated by National Institutes of Health image analysis, and each value was normalized against that of the internal control in the corresponding lane.

In situ hybridization
Uteri harvested from rats on d 3–7 were directly fixed for 8 h in 0.15 M phosphate buffer (pH 7.45, room temperature) containing 4% paraformaldehyde. Paraffin-embedded sections (6 µm) were prepared for in situ hybridization and immunocytochemistry. As described previously (12), hybridization was performed in a humidified chamber at 40 C for 18 h, and bound probes were visualized using an alkaline phosphatase-conjugated anti-DIG antibody and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega) as substrates.

Preparation of expression vector and production of recombinant IGFBP-rP1
IGFBP-rP1 cDNA was amplified using the vector pBKMVmmac25 (donated by Dr. M. V. Kato, Kyoto Prefectural University of Medicine, Kyoto, Japan), which contains the full-length IGFBP-rP1 (GenBank accession no. L75822), and subcloned into a mammalian expression vector to create pSR{alpha}CHFX (13 and references herein). pSR{alpha}CHFX encodes a protein that has a CD8{alpha} signal peptide linked with the FLAG epitope and six histidine tags at the N terminus of IGFBP-rP1. This plasmid was transfected into COS7 cells at 60% confluency using a standard diethylaminoethyl-dextran transfection procedure, as previously reported (13). Five hundred milliliters of the culture media were collected and purified with a Ni-NTA column. Bound material was eluted on a 20- to 500-mM imidazole buffer gradient (pH 6.4), and 20 ml of solution eluted with 50 and 75 mM imidazole was further purified with anti-Flag M2 affinity resin. Based on silver staining (see Fig. 6Go), the eluate (fractions 4–6) was concentrated with a Centricon (Millipore Corp., Bedford, MA) and assayed for protein concentration (protein assay kit; Bio-Rad Laboratories, Hercules, CA).



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  		FIG. 6. Expression of recombinant IGFBP-rP1 in COS cells and confirmation of bioactivity in BALBc/3T3 cells. A, N-tag IGFBP-rP1 in conditioned media was purified by Ni-nitrilotriacetic acid (NTA) column chromatography and subsequently by anti-FLAG M2 affinity chromatography. The protein was eluted with sodium citrate (pH 3.5), and the eluate was subjected to SDS-PAGE and silver staining. Sup, Media before purification on NTA and anti-FLAG M2 affinity columns; M, molecular marker. B, Effect of IGFBP-rP1 protein on the proliferation of BALBc/3T3 cells. Cells (104/well) were cultured with DMEM containing 0.5% FBS and N-tag (100 ng/ml) or no-tag (1:100 dilution of conditioned media) IGFBP-rP1 in the absence (open column) or presence (closed column) of IGF-I (50 ng/ml). After 3 d of culture, cell growth was monitored by a cell counting kit. Each column represents the mean ± SD of three cultures. *, P < 0.05 vs. IGF-I alone (open column).

 
Preparation and culture of ESCs and myometrial cells (MCs)
Uteri were removed from d 4 pregnant rats. ESC-enriched uterine cells were separated from MC-enriched uterine cells by minor modification of a method of sequential enzymatic dissociation (14, 15). In brief, the uteri were slit longitudinally and transferred to 15-ml conical tubes (Asahi Techno Glass Co., Tokyo, Japan). The tissues were incubated with Ca2+- and Mg2+-free balanced PBS containing trypsin (0.5%, type II, Sigma Chemical Co., St. Louis, MO) and pancreatin (0.25%, WACO, Tokyo, Japan) at 4 C for 40 min. Digestion was followed by incubation at room temperature for 40 min and then vortexing for 10 sec on a Vortex mixer (Asahi Techno Glass Co.). The supernatant containing luminal epithelial cells was discarded. ESCs were isolated by digestion at 37 C for 30 min with collagenase (0.3%, type I, II, IV; Sigma) and 250 µg/ml DNase I (Sigma). The purity of stroma cells obtained by this method was greater than 85%, as determined by immunocytochemical detection of the stromal cell markers vimentin and desmin. To collect MCs, the above uterine tissues were incubated for a further 60 min with the fresh collagenase solution. Each cell suspension was washed, filtered through lens-cleaning paper (Kodak) to remove clumps, and transferred at 5 x 105 cells/0.5 ml (24-well plates) in DMEM supplemented with 10% fetal bovine serum (FBS), 100 µg/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamicin in a CO2 incubator.

Evaluation of cell proliferation, cell cycle status, and prostacyclin levels in cultured uterine cells
Effects of IGFBP-rP1 on the proliferation of ESCs and MCs were assessed after 72 h of treatment using the WST-1 [2-(4-iodophenyl)-3(4-nitrophenyl)-5-(24-disulfophenyl)-2H-tetrazolium, monosodium salt] cell counting kit (Dojindo, Tokyo, Japan). Cells were grown to 40% confluency, and culture medium was changed to serum-free DMEM (Life Technologies, Inc., Invitrogen, Carlsbad, CA). After 24 h of culture, the medium was changed again to DMEM containing 1% FBS with or without IGFBP-rP1. Twenty-four hours later, the cells were stained with 125 µg/ml propidium iodide using the Cycle Test Plus DNA reagent kit (Becton Dickinson, Lincoln Park, NJ) and loaded in a FACScaliber (Becton Dickinson). Data were collected and analyzed using CellQuest and ModiFit software (BD Biosciences, Franklin Lakes, NJ). PGI2 levels in culture media were measured after 16 h of treatment using an enzyme immunoassay kit (Correlate-EIA; Assay Designs, Inc., Ann Arbor, MI).

RT-PCR analysis of cyclooxygenase (COX) in cultured uterine cells
Poly(A)+ RNA was isolated from cultured cells using a Quick Prep Micro mRNA purification kit (Amersham Pharmacia Biotech, Little Chalfont, UK). RT-PCR was performed with primers specific for COX-I (sense primer, 5'-CCTTCTCCAACGTGAGC-3' and antisense, 5'-TCCTTCTCTCCTGTGAACTCCT-3') and COX-II (sense primer, 5'-AGACAGATCATAAGCGAGGACC-3' and antisense, 5'-CAGTTGACATTGATGGTG GCTGT-3') mRNA levels using a one-step RNA kit (AMV) (TaKaRa, Siga, Japan). The predicted lengths of the COX-I and COX-II mRNA fragments amplified by RT-PCR are 1036 and 1158 bp, respectively. G3PDH was used as an internal control for standardization. The PCR amplification program optimized for a iCycler (Bio-Rad) was as follows: denaturation for 5 min, 30 cycles of 45 sec at 94 C, 45 sec at 56 C, and 2 min at 72 C. The amplification products were separated on a 1.5% agarose gel containing 1 µg/ml ethidium bromide, and the gels were photographed under UV transillumination.

Statistical analysis
The results of densitometric analysis (see Fig. 4Go) obtained from three independent experiments are expressed as the mean ± SEM. All experiments (proliferation and PGI2 assay) using cultured cell preparations were repeated at least twice. Representative data are shown as the mean ± SD. Statistical significance (P < 0.05) was determined using Student’s t test and ANOVA. The representative raw data of Northern, RT-PCR, and FACS analyses are shown in these figures.



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  		FIG. 4. Effect of E2 on the expression of IGFBP-rP1 mRNA in the delayed implantation model. Pregnancy was maintained in hypophysectomized rats by the administration of progesterone (3 mg) with or without E2 (0.5 µg). The uterus was removed 24 h after the injection of E2. A, Each lane shows representative data obtained from two to three animals. B, IGFBP-rP1 mRNA levels (three experiments) were quantified with a NIH image analysis program, and the values obtained were normalized against G3PDH mRNA levels. The relative levels are expressed as a ratio of these values to the value obtained for mRNA from the whole uterus (W) of non-E2-treated rats.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in uterine IGFBP-rP1mRNA during early pregnancy
The pattern of expression of IGFBP-rP1 before and after the initiation of implantation was quantitatively determined by Northern analysis between d 3 and 7 in gestation (Fig. 1Go). The 1.0-kb IGFBP-rP1 transcript was strongly expressed from d 5–7, with an increase between d 4 and 6 followed by a slight decrease on d 7. Weak signals were found in uteri from nonpregnant rats and from d 8 to 14 of pregnancy, similar to the level observed on d 3 (data not shown). Furthermore, on d 6 and 7, the intensity of IGFBP-rP1 mRNA signals was much higher at interimplantation sites than at implantation sites (7IS vs. 7Inter, Fig. 1AGo; 6IS vs. 6Inter, Fig. 1BGo).



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  		FIG. 1. Uterine IGFBP-rP1 mRNA expression in the periimplantation period in the rat. A and B, Two representative Northern blot data of uterine IGFBP-rP1 mRNA expression during the early stage of pregnancy. Each lane contains poly(A)+ RNA (0.5 µg) pooled from two to three animals (n = 3–6 each day). Implantation sites were isolated on d 6 and 7 of pregnancy (6IS or 7IS) for RNA extraction. Numbers in the figure indicate the day of pregnancy. 6IS or 7IS, Implantation sites on d 6 or 7; 6Inter, 7Inter, nonimplantation sites (interimplantation sites) on d 6 or 7.

 
Localization of IGFBP-rP1 mRNA during early pregnancy
The distribution of IGFBP-rP1 mRNA in the rat uterus on d 4–14 of pregnancy was determined by in situ hybridization (Figs. 2Go and 3Go);F2 F3. Low levels of IGFBP-rP1 were seen in the endometrium on d 4. On d 5, just before implantation, a modest signal was seen primarily in ESCs underneath smooth muscle cells (Fig. 2Go, B and C). The specificity of hybridization was confirmed by the lack of signal in tissue sections hybridized with a IGFBP-rP1 sense probe (Fig. 2DGo). A similar pattern of IGFBP-rP1 expression was seen in uterine interimplantation sites on d 6 (Fig. 2EGo). When longitudinal sections of d 6 uteri were analyzed (Fig. 2FGo), the IGFBP-rP1 mRNA signal was found to be limited to a specific region of ESCs beneath the smooth muscle cells. No similar localization was detected in subluminal stromal cells, the glandular epithelium, or the area surrounding the implanting blastocyst. The same pattern was observed in the endometrium on d 7 (Fig. 3Go, A and B), with low levels of signal in the decidual tissues (Fig. 3Go, A and B). The lack of signal in decidual cells made the difference clear in the pattern of IGFBP-rP1 signals between the interimplantation sites and the implantation sites. The signals were detected in theca cells in follicles and corpora lutea but lower than those in uterine stroma. IGFBP-rP1 signal was also absent from decidual tissues and the embryo on d 8.5 and 14 (Fig. 3Go, C and D). On d 14, IGFBP-rP1 mRNA was detected in the endometrium but not in the decidual zone.



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  		FIG. 2. Localization of uterine IGFBP-rP1 mRNA during preimplantation (d 4, 5) and just after implantation (d 6). A, Day 4. B and C, Day 5. D and E, Day 6 (interimplantation sites). F, Day 6. A, B, D, and E, Cross-sections. C and F, Longitudinal sections. The section in D (control) was hybridized with a sense cRNA probe. Bars, 500 µm. ge, Glandular epithelium; le, luminal epithelium; s, stroma; sm, smooth muscle; dz, decidual zone; IS (bold arrow), implantation sites.

 


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  		FIG. 3. Localization of uterine IGFBP-rP1 mRNA during the postimplantation period (d 7–14). A and B, Day 7. C, Day 8. D, Day 14, A–C, Longitudinal sections. D, Cross-sections. Bars, 500 µm. dz, Decidual zone; IS, implantation sites, interimplantation sites (nonimplantation sites); ov, ovary; ge, glandular epithelium; s, stroma; sm, smooth muscle; le, luminal epithelium; em, embryo; am, amnion; pl, placenta; dc, decidual cells.

 
Expression of IGFBP-rP1 mRNA in the delayed implantation uterus and the decidualization model
To further explore whether IGFBP-rP1 expression is associated with the induction of implantation, Northern blot analyses were carried out using the delayed implantation model. After the termination of delayed implantation by the injection of E2, the level of IGFBP-rP1 transcript increased at uterine interimplantation sites, compared with that at implantation sites (Fig. 4AGo). No change in IGFBP-rP1 expression levels was detected between the whole uterus in intact pregnant rats and E2-injected rats in the delayed implantation model. However, IGFBP-rP1 mRNA levels were significantly higher at interimplantation sites than at implantation sites in uteri with delayed implantation (Fig. 4BGo), indicating the down-regulation of IGFBP-rP1 expression at implantation sites during the early phase of the decidualization process. The effect of decidual stimulation on IGFBP-rP1 expression in the pseudopregnant uterus was further examined using the artificial decidualization model (Fig. 5Go). The infusion of oil to induce decidualization caused decreases in IGFBP-rP1 mRNA expression after 24–96 h, whereas there was no decrease in IGFBP-rP1 expression in the intact uterine horn.



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  		FIG. 5. Effect of artificial decidualization on uterine IGFBP-rP1 mRNA expression in pseudopregnant rats. The uterus was removed 0–96 h after sesame oil infusion into a uterine horn on d 5 of pseudopregnancy (oil: +). The intact horn was subjected to the same analysis as a control (oil: –). Each lane shows the Northern analysis of uterine poly(A)+ RNA (0.5 µg) pooled from two to three animals.

 
Preparation of IGFBP-rP1 protein and confirmation of bioactivity
N-terminal FLAG fusion IGFBP-rP1 (N tag) was expressed in COS-7 cells transformed with pSR{alpha}CHFX and subjected to purification using a histidine tag system. A recombinant fusion protein-lacking tag (no tag) and a C-terminal FLAG fusion IGFBP-rP1 (C tag) were also constructed. After purification of N-tag IGFBP-rP1 from conditioned medium, the protein was identified as a single band on silver stained gels (Fig. 6AGo). To examine the biological activity of IGFBP-rP1, an in vitro proliferation assay was carried out using BALBc/3T3 cells. Consistent with a report showing that cellular proliferation is stimulated by IGFBP-rP1 (16), the assay revealed that IGF-I significantly stimulated the proliferation of BALBc/3T3 cells. Two forms of IGFBP-rP1 (N tag and no tag) enhanced IGF-I-stimulated proliferation (Fig. 6BGo), although addition of IGFBP-rP1 (100 ng/ml) had no effect on the growth of BALBc/3T3 cells in the absence of IGF-I.

Effects of IGFBP-rP1 on the proliferation of rat uterine endometrial cells
Primary rat uterine endometrial cells were prepared from d 4 of pregnant rat uterus, and exogenous IGFBP-rP1 was added to chemically defined serum-free medium at final concentrations ranging from 20 to 100 ng/ml (Fig. 7Go). In contrast to what was observed for BALBc/3T3 cells, the proliferation of uterine endometrial cells was significantly suppressed in the presence of 100 ng/ml of N-tag IGFBP-rP1 (31% inhibition, compared with untreated cells). When the cells were treated with IGF-I, IGFBP-rP1 also inhibited cellular proliferation in a dose-dependent manner. To further examine the basis of the inhibitory effect of IGFBP-rP1 on proliferation, cell cycle status was examined in cells that had been cultured for 24 h in the presence or absence of IGFBP-rP1. Among control cells, almost equal numbers (about 30% each) were in the G1 and S phases. Treatment with IGFBP-rP1 (100 ng/ml) slightly increased the frequency of cells in the G1 phase (by 11% relative to the control) and lowered the frequencies of those in the G2 and S phases, by 8 and 4%, respectively. Higher levels of IGFBP-rP1 (200 ng/ml) further increased the frequency of G1 phase cells and reduced the number of G2 phase cells (22% decrease vs. control). Accordingly, the number of cells in the S phase decreased by 15%, compared with control cells.



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  		FIG. 7. Effect of IGFBP-rP1 on the proliferation of ESCs. A, Cells (104 cells/well) were cultured with DMEM containing 0.1% FBS and various doses of IGFBP-rP1 in the absence (open column) or presence (closed column) of IGF-I (50 ng/ml). After 3 d of culture, cell growth was monitored by a cell counting kit. Representative data show the mean ± SD of three cultures. *, P < 0.05; *, P < 0.01 vs. control without IGF-I; #, P < 0.05 vs. control with IGF-I. B, Cells (approximately 7.5 x 105 cells/well) were treated for 24 h with IGFBP-rP1 (100 or 200 ng/ml) and subjected to the analysis for the cell cycle using a Becton Dickinson FACScaliber.

 
Effects of IGFBP-rP1 on the production of prostacyclin in rat MCs
The influence of IGFBP-rP1 on PGI2 production was determined using the MC preparation (Fig. 8Go). PGI2 production was increased in a dose-dependent manner by the addition of 20–100 ng/ml N-tag IGFBP-rP1 to the culture system (Fig. 8AGo). Phobol 12-myristate 13-acetate also markedly stimulated PGI2 secretion, consistent with a previous report (17). The stimulatory effect was slightly enhanced in the presence of IGF-I (100 ng/ml) (Fig. 8BGo). The roles of COX-II in implantation and decidualization have been extensively investigated so far (18, 19, 20). When the effects of IGFBP-rP1 on COX mRNA expression were evaluated, IGFBP-rP1 (20 or 50 ng/ml) was found to increase the level of COX-II transcript. (Fig. 8CGo).



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  		FIG. 8. Effect of IGFBP-rP1 on prostacyclin production (A) and expressions of COX-I and COX-II mRNA (B) in rat MCs. A and B, Subconfluent cells were cultured for 16 h in serum-free DMEM containing IGFBP-rP1 (20–100 ng/ml) or phorbol ester (PMA; 100 ng/ml) in a 24-well dish. PGI2 levels in the culture medium were measured by enzyme immunoassay. Representative data show the mean ± SD of three cultures. *, P < 0.01 vs. IGFBP-rP1 (0 ng/ml) without IGF-I; #, P < 0.01 vs. IGFBP-rP1 (50 ng/ml) without IGF-I. C, Subconfluent cells were treated with IGFBP-rP1 (20, 50 ng/ml) as shown above and subjected to RT-PCR analysis of COX-I and COX-II mRNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a previous study, we found that IGFBP-rP1 is expressed preferentially in the uterus on the day of implantation (d 6), compared with what is observed before the initiation of implantation (4). The present study was undertaken to determine the expression pattern and localization of endogenous IGFBP-rP1 in the rat uterus, and we further explored the biological activities of recombinant IGFBP-rP1 protein on uterine cells to clarify its physiological roles in uterine functions. At an early stage (d 3: data not shown, and d 4) of pregnancy, IGFBP-rP1 expression was low, but it increased on d 5 and 6. The levels of mRNA in the whole uterine preparation were higher on d 6 (when implantation sites can be recognized as blue bands by Chicago sky blue infusion) than on d 5. However, expression was low at implantation sites despite high levels at interimplantation sites. After implantation was initiated, IGFBP-rP1 expression was restricted to ESCs close to uterine smooth muscle, except in the decidual zone, i.e. in ESCs adjacent to the sites of blastocyst attachment. Thus, IGFBP-rP1 mRNA levels were enhanced at interimplantation sites but not at implantation sites, around the time of implantation. These results imply that IGFBP-rP1 is not directly involved in blastocyst attachment to the endometrium or endometrial decidualization but that it might be involved in preparing the uterus for successful implantation by affecting the luminal and subluminal epithelium in a paracrine fashion and/or acting on ESCs. The decreased expression of IGFBP-rP1 observed at attachment sites suggests that it might be associated with the initiation of implantation and decidualization.

Several lines of evidence have supported critical role for IGFs in the growth and development of the uterus (21). IGF mRNA is localized in the rat uterus during implantation (22). Both IGF-I and IGF-II receptor mRNAs localize to endometrial cells and MCs and are expressed in the embryo as well (23). IGF-I stimulates the mitogenesis of endometrial cells and acts synergistically with estrogen to induce DNA synthesis in vitro (22). A role for IGF-I in embryonic growth and development has been gleaned from studies of IGF-I null mice (24). Female IGF-I knockout mice have characteristic hypoplastic uteri, and their neonates have body weights that are only 30% those of wild-type mice 8 wk after birth (25). IGF-I is essential for normal embryonic growth and development (26). The modulation of IGF signaling pathways may be requisite for proper uterine. Disruption of the Igf2 gene also demonstrates its importance in embryonic and placental growth (27). IGFBPs are known to be modulators of IGF actions. The levels of several IGFBPs (IGFBP-1, -3, -4, -5) increase in rat uterine tissues during periimplantation (21). IGFBP-rP1 stimulates the growth of human colon carcinoma cells in synergy with IGF-I or insulin, although IGFBP-rP1 binds IGF-I and IGF-II with a 5- and 20-fold lower affinity, respectively, than does IGFBP1–5 (8). In the present study, we found that the stimulatory effect of IGFBP-rP1 on PGI2 production in rat uterine cells was enhanced by the presence of IGF-I. These data indicate that IGFBP-rP1 may interact cooperatively with IGF-I in the endometrium. In contrast, IGFBP-rP1 inhibited the proliferation of endometrial cells in the presence and absence of IGF-I. This result indicates that IGFBP-rP1 can interfere with the actions of endogenous IGF-I or that the activity of IGFBP-rP1 does not depend only on the regulation of IGF-I actions.

There has been little evidence for a role for IGFBP-rP1 in physiological uterine functions, except for a report by Dominguez et al. (10) showing the involvement of IGFBP-rP1 in endometrial receptivity. Decreased expression of IGFBP-rP1 has been detected in the uterine leiomyomata, compared with the adjacent myometrium (28). The suppressive effect of IGFBP-rP1 on endometrial cell proliferation was confirmed here by flow cytometry analysis. The proportion of cells remaining in G1 phase increased, whereas the proportion in G2/M phase decreased. These results agree with observations made with a human prostate cancer cell line (29) and suggest that IGFBP-rP1 inhibits the proliferation of endometrial cells by arresting them in G1 phase. IGFBP-rP1 mRNA expression was low in decidual cells and high at interimplantation sites during the implantation process. The expression of IGFBP-rP1 gradually decreased with the completion of decidualization in an artificial decidualization model in vivo. If endogenous IGFBP-rP1 exerts its inhibitory action on cell cycle progression, it is possible that the enhanced expression of IGFBP-rP1 may block stromal cell proliferation and the differentiation of decidual cells. The down-regulation of IGFBP-rP1 expression, which occurs at implantation sites, might then prompt endometrial cells to proliferate and differentiate into decidual cells, whereas continuous expression at interimplantation sites might inhibit proliferation. Treatment with IGFBP-rP1 inhibits the proliferation of HeLa, P19, and Saos-2 cells (6). Stable transformation of IGFBP-rP1 cDNA into malignant prostate epithelial cells results in the suppression of tumor formation in nude athymic mice (29). In contrast to the inhibitory effect of IGFBP-rP1 on endometrial cell proliferation, the growth of BALBc/3T3 cells was enhanced by IGFBP-rP1 and potentiated by the combination of IGFBP-rP1 and IGF-I (15 and the data in the present study). Thus, IGFBP-rP1 seems to have a role as a cell cycle regulator that varies in different cell types.

Several factors that are good uterine markers and essential for the initiation of implantation have been identified (20, 30). COX-II-deficient female mice are infertile (18). A temporal and histologically restricted expression of COX-II around the time of implantation is induced in luminal epithelial and ESCs. The uterine COX-II gene is regulated by the implanting blastocyst, whereas the COX-I gene is influenced by ovarian steroids during early pregnancy. Prostaglandins (PGs) may be related to increased endometrial capillary permeability and decidualization for the establishment of implantation and placental development. Leukemia inhibitory factor, which permits uterine heparin-binding epidermal growth factor to interact with its blastocyst receptor to direct blastocyst attachment, is essential to transform the ovarian steroid-primed uterus into a receptive state (31, 32). The interaction of the blastocyst with the uterine epithelium leads to an elevation of uterine COX-II-derived PG required for the implantation process. Hoxa-10 also modulates transformation in ESCs and in cooperation with PGs regulates the process of decidualization (33). PGI2, which is produced in response to increased COX-II activity, may induce the expression of genes that are involved in the induction of implantation via peroxisomal proliferator-activated receptor-{delta} (34), although we did not detect PGI2 in the conditioned media of ESCs. PGI2 synthase is expressed in the endometrial stroma and myometrium on the day of implantation (34). These finding may imply that IGFBP-rP1 renders the uterine environment suitable for successful implantation by enhancing PGI2 production in a paracrine and/or autocrine fashion. Furthermore, our data demonstrate that IGFBP-rP1 stimulates PGI2 production in cultured uterine cells in part via the increase in COX-II mRNA.

In conclusion, our results indicate that endometrial IGFBP-rP1 is expressed in the stromal cells of interimplantation sites and that the elevation of IGFBP-rP1 levels in stromal cells is restricted at the time of implantation in rats. The functions of IGFBP-rP1 in inhibiting uterine proliferation and stimulating PGI2 may be associated with implantation and with the initiation of decidualization in rats. However, further studies must be conducted to understand the role of IGFBP-rP1 in molecular mechanisms of implantation.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research (13470355) from the Ministry of Education, Science, Culture, and Sports of Japan.

Current address for K.I.: Department of Physiology and Biophysics, The Mount Sinai School of Medicine, 1425 Madison Avenue, New York, New York 10029.

Abbreviations: COX, Cyclooxygenase; DIG, digoxigenin; E2, 17ß-estradiol; ESC, endometrial stromal cell; FBS, fetal bovine serum; G3PDH, glyceraldehyde phosphate dehydrogenase; IGFBP, IGF binding protein; IGFBP-rP1, IGF binding protein-related protein 1; MC, myometrial cell; PG, prostaglandin; PGI2, prostacyclin.

Received March 31, 2004.

Accepted for publication July 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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