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Journal of Molecular Endocrinology (2006) 36 121-130    DOI: 10.1677/jme.1.01910
© 2006 Society for Endocrinology
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Tetraspanin CD9 regulates invasion during mouse embryo implantation

W M Liu1,2, Y J Cao1, Y J Yang1,2, J Li1,2, Z Hu1,2 and E-K Duan1

1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Science, Beijing, People’s Republic of China 100080
2 Graduate School of the Chinese Academy of Sciences, 19 Yu-quan Road, Beijing, People’s Republic of China 100039

(Requests for offprints should be addressed to E-K Duan, Institute of Zoology, Chinese Academy of Science, 25 Beisihuanxi Lu, Haidian, Beijing 100080, People’s Republic of China; Email: duane{at}ioz.ac.cn)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression of tetraspanin CD9 was found on blastocysts in mice and endometrium epithelial cells in human and bovine. However, it remains unknown how CD9 is involved in the precise dialogue between embryo and uterus during early pregnancy. This study was designed to investigate the functional roles of CD9 in the embryo implantation with monoclonal antibody against CD9 protein (anti-CD9 mAb) and antisense oligonucleotide against CD9 gene (AS-CD9). Our results showed that intrauterine injection of anti-CD9 mAb on day 4 of pregnancy significantly increased the number of embryos implanted (7.24±0.39 versus 4.04±0.38). In vitro, anti-CD9 mAb or AS-CD9 significantly enhanced embryo-outgrowth ability on the monolayer of uterus epithelial cells in a dose-dependent manner. However, the attachment of blastocysts to epithelial cells was unaffected. Furthermore, we found that anti-CD9 mAb or AS-CD9 stimulated matrix metalloproteinase 2 (MMP-2) production of blastocysts on Fibronectin. LY294002, a specific inhibitor of phosphoinositide 3-kinase, was able to counteract the effect of anti-CD9 mAb and AS-CD9 on outgrowth ability and production of MMP-2. Our results indicated that CD9 played a role of inhibiting embryo implantation. CD9 was able to impair embryo invasion and the production of MMP-2 through the phosphoinositide 3-kinase signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
CD9, a member of the tetraspanin family, is a 24–27 kDa cell-surface protein with four predicted transmembrane domains. CD9 has been implicated in the regulation of cell-biological functions, including cell adhesion, motility, proliferation, differentiation, and signaling (Ikeyama et al. 1993, Anton et al. 1995, Aoyama et al. 1999, Clay et al. 2001). Regarding tumor progression, previous experimental and clinical studies have demonstrated that CD9 had a functional role as a tumor metastasis suppressor. CD9 transfection into Chinese hamster ovary, MHO10, ARH77, and MAC 10 cells, for example, inhibited cell motility (Ikeyama et al. 1993, Miyake et al. 2000). Many clinical studies have revealed that reduced CD9 expression is associated with a poor prognosis in various cancers, including non-small-cell lung cancers, breast cancers, and colon cancers (Huang et al. 1998, Funakoshi et al. 2003, Hashida et al. 2003). Although originally identified as a surface antigen on a pre-B cell line (Kersey et al. 1981), CD9 is also expressed on blastocysts in mice and endometrium epithelial cells in human and cattle (Le Naour et al. 2000, Park et al. 2000, Xiang & MacLaren 2002). Given the striking similarities between embryogenesis and the biology of cancer cells, especially in the process of the invasion, CD9 might be involved in embryo-invasive behaviors.

Embryo implantation is an important step in the establishment of pregnancy. Successful invasiveness or migration into the extracellular matrix environment is a fundamental property of embryo implantation. Matrix metalloproteinases (MMPs), one of the well-known families of molecule involved in embryo invasion, were expressed by trophoblast cells and endometrium during embryo implantation (Salamonsen 1999). In vitro, MMP-2 was secreted by human embryos (Graham et al. 1993, Puistola et al. 1989). Maximal enzyme concentrations occur at days 4 and 5 of culture, corresponding with the time of implantation in vivo (Turpeenniemi-Hujanen et al. 1992). Treatment with monoclonal antibodies to various tetraspanin proteins specifically stimulates production of MMP-2 and induced formation of long invasive protrusions of the MDA-MB-231 cells in the Matrigel assay (Sugiura & Berditchevski 1999). However, it is still not clearly known whether there are the similar effects of tetraspanin proteins on MMP-2 in the process of embryo implantation.

Attachment of epithelial cells to the underlying basement membrane via integrins activates the phosphoinositide 3-kinase (PI3K)/Akt/protein kinase B survival signal. However, upon detachment, these cells undergo anoikis (Lee & Juliano 2000). Constitutive activation of the PI3K signaling pathway, which is one of major regulators of cell survival, is found in various human cancers (Bin et al. 2002, Samuels et al. 2004). In addition, the role for PI3K-mediated MMP-2 activity in malignant gliomas and effects of CD9 on MDA-MB-231 cells have recently been demonstrated (Sugiura & Berditchevski 1999, Hess et al. 2003). Although significant progress has been made recently towards identifying key elements within the CD9 signaling network, relatively little is known about its involvement in embryo invasion.

In this study we analyzed the effects CD9 on embryo implantation by in vivo and in vitro experimental models. Our results indicated that CD9 played an important role in regulating embryo implantation and contributing to the production of MMP-2.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents

Rat anti-mouse CD9 (KMC8) monoclonal antibody (mAb) was purchased from BD Bioscience Pharmigen (catalog no. 553758). PI3K inhibitor LY294002 (10 µM) was from Sigma, and mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase kinase (MEK) inhibitor PD98059 (50 µM), Janus kinase 2 (JAK2) inhibitor AG490 (50 µM) and p38 inhibitor SB203580 (50 µM) were obtained from Calbiochem.

Animals

Kunming white strain mice (Cai et al. 2000, Liu et al. 2002; Experimental Animal Center, Institute of Genetic Science, Chinese Academy of Sciences, Beijing, People’s Republic of China) were housed in the animal facility of the State Key Laboratory of Reproductive Biology. Adult female mice (25–30 g, 5–8 weeks old) were mated with males of the same strain at room temperature and with a constant photoperiod (light:dark cycle, 14 h/10 h). Food and water were freely available. Guidelines for the care and use of the animals were followed. Mice were killed by cervical dislocation after deep anaesthesia with isoflurane by inhalation. All experiments were approved by the local Animal Care Committee (Beijing).

Indirect immunofluorescence and laser scanning confocal microscopy

After being flushed, pre-implantation embryos were washed with Dulbecco’s PBS (DPBS) containing CaCl2 and MgCl2 (1 mM each) and fixed in 4% paraformaldehyde (15 min, room temperature). Embryos were permeabilized with 0.1% Triton X-100 in DPBS for 4 min, incubated for 1.5 h in DPBS containing 10% goat serum at room temperature, and reacted with anti-CD9 mAb (1:100) at 4 °C overnight followed by secondary antibody (fluorescein isothiocyanate (FITC)-labeled anti-rat IgG) for 1 h at 37 °C. Nuclei were stained with 5 µg/ml propidium iodide (Sigma) for 5 min. Finally, the blastocysts were rinsed in DPBS to remove excess fluorescence agents, and viewed under laser scanning confocal microscope (Leica).

Treatment with antisense oligonucleotides

Phosphorothionated antisense oligonucleotide to mouse CD9 mRNA (5'-GAGCAGGTATTTGATGCA-3') and control complementary phosphorothionated sense oligonucleotide (5'-TGCATCAAATACCTGCTC-3') were synthesized. The efficacy of antisense oligonucleotide against CD9 gene (AS-CD9) has been described previously (Shallal & Kornbluth 2000). We modified three bases in order to match completely the mouse CD9 gene. After being washed and incubated as described by Shallal & Kornbluth (2000), blastocysts were transferred to 0.4% BSA (Sigma) in Ham’ F-12 medium with antisense or sense CD9 oligonucleotide at a concentration of 0.2–20 µM and incubated for 72 h.

Intrauterine injection

Each female mouse was caged with one male and allowed to mate naturally overnight. Day 1 of pregnancy was designated as the next morning when a vaginal plug was formed. Pregnant mice on day 4 were injected with 1.5 µg anti-CD9 mAb into the lumen of one uterine horn adjacent to the ovary, while another horn was injected with 1.5 µg isotype rat IgG as a control. On day 7, the treated animals were killed to count the number of implanted embryos.

Preparation of the monolayer of uterine epithelial cells

On day 4 of pregnancy, preparation of a monolayer of uterine epithelial cells was carried out using the method reported by Zeng & Cao (1996). Harvested epithelial cells were placed in 24-well sterile plastic plates and cultured in the medium of Ham’s F-12 medium supplemented with 10% fetal calf serum. After 24 h, the monolayer was rinsed three times in Ham’s F-12 medium, and then co-culture medium was added containing Ham’s F-12 medium supplement with 0.5% BSA.

Mouse embryo attachment and outgrowth assay

Blastocysts were obtained by flushing the uterine horns of day-4 pregnant mice with Hank’s solution and rinsed three times in medium Ham’s F-12 medium, then transferred in droplets of preheated medium Ham’s F-12 medium supplemented with 0.5% BSA. For the CD9 block assay, the blastocysts were incubated with anti-CD9 mAb at concentrations of 10–1000 ng/ml or AS-CD9 at concentrations of 0.2–20 µM. In control cultures, blastocysts were incubated with a purified isotype rat IgG or sense oligonucleotide against CD9 gene (S-CD9) at the same concentration as the treated blastocysts. To determine embryo attachment, the plate was shaken for 20 s with one rotation/s. If the blastocyst was found to stay at the same place, this blastocyst was designated as attachment; if not, it was designated as non-attachment. After attachment, blastocysts began to outgrow outwards. When primary giant trophoblast cells were visible around the attachment site of the attached blastocysts, we designated the blastocysts as outgrowth (Sherman & Atienza-Samols 1978). The outgrowth area, which is occupied by primary giant trophoblast cells, was recorded and measured by a digital camera. Each experiment was repeated three times.

Gelatin zymography

The culture medium (6 µl) at 48 h was mixed with 4 x 5 sample buffer (8% SDS (w/v), 0.04% Bromophenol Blue (w/v), 40% glycerol (v/v) and 0.25 M Tris) and then subjected to electrophoresis in a 10% polyacrylamine gel containing 0.5 mg/ml gelatin (Sigma). The gel was washed in 2.5% Triton X-100 and 50 mM Tris/HCl, at pH 7.5 for 1 h to remove the SDS and incubated for 18 h in calcium assay buffer (50 mM Tris, 200 mM NaCl and 10 mM CaCl2, pH 7.5) at 37 °C. After staining with 0.2% Coomassie Brilliant Blue R250 in 50% methanol and 10% acetic acid, the gel was destained with 10% acetic acid. The lytic bands were quantified by computer-aided densitometry. Each experiment was repeated three times.

Statistical analysis

All the results are shown as means ± S.E.M. All the data were analyzed using one-way analysis of variance (ANOVA) on SPSS software. The value of P<0.05 was considered to represent statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Expression of CD9 in mouse pre-implantation embryos

By using immunofluorescence staining, CD9 antigen was detected to express strongly on the surface of blastomeres of two-, four-, and eight-cell embryos and morulae, as well as on the trophoblast cells of blastocysts (Fig. 1Go). This expression pattern suggested that CD9 might play a role in mouse embryo development and embryo implantation.


Figure 1
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  		Figure 1 Localization of CD9 protein on mouse pre-implantation embryos. CD9 protein was localized immunohistologically using a FITC-conjugated secondary antibody (green). Nuclei were labeled with propidium iodide (red). (A) Two-cell embryo. (B) Four-cell embryo. (C) Eight-cell embryo. (D) Morula. (E) Blastocyst. (F) Immunofluorescence with isotype rat IgG. Note the positive signal for CD9 protein in the trophoblast cells of blastocysts and blastomeres of two-, four-, and eight-cell embryos and morulae. ICM, inner cell mass; TC, trophoblast cells. Scale bars, 20 µm.

 
Anti-CD9 mAb increases the number of implanted embryo

Given the abundant presence of CD9 on trophoblast cells of blastocysts, we addressed the question as to whether the CD9 has the potential to affect embryo implantation. Thus we further tested the requirement for CD9 protein in the mouse embryo implantation by injecting the anti-CD9 mAb into the lumen of the uterus on day 4 of pregnancy (the ‘implantation window’). As shown in Fig. 2Go, injection of the anti-CD9 mAb significantly increased the number of embryos developed to day 7 (the mean numbers of implanted embryos were 7.24 ± 0.39 versus 4.02 ± 0.38, P<0.01). This result indicates that CD9 protein may have an effect on embryo implantation or development in vivo.


Figure 2
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  		Figure 2 Effect of intrauterine injection of anti-CD9 mAb on embryo implantation in the mouse. The uterine horn of day-4 (14:00 h) mice was injected with 1.5 µg (3 µl) anti-CD9 mAb and isotype rat IgG in another uterine horn. Day-7 animals (n=50) were killed to count the number of embryos. Kunming strain mice yield nine or so offspring per litter at one time under physiological conditions (**P < 0.01 versus control).

 
Anti-CD9 mAb and AS-CD9 promote mouse blastocyst spreading ability

Furthermore, we utilized a widely used blastocyst/uterine epithelial cell co-culture system to study the effects of CD9 on aspects of implantation. Indeed, the data accumulated in this in vitro model provided important insights into the molecular mechanism that might control embryo implantation. In the course of this study we investigated the effects of anti-CD9 mAb and AS-CD9 on blastocyst implantation by examining attachment, outgrowth, and the outgrowth area of blastocysts on uterine epithelial cells. Data were collected at different times (24, 48, and 72 h) after embryos hatched and transferred onto the monolayer of the uterine epithelial cells. Results showed that, compared with the control group, both anti-CD9 mAb and AS-CD9 had little effect on blastocyst attachment to uterine epithelial cells (data not shown). However, anti-CD9 mAb (0.1 and 1 µg/ml) and the AS-CD9 (2 and 20 µM) had significantly promoted the percentage of outgrowth at 48 h (Fig. 3BGo) and the blastocyst outgrowth area at 72 h (Fig. 3CGo). Taken together, these results indicate that CD9 may regulate embryo implantation, probably by inhibiting embryo invasion.


Figure 3
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  		Figure 3 Effects of anti-CD9 mAb and AS-CD9 on percentage and area of outgrowth of the blastocyst. Hatched blastocysts were incubated on the monolayer of uterine epithelial cells in serum-free media supplemented with BSA plus 0.01, 0.1, or 1 µg/ml anti-CD9 mAb or 0.2, 2, or 20 µM antisense oligonucleotide against CD9, or the same concentrations of isotype rat IgG or S-CD9 as a control. (A) Efficacy of 2 µM AS-CD9 in downregulating the expression of CD9 on blastocysts after 24 h culture. (a) S-CD9; (b) AS-CD9. (B) After hatched blastocysts were incubated for 48 h, the percentage of blastocyst outgrowth was calculated. (C) After hatched blastocysts were incubated for 72 h, the area of blastocyst outgrowth was calculated (n=40 blastocysts per well). *P < 0.05 versus control; **P < 0.01 versus control.

 
Effects of anti-CD9 mAb and AS-CD9 on production of MMP-2

It is well established that MMP-2 may play an important role in controlling embryo invasion in the process of the embryo implantation. In particular, embryos in vitro secreted a large amount of MMP-2 onto fibronetin-coated Petri dishes. Given these results, we next wanted to know whether CD9 is able to regulate MMP-2 production. Hatched embryos were incubated with anti-CD9 mAb or AS-CD9 for 72 h on FN-coated Petri dishes, and the conditioned media were analyzed by gelatin zymography. As illustrated in Fig. 4Go, treatment with anti-CD9 mAb and AS-CD9 enhanced the production of MMP 2 by 2–3 fold.


Figure 4
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  		Figure 4 Anti-CD9 mAb and AS-CD9 induce production of MMP-2. Blastocysts were incubated for 72 h on petri dishes coated with FN in serum-free media supplemented with different concentrations of anti-CD9 mAb or AS-CD9 as shown. Controls were treated with 0.1 µg/ml isotype rat IgG (lane 1) or 2 µM S-CD9 (lane 7). Conditioned media were analyzed by gelatin zymography. Gelatinolytic activities of the samples were quantified by scanning densitometry using the AlphaImager V5.5 software package. IDV indicates a relative scanning value (n=50 blastocysts per well). *P < 0.05, **P < 0.01, ***P < 0.001 versus control.

 
Effects of pharmacological inhibitors on anti-CD9 mAb- or AS-CD9-induced blastocyst invasion ability

In an attempt to determine which signaling pathways were involved in the regulation of anti-CD9 mAb- and AS-CD9-induced blastocyst invasion, blastocysts were cultured on the monolayer of uterine epithelial cells in the presence of various pharmacological inhibitors, which specifically inhibit key regulators of different signaling pathways. The results of these experiments are shown in Fig. 5Go. Notably, a specific inhibitor to PI3K, LY294002, inhibited the CD9-mediated percentage of outgrowth and blastocyst outgrowth area. In contrast, similar treatments with MEK, p38, or JAK2 inhibitors had modest effects on CD9-mediated outgrowth ability of blastocysts. In addition, we investigated the effects of inhibitors such as PD98059, AG490, SB203580, and LY294002 on JAR cells (human trophoblastic cell line) and testified that they were working. Under normal culture conditions, LY294002 had little role in blastocyst outgrowth (data not shown). Collectively, these results demonstrated that CD9-mediated outgrowth ability of blastocysts involves the PI3K signaling pathway.


Figure 5
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  		Figure 5 Effect of the pharmacological inhibitors on the anti-CD9 mAb- and AS-CD9-mediated spreading ability of blastocysts. The hatched blastocysts were incubated for 48 or 72 h on the monolayer of uterine epithelial cells in media containing various pharmacological inhibitors as shown. Control indicates anti-CD9 mAb or AS-CD9 only. (–) Indicates isotype rat IgG or S-CD9. Concentrations of anti-CD9 mAb and AS-CD9 were 0.1 µg/ml and 2 µM, respectively. (The concentration of LY294002 was 10 µM and the concentrations of PD98059, AG490 and SB203580 were 50 µM) (A) The effect on percentage of outgrowth. (B) The effect on outgrowth area. *P < 0.05, **P < 0.01 versus control.

 
Next we examined the effects of different inhibitors on production of MMP-2. Of the various inhibitors tested, we found that only LY294002 (inhibitor of PI3K) had a negative effect on MMP-2 production induced by anti-CD9 mAb and AS-CD9 (Fig. 6Go). Thus we concluded that PI3K-dependent signaling pathway makes an essential contribution to the production of MMP-2 triggered by CD9 protein.


Figure 6
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  		Figure 6 Effect of pharmacological inhibitors on anti-CD9 mAb- or AS-CD9-induced production of MMP-2. The blastocysts were incubated for 72 h on a petri dishes coated with FN in serum-free growth media supplemented with anti-CD9 mAb or AS-CD9 and different pharmacological inhibitors as shown. Concentrations of anti-CD9 mAb and AS-CD9 were 0.1 µg/ml and 2 µM, respectively. The concentrations of LY294002 was 10 µM, PD98059 and AG490, SB203580 were 50 µM). **P < 0.01 versus control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Blastocyst adhesion and invasion into uterine endometrium are two important steps of embryo successful implantation. To gain an insight into the cellular and molecular mechanisms that control this process, we investigated the attachment and outgrowth behavior of blastocysts in vitro on a monolayer of uterine epithelial cells, a widely used mimic of uterine conditions. In this study, for the first time we have demonstrated the expression profile of CD9 in pre-implantation embryos and the functional role that CD9 plays in embryo implantation in the mouse. Results showed that CD9 was expressed on blastomeres of two-, four-, and eight-cell and morula embryos and on trophoblast cells of blastocysts, and that CD9 is involved in the regulation of embryo invasion and MMP-2 production through the PI3K signaling pathway.

Although CD9 protein was expressed in endometrium epithelial cells and pre-implantation embryos, it played an inhibitory role in embryo implantation. At present, the ligands or mediators for CD9 have not been confirmed, but there is evidence to suggest that morphological or functional alteration of the CD9 molecule by anti-CD9 mAb can lead to significant biological changes in various cells, such as anti-CD9 mAb (KMC8) activating mouse macrophages; inducing cell aggregation in the mouse macrophage cell line J774 and causing hematopoietic cells to migrate (Forsyth 1991, Jennings et al. 1990, Kaji et al. 2001, Aoyama et al. 1999). We showed that expression of CD9 on blastocysts was too low to be detected after blastocysts were transferred to culture medium containing AS-CD9 for 24 h. At the same time, AS-CD9 and anti-CD9 mAb had similar effects on embryo outgrowth and MMP-2 production, so we assumed that anti-CD9 mAb played an important role in embryo implantation by blocking the CD9 extracellular domain, although the mechanism of which ligands contact with CD9 is not fully understood.

In addition, CD9 seems to act as a suppressor of metastasis because its transfection into melanoma cells reduces their metastatic potential and an inverse correlation between expression of CD9 and appearance of metastasis in melanoma, colon, lung, and breast cancers has been described (Gutierrez-Lopez et al. 2003, Higashiyama et al. 1995, Miyake et al. 1995, 1996, Mori et al. 1998). However, when CD9–/–or CD9+/+ two-cell embryos were transferred into wild-type pseudopregnant females, the rate of embryos developing to term was not significantly different (42 and 24%, respectively; Miyado et al. 2000). There exist some discrepancies between their results and ours, which may be due to anti-CD9 mAb blocking simultaneously CD9 antigen in blastocyst and uterus epithelium. Although similarities between trophoblast invasion and tumor cell invasion have been investigated extensively, the factors which limit trophoblast invasion within the uterus and which cause these invading trophoblasts to revert into their quiescent, non-invasive state are largely unknown. Given the expression of CD9 in the uterus epithelium and embryo and the effect of anti-CD9 mAb on embryo invasion, we assumed that this role might be related to avoidance of excessive trophoblast invasion during embryo implantation by blocking CD9 of blastocyst and uterus epithelium.

Several earlier studies have shown that the contribution of CD9 to migration or adhesion seems to be linked to signaling pathway components such as signal transduction and activators of transcription (STAT), PI3K, or MAPK in cancer cells (Hemler 1998, Wang et al. 2002). Sugiura & Berditchevski (1999) have reported that CD9 controlled invasive migration of tumor cells through two distinct PI3K-dependent mechanisms. As a part of the current study, we specifically addressed the question as to whether or not the CD9 molecule is also linked to the PI3K signaling pathway in the process of embryo implantation. In agreement with Sugiura & Berditchevski’s reports in human breast cancer MDA-MB-231 cells, our data indicated that this link was possible. We found that LY29004, a specific inhibitor of PI3K, completely abolished the effect of anti-CD9 mAb-or AS-CD9-induced blastocyst outgrowth. These data illustrated a functional connection between CD9 and the PI3K-dependent pathway in embryo implantation.

Embryo implantation is a balanced process of extracellular matrix degradation and re-establishment. Although different groups of extracellular matrix-degrading enzymes may be potentially involved, previous reports indicated that MMPs have a major role in embryo degradation of the basement membrane of the uterine epithelium and invasion of the uterine stroma (Alexander et al. 1996, Behrendtsen et al. 1992). Among these MMPs, MMP-2 is a major enzyme secreted by in vitro-cultured embryos. In addition, production of MMP-2 is clearly regulated in both cultured embryos and cancer tissue. However, surprisingly little is known about the signaling pathways that control this process. Our data indicated that both anti-CD9 mAb and AS-CD9 enhanced the production of MMP-2 of blastocysts cultured on FN. Notably, this role did not involve the activation of cAMP-dependent protein kinase, but instead required the activity of PI3K. These data suggested that CD9 modulated MMP-2 production of blastocysts cultured on FN via the PI3K signaling pathway. This result is in agreement with studies of MMP-2 in NIH3T3 and MDA-MB-231 cells (Sugiura & Berditchevski 1999, Choi et al. 2004).

Taken together, the results obtained in these four different functional assays showed that CD9 is a suppressor of embryo implantation and MMP-2 production in the mouse and that this inhibitory effect is mediated by the PI3K signaling pathway. In addition, the role of CD9 occurred at the embryo invasion stage, not the adhesion stage.


   Acknowledgements
 
This work was supported by grants from the NSFC (30400042), from the Chinese Academy of Sciences (KSCX2-SW-322) and the Special Funds for Major State Basic Research Project (G1999055903). The authors appreciate careful revision by Dr Frank Yelian at Wayne State University School of Medicine. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alexander CM, Hansell EJ, Behrendtsen O, Flannery ML, Kishnani NS, Hawkes SP & Werb Z 1996 Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation. Development 122 1723–1736.[Abstract]

Anton ES, Hadjiargyrou M, Patterson PH & Matthew WD 1995 CD9 plays a role in Schwann cell migration in vitro. Journal of Neuroscience 15 584–595.[Abstract]

Aoyama K, Oritani K, Yokota T, Ishikawa J, Nishiura T, Miyake K, Kanakura Y, Tomiyama Y, Kincade PW & Matsuzawa Y 1999 Stromal cell CD9 regulates differentiation of hematopoietic stem/progenitor cells. Blood 93 2586–2594.[Abstract/Free Full Text]

Behrendtsen O, Alexander CM & Werb Z 1992 Metalloproteinases mediate extracellular matrix degradation by cells from mouse blastocyst outgrowths. Development 114 447–456.[Abstract]

Bin T, Khashayar L, Judy K, Jill K & Craig H 2002 ß1 integrin regulates fibroblast viability during collagen matrix contraction through a phosphatidylinositol 3-kinase/Akt/protein kinase B signaling pathway. Jouranl of Biological Chemistry 277 24667–24675.

Cai LQ, Cao YJ & Duan EK 2000 Effects of leukemia inhibitory factor on embryo implantation in the mouse. Cytokine 12 1676–1682.[CrossRef][Web of Science][Medline]

Choi YA, Lim HK, Kim JR, Lee CH, Kim YJ, Kang SS & Baek SH 2004 Group IB secretory phospholipase A2 promotes matrix metalloproteinase-2-mediated cell migration via the phosphatidylinositol 3-kinase and Akt pathway. Journal of Biological Chemistry 279 36579–36585.[Abstract/Free Full Text]

Clay D, Rubinstein E, Mishal Z, Anjo A, Prenant M, Jasmin C, Boucheix C & Le Bousse-Kerdiles MC 2001 CD9 and megakaryocyte differentiation. Blood 97 1982–1989.[Abstract/Free Full Text]

Forsyth KD 1991 Anti-CD9 antibodies augment neutrophil adherence to endothelium. Immunology 72 292–296.[Web of Science][Medline]

Funakoshi T, Tachibana I, Hoshida Y, Kimura H, Takeda Y, Kijima T, Nishino K, Goto H, Yoneda T, Kumagai T et al. 2003 Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer. Oncogene 22 674–687.[CrossRef][Medline]

Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N & Lala PK 1993 Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Experimental Cell Research 206 204–211.[CrossRef][Web of Science][Medline]

Gutierrez-Lopez MD, Ovalle S, Yanez-Mo M, Sanchez-Sanchez N, Rubinstein E, Olmo N, Lizarbe MA, Sanchez-Madrid F & Cabanas C 2003 A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin. Journal of Biological Chemistry 278 208–218.[Abstract/Free Full Text]

Hashida H, Takabayashi A, Tokuhara T, Hattori N, Taki T, Hasegawa H, Satoh S, Kobayashi N, Yamaoka Y & Miyake M 2003 Clinical significance of transmembrane 4 superfamily in colon cancer. British Journal of Cancer 89 158–167.[CrossRef][Medline]

Hemler ME 1998 Integrin associated proteins. Current Opinion in Cell Biology 10 578–585.[CrossRef][Web of Science][Medline]

Hess AR, Seftor EA, Seftor RE & Hendrix MJ 2003 Phosphoinositide 3-kinase regulates membrane Type 1-matrix metalloproteinase (MMP) and MMP-2 activity during melanoma cell vasculogenic mimicry. Cancer Research 63 4757–4762.[Abstract/Free Full Text]

Higashiyama M, Taki T, Ieki Y, Adachi M, Huang CL, Koh T, Kodama K, Doi O & Miyake M. 1995 Reduced motility related protein-1 (MRP-1/CD9) gene expression as a factor of poor prognosis in non-small cell lung cancer. Cancer Research 55 6040–6044.[Abstract/Free Full Text]

Huang CI, Kohno N, Ogawa E, Adachi M, Taki T & Miyake M 1998 Correlation of reduction in MRP-1/CD9 and KAI1/CD82 expression with recurrences in breast cancer patients. American Journal of Pathology 153 973–983.[Abstract/Free Full Text]

Ikeyama S, Koyama M, Yamaoko M, Sasada R & Miyake M 1993 Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA. Journal of Experimental Medicine 177 1231–1237.[Abstract/Free Full Text]

Jennings LK, Fox CF, Kouns WC, McKay CP, Ballou LR & Schultz HE 1990 The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies. Journal of Biological Chemistry 265 3815–3822.[Abstract/Free Full Text]

Kaji K, Takeshita S, Miyake K, Takai T & Kudo A 2001 Functional association of CD9 with the Fc gamma receptors in macrophages. Journal of Immunology 166 3256–3265.[Abstract/Free Full Text]

Kersey JH, LeBien TW, Abramson CS, Newman R, Sutherland R & Greaves M 1981 P-24: a human leukemia-associated and lymphohemopoietic progenitor cell surface structure identified with monoclonal antibody. Journal of Experimental Medicine 153 726–731.[Abstract/Free Full Text]

Lee JW & Juliano RL 2000 Alpha5 beta1 integrin protects intestinal epithelial cells from apoptosis through a phosphatidylinositol 3-kinase and protein kinase B-dependent pathway. Molecular Biology of the Cell 11 1973–1987.[Abstract/Free Full Text]

Le Naour F, Rubinstein E, Jasmin C, Prenant M & Boucheix C 2000 Severely reduced female fertility in CD9-deficient mice. Science 287 319–321.[Abstract/Free Full Text]

Liu WM, Duan EK & Cao YJ 2002 Effects of anandamide on embryo implantation in the mouse. Life Science 71 1623–1632.[CrossRef][Web of Science][Medline]

Miyado K, Yamada G, Yamada S, Hasuwa H, Nakamura Y, Ryu F, Suzuki K, Kosal K, Inoue K, Ogura A, et al. 2000 Requirement of CD9 on the egg plasma membrane for fertilization. Science 287 321–324.[Abstract/Free Full Text]

Miyake M, Nakano K, Ieki Y, Adachi M, Huang CL, Itoi S, Koh T & Taki T 1995 Motility related protein 1 (MRP-1/CD9) expression: inverse correlation with metastases in breast cancer. Cancer Research 55 4127–4131.[Abstract/Free Full Text]

Miyake M, Nakano K, Itoi SI, Koh T & Taki T 1996 Motility-related protein-1 (MRP-1/CD9) reduction as a factor of poor prognosis in breast cancer. Cancer Research 56 1244–1249.[Abstract/Free Full Text]

Miyake M, Inufusa H, Adachi M, Ishida H, Hashida H, Tokuhara T & Kakehi Y 2000 Suppression of pulmonary metastasis using adenovirally motility related protein-1 (MRP-1/CD9) gene delivery. Oncogene 19 5221–5226.[CrossRef][Medline]

Mori M, Mimori K, Shiraishi T, Haraguchi M, Ueo H, Barnard GF & Akiyoshi T 1998 Motility related protein 1 (MRP1/CD9) expression in colon cancer. Clinical Cancer Research 4 1507–1510.[Abstract]

Park KR, Inoue T, Ueda M, Hirano T, Higuchi T, Maeda M, Konishi I, Fujiwara H & Fujii S 2000 CD9 is expressed on human endometrial epithelial cells in association with integrins alpha(6), alpha(3) and beta(1). Molecular Human Reproduction 6 252–257.[Abstract/Free Full Text]

Puistola U, Ronnberg L, Martikainen H & Turpeenniemi-Hujanen T 1989 The human embryo produces basement membrane collagen (type IV collagen)-degrading protease activity. Human Reproduction 4 309–311.[Abstract/Free Full Text]

Salamonsen LA 1999 Role of proteases in implantation. Reviews of Reproduction 4 11–22.[Abstract]

Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, Yan H, Gazdar A, Powell SM, Riggins GJ et al. 2004 High frequency of mutations of the PIK3CA gene in human cancers. Science 304 554.[Free Full Text]

Shallal S & Kornbluth J 2000 CD9 expression enhances the susceptibility of myeloma cell lines to cell-mediated cytolysis. Blood 96 224–233.[Abstract/Free Full Text]

Sherman MI & Atienza-Samols SB 1978 In vitro studies on the adhesiveness of mouse blastocysts. In Human Fertilization, pp 179–183. Eds H Ludwig & P Tauber. Boston: PSG.

Sugiura T & Berditchevski F 1999 Function of alpha3 beta1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2). Journal of Cell Biology 146 1375–1389.[Abstract/Free Full Text]

Turpeenniemi-Hujanen T, Ronnberg L, Kauppila A & Puistola U 1992 Laminin in the human embryo implantation: analogy to the invasion by malignant cells. Fertility and Sterility 58 105–113.[Web of Science][Medline]

Wang XQ, Evans GF, Alfaro ML & Zuckerman SH 2002 Down-regulation of macrophage CD9 expression by interferon-{gamma}. Biochemical and Biophysical Research Communications 290 891–897.[CrossRef][Medline]

Xiang W & MacLaren LA 2002 Expression of fertilin and CD9 in bovine trophoblast and endometrium during implantation. Biology of Reproduction 66 1790–1796.[Abstract/Free Full Text]

Zeng GQ & Cao YJ 1996 Co-culture of mouse embryos with monolayer of uterine epithelial cells. Acta Zoologica Sinica 42 212–217.

Received 29 September 2005
Accepted 3 November 2005
Made available online as an Accepted Preprint 21 November 2005




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