- Email: [email protected]
CCR2 interaction in human early pregnancy
Placenta 34 (2013) 663e671
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
RANKL promotes the growth of decidual stromal cells in an autocrine manner via CCL2/CCR2 interaction in human early pregnancy Y.-H. Meng, H. Li, X. Chen, L.-B. Liu, J. Shao, K.-K. Chang, M.-R. Du, L.-P. Jin, M.-Q. Li*, D.-J. Li* Laboratory for Reproductive Immunology, Hospital & Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, 413 Zhaozhou Rd., Shanghai 200011, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 29 April 2013
Objective: Receptor-activator of NF-kB ligand (TNFSF11, also known as RANKL) and its receptor RANK are essential regulators on bone remodeling, mammary gland development and hormone-associated breast cancer development. However, the expression pattern and role of RANKL/RANK axis in decidual stromal cells (DSCs) are unclear in human early pregnancy. Study design: We analyzed RANKL/RANK expression in DSCs by real-time PCR, immunhistochemistry, enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. Then BrdU cell proliferation assay, flow cytometry assay and ELISA were performed to investigate the effect of recombinant human RANKL and DSCs-derived RANKL on the proliferation, apoptosis, chemokine (C-C motif) ligand 2 (CCL2) secretion, CC chemokine receptor type 2 (CCR2) and other target proteins expression in DSCs in vitro, respectively. Results: Here we show that DSCs co-express RANKL/RANK. Not only recombinant human (rh) RANKL but also the DSC-secreted RANKL stimulate proliferation and anti-apoptosis, and elevate CCL2 secretion and CCR2 expression of DSCs. Furthermore, the stimulatory effects on the proliferation, anti-apoptosis and the expression of Bcl-2 and Ki67 and inhibitory signaling on Fas ligand (FasL) in DSCs induced by RANKL can be partly reversed by the way of blocking CCL2 and or CCR2. Conclusions: Our results have revealed that RANKL/RANK signal promotes Bcl-2 and Ki67 and decreases FasL expression, and further as a positive regulator for stimulating the proliferation and growth of DSCs through up-regulating CCL2/CCR2 signal, which finally contributes to the establishment and maintenance of physiological pregnancy. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: RANKL RANK DSCs CCR2 Proliferation Early pregnancy
1. Introduction The embryo implantation into the maternal endometrium is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormonedependent decidualization. During decidualization, the endometrial stromal cells (ESCs) undergo proliferation and differentiation into decidual cells [1e3]. This process ultimately results in the formation of the maternal component in the placentation. Decidual cells are thought to be involved in embryo implantation and in the maintenance of pregnancy through the regulation of trophoblast invasion [4], the development of the blastocyst [5], hormonal secretions [6], and the protection of the embryo from maternal immune rejection [7].
* Corresponding authors. Tel./fax: þ86 21 63457331. E-mail addresses: [email protected] (M.-Q. Li), [email protected], lidajin@ fudan.edu.cn (D.-J. Li). 0143-4004/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.04.020
It has been found that several ovarian steroid hormones are involved in the proliferation and differentiation of DSCs. Endometrial stromal cells can differentiate in vitro into decidual cells in response to exogenous factors such as estrogen (E) and progesterone (P) and to analogs of cyclic adenosine monophosphate (cAMP), exhibiting morphological and biochemical changes similar to those observed in vivo [8e10]. In addition, molecules such as relaxin [9], activin A [11,12], prostaglandin E2 (PGE2) [12], epidermal growth factor (EGF) [13] and corticotropin releasing hormone (CRH) [14] that are expressed in the human endometrium in vivo have been shown to facilitate the process of endometrial stromal cell decidualization in vitro. However, the molecular mechanisms and the signaling cascades of the proliferation and differentiation of DSCs need to be fully elucidated. Receptor-activator of NF-kB ligand (TNFSF11, also known as RANKL, OPGL, TRANCE, and ODF) and its tumor necrosis factor (TNF)-family receptor RANK (also known as TNFRSF11A, OFE, PDFR, TRANCE-R, ODAR, CD265) are essential regulators of osteoclast differentiation and thereby fundamental aspects of bone physiology,
664
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
bone remodeling [15e18], lymph node formation [19,20], establishment of the thymic microenvironment [21], mammary gland development during pregnancy [22e24], and bone metastasis of cancer [25]. Osteoprotegerin (OPG) is a decoy receptor for RANKL. By binding RANKL, OPG blocks the RANKLeRANK ligand interaction. Interestingly, these hormones involved in regulating decidualization (such as E, P, PGE2 and EGF) can also regulate the RANKL/RANK system and further affect osteoclastogenesis at distinct stages of development [26,27]. It was recently reported that the RANKL/RANK system controls the incidence and onset of sex hormone, progestindriven breast cancer [24]. Moreover, the RANKL/RANK system also controls the physiological thermoregulation in females under the control of sex hormones [28]. Therefore, it can be speculated that the RANKL/RANK axis may also be involved in the modulating growth of DSCs. This process may involve a variety of proliferation and apoptosis related molecules, such as Proliferating Cell Nuclear Antigen (PCNA), Ki67 (a cellular marker for proliferation) and apoptosis regulator Bcl-2 (Bcell lymphoma 2). The present study was undertaken to investigate the expression pattern and possible regulation of the RANKL/RANK system on DSCs in human early pregnancy. 2. Materials and methods 2.1. Tissue collection, and cell isolation and culture All tissue samples were obtained with informed consent in accordance with the requirements of the Research Ethics Committee in the Obstetrics and Gynecology, Fudan University Shanghai Medical College, and all subjects have completed an
informed consent to collect tissue samples. Decidual tissues (n ¼ 64) were from elective termination of the first-trimester pregnancies (gestational age, 7e9 weeks) for no medical reason. Among them, 10 specimens were used for immunohistochemical staining. The tissues (n ¼ 54) from the first-trimester pregnancy were put immediately into ice-cold Dulbecco’s modified Eagle’s medium (DMEM high D-glucose; Gibco, Grand Island, NY, USA), transported to the laboratory within 30 min after surgery, and washed with Hank’s balanced salt solution for isolation of DSCs. The DSCs were isolated according to our previous procedures [29]. These methods supplied >98% vimentin-positive and cytokeratin-negative DSCs. 2.2. Immunohistochemistry Paraffin sections (5 mm) of human decidua from the early pregnancy (n ¼ 10) were dehydrated in grade ethanol, and incubated with hydrogen peroxide and 1% bovine serum albumin (BSA)/TBS to block endogenous peroxidase. The samples were then incubated with mouse anti-human RANKL monoclonal antibody (25 mg/ ml, R&D Systems, USA), RANK antibody (15 mg/ml, R&D Systems, USA), or mouse IgG isotype overnight at 4 C in a humid chamber. After washing three times with TBS, the sections were overlaid with peroxidase-conjugated goat anti-mouse IgG (SP9002; Golden Bridge International, Inc., China), and the reaction was developed with 3,3-diaminobenzidine (DAB), and counterstained with hematoxylin. The experiments were repeated five times. 2.3. RT-PCR The total RNA was extracted from human DSCs (n ¼ 6) with Tri reagent (Molecular Research Center, USA). The complementary DNA (cDNA) was generated with oligo (dT)18 primers by using Revert AidÔ First Strand cDNA Synthesis Kit (Fermentas Life Science, USA). The 50 ml PCR amplification of the single strand cDNA was performed by 28 cycles of 5 min precycle at 95 C, then denaturation (94 C) for 45 s, annealing (59 C) for 45 s, and elongation (72 C) for 45 s using 2.5 U Taq polymerase (Fermentas Life Science, USA). The primer sequences are: for RANKL (243 bp), sense, 50 -GTC GCC CTG TTC TTC TAT TTC A-30 ; antisense, 50 -TTT CTC TGC TCT GAT GTG CTG
Fig. 1. RANKL/RANK is co-expressed by DSCs in human first-trimester pregnancy. (a) The expression of RANKL and its receptor RANK in deciduas (n ¼ 10) of human early pregnancy was analyzed by immunohistochemistry. Original magnification: 200. The transcription of RANKL/RANK (b), the secretion of soluble RANKL (c) and the expression of member RANKL/RANK (d) in primary DSCs (n ¼ 6) were determined by RT-PCR, ELISA and flow cytometry, respectively. Results are highly reproducible in three independent experiments. Error bars depict the standard error of the mean. **P < 0.01 compared to the 24 h time point.
Y.-H. Meng et al. / Placenta 34 (2013) 663e671 T-30 ; for RANK (137 bp), sense, 50 -GTC AGC AAG ACC GAG ATA GAG G-30 ; antisense, 50 -TCA GAG AAA GGA GGT GTG GAT T-30 ; for GAPDH (258 bp), sense, 50 -AGA AGG CTG GGG CTC ATT TG-30 ; antisense, 50 -AGG GGC CAT CCA CAG TCT TC-30 . The amplified DNA was fractionated by 2% agarose gel (Oxiod, UK) electrophoresis, and ethidium bromide-stained bands were photographed. The experiments were repeated three times. 2.4. Enzyme-linked immunosorbent assay for the determination of RANKL and CCL2 In order to evaluate the secretion level of RANKL, DSCs (1 105 cells/well, 5 105 cells/well or 1 106 cells/well) (n ¼ 6) were cultured in 24-well plates for 24, 48 and 72 h, and then the culture supernatants were harvested, centrifuged to remove cellular debris, and stored at 80 C until being assayed by enzyme-linked immunosorbent assay (ELISA) for RANKL determination (BioVendor Laboratories Ltd., GuangZhou, China). In addition, DSCs (1 105 cells/well) (n ¼ 6) in 24-well plates were treated with recombinant human (rh) RANKL (1, 10 or 100 ng/ml, R&D Systems, USA), rhOPG (100 ng/ml, R&D Systems, USA), anti-RANKL neutralizing antibody (15 mg/ml, R&D Systems, USA), rhRANKL plus rhOPG, or rhRANKL plus anti-RANKL neutralizing antibody for 48 h, and then we detected CCL2 levels in the supernatant of DSCs by ELISA (Dakewe Biotech Co., Ltd., Shen Zhen, China) and CCR2 expression (with mouse anti-human CCR2-FITC monoclonal antibody, Biolegend, USA) on DSCs by flow cytometry. At the same time, we detected the protein concentration, and the
665
CCL2 level of each group was calculated as the ratio of the CCL2 concentration of supernatant to the protein concentration. 2.5. RANKL/RANK expression by first-trimester human DSCs using flow cytometry DSCs (2 105 cells/well) (n ¼ 6) were seeded in 12-well plates for 48 h, and then digested with 0.25% trypsin only for 30e50 s, and blown off gently and washed with phosphate-buffered saline (PBS). After blocking with 10% FBS, the recovered cells were mixed with mouse anti-human RANKL-PE monoclonal antibody (Biolegend, USA) or RANK-PE monoclonal antibody (R&D Systems, USA), meanwhile, the isotypic control was used. After incubation in darkness for 30 min at room temperature, the cells were analyzed immediately by a flow cytometer (FACSCalibur, BD). The experiments were repeated five times. 2.6. BrdU cell proliferation assay, apoptosis assay and flow cytometry The BrdU cell proliferation and apoptosis assays were utilized to evaluate the effects of RANKL on cell proliferation and apoptosis, respectively. DSCs (n ¼ 6) were re-suspended in DMEM/F-12 supplemented with 10% FBS, and seeded at a density of 1 104 cells/well in 96-well flat-bottom microplates for the BrdU cell proliferation assay, or 1 105 cells/well in 24-well flat-bottom microplates for the apoptosis assay. Thereafter, the cells were starved with DMEM containing 1% FBS for 12 h before treatment, and then were stimulated with rhRANKL protein (1, 10, or 100 ng/
Fig. 2. RANKL stimulates proliferation and suppresses apoptosis of DSCs. We treated DSCs (n ¼ 6) with recombinant human RANKL (rhRANKL) (0e100 ng/ml), rhOPG (0e100 ng/ ml), anti-RANKL neutralizing antibody (0e15 mg/ml), rhRANKL (100 ng/ml) plus rhOPG (100 ng/ml), or rhRANKL (100 ng/ml) plus anti-RANKL neutralizing antibody (15 mg/ml) for 48 h, then analyzed the proliferation and apoptosis of DSCs by BrdU proliferation assay (aed) and annexin V-FITC apoptosis detection assay (e, f), respectively. a-RANKL: treatment with anti-human RANKL neutralizing antibody. Results are highly reproducible in three independent experiments. Error bars depict the standard error of the mean. **P < 0.05, **P < 0.01 compared to the vehicle control. #P < 0.05 or ##P < 0.01 compared to rhRANKL alone.
666
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
ml), rhOPG protein (1, 10, or 100 ng/ml), anti-RANKL neutralizing antibody (0.15, 1.5 or 15 mg/ml), rhRANKL (100 ng/ml) plus rhOPG (100 ng/ml), or rhRANKL (100 ng/ml) plus anti-RANKL neutralizing antibody (15 mg/ml) for 48 h. In addition, vehicle was added to some wells as a negative control. Moreover, in order to analyze whether the effect of RANKL on proliferation and apoptosis is dependent on CCL2/CCR2, we treated DSCs (n ¼ 6) with or without rhRANKL, anti-CCL2 neutralizing antibody (1 mg/ml, R&D Systems, USA) and or RS102895 (a CCR2 antagonist, 100 ng/ml, Sigma) for 48 h, with mouse isotype (20 mg/ml) (Sino-America Co. Ltd.) or media as a control. The ability of DSCs to proliferate was detected with BrdU cell proliferation assay (Millipore, USA) according to the manufacturer’s instruction. Each experiment was performed in triplicate, and repeated four times. Phosphatidylserine externalization was quantified by flow cytometry with a commercially available annexin V-FITC apoptosis detection kit (Invitrogen,
USA) according to the manufacturer’s guideline and our previous procedure [30]. The experiments were performed in triplicate, and repeated three times. Finally, flow cytometry was performed to analyze the expression of Bcl-2 (BD, USA), Ki67 (Biolegend, San Diego, USA), PCNA (BD, USA), Fas (Biolegend, San Diego, USA) and FasL (Biolegend, San Diego, USA) in DSCs. Samples were analyzed in a FACS Calibur flow cytometer (FACSCalibur, BD). Statistical analysis was conducted by using isotype matched controls. 2.7. Statistics All values are shown as mean SD. Data were analyzed by using one-way analysis of variance and least significant difference (equal variances assumed), or Tamhane’s test (equal variances not assumed) was used post-hoc for multiple
Fig. 3. DSCs-derived RANKL promotes CCL2 secretion and CCR2 expression. DSCs (n ¼ 6) were treated respectively with rhRANKL (1e100 ng/ml), rhOPG (100 ng/ml), anti-human RANKL neutralizing antibody (15 mg/ml), rhRANKL (100 ng/ml) plus rhOPG (100 ng/ml), or rhRANKL (100 ng/ml) plus anti-RANKL neutralizing antibody (15 mg/ml) for 48 h, with vehicle as a control. Then ELISA and flow cytometry were performed, respectively, to analyze the secretion level of RANKL (a, d) and the expression of CCR2 (b, c and e) on DSCs. aRANKL: treatment with anti-human RANKL neutralizing antibody. Data represent three individual experiments. Error bars depict the standard error of the mean. **P < 0.05, **P < 0.01 compared to the vehicle control. #P < 0.05 or ##P < 0.01 compared to rhRANKL alone.
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
667
Fig. 4. RANKL increases the expression of Ki67 through stimulating CCL2/CCR2 expression. DSCs (n ¼ 6) were incubated respectively with rhRANKL (100 ng/ml), anti-CCL2 neutralizing antibody (1 mg/ml) and or RS102895 (a CCR2 antagonist) (100 ng/ml) for 48 h, with vehicle as a control. The expression of Ki67 (a, b) and PCNA (c) in DSCs was then detected by flow cytometry. a-CCR2: treatment with RS102895. These pictures are representatives of three individual experiments. Error bars depict the standard error of the mean. **P < 0.05, **P < 0.01 compared to the vehicle control. ##P < 0.01 compared to rhRANKL alone.
668
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
comparisons with Statistical Package for the Social Sciences software version 11.5. Differences were considered as statistically significant at P < 0.05.
3. Results 3.1. RANKL/RANK is co-expressed by DSCs in human first-trimester pregnancy Immunohistochemistry was performed on paraffin-embedded decidua to localize RANKL and RANK proteins at the maternale fetal interface. As shown in Fig. 1a, DSCs and decidual epithelial cells (DECs) were moderately stained for RANKL and RANK in all the samples (n ¼ 10). Thereafter we investigated the mRNA level of RANKL/RANK, the secretion of RANKL and the expression of RANKL/RANK proteins in primary DSCs by RT-PCR, ELISA and flow cytometry, respectively. It was found that DSCs could transcribe RANKL/RANK (Fig. 1b),
secrete soluble RANKL in a time dependent manner (Fig. 1c). Membrane RANKL and RANK proteins could be detected in all of the analyzed DSCs with the percentage of RANKL-positive and RANK-positive cells varying from 12.24% to 50.68% and from 11.54% to 30.34%, respectively (Fig. 1d). These findings suggest that DSCs-derived RANKL may regulate the function of DSC and other RANK-positive cells in an autocrine manner at the maternalefetal interface. 3.2. RANKL stimulates proliferation and suppresses apoptosis of DSCs To investigate the influence of RANKL on the proliferation and apoptosis of DSCs, we treated DSCs with rhRANKL (0e100 ng/ml), rhOPG (0e100 ng/ml), anti-RANKL neutralizing antibody (0e15 mg/ ml), rhRANKL (100 ng/ml) plus rhOPG (100 ng/ml), or rhRANKL (100 ng/ml) plus anti-RANKL neutralizing antibody (15 mg/ml) for
Fig. 5. RANKL promotes Bcl-2 and suppresses FasL level by up-regulating CCL2/CCR2. After treatment with rhRANKL (100 ng/ml), anti-CCL2 neutralizing antibody (1 mg/ml) and or RS102895 (100 ng/ml) for 48 h, flow cytometry were performed to analyze the expression of Bcl-2 (a, b), FasL (c, d) and Fas (e) on DSCs (n ¼ 6). a-CCR2: treatment with RS102895. Results are highly reproducible in three independent experiments. Error bars depict the standard error of the mean. **P < 0.05, **P < 0.01 compared to the vehicle control. ## P < 0.01 compared to rhRANKL alone.
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
48 h in vitro. Data presented in Fig. 2 showed that rhRANKL could promote the proliferation of DSCs in a dosage dependent manner, especially at the concentration of 100 ng/ml (Fig. 2a and d, P < 0.05 or P < 0.01), and reduced apoptosis (Fig. 2e and f, P < 0.01). On the contrary, blocking RANKL with rhOPG (especially for 100 ng/ml) or anti-RANKL neutralizing antibody (especially for 15 mg/ml) decreased proliferation (Fig. 2bed, P < 0.05 or P < 0.01) and increased apoptosis of DSCs (Fig. 2e and f, P < 0.01), Moreover, the increase of proliferation and anti-apoptosis mediated by rhRANKL can be reversed by rhOPG or anti-RANKL neutralizing antibody. These results indicate that RANKL/RANK axis promotes the proliferation and growth of DSCs in an autocrine manner. 3.3. DSCs-derived RANKL promotes CCL2 secretion and CCR2 expression Kim et al. have shown that RANKL can induce the secretion of several chemokines, such as CCL2 and RANTES, which are involved in osteoclast differentiation [31]. The RANKL can synergistically stimulate the CCL2 secretion and increase CCR2 expression in human osteoclasts [32]. According to our previous study, CCL2 can promote the proliferation of DSCs [33] and endometrium stromal cells (ESCs) [34] in an autocrine manner. In order to probe into whether the regulatory action of DSCs-derived RANKL is dependent on CCL2/CCR2, we first analyzed CCL2 production and CCR2 expression in DSCs after treatment with rhRANKL, rhOPG or antiRANKL neutralizing antibody. Data presented in Fig. 3 showed that rhRANKL only significantly up-regulated CCR2 expression (Fig. 3b and c, P < 0.01), but not changed CCL2 secretion (Fig. 3a and d, P > 0.01). The different effect indicates that sensitivity of CCL2 to exogenous RANKL may be lower than that of CCR2. On the contrast, both rhOPG and anti-RANKL neutralizing antibody not only suppressed CCL2 production (Fig. 3d, P < 0.05) and CCR2 protein expression (Fig. 3e, P < 0.01), but also inhibited the stimulatory effect on CCR2 induced by rhRANKL (Fig. 3e, P < 0.05 or P < 0.01). Our observations suggest that the DSC-secreted RANKL may regulate the biological behavior of DSCs through modulating CCL2 and CCR2 interaction, which may benefit the establishment and maintenance of pregnancy.
669
treated DSCs with rhRANKL and anti-CCL2 neutralizing antibody or RS102895 for 48 h. As shown, the anti-CCL2 neutralizing antibody and/or RS102895 significantly inhibited the growth and accelerated the apoptosis of DSCs (P < 0.05 or P < 0.01) (Fig. 6a and b). RANKL could stimulate the proliferation (P < 0.01) (Fig. 6a) and reduce the apoptosis of DSCs (P < 0.01) (Fig. 6b), and these effects can be abolished by blocking CCL2 and/or CCR2 (P < 0.05 or P < 0.01) (Fig. 6a and b). Our observations suggest that RANKL can not only promote the proliferation but also inhibit the apoptosis of DSCs through CCL2/CCR2 interaction, which finally leads to growth acceleration of DSCs. 4. Discussion DSCs, comprising 75% of the cells in decidua, are the major cellular component at the maternalefetal interface [35] that regulates uterine remodeling, maternal immune response, uterine angiogenesis and early embryonic growth [36e38]. The process of decidualization is essential for a successful pregnancy and is likely to be regulated by locally acting soluble, extracellular matrixincorporated and signaling molecules. The mechanisms underlying embryo implantation and steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood.
3.4. RANKL up-regulates Ki67 and Bcl-2 and down-regulates FasL through increasing CCL2/CCR2 interaction To determine whether RANKL regulates proliferation and apoptosis-related molecules via the CCL2/CCR2 axis, we treated DSCs with anti-CCL2 neutralizing antibody, RS102895 (a CCR2 antagonist) and/or rhRANKL, and then analyzed the expression of Ki67, PCNA, Bcl-2, FasL and Fas in DSCs. It was shown in Figs. 4 and 5 that blocking CCL2 and or CCR2 not only decreased Ki67 expression, but also reversed the up-regulated action on Ki67 induced by rhRANKL (Fig. 4a and b, P < 0.05 or P < 0.01). In contrast, rhRANKL markedly promoted Bcl-2 expression and inhibited FasL expression, blocking CCL2 and/or CCR2 could also abolish these action induced by rhRANKL (Fig. 5aed, P < 0.05 or P < 0.01). However, these treatments have no influence on the PCNA and Fas translation (Figs. 4c and 5e, P > 0.05). The results above indicate that the DSCsecreted RANKL promotes Ki67, Bcl-2 and FasL expression in themselves, which may further modulate the proliferation and apoptosis of DSCs with aid of CCL2/CCR2 interaction. 3.5. The increased proliferation and anti-apoptosis of DSCs induced by RANKL are dependent on CCL2/CCR2 interaction In order to evaluate whether CCL2/CCR2 signaling is involved in regulating the RANKL-mediated growth and apoptosis of DSCs, we
Fig. 6. The increased proliferation and anti-apoptotic action of DSCs induced by RANKL are dependent on the CCL2/CCR2 interaction. DSCs (n ¼ 6) were incubated with rhRANKL (100 ng/ml), anti-CCL2 neutralizing antibody (1 mg/ml) and or RS102895 (100 ng/ml) for 48 h, with vehicle as controls. Thereafter, the proliferation (a) and apoptosis (b) of DSCs were detected by BrdU proliferation assay and annexin V-FITC apoptosis detection assay, respectively. a-CCR2: treatment with RS102895. Results are highly reproducible in three independent experiments. Error bars depict the standard error of the mean. *P < 0.05, **P < 0.01 compared to the vehicle control. #P < 0.05 or ## P < 0.01 compared to rhRANKL alone.
670
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
Fig. 7. Schematic roles of RANKL in regulating biological behavior of DSCs. RANKL secreted by DSCs can stimulate CCL2 production and CCR2 expression, hereafter increase Ki67, Bcl2 and decrease FasL level on DSCs and further stimulates proliferation and reduces apoptosis of DSCs through binding RANK in an autocrine manner, which will be conducive to decidualization and finally contribute to the growth of DSCs in human early pregnancy.
The present work has first found that DSCs highly co-express RANKL and it receptor RANK in human early pregnancy. As we have known, the regulatory mechanisms of RANKL/RANK expression are extremely complicated. It has been proposed that the soluble forms of RANKL (sRANKL) have more potent activity [39], and play important roles in osteolysis induced by tumors [40]. sRANKL derives from the membrane form as a result of either proteolytic cleavage or alternative splicing [41], and the proteolytic cleavage of RANKL requires disintegrin and metalloprotease domain (ADAM) family members and matrix metalloproteases (MMPs). In addition to the regulatory role of hormones in RANKL/ RANK, hypoxia also induces RANK and RANKL expression by activating HIF-1a in breast cancer cells [42]. It has been reported that MMPs levels are rich at the maternalefetal interface which not only regulate the trophoblast invasion but also participate in the decidualization [43]. Furthermore, the hypoxic environment at the maternalefetal interface has been widely proved during the first trimester pregnancy. Therefore, such a high level of RANKL/RANK expression may be closely related to the MMPs expression and hypoxia environment. Accumulating evidence has shown that the RANKL/RANK system is not only restricted to bone turnover but also appears to play pleiotropic roles in multiple organ systems [26]. It has been shown that the RANKL-induced mammary epithelial proliferation even results in the formation of hyperplasias within the mammary epithelium of aged females [44]. Moreover, RANKL is crucial during tumor genesis, and RANKL expression is induced in response to progestins [24]. It is generally accepted that there is a complicated hormoneecytokine network at the maternalefetal interface [36]. As shown in Fig. 7, the key finding in the present work is that RANKL secreted by DSCs can stimulate CCL2 production and CCR2 expression, and further promote the proliferation through increasing Ki67 level and suppress the apoptosis by up-regulating Bcl-2 expression and down-regulating FasL expression in an autocrine manner, respectively, which leads to the growth of DSCs in human early pregnancy. Membrane-bound RANKL or soluble RANKL (sRANKL) cleaved by MMPs or ADAMs binds and activates the receptor RANK, and then induces the NF-kB, MAPK, and PI3K/AKT pathways to control osteoclastogenesis through adaptor molecules such as TRAFs and Gab2 [45]. It has been reported that NF-kB [46], MAPK [47] and PI3K/AKT [48] pathways are involved in driving CCL2 and or CCR2 expression. Therefore, activation of these signal pathways may lead
to the increased level of the RANKL-mediated CCL2, CCR2, proliferation and anti-apoptosis related genes in DSCs. Our previous studies have found integrating roles of multiple chemokines, such as CXCL12/CXCR4 [49e52] and CCL2/CCR2 [33,38], participate in the coordinated relationship between trophoblasts and decidual cells (such as DSCs and immune cells). Taking into account the role of RANKL in the regulation of tumor cell invasion [25], we propose that RANKL derived from DSCs at the maternalefetal interface may directly promote DSCs and trophoblast invasion in autocrine and paracrine dependent manner, respectively; and RANKL/RANK signal may indirectly regulate trophoblast behaviors and decidual immune cells (DICs) functions through regulating cytokines, such as CCL2 inducing Th2-type bias at the maternalefetal interface [38], which is beneficent to formation of maternalefetal immune tolerance. In conclusion, our findings suggest that RANKL/RANK signal in DSCs can up-regulate CCL2 production and CCR2 expression, which further increases Ki67 and Bcl-2, decreases FasL expression in DSCs. These integral effects will promote survival and growth of DSCs in an autocrine manner. Therefore, RANKL/RANKeCCL2/CCR2 crosstalking loops may represent novel therapeutic targets for strategies to ameliorate diseases associated with defective proliferation and differentiation of DSCs. Acknowledgments This study was supported by Major International Joint Research Project of National Natural Science Foundation of China NSFC 30910103909 and NSFC 31270969 to Da-Jin Li; National and Shanghai Leading Academic Discipline Project (211XK22) to Da-Jin Li; Program for Outstanding Medical Academic Leader of Shanghai to Da-Jin Li; NSFC 31101064 to Ming-Qing Li; Research Program of Shanghai Health Bureau (2011Y080) to Ming-Qing Li; Ministry of Education Research Fund for Doctoral Program (20110071120092) to Ming-Qing Li and Program for ZhuoXue of Fudan University to Ming-Qing Li. References [1] Tang B, Guller S, Gurpide E. Mechanisms of human endometrial stromal cell decidualization. Ann N Y Acad Sci 1994;734:19e25. [2] Maruyama T, Yoshimura Y. Molecular and cellular mechanisms for differentiation and regeneration of the uterine endometrium. Endocr J 2008;55: 795e810.
Y.-H. Meng et al. / Placenta 34 (2013) 663e671 [3] Salamonsen LA, Dimitriadis E, Jones RL, Nie G. Complex regulation of decidualization: a role for cytokines and proteasesda review. Placenta 2003;24:S76e85. [4] Pijnenborg R, Dixon G, Robertson WB, Brosens I. Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy. Placenta 1980;1:3e19. [5] Edwards RG. Implantation, interception and contraception. Hum Reprod 1994;9:985e95. [6] Maslar IA, Ansbacher R. Effect of short-duration progesterone treatment on decidual prolactin production by cultures of proliferative human endometrium. Fertil Steril 1988;50:250e4. [7] Golander A, Zakuth V, Shechter Y, Spirer Z. Suppression of lymphocyte reactivity in vitro by a soluble factor secreted by explants of human decidua. Eur J Immunol 1981;11:849e51. [8] Daly DC, Maslar IA, Riddick DH. Prolactin production during in vitro decidualization of proliferative endometrium. Am J Obstet Gynecol 1983;145:672e8. [9] Huang JR, Tseng L, Bischof P, Janne OA. Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells. Endocrinology 1987;121:2011e7. [10] Karpovich N, Klemmt P, Hwang JH, McVeigh JE, Heath JK, Barlow DH, et al. The production of interleukin-11 and decidualization are compromised in endometrial stromal cells derived from patients with infertility. J Clin Endocrinol Metab 2005;90:1607e12. [11] Jones RL, Salamonsen LA, Findlay JK. Activin A promotes human endometrial stromal cell decidualization in vitro. J Clin Endocrinol Metab 2002;87:4001e4. [12] Dimitriadis E, Stoikos C, Baca M, Fairlie WD, McCoubrie JE, Salamonsen LA. Relaxin and prostaglandin E2 regulate interleukin 11 during human endometrial stromal cell decidualization. J Clin Endocrinol Metab 2005;90: 3458e65. [13] Irwin JC, de las Fuentes L, Giudice LC. Growth factors and decidualization in vitro. Ann N Y Acad Sci 1994;734:7e18. [14] Zoumakis E, Margioris AN, Stournaras C, Dermitzaki E, Angelakis E, Makrigiannakis A, et al. Corticotrophin-releasing hormone (CRH) interacts with inflammatory prostaglandins and interleukins and affects the decidualization of human endometrial stroma. Mol Hum Reprod 2000;6:344e51. [15] Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165e76. [16] Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 1997;390:175e9. [17] Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, et al. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 1997;272:25190e4. [18] Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/ osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597e602. [19] Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315e23. [20] Dougall WC, Glaccum M, Charrier K, Rohrbach K, Brasel K, De Smedt T, et al. RANK is essential for osteoclast and lymph node development. Genes Dev 1999;13:2412e24. [21] Rossi SW, Kim MY, Leibbrandt A, Parnell SM, Jenkinson WE, Glanville SH, et al. RANK signals from CD4(þ)3() inducer cells regulate development of Aireexpressing epithelial cells in the thymic medulla. J Exp Med 2007;204: 1267e72. [22] Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, et al. The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 2000;103:41e50. [23] Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV, et al. IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 2001;107:763e75. [24] Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, et al. Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature 2010;468:98e102. [25] Jones DH, Nakashima T, Sanchez OH, Kozieradzki I, Komarova SV, Sarosi I, et al. Regulation of cancer cell migration and bone metastasis by RANKL. Nature 2006;440:692e6. [26] Hanada R, Hanada T, Sigl V, Schramek D, Penninger JM. RANKL/RANK-beyond bones. J Mol Med 2011;89:647e56. [27] Theill LE, Boyle WJ, Penninger JM. RANK-L and RANK: T cells, bone loss, and mammalian evolution. Annu Rev Immunol 2002;20:795e823. [28] Hanada R, Leibbrandt A, Hanada T, Kitaoka S, Furuyashiki T, Fujihara H, et al. Central control of fever and female body temperature by RANKL/RANK. Nature 2009;462:505e9. [29] Li MQ, Hou XF, Shao J, Tang CL, Li DJ. DSCs-expressed CD82 promoted the expression of TIMP1 and inhibited the invasion of trophoblast cells by integrinb1/MAPK/Erk1/2 signaling pathway at materno-fetal interface of human first-trimester pregnancy. Biol Reprod 2010;82:968e79.
671
[30] Li MQ, Luo XZ, Meng YH, Mei J, Zhu XY, Jin LP, et al. CXCL8 enhances proliferation and growth and reduces apoptosis in endometrial stromal cells in an autocrine manner via a CXCR1-triggered PTEN/AKT signal pathway. Hum Reprod 2012;27:2107e16. [31] Kim MS, Day CJ, Morrison NA. MCP-1 is induced by receptor activator of nuclear factor-{kappa}B ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony-stimulating factor suppression of osteoclast formation. J Biol Chem 2005;280:16163e9. [32] Kim MS, Magno CL, Day CJ, Morrison NA. Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP-1 and RANTES. J Cell Biochem 2006;97:512e8. [33] He YY, Du MR, Gou PF, He XJ, Zhou WH, Zhu XY, et al. Regulation of C-C motif chemokine ligand2 and its receptor in human decidual stromal cells by pregnancy-associated hormones in early gestation. Hum Reprod 2007;22: 2733e42. [34] Li MQ, Li HP, Meng YH, Wang XQ, Zhu XY, Mei J, et al. Chemokine CCL2 enhances survival and invasiveness of endometrial stromal cells in an autocrine manner by activating Akt and MAPK/Erk1/2 signal pathway. Fertil Steril 2012;97:919e29. [35] Ren L, Liu YQ, Zhou WH, Zhang YZ. Trophoblast-derived chemokine CXCL12 promotes CXCR4 expression and invasion of human first-trimester decidual stromal cells. Hum Reprod 2012;27:366e74. [36] Dimitriadis E, White CA, Jones RL, Salamonsen LA. Cytokines, chemokines and growth factors in endometrium related to implantation. Hum Reprod Update 2005;11:613e30. [37] Lockwood CJ, Krikun G, Hickey M, Huang SJ, Schatz F. Decidualized human endometrial stromal cells mediate hemostasis, angiogenesis, and abnormal uterine bleeding. Reprod Sci 2009;16:162e70. [38] He YY, He XJ, Guo PF, Du MR, Shao J, Li MQ, et al. The decidual stromal cellssecreted CCL2 induces and maintains decidual leukocytes into Th2 bias in human early pregnancy. Clin Immunol 2012;145:161e73. [39] Nakashima T, Kobayashi Y, Yamasaki S, Kawakami A, Eguchi K, Sasaki H, et al. Protein expression and functional difference of membrane-bound and soluble receptor activator of NFkappaB ligand: modulation of the expression by osteotropic factors and cytokines. Biochem Biophys Res Commun 2000;275: 768e75. [40] Lynch CC, Hikosaka A, Acuff HB, Martin MD, Kawai N, Singh RK, et al. MMP-7 promotes prostate cancer-induced osteolysis via the solubilization of RANKL. Cancer Cell 2005;7:485e96. [41] Ikeda T, Kasai M, Utsuyama M, Hirokawa K. Determination of three isoforms of the receptor activator of nuclear factor-kappaB ligand and their differential expression in bone and thymus. Endocrinology 2001;142:1419e26. [42] Tang ZN, Zhang F, Tang P, Qi XW, Jiang J. Hypoxia induces RANK and RANKL expression by activating HIF-1a in breast cancer cells. Biochem Biophys Res Commun 2011;408:411e6. [43] Jones RL, Findlay JK, Farnworth PG, Robertson DM, Wallace E, Salamonsen LA. Activin A and inhibin A differentially regulate human uterine matrix metalloproteinases: potential interactions during decidualization and trophoblast invasion. Endocrinology 2006;147:724e32. [44] Fernandez-Valdivia R, Mukherjee A, Ying Y, Li J, Paquet M, DeMayo FJ, et al. The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse. Dev Biol 2009;328: 127e39. [45] Wada T, Nakashima T, Hiroshi N, Penninger JM. RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends Mol Med 2006;12:17e25. [46] Khorooshi R, Babcock AA, Owens T. NF-kappaB-driven STAT2 and CCL2 expression in astrocytes in response to brain injury. J Immunol 2008;181: 7284e91. [47] Li MQ, Hou XF, Lv SJ, Meng YH, Wang XQ, Tang CL, et al. CD82 gene suppression in endometrial stromal cells leads to increase of the cell invasiveness in the endometriotic milieu. J Mol Endocrinol 2011;47:195e208. [48] Chen TA, Wang JL, Hung SW, Chu CL, Cheng YC, Liang SM. Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production. PLoS One 2011;6: e23317. [49] Zhou WH, Du MR, Dong L, Yu J, Li DJ. Chemokine CXCL12 promotes the crosstalk between trophoblasts and decidual stromal cells in human first-trimester pregnancy. Hum Reprod 2008;23:2669e79. [50] Li MQ, Tang CL, Du MR, Fan DX, Zhao HB, Xu B, et al. CXCL12 controls overinvasion of trophoblasts via upregulating CD82 expression in DSCs at maternal-fetal interface of human early pregnancy in a paracrine manner. Int J Clin Exp Pathol 2011;4:276e86. [51] Wu X, Jin LP, Yuan MM, Zhu Y, Wang MY, Li DJ. Human first-trimester trophoblast cells recruit CD56brightCD16- NK cells into decidua by way of expressing and secreting of CXCL12/stromal cell-derived factor 1. J Immunol 2005;175:61e8. [52] Piao HL, Tao Y, Zhu R, Wang SC, Tang CL, Fu Q, et al. The CXCL12/CXCR4 axis is involved in the maintenance of Th2 bias at the maternal/fetal interface in early human pregnancy. Cell Mol Immunol 2012;9:423e30.
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
RANKL promotes the growth of decidual stromal cells in an autocrine manner via CCL2/CCR2 interaction in human early pregnancy Y.-H. Meng, H. Li, X. Chen, L.-B. Liu, J. Shao, K.-K. Chang, M.-R. Du, L.-P. Jin, M.-Q. Li*, D.-J. Li* Laboratory for Reproductive Immunology, Hospital & Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, 413 Zhaozhou Rd., Shanghai 200011, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 29 April 2013
Objective: Receptor-activator of NF-kB ligand (TNFSF11, also known as RANKL) and its receptor RANK are essential regulators on bone remodeling, mammary gland development and hormone-associated breast cancer development. However, the expression pattern and role of RANKL/RANK axis in decidual stromal cells (DSCs) are unclear in human early pregnancy. Study design: We analyzed RANKL/RANK expression in DSCs by real-time PCR, immunhistochemistry, enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. Then BrdU cell proliferation assay, flow cytometry assay and ELISA were performed to investigate the effect of recombinant human RANKL and DSCs-derived RANKL on the proliferation, apoptosis, chemokine (C-C motif) ligand 2 (CCL2) secretion, CC chemokine receptor type 2 (CCR2) and other target proteins expression in DSCs in vitro, respectively. Results: Here we show that DSCs co-express RANKL/RANK. Not only recombinant human (rh) RANKL but also the DSC-secreted RANKL stimulate proliferation and anti-apoptosis, and elevate CCL2 secretion and CCR2 expression of DSCs. Furthermore, the stimulatory effects on the proliferation, anti-apoptosis and the expression of Bcl-2 and Ki67 and inhibitory signaling on Fas ligand (FasL) in DSCs induced by RANKL can be partly reversed by the way of blocking CCL2 and or CCR2. Conclusions: Our results have revealed that RANKL/RANK signal promotes Bcl-2 and Ki67 and decreases FasL expression, and further as a positive regulator for stimulating the proliferation and growth of DSCs through up-regulating CCL2/CCR2 signal, which finally contributes to the establishment and maintenance of physiological pregnancy. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: RANKL RANK DSCs CCR2 Proliferation Early pregnancy
1. Introduction The embryo implantation into the maternal endometrium is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormonedependent decidualization. During decidualization, the endometrial stromal cells (ESCs) undergo proliferation and differentiation into decidual cells [1e3]. This process ultimately results in the formation of the maternal component in the placentation. Decidual cells are thought to be involved in embryo implantation and in the maintenance of pregnancy through the regulation of trophoblast invasion [4], the development of the blastocyst [5], hormonal secretions [6], and the protection of the embryo from maternal immune rejection [7].
* Corresponding authors. Tel./fax: þ86 21 63457331. E-mail addresses: [email protected] (M.-Q. Li), [email protected], lidajin@ fudan.edu.cn (D.-J. Li). 0143-4004/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.04.020
It has been found that several ovarian steroid hormones are involved in the proliferation and differentiation of DSCs. Endometrial stromal cells can differentiate in vitro into decidual cells in response to exogenous factors such as estrogen (E) and progesterone (P) and to analogs of cyclic adenosine monophosphate (cAMP), exhibiting morphological and biochemical changes similar to those observed in vivo [8e10]. In addition, molecules such as relaxin [9], activin A [11,12], prostaglandin E2 (PGE2) [12], epidermal growth factor (EGF) [13] and corticotropin releasing hormone (CRH) [14] that are expressed in the human endometrium in vivo have been shown to facilitate the process of endometrial stromal cell decidualization in vitro. However, the molecular mechanisms and the signaling cascades of the proliferation and differentiation of DSCs need to be fully elucidated. Receptor-activator of NF-kB ligand (TNFSF11, also known as RANKL, OPGL, TRANCE, and ODF) and its tumor necrosis factor (TNF)-family receptor RANK (also known as TNFRSF11A, OFE, PDFR, TRANCE-R, ODAR, CD265) are essential regulators of osteoclast differentiation and thereby fundamental aspects of bone physiology,
664
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
bone remodeling [15e18], lymph node formation [19,20], establishment of the thymic microenvironment [21], mammary gland development during pregnancy [22e24], and bone metastasis of cancer [25]. Osteoprotegerin (OPG) is a decoy receptor for RANKL. By binding RANKL, OPG blocks the RANKLeRANK ligand interaction. Interestingly, these hormones involved in regulating decidualization (such as E, P, PGE2 and EGF) can also regulate the RANKL/RANK system and further affect osteoclastogenesis at distinct stages of development [26,27]. It was recently reported that the RANKL/RANK system controls the incidence and onset of sex hormone, progestindriven breast cancer [24]. Moreover, the RANKL/RANK system also controls the physiological thermoregulation in females under the control of sex hormones [28]. Therefore, it can be speculated that the RANKL/RANK axis may also be involved in the modulating growth of DSCs. This process may involve a variety of proliferation and apoptosis related molecules, such as Proliferating Cell Nuclear Antigen (PCNA), Ki67 (a cellular marker for proliferation) and apoptosis regulator Bcl-2 (Bcell lymphoma 2). The present study was undertaken to investigate the expression pattern and possible regulation of the RANKL/RANK system on DSCs in human early pregnancy. 2. Materials and methods 2.1. Tissue collection, and cell isolation and culture All tissue samples were obtained with informed consent in accordance with the requirements of the Research Ethics Committee in the Obstetrics and Gynecology, Fudan University Shanghai Medical College, and all subjects have completed an
informed consent to collect tissue samples. Decidual tissues (n ¼ 64) were from elective termination of the first-trimester pregnancies (gestational age, 7e9 weeks) for no medical reason. Among them, 10 specimens were used for immunohistochemical staining. The tissues (n ¼ 54) from the first-trimester pregnancy were put immediately into ice-cold Dulbecco’s modified Eagle’s medium (DMEM high D-glucose; Gibco, Grand Island, NY, USA), transported to the laboratory within 30 min after surgery, and washed with Hank’s balanced salt solution for isolation of DSCs. The DSCs were isolated according to our previous procedures [29]. These methods supplied >98% vimentin-positive and cytokeratin-negative DSCs. 2.2. Immunohistochemistry Paraffin sections (5 mm) of human decidua from the early pregnancy (n ¼ 10) were dehydrated in grade ethanol, and incubated with hydrogen peroxide and 1% bovine serum albumin (BSA)/TBS to block endogenous peroxidase. The samples were then incubated with mouse anti-human RANKL monoclonal antibody (25 mg/ ml, R&D Systems, USA), RANK antibody (15 mg/ml, R&D Systems, USA), or mouse IgG isotype overnight at 4 C in a humid chamber. After washing three times with TBS, the sections were overlaid with peroxidase-conjugated goat anti-mouse IgG (SP9002; Golden Bridge International, Inc., China), and the reaction was developed with 3,3-diaminobenzidine (DAB), and counterstained with hematoxylin. The experiments were repeated five times. 2.3. RT-PCR The total RNA was extracted from human DSCs (n ¼ 6) with Tri reagent (Molecular Research Center, USA). The complementary DNA (cDNA) was generated with oligo (dT)18 primers by using Revert AidÔ First Strand cDNA Synthesis Kit (Fermentas Life Science, USA). The 50 ml PCR amplification of the single strand cDNA was performed by 28 cycles of 5 min precycle at 95 C, then denaturation (94 C) for 45 s, annealing (59 C) for 45 s, and elongation (72 C) for 45 s using 2.5 U Taq polymerase (Fermentas Life Science, USA). The primer sequences are: for RANKL (243 bp), sense, 50 -GTC GCC CTG TTC TTC TAT TTC A-30 ; antisense, 50 -TTT CTC TGC TCT GAT GTG CTG
Fig. 1. RANKL/RANK is co-expressed by DSCs in human first-trimester pregnancy. (a) The expression of RANKL and its receptor RANK in deciduas (n ¼ 10) of human early pregnancy was analyzed by immunohistochemistry. Original magnification: 200. The transcription of RANKL/RANK (b), the secretion of soluble RANKL (c) and the expression of member RANKL/RANK (d) in primary DSCs (n ¼ 6) were determined by RT-PCR, ELISA and flow cytometry, respectively. Results are highly reproducible in three independent experiments. Error bars depict the standard error of the mean. **P < 0.01 compared to the 24 h time point.
Y.-H. Meng et al. / Placenta 34 (2013) 663e671 T-30 ; for RANK (137 bp), sense, 50 -GTC AGC AAG ACC GAG ATA GAG G-30 ; antisense, 50 -TCA GAG AAA GGA GGT GTG GAT T-30 ; for GAPDH (258 bp), sense, 50 -AGA AGG CTG GGG CTC ATT TG-30 ; antisense, 50 -AGG GGC CAT CCA CAG TCT TC-30 . The amplified DNA was fractionated by 2% agarose gel (Oxiod, UK) electrophoresis, and ethidium bromide-stained bands were photographed. The experiments were repeated three times. 2.4. Enzyme-linked immunosorbent assay for the determination of RANKL and CCL2 In order to evaluate the secretion level of RANKL, DSCs (1 105 cells/well, 5 105 cells/well or 1 106 cells/well) (n ¼ 6) were cultured in 24-well plates for 24, 48 and 72 h, and then the culture supernatants were harvested, centrifuged to remove cellular debris, and stored at 80 C until being assayed by enzyme-linked immunosorbent assay (ELISA) for RANKL determination (BioVendor Laboratories Ltd., GuangZhou, China). In addition, DSCs (1 105 cells/well) (n ¼ 6) in 24-well plates were treated with recombinant human (rh) RANKL (1, 10 or 100 ng/ml, R&D Systems, USA), rhOPG (100 ng/ml, R&D Systems, USA), anti-RANKL neutralizing antibody (15 mg/ml, R&D Systems, USA), rhRANKL plus rhOPG, or rhRANKL plus anti-RANKL neutralizing antibody for 48 h, and then we detected CCL2 levels in the supernatant of DSCs by ELISA (Dakewe Biotech Co., Ltd., Shen Zhen, China) and CCR2 expression (with mouse anti-human CCR2-FITC monoclonal antibody, Biolegend, USA) on DSCs by flow cytometry. At the same time, we detected the protein concentration, and the
665
CCL2 level of each group was calculated as the ratio of the CCL2 concentration of supernatant to the protein concentration. 2.5. RANKL/RANK expression by first-trimester human DSCs using flow cytometry DSCs (2 105 cells/well) (n ¼ 6) were seeded in 12-well plates for 48 h, and then digested with 0.25% trypsin only for 30e50 s, and blown off gently and washed with phosphate-buffered saline (PBS). After blocking with 10% FBS, the recovered cells were mixed with mouse anti-human RANKL-PE monoclonal antibody (Biolegend, USA) or RANK-PE monoclonal antibody (R&D Systems, USA), meanwhile, the isotypic control was used. After incubation in darkness for 30 min at room temperature, the cells were analyzed immediately by a flow cytometer (FACSCalibur, BD). The experiments were repeated five times. 2.6. BrdU cell proliferation assay, apoptosis assay and flow cytometry The BrdU cell proliferation and apoptosis assays were utilized to evaluate the effects of RANKL on cell proliferation and apoptosis, respectively. DSCs (n ¼ 6) were re-suspended in DMEM/F-12 supplemented with 10% FBS, and seeded at a density of 1 104 cells/well in 96-well flat-bottom microplates for the BrdU cell proliferation assay, or 1 105 cells/well in 24-well flat-bottom microplates for the apoptosis assay. Thereafter, the cells were starved with DMEM containing 1% FBS for 12 h before treatment, and then were stimulated with rhRANKL protein (1, 10, or 100 ng/
Fig. 2. RANKL stimulates proliferation and suppresses apoptosis of DSCs. We treated DSCs (n ¼ 6) with recombinant human RANKL (rhRANKL) (0e100 ng/ml), rhOPG (0e100 ng/ ml), anti-RANKL neutralizing antibody (0e15 mg/ml), rhRANKL (100 ng/ml) plus rhOPG (100 ng/ml), or rhRANKL (100 ng/ml) plus anti-RANKL neutralizing antibody (15 mg/ml) for 48 h, then analyzed the proliferation and apoptosis of DSCs by BrdU proliferation assay (aed) and annexin V-FITC apoptosis detection assay (e, f), respectively. a-RANKL: treatment with anti-human RANKL neutralizing antibody. Results are highly reproducible in three independent experiments. Error bars depict the standard error of the mean. **P < 0.05, **P < 0.01 compared to the vehicle control. #P < 0.05 or ##P < 0.01 compared to rhRANKL alone.
666
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
ml), rhOPG protein (1, 10, or 100 ng/ml), anti-RANKL neutralizing antibody (0.15, 1.5 or 15 mg/ml), rhRANKL (100 ng/ml) plus rhOPG (100 ng/ml), or rhRANKL (100 ng/ml) plus anti-RANKL neutralizing antibody (15 mg/ml) for 48 h. In addition, vehicle was added to some wells as a negative control. Moreover, in order to analyze whether the effect of RANKL on proliferation and apoptosis is dependent on CCL2/CCR2, we treated DSCs (n ¼ 6) with or without rhRANKL, anti-CCL2 neutralizing antibody (1 mg/ml, R&D Systems, USA) and or RS102895 (a CCR2 antagonist, 100 ng/ml, Sigma) for 48 h, with mouse isotype (20 mg/ml) (Sino-America Co. Ltd.) or media as a control. The ability of DSCs to proliferate was detected with BrdU cell proliferation assay (Millipore, USA) according to the manufacturer’s instruction. Each experiment was performed in triplicate, and repeated four times. Phosphatidylserine externalization was quantified by flow cytometry with a commercially available annexin V-FITC apoptosis detection kit (Invitrogen,
USA) according to the manufacturer’s guideline and our previous procedure [30]. The experiments were performed in triplicate, and repeated three times. Finally, flow cytometry was performed to analyze the expression of Bcl-2 (BD, USA), Ki67 (Biolegend, San Diego, USA), PCNA (BD, USA), Fas (Biolegend, San Diego, USA) and FasL (Biolegend, San Diego, USA) in DSCs. Samples were analyzed in a FACS Calibur flow cytometer (FACSCalibur, BD). Statistical analysis was conducted by using isotype matched controls. 2.7. Statistics All values are shown as mean SD. Data were analyzed by using one-way analysis of variance and least significant difference (equal variances assumed), or Tamhane’s test (equal variances not assumed) was used post-hoc for multiple
Fig. 3. DSCs-derived RANKL promotes CCL2 secretion and CCR2 expression. DSCs (n ¼ 6) were treated respectively with rhRANKL (1e100 ng/ml), rhOPG (100 ng/ml), anti-human RANKL neutralizing antibody (15 mg/ml), rhRANKL (100 ng/ml) plus rhOPG (100 ng/ml), or rhRANKL (100 ng/ml) plus anti-RANKL neutralizing antibody (15 mg/ml) for 48 h, with vehicle as a control. Then ELISA and flow cytometry were performed, respectively, to analyze the secretion level of RANKL (a, d) and the expression of CCR2 (b, c and e) on DSCs. aRANKL: treatment with anti-human RANKL neutralizing antibody. Data represent three individual experiments. Error bars depict the standard error of the mean. **P < 0.05, **P < 0.01 compared to the vehicle control. #P < 0.05 or ##P < 0.01 compared to rhRANKL alone.
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
667
Fig. 4. RANKL increases the expression of Ki67 through stimulating CCL2/CCR2 expression. DSCs (n ¼ 6) were incubated respectively with rhRANKL (100 ng/ml), anti-CCL2 neutralizing antibody (1 mg/ml) and or RS102895 (a CCR2 antagonist) (100 ng/ml) for 48 h, with vehicle as a control. The expression of Ki67 (a, b) and PCNA (c) in DSCs was then detected by flow cytometry. a-CCR2: treatment with RS102895. These pictures are representatives of three individual experiments. Error bars depict the standard error of the mean. **P < 0.05, **P < 0.01 compared to the vehicle control. ##P < 0.01 compared to rhRANKL alone.
668
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
comparisons with Statistical Package for the Social Sciences software version 11.5. Differences were considered as statistically significant at P < 0.05.
3. Results 3.1. RANKL/RANK is co-expressed by DSCs in human first-trimester pregnancy Immunohistochemistry was performed on paraffin-embedded decidua to localize RANKL and RANK proteins at the maternale fetal interface. As shown in Fig. 1a, DSCs and decidual epithelial cells (DECs) were moderately stained for RANKL and RANK in all the samples (n ¼ 10). Thereafter we investigated the mRNA level of RANKL/RANK, the secretion of RANKL and the expression of RANKL/RANK proteins in primary DSCs by RT-PCR, ELISA and flow cytometry, respectively. It was found that DSCs could transcribe RANKL/RANK (Fig. 1b),
secrete soluble RANKL in a time dependent manner (Fig. 1c). Membrane RANKL and RANK proteins could be detected in all of the analyzed DSCs with the percentage of RANKL-positive and RANK-positive cells varying from 12.24% to 50.68% and from 11.54% to 30.34%, respectively (Fig. 1d). These findings suggest that DSCs-derived RANKL may regulate the function of DSC and other RANK-positive cells in an autocrine manner at the maternalefetal interface. 3.2. RANKL stimulates proliferation and suppresses apoptosis of DSCs To investigate the influence of RANKL on the proliferation and apoptosis of DSCs, we treated DSCs with rhRANKL (0e100 ng/ml), rhOPG (0e100 ng/ml), anti-RANKL neutralizing antibody (0e15 mg/ ml), rhRANKL (100 ng/ml) plus rhOPG (100 ng/ml), or rhRANKL (100 ng/ml) plus anti-RANKL neutralizing antibody (15 mg/ml) for
Fig. 5. RANKL promotes Bcl-2 and suppresses FasL level by up-regulating CCL2/CCR2. After treatment with rhRANKL (100 ng/ml), anti-CCL2 neutralizing antibody (1 mg/ml) and or RS102895 (100 ng/ml) for 48 h, flow cytometry were performed to analyze the expression of Bcl-2 (a, b), FasL (c, d) and Fas (e) on DSCs (n ¼ 6). a-CCR2: treatment with RS102895. Results are highly reproducible in three independent experiments. Error bars depict the standard error of the mean. **P < 0.05, **P < 0.01 compared to the vehicle control. ## P < 0.01 compared to rhRANKL alone.
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
48 h in vitro. Data presented in Fig. 2 showed that rhRANKL could promote the proliferation of DSCs in a dosage dependent manner, especially at the concentration of 100 ng/ml (Fig. 2a and d, P < 0.05 or P < 0.01), and reduced apoptosis (Fig. 2e and f, P < 0.01). On the contrary, blocking RANKL with rhOPG (especially for 100 ng/ml) or anti-RANKL neutralizing antibody (especially for 15 mg/ml) decreased proliferation (Fig. 2bed, P < 0.05 or P < 0.01) and increased apoptosis of DSCs (Fig. 2e and f, P < 0.01), Moreover, the increase of proliferation and anti-apoptosis mediated by rhRANKL can be reversed by rhOPG or anti-RANKL neutralizing antibody. These results indicate that RANKL/RANK axis promotes the proliferation and growth of DSCs in an autocrine manner. 3.3. DSCs-derived RANKL promotes CCL2 secretion and CCR2 expression Kim et al. have shown that RANKL can induce the secretion of several chemokines, such as CCL2 and RANTES, which are involved in osteoclast differentiation [31]. The RANKL can synergistically stimulate the CCL2 secretion and increase CCR2 expression in human osteoclasts [32]. According to our previous study, CCL2 can promote the proliferation of DSCs [33] and endometrium stromal cells (ESCs) [34] in an autocrine manner. In order to probe into whether the regulatory action of DSCs-derived RANKL is dependent on CCL2/CCR2, we first analyzed CCL2 production and CCR2 expression in DSCs after treatment with rhRANKL, rhOPG or antiRANKL neutralizing antibody. Data presented in Fig. 3 showed that rhRANKL only significantly up-regulated CCR2 expression (Fig. 3b and c, P < 0.01), but not changed CCL2 secretion (Fig. 3a and d, P > 0.01). The different effect indicates that sensitivity of CCL2 to exogenous RANKL may be lower than that of CCR2. On the contrast, both rhOPG and anti-RANKL neutralizing antibody not only suppressed CCL2 production (Fig. 3d, P < 0.05) and CCR2 protein expression (Fig. 3e, P < 0.01), but also inhibited the stimulatory effect on CCR2 induced by rhRANKL (Fig. 3e, P < 0.05 or P < 0.01). Our observations suggest that the DSC-secreted RANKL may regulate the biological behavior of DSCs through modulating CCL2 and CCR2 interaction, which may benefit the establishment and maintenance of pregnancy.
669
treated DSCs with rhRANKL and anti-CCL2 neutralizing antibody or RS102895 for 48 h. As shown, the anti-CCL2 neutralizing antibody and/or RS102895 significantly inhibited the growth and accelerated the apoptosis of DSCs (P < 0.05 or P < 0.01) (Fig. 6a and b). RANKL could stimulate the proliferation (P < 0.01) (Fig. 6a) and reduce the apoptosis of DSCs (P < 0.01) (Fig. 6b), and these effects can be abolished by blocking CCL2 and/or CCR2 (P < 0.05 or P < 0.01) (Fig. 6a and b). Our observations suggest that RANKL can not only promote the proliferation but also inhibit the apoptosis of DSCs through CCL2/CCR2 interaction, which finally leads to growth acceleration of DSCs. 4. Discussion DSCs, comprising 75% of the cells in decidua, are the major cellular component at the maternalefetal interface [35] that regulates uterine remodeling, maternal immune response, uterine angiogenesis and early embryonic growth [36e38]. The process of decidualization is essential for a successful pregnancy and is likely to be regulated by locally acting soluble, extracellular matrixincorporated and signaling molecules. The mechanisms underlying embryo implantation and steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood.
3.4. RANKL up-regulates Ki67 and Bcl-2 and down-regulates FasL through increasing CCL2/CCR2 interaction To determine whether RANKL regulates proliferation and apoptosis-related molecules via the CCL2/CCR2 axis, we treated DSCs with anti-CCL2 neutralizing antibody, RS102895 (a CCR2 antagonist) and/or rhRANKL, and then analyzed the expression of Ki67, PCNA, Bcl-2, FasL and Fas in DSCs. It was shown in Figs. 4 and 5 that blocking CCL2 and or CCR2 not only decreased Ki67 expression, but also reversed the up-regulated action on Ki67 induced by rhRANKL (Fig. 4a and b, P < 0.05 or P < 0.01). In contrast, rhRANKL markedly promoted Bcl-2 expression and inhibited FasL expression, blocking CCL2 and/or CCR2 could also abolish these action induced by rhRANKL (Fig. 5aed, P < 0.05 or P < 0.01). However, these treatments have no influence on the PCNA and Fas translation (Figs. 4c and 5e, P > 0.05). The results above indicate that the DSCsecreted RANKL promotes Ki67, Bcl-2 and FasL expression in themselves, which may further modulate the proliferation and apoptosis of DSCs with aid of CCL2/CCR2 interaction. 3.5. The increased proliferation and anti-apoptosis of DSCs induced by RANKL are dependent on CCL2/CCR2 interaction In order to evaluate whether CCL2/CCR2 signaling is involved in regulating the RANKL-mediated growth and apoptosis of DSCs, we
Fig. 6. The increased proliferation and anti-apoptotic action of DSCs induced by RANKL are dependent on the CCL2/CCR2 interaction. DSCs (n ¼ 6) were incubated with rhRANKL (100 ng/ml), anti-CCL2 neutralizing antibody (1 mg/ml) and or RS102895 (100 ng/ml) for 48 h, with vehicle as controls. Thereafter, the proliferation (a) and apoptosis (b) of DSCs were detected by BrdU proliferation assay and annexin V-FITC apoptosis detection assay, respectively. a-CCR2: treatment with RS102895. Results are highly reproducible in three independent experiments. Error bars depict the standard error of the mean. *P < 0.05, **P < 0.01 compared to the vehicle control. #P < 0.05 or ## P < 0.01 compared to rhRANKL alone.
670
Y.-H. Meng et al. / Placenta 34 (2013) 663e671
Fig. 7. Schematic roles of RANKL in regulating biological behavior of DSCs. RANKL secreted by DSCs can stimulate CCL2 production and CCR2 expression, hereafter increase Ki67, Bcl2 and decrease FasL level on DSCs and further stimulates proliferation and reduces apoptosis of DSCs through binding RANK in an autocrine manner, which will be conducive to decidualization and finally contribute to the growth of DSCs in human early pregnancy.
The present work has first found that DSCs highly co-express RANKL and it receptor RANK in human early pregnancy. As we have known, the regulatory mechanisms of RANKL/RANK expression are extremely complicated. It has been proposed that the soluble forms of RANKL (sRANKL) have more potent activity [39], and play important roles in osteolysis induced by tumors [40]. sRANKL derives from the membrane form as a result of either proteolytic cleavage or alternative splicing [41], and the proteolytic cleavage of RANKL requires disintegrin and metalloprotease domain (ADAM) family members and matrix metalloproteases (MMPs). In addition to the regulatory role of hormones in RANKL/ RANK, hypoxia also induces RANK and RANKL expression by activating HIF-1a in breast cancer cells [42]. It has been reported that MMPs levels are rich at the maternalefetal interface which not only regulate the trophoblast invasion but also participate in the decidualization [43]. Furthermore, the hypoxic environment at the maternalefetal interface has been widely proved during the first trimester pregnancy. Therefore, such a high level of RANKL/RANK expression may be closely related to the MMPs expression and hypoxia environment. Accumulating evidence has shown that the RANKL/RANK system is not only restricted to bone turnover but also appears to play pleiotropic roles in multiple organ systems [26]. It has been shown that the RANKL-induced mammary epithelial proliferation even results in the formation of hyperplasias within the mammary epithelium of aged females [44]. Moreover, RANKL is crucial during tumor genesis, and RANKL expression is induced in response to progestins [24]. It is generally accepted that there is a complicated hormoneecytokine network at the maternalefetal interface [36]. As shown in Fig. 7, the key finding in the present work is that RANKL secreted by DSCs can stimulate CCL2 production and CCR2 expression, and further promote the proliferation through increasing Ki67 level and suppress the apoptosis by up-regulating Bcl-2 expression and down-regulating FasL expression in an autocrine manner, respectively, which leads to the growth of DSCs in human early pregnancy. Membrane-bound RANKL or soluble RANKL (sRANKL) cleaved by MMPs or ADAMs binds and activates the receptor RANK, and then induces the NF-kB, MAPK, and PI3K/AKT pathways to control osteoclastogenesis through adaptor molecules such as TRAFs and Gab2 [45]. It has been reported that NF-kB [46], MAPK [47] and PI3K/AKT [48] pathways are involved in driving CCL2 and or CCR2 expression. Therefore, activation of these signal pathways may lead
to the increased level of the RANKL-mediated CCL2, CCR2, proliferation and anti-apoptosis related genes in DSCs. Our previous studies have found integrating roles of multiple chemokines, such as CXCL12/CXCR4 [49e52] and CCL2/CCR2 [33,38], participate in the coordinated relationship between trophoblasts and decidual cells (such as DSCs and immune cells). Taking into account the role of RANKL in the regulation of tumor cell invasion [25], we propose that RANKL derived from DSCs at the maternalefetal interface may directly promote DSCs and trophoblast invasion in autocrine and paracrine dependent manner, respectively; and RANKL/RANK signal may indirectly regulate trophoblast behaviors and decidual immune cells (DICs) functions through regulating cytokines, such as CCL2 inducing Th2-type bias at the maternalefetal interface [38], which is beneficent to formation of maternalefetal immune tolerance. In conclusion, our findings suggest that RANKL/RANK signal in DSCs can up-regulate CCL2 production and CCR2 expression, which further increases Ki67 and Bcl-2, decreases FasL expression in DSCs. These integral effects will promote survival and growth of DSCs in an autocrine manner. Therefore, RANKL/RANKeCCL2/CCR2 crosstalking loops may represent novel therapeutic targets for strategies to ameliorate diseases associated with defective proliferation and differentiation of DSCs. Acknowledgments This study was supported by Major International Joint Research Project of National Natural Science Foundation of China NSFC 30910103909 and NSFC 31270969 to Da-Jin Li; National and Shanghai Leading Academic Discipline Project (211XK22) to Da-Jin Li; Program for Outstanding Medical Academic Leader of Shanghai to Da-Jin Li; NSFC 31101064 to Ming-Qing Li; Research Program of Shanghai Health Bureau (2011Y080) to Ming-Qing Li; Ministry of Education Research Fund for Doctoral Program (20110071120092) to Ming-Qing Li and Program for ZhuoXue of Fudan University to Ming-Qing Li. References [1] Tang B, Guller S, Gurpide E. Mechanisms of human endometrial stromal cell decidualization. Ann N Y Acad Sci 1994;734:19e25. [2] Maruyama T, Yoshimura Y. Molecular and cellular mechanisms for differentiation and regeneration of the uterine endometrium. Endocr J 2008;55: 795e810.
Y.-H. Meng et al. / Placenta 34 (2013) 663e671 [3] Salamonsen LA, Dimitriadis E, Jones RL, Nie G. Complex regulation of decidualization: a role for cytokines and proteasesda review. Placenta 2003;24:S76e85. [4] Pijnenborg R, Dixon G, Robertson WB, Brosens I. Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy. Placenta 1980;1:3e19. [5] Edwards RG. Implantation, interception and contraception. Hum Reprod 1994;9:985e95. [6] Maslar IA, Ansbacher R. Effect of short-duration progesterone treatment on decidual prolactin production by cultures of proliferative human endometrium. Fertil Steril 1988;50:250e4. [7] Golander A, Zakuth V, Shechter Y, Spirer Z. Suppression of lymphocyte reactivity in vitro by a soluble factor secreted by explants of human decidua. Eur J Immunol 1981;11:849e51. [8] Daly DC, Maslar IA, Riddick DH. Prolactin production during in vitro decidualization of proliferative endometrium. Am J Obstet Gynecol 1983;145:672e8. [9] Huang JR, Tseng L, Bischof P, Janne OA. Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells. Endocrinology 1987;121:2011e7. [10] Karpovich N, Klemmt P, Hwang JH, McVeigh JE, Heath JK, Barlow DH, et al. The production of interleukin-11 and decidualization are compromised in endometrial stromal cells derived from patients with infertility. J Clin Endocrinol Metab 2005;90:1607e12. [11] Jones RL, Salamonsen LA, Findlay JK. Activin A promotes human endometrial stromal cell decidualization in vitro. J Clin Endocrinol Metab 2002;87:4001e4. [12] Dimitriadis E, Stoikos C, Baca M, Fairlie WD, McCoubrie JE, Salamonsen LA. Relaxin and prostaglandin E2 regulate interleukin 11 during human endometrial stromal cell decidualization. J Clin Endocrinol Metab 2005;90: 3458e65. [13] Irwin JC, de las Fuentes L, Giudice LC. Growth factors and decidualization in vitro. Ann N Y Acad Sci 1994;734:7e18. [14] Zoumakis E, Margioris AN, Stournaras C, Dermitzaki E, Angelakis E, Makrigiannakis A, et al. Corticotrophin-releasing hormone (CRH) interacts with inflammatory prostaglandins and interleukins and affects the decidualization of human endometrial stroma. Mol Hum Reprod 2000;6:344e51. [15] Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165e76. [16] Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 1997;390:175e9. [17] Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, et al. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 1997;272:25190e4. [18] Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/ osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597e602. [19] Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315e23. [20] Dougall WC, Glaccum M, Charrier K, Rohrbach K, Brasel K, De Smedt T, et al. RANK is essential for osteoclast and lymph node development. Genes Dev 1999;13:2412e24. [21] Rossi SW, Kim MY, Leibbrandt A, Parnell SM, Jenkinson WE, Glanville SH, et al. RANK signals from CD4(þ)3() inducer cells regulate development of Aireexpressing epithelial cells in the thymic medulla. J Exp Med 2007;204: 1267e72. [22] Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, et al. The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 2000;103:41e50. [23] Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV, et al. IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 2001;107:763e75. [24] Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, et al. Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature 2010;468:98e102. [25] Jones DH, Nakashima T, Sanchez OH, Kozieradzki I, Komarova SV, Sarosi I, et al. Regulation of cancer cell migration and bone metastasis by RANKL. Nature 2006;440:692e6. [26] Hanada R, Hanada T, Sigl V, Schramek D, Penninger JM. RANKL/RANK-beyond bones. J Mol Med 2011;89:647e56. [27] Theill LE, Boyle WJ, Penninger JM. RANK-L and RANK: T cells, bone loss, and mammalian evolution. Annu Rev Immunol 2002;20:795e823. [28] Hanada R, Leibbrandt A, Hanada T, Kitaoka S, Furuyashiki T, Fujihara H, et al. Central control of fever and female body temperature by RANKL/RANK. Nature 2009;462:505e9. [29] Li MQ, Hou XF, Shao J, Tang CL, Li DJ. DSCs-expressed CD82 promoted the expression of TIMP1 and inhibited the invasion of trophoblast cells by integrinb1/MAPK/Erk1/2 signaling pathway at materno-fetal interface of human first-trimester pregnancy. Biol Reprod 2010;82:968e79.
671
[30] Li MQ, Luo XZ, Meng YH, Mei J, Zhu XY, Jin LP, et al. CXCL8 enhances proliferation and growth and reduces apoptosis in endometrial stromal cells in an autocrine manner via a CXCR1-triggered PTEN/AKT signal pathway. Hum Reprod 2012;27:2107e16. [31] Kim MS, Day CJ, Morrison NA. MCP-1 is induced by receptor activator of nuclear factor-{kappa}B ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony-stimulating factor suppression of osteoclast formation. J Biol Chem 2005;280:16163e9. [32] Kim MS, Magno CL, Day CJ, Morrison NA. Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP-1 and RANTES. J Cell Biochem 2006;97:512e8. [33] He YY, Du MR, Gou PF, He XJ, Zhou WH, Zhu XY, et al. Regulation of C-C motif chemokine ligand2 and its receptor in human decidual stromal cells by pregnancy-associated hormones in early gestation. Hum Reprod 2007;22: 2733e42. [34] Li MQ, Li HP, Meng YH, Wang XQ, Zhu XY, Mei J, et al. Chemokine CCL2 enhances survival and invasiveness of endometrial stromal cells in an autocrine manner by activating Akt and MAPK/Erk1/2 signal pathway. Fertil Steril 2012;97:919e29. [35] Ren L, Liu YQ, Zhou WH, Zhang YZ. Trophoblast-derived chemokine CXCL12 promotes CXCR4 expression and invasion of human first-trimester decidual stromal cells. Hum Reprod 2012;27:366e74. [36] Dimitriadis E, White CA, Jones RL, Salamonsen LA. Cytokines, chemokines and growth factors in endometrium related to implantation. Hum Reprod Update 2005;11:613e30. [37] Lockwood CJ, Krikun G, Hickey M, Huang SJ, Schatz F. Decidualized human endometrial stromal cells mediate hemostasis, angiogenesis, and abnormal uterine bleeding. Reprod Sci 2009;16:162e70. [38] He YY, He XJ, Guo PF, Du MR, Shao J, Li MQ, et al. The decidual stromal cellssecreted CCL2 induces and maintains decidual leukocytes into Th2 bias in human early pregnancy. Clin Immunol 2012;145:161e73. [39] Nakashima T, Kobayashi Y, Yamasaki S, Kawakami A, Eguchi K, Sasaki H, et al. Protein expression and functional difference of membrane-bound and soluble receptor activator of NFkappaB ligand: modulation of the expression by osteotropic factors and cytokines. Biochem Biophys Res Commun 2000;275: 768e75. [40] Lynch CC, Hikosaka A, Acuff HB, Martin MD, Kawai N, Singh RK, et al. MMP-7 promotes prostate cancer-induced osteolysis via the solubilization of RANKL. Cancer Cell 2005;7:485e96. [41] Ikeda T, Kasai M, Utsuyama M, Hirokawa K. Determination of three isoforms of the receptor activator of nuclear factor-kappaB ligand and their differential expression in bone and thymus. Endocrinology 2001;142:1419e26. [42] Tang ZN, Zhang F, Tang P, Qi XW, Jiang J. Hypoxia induces RANK and RANKL expression by activating HIF-1a in breast cancer cells. Biochem Biophys Res Commun 2011;408:411e6. [43] Jones RL, Findlay JK, Farnworth PG, Robertson DM, Wallace E, Salamonsen LA. Activin A and inhibin A differentially regulate human uterine matrix metalloproteinases: potential interactions during decidualization and trophoblast invasion. Endocrinology 2006;147:724e32. [44] Fernandez-Valdivia R, Mukherjee A, Ying Y, Li J, Paquet M, DeMayo FJ, et al. The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse. Dev Biol 2009;328: 127e39. [45] Wada T, Nakashima T, Hiroshi N, Penninger JM. RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends Mol Med 2006;12:17e25. [46] Khorooshi R, Babcock AA, Owens T. NF-kappaB-driven STAT2 and CCL2 expression in astrocytes in response to brain injury. J Immunol 2008;181: 7284e91. [47] Li MQ, Hou XF, Lv SJ, Meng YH, Wang XQ, Tang CL, et al. CD82 gene suppression in endometrial stromal cells leads to increase of the cell invasiveness in the endometriotic milieu. J Mol Endocrinol 2011;47:195e208. [48] Chen TA, Wang JL, Hung SW, Chu CL, Cheng YC, Liang SM. Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production. PLoS One 2011;6: e23317. [49] Zhou WH, Du MR, Dong L, Yu J, Li DJ. Chemokine CXCL12 promotes the crosstalk between trophoblasts and decidual stromal cells in human first-trimester pregnancy. Hum Reprod 2008;23:2669e79. [50] Li MQ, Tang CL, Du MR, Fan DX, Zhao HB, Xu B, et al. CXCL12 controls overinvasion of trophoblasts via upregulating CD82 expression in DSCs at maternal-fetal interface of human early pregnancy in a paracrine manner. Int J Clin Exp Pathol 2011;4:276e86. [51] Wu X, Jin LP, Yuan MM, Zhu Y, Wang MY, Li DJ. Human first-trimester trophoblast cells recruit CD56brightCD16- NK cells into decidua by way of expressing and secreting of CXCL12/stromal cell-derived factor 1. J Immunol 2005;175:61e8. [52] Piao HL, Tao Y, Zhu R, Wang SC, Tang CL, Fu Q, et al. The CXCL12/CXCR4 axis is involved in the maintenance of Th2 bias at the maternal/fetal interface in early human pregnancy. Cell Mol Immunol 2012;9:423e30.