Cell Communication & Adhesion, 21: 221–228, 2014 © 2014 Informa Healthcare USA, Inc. ISSN: 1541-9061 print / 1543-5180 online DOI: 10.3109/15419061.2013.876013
RESEARCH ARTICLE
CXCL12–CXCR7 Signaling Activates ERK and Akt Pathways in Human Choriocarcinoma Cells Vishwas Tripathi1, Romsha Kumar1, Amit K. Dinda2, Jagdeep Kaur3, and Kalpana Luthra1 1
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India, 2Department of Pathology, All India Institute of Medical Sciences, New Delhi, India, and 3Department of Biotechnology, Panjab University, Chandigarh, India
Abstract CXCL12 acts as a physiological ligand for the chemokine receptor CXCR7. Chemokine receptor expression by human trophoblast and other placental cells have important implications for understanding the regulation of placental growth and development. We had previously reported the differential expression of CXCR7 in different stages of the human placenta suggesting its possible role in regulation of placental growth and development. In this study, we determined the expression of CXCR7 in human choriocarcinoma JAR cells at the mRNA level and protein level and the downstream signaling pathway mediated by CXCL12–CXCR7 interaction. We observed that binding of CXCL12 to CXCR7 activates the ERK and Akt cell-survival pathways in JAR cells. Inhibition of the ERK and Akt pathways using specific inhibitors (Wortmanin & PD98509) led to the activation of the p38 pathway. Our findings suggest a possible role of CXCR7 in activating the cell survival pathways ERK and Akt in human choriocarcinoma JAR cells. Keywords: RDC1, CXCR7, SDF-1, human placental cells, chemokine receptor, Chemokines
INTRODUCTION
apoptosis, and tumorigenesis. CXCL12 is a potent chemotactic factor for T cells (Kantele et al., 2000), monocytes (Bleul et al., 1996), B cells (D’Apuzzo et al., 1997; Bleul et al., 1998; Corcione et al., 2000), dendritic cells (Sozzani et al., 1997), mast cells (Lin et al., 2000), eosinophils (Nagase et al., 2001), and CD34þ hematopoietic progenitors (Aiuti et al., 1997; Mohle et al., 1998; Wang et al., 1998). CXCL12 regulates homing of hematopoietic stem cells to the bone marrow (Kawabata et al., 1999; Naiyer et al., 1999), megakaryocyte transepithelial migration (Hamada et al., 1998; Wang et al., 1998), platelet aggregation, and differentiation of early B cells, megakaryocytic-, and erythroid lineages (Nagasawa et al., 1996; Ma et al., 1998; Kowalska et al., 1999; Majka et al., 2000). CXCL12 binds to its cognate receptor CXCR4 and thus regulates the trafficking of normal and malignant cells. For many years, it was believed that CXCR4 was the only receptor for CXCL12. It has been recently observed that CXCL12 also binds to another transmembrane receptor called CXCR7 and this binding is very specific with high affinity (Balabanian et al., 2005). The affinity of CXCL12 to CXCR7 is approximately ten fold higher than the affinity of CXCL12 to CXCR4 (Balabanian et al., 2005; Burns et al., 2006). CXCR7, previously known as RDC1, is a chemokine receptor and is expressed in a number of mammalian tissues (Thelen & Thelen, 2008). Based on its binding to inflammatory and homing chemokines IFN-inducible T cell chemo-attractant (CXCL11) and CXCL12, CXCR7 is now classified as a deorphanized G-protein-coupled receptor (Balabanian et al., 2005; Burns et al., 2006).
Chemokines and their receptors have been implicated as pivotal players in many physiological and pathological conditions, but the expression and signaling pathways mediated by the interaction of chemokine and chemokine receptors at the materno–fetal interface is poorly understood. Chemokine receptor expression by human trophoblast and other placental cells have important implications for understanding the regulation of placental growth, development, and their role in materno–fetal HIV transmission. Chemokines belong to a superfamily of chemoattracting, cytokine-like proteins that bind to chemokine receptors and activate a number of distinct intracellular signaling pathways leading to functional responses such as chemotaxis, secretion, and transcriptional activation. (Thelen, 2001) Over 50 chemokines have been identified, and divided into four families (CXC, CX3C, CC, and C) on the basis of the positions of four conserved cysteine residues (Bromley et al., 2008). The chemokine CXCL12 (also called stromal cellderived factor-1, SDF-1) belongs to the family of CXC chemokines. It regulates many essential biological processes, including cardiac and neuronal development, stem cell motility, neovascularization, angiogenesis, Received 1 October 2013; accepted 12 December 2013. Address correspondence to Kalpana Luthra, Department of Biochemistry, All India Institute of Medical Sciences, Room No-3002, Ansari Nagar East, Gautam Nagar, New Delhi, Delhi 110029, India. E-mail: kalpanaluthra@ gmail.com
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Recent reports suggest that CXCR7 does not mediate a typical chemokine receptor response and is probably involved in CXCL12-mediated cell growth and survival (Thelen & Thelen, 2008). CXCR7 has been suggested to mediate CXCR4-like functions in subsets of leukocytes (Balabanian et al., 2005). Like CXCR4, CXCR7 is highly conserved between human and mouse (Heesen et al., 1998; Shimizu et al., 2000) and CXCR7/ mice die rapidly after birth (Sierro et al., 2007). In our previous study, we had assessed the expression of CXCR4 in the human placenta at different stages and observed its lower expression in the term as compared with the early placenta (Kumar et al., 2004). Having observed the expression of CXCR7 for the first time in the human placenta in a recently conducted study (Tripathi et al., 2009), we undertook to study the CXCL12 signaling mediated via CXCR7 in the human choriocarcinoma JAR cells.
MATERIALS AND METHODS Cell culture The human choriocarcinoma cell line JAR was obtained from National centre for cell sciences, India. Cells were plated in six-well plates at a density of 1.0–1.5 107 cells/ dish in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Rockville, MD,USA) containing 25 mM glucose, 20% fetal bovine serum, 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate; and were cultured at 37°C in 5% CO2. RNA isolation and RT-PCR To observe the expression of CXCR7 at the mRNA level in JAR cells, RT–PCR was performed after isolating total RNA from JAR cells by using the phenol–chloroform method. RNA was quantitated and checked for purity by determining the absorbance ratio at 260 and 280 nm. Two hundred nanogram of RNA was reverse transcribed in a total volume of 20 μl containing DTT (10 mM), dNTPs (1 mM each), random hexamers (5 mM; Promega, USA), RNasin (40 U/μl; Promega, USA) and 5X RT buffer with the addition of 200 U Superscript II Reverse Transcriptase (Promega, USA) for 2 h at 42°C. PCR was performed using 2.5 μl of the resultant cDNA in a 25 μl reaction volume containing 1X PCR buffer, 1.5 mM MgCl2, 2.5 mM dNTPs each, 1.25 U of Taq DNA polymerase (GIBCO BRL, USA) and 10 pmoles of CXCR7-specific forward and reverse primers, amplified at 67°C annealing temperature using following primer sequence (Microsynth, Switzerland) CXCR7: forward CTACA 3¢ CXCR7: reverse ACT 3¢
5¢
GGCTATGACACGCACTG
5¢
TGGTTGTGCTGCACGAG
Expression of CXCR7 gene in MCF-7 cells was used as a positive control (PC) and expression of β-actin as an internal control. Immunocytochemistry To determine the expression of CXCR7 at the protein level in JAR cells, immunocytochemistry experiments were performed. JAR cells were grown on cover slips and fixed using chilled methanol, followed by staining with polyclonal anti human CXCR7 antibody (OPA115173, Affinity Bioreagent, USA) at concentration of 1:50 for 4 h at 37°C. Slides were then washed with PBS (pH 7.4) and treated with R.T.U. Vecta stain kit, (Vector universal quick kit, Vector Laboratories, U.S.A.). Slides were visualized after staining with FITC-conjugated secondary antibody. Two controls were taken, primary antibody (anti-human CXCR7) substituted with normal saline and MCF-7 cells were taken as a PC23 using polyclonal anti-human CXCR7 primary antibody (OPA115173, Affinity Bioreagent, USA) and FITC-conjugated secondary antibody (Vector Laboratories, USA). Expression of CXCR7 in all the samples were scored by five independent observers (three pathologists and two biochemists) blindly by a semi-quantitative scoring method as – negative, faintly or weakly positive, moderately positive, and strongly positive. Flow cytometry To determine the surface expression of CXCR4 and CXCR7, JAR cells were serum starved (to synchronize the cells) for 24 h in DMEM medium without serum containing 2% penicillin and streptomycin. Cells (1X106) were incubated at 4°C for 1 h with nonspecific isotypematched controls, mouse human IgG2A (MAB0031) (R&D systems, USA), and rabbit polyclonals to human IgG (Abcam Inc., USA), and 20 μg/ml of each of the following: anti-human CXCR4 (12 G5, R&D Systems, USA) (Castellone et al., 2004; Ottaiano et al., 2005; Mazzinghi et al., 2008; Taichman et al., 2002; Weisel et al., 2009; Zheng et al., 1999) and anti-human CXCR7/RDC1 (Abcam Inc., USA) (Cat no-ab12870) antibodies. The mouse or rabbit primary antibodies were then detected by incubating the cells at 25°C for 45 min with the respective fluorescein isothiocyanate (FITC)conjugated goat (Fab¢) 2 anti-mouse or rabbit IgG (R&D systems, USA) secondary antibodies. The cells were washed twice with phosphate-buffered saline, re-suspended, and fixed in 1% (w/v) paraformaldehyde for analysis. Ten thousand cells from each sample were evaluated for fluorescence detection using FACScan (BD Biosciences, USA), and the data were analyzed with Diva software (BD Biosciences, USA). Each of the experiments were performed in triplicate and repeated thrice.
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Western blotting To elucidate the signaling pathways triggered by the CXCL12–CXCR7 interaction, JAR cells were cultured to confluence, washed, and then serum starved in DMEM for 24 h. One million cells were pre-incubated for 1 h at 4°C with 20 μg/ml of anti-CXCR4 blocking antibody (12G5 R&D systems, USA) to block the CXCR4 receptor. Thereafter the cells were stimulated by 200 nM CXCL12 (R&D system, USA) at different selected time points followed by lysis in ice-cold RIPA buffer. Protein concentration was determined in the cell lysate using BCA estimation kit (Banglore genei, India). The lysate was clarified by centrifugation at 14,000 rpm for 10 min. Normalized lysate (30 μg) was resuspended in loading buffer and electrophoresed in 10% polyacrylamide gel under reducing conditions and transferred to nitrocellulose membrane. Another set of human choriocarcinoma JAR cells were preincubated at 37°C for 1 h with, 100 nM of wortmannin and 10 μM of PD98509 (Calbiochem, San Diego, CA) before adding CXCL12. Wortmannin is a selective, cell-permeable, and irreversible inhibitor of PI 3-kinase (Vlahakis et al., 2002; Cross et al., 1995) and PD98509 is a selective and cell-permeable inhibitor of MAP kinase kinase (MEK) and acts by inhibiting the activation of MAP kinase and subsequent phosphorylation of MAP kinase substrates (Vlahakis et al., 2002; Means et al., 2000). The blots were probed with antibodies specific for ERK phosphorylation at Thr202 and Tyr204, Akt phosphorylation at Ser473, and p38 phosphorylation at Thr180/Tyr182 (Cell signaling, USA). Membranes were stripped with stripping buffer (314 μl of BME, 0.5 mM Tris pH 6.8) for 30 min at 37°C, washed thoroughly and then reblotted with antibodies to ERK2 (Santa Cruz CA), Akt, and p38, (Cell signaling, USA). The protein bands were visualized using the ECL detection system (Lumi Glo, Cell signaling, USA), after incubating the membrane with anti-biotin HRP-linked (1:1000), antiHRP-linked (1:2000) secondary antibody. All Western blotting experiments were performed in duplicate and repeated thrice. In all the experiments, the respective PC (Cell signaling, USA) and lysate from Control (Ctrl) without blocking the CXCR4 receptor were also loaded.
Figure 1. Expression of CXCR7 at the RNA level in JAR cells (Lane 1 showing expression of β-actin, (172 bp), Lanes 2 and 3 showing expression of CXCR7 (100 bp) in JAR and MCF-7 cells, respectively.
the expression of CXCR7 in MCF-7 cells (a PC, Figure 1, Lane 3). Expression of CXCR7 in JAR cells was evaluated at the protein level by immunocytochemistry and flow cytometry using specific polyclonal anti-human CXCR7 antibody (OPA-15173, Affinity Bioreagent, USA). A distinct surface expression of CXCR7 protein was observed in JAR cells on immunocytochemical staining (Figure 2B). Treatment of the JAR human choriocarcinoma cells with different concentrations of the anti-CXCR4 blocking monoclonal antibody 12G5, demonstrated maximal expression of CXCR4 receptors at 20 μg/ml of the mAb (Figure 3A). Thereafter, the expression of CXCR7/RDC1 was determined (by flow cytometry) using anti-CXCR7 antibody after treating cells with the anti-CXCR4 (12G5) blocking antibody (20 μg/ml) to completely block the
RESULTS JAR cells expressed CXCR7 both at the mRNA (RT-PCR) and at the protein level (by immunocytochemistry and flowcytometry). The expression of CXCR7 was confirmed, both at the mRNA and protein level in MCF-7 cells (known to express CXCR7 (Burns et al., 2006), which served as a PC. The expression of mRNA after RT-PCR was obtained in JAR cells. A 100 base pair amplified product of CXCR7 was obtained and the results were reproducible. A representative gel photograph reveals the expression of CXCR7 mRNA in JAR cells (Figure 1, Lane 2) and
Figure 2. (A) Negative control (without primary antibody). (B) Surface expression of CXCR7 protein in JAR cells (using FITC staining). (C) PC (MCF-7 cells showing expression of RDC1).
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Figure 3. (A) Showing expression of CXCR4 receptors in JAR cells (for identification of dosage of anti-CXCR4 antibody required to block the receptor). (B) Showing expression of CXCR7 protein on JAR cells by flow cytometric analysis.
CXCR4 receptors expressed on human choriocarcinoma JAR cells. More than 75% cells were found to express CXCR7 (Figure 3B). Anti-CXCR4 mAb 12G5, at a concentration of 20 μg/ml, was then used as a blocking antibody in further experiments to study the signaling via CXCR7 after blocking the CXCR4 receptors.
CXCL12 on interaction with CXCR7 in JAR cells (after blocking CXCR4 receptors) triggered phosphorylation of ERK1/2 and the extent of phosphorylation was maximal at 10 min on incubation of the cells with CXCL12 at different time periods followed by Western blotting (Figure 4A). In order to determine the activation of MAPK pathway in JAR cells, MAP kinase, the upstream regulator of ERK 1/2, was inhibited by incubating the cells with the inhibitor PD98509 before CXCL12 treatment. The specificity and efficacy of PD98509 as an inhibitor was checked by determining ERK 1/2 phosphorylation following CXCL12 treatment. CXCL12 triggered phosphorylation of ERK 1/2 was abrogated in the presence of MEK inhibitor (Figure 4B). Furthermore PD98509 did not affect the cell surface expression of CXCR4 and CXCR7 when analyzed by flow cytometry (P ns, data not shown). These results demonstrate the activation of the MAPK pathway (ERK 1/2) by CXCL12–CXCR7 interaction in JAR cells. Another important kinase is Akt which is a serine/ threonine Kinase. It is involved in mediating various physiological responses, such as inhibition of apoptosis and stimulation of cell proliferation. Further, to assess the involvement of Akt, Western blotting was performed after stimulating JAR cells (pretreated with anti-CXCR4 antibody 12G5) with CXCL12. The results of Western blotting showed that CXCL12, on interaction with CXCR7, triggered activation of AKT (Figure 5A). CXCL12-triggered phosphorylation of AKT was completely abrogated in the presence of wortmannin inhibitor (Figure 5B) which is a selective inhibitor of PI 3-kinase (Vlahakis et al., 2002; Cross et al., 1995). Pre-treating the cells with wortmannin before activation of CXCL12 did not alter the cell surface expression of CXCR7 (data not shown). The specificity of the wortmannin was demonstrated by its ability to inhibit Akt phosphorylation following CXCL12 treatment. To determine the kinetics of CXCL12 activation of Akt via the CXCR7 receptor, cells were incubated with CXCL12 for progressively longer duration (Figure 5A). Akt phosphorylation was maximal and peaked at 10 min after which it vanished slowly. To investigate whether CXCR7 receptors signals through the cell survival pathways similar to the CXCR4
Figure 4. CXCL12–CXCR7 interaction-mediated ERK1/2 phosphorylation in the JAR placental cells. (A) JAR cells pre-treated with anti-CXCR4 antibody followed by incubation with 200 nM CXCL12 for various time points (0, 5, 10, 15, and 20 min) lysed and blotted using anti-phospho ERK1/2 and anti-ERK2 antibodies. (B) JAR cells pre-treated with anti-CXCR4 antibody followed by incubation with 200 nM CXCL12, untreated or pre-incubated with ERK1/2 inhibitor PD98509. Membranes were blotted using anti-phospho ERK1/2 and anti-ERK2 antibodies. Lane 1, Untreated; Lane 2, Treated with CXCL12; Lane 3, PD98509 CXCL12.
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Figure 5. CXCL12–CXCR7 interaction-mediated Akt phosphorylation placental cells JAR. (A) JAR cells pre-treated with anti-CXCR4 antibody, were incubated with 200 nM CXCL12 for various time points, and then lysed and blotted using anti phospho Akt and anti-Akt antibodies. (B) JAR cells pre-treated with anti-CXCR4 antibody or pre-incubated with PI 3-kinase inhibitor Wortmannin, and then treated with 200 nM CXCL12. PC (Cat no-9273, Cell signaling, USA) was loaded in the gel to check for specificity. Membranes were blotted using anti-phospho Akt and anti-Akt antibodies. Lane 1, Pretreated with anti-CXCR4 antibody then treated with CXCL12; Lane 2, JAR cells treated with CXCL12 without blocking the CXCR4 receptor; Lane 3, PC (Cat no-9273, Cell signaling, USA); Lane 4, JAR cells pretreated with Wortmannin then treated with CXCL12.
receptor via the Akt-p38 balance in JAR placental cells, we determined the activation of phosphorylated p38 at different time periods (1–30 min), after treating the cells with CXCL12. We did not observe p38 phosphorylation (Figure 6A) suggesting that unlike CXCR4, CXCR7 on CXCL12 interaction (after blocking the CXCR4 receptors) does not activate the phosphorylation of p38. This unique feature of CXCR7 receptor that it does not activate p38, suggests its exclusive role in activating the ERK and Akt cell survival pathways. The p38 kinase is stimulated by different types of cellular stress such as UV radiation, heat shock, inflammatory cytokines including TNFa-1 and IL-1 etc. On investigation we observed that on blocking the cell survival pathways using Akt inhibitor wortmannin
and ERK inhibitor PD98509, CXCR7–CXCL12 interaction activates the p38 pathway (Figure 6B).
DISCUSSION The present study was undertaken to determine the downstream signaling of CXCL12 mediated via CXCR7 in the human choriocarcinoma JAR cells JAR. Our study reveals that CXCL12–CXCR7 interaction activates ERK and Akt pathways in the human choriocarcinoma JAR cells. CXCR7, also known as RDC1, is a recently deorphanized chemokine receptor which shares the ligand
Figure 6. CXCL12–CXCR7 interaction does not activate the p38 phosphorylation. On blocking the survival pathways by pre incubating the cells with the Akt inhibitor wortmannin and ERK inhibitor PD98509, CXCR7–CXCL12 interaction activated the phosphorylation of p38. JAR cells, incubated with 200 nM CXCL12 for various time points, and then lysed and blotted using anti-phospho p38 and anti-p38 antibodies. PC (Cat no-9213) was loaded in the gel to check for specificity. Membranes were blotted using anti-phospho p38 and anti-p38 antibodies. (A) Lane 1, Untreated; Lane 2, without blocking the CXCR4 receptor CXCL12 for 2 min; Lane 3, PC; Lane 4, Treated with anti CXCR4 CXCL12 for 2 min; Lane 5, Treated with anti CXCR4 CXCL12 for 5 min; Lane 6, Treated with anti CXCR4 CXCL12 for 10 min; Lane 7, Treated with anti CXCR4 CXCL12 for 15 min. (B) JAR cells were untreated or preincubated with Wortmannin (100 nM), PD98509 (10 μM) and without blocking the CXCR4 receptor followed by stimulation with 200 nM CXCL12.Western blotting was done using specific antibodies. Lane 1, Treated with anti CXCR4 CXCL12 (without any inhibitor); Lane 2, Treated with anti CXCR4 Wortmannin CXCL12; Lane 3, Treated with anti CXCR4 PD98509 CXCL12; Lane 4, Cells without blocking the CXCR4 receptor CXCL12.
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CXCL12 with another chemokine receptor CXCR4 and plays a crucial role in regulating immunity, angiogenesis, stem cell trafficking, and mediating organ-specific metastases of cancer (Xueqing et al., 2010). In our previous study, we reported for the first time that CXCR7 is differentially expressed and its expression increases at the term stage of the human placenta unlike the CXCR4 (Tripathi et al., 2009) The interaction between CXCR4 and CXCL12 was previously thought to be exclusive as CXCR4 was the only known receptor of CXCL12. However, Balabanian et al. (2005) observed that CXCL12 binds and signals through CXCR7 expressed on the surface of primary T cells and participates in lymphocyte motility. This breakthrough opened a new question about the role of CXCL12-mediated signaling pathways in those cells where CXCR4 is less expressed. The precise role of CXCR7 in the human placenta needs to be elucidated. We therefore undertook to study CXCL12 signaling mediated via CXCR7 in placental cells (JAR). CXCL12–CXCR7 interaction led to activation of the ERK and Akt pathways in JAR cells. Further, the inhibition of Akt and ERK using specific inhibitors (wortmannin & PD98509) abrogated the phosphorylation of ERK and Akt. Moreover, our study revealed that the CXCL12–CXCR7 interaction did not activate p38 pathway and therefore did not favor cell death. An interesting observation was the activation of the p38 pathway after blocking the ERK and Akt pathways using pathway-specific inhibitors. One probable reason could be that the Akt and/or ERK pathway/s may have a negative regulatory effect on the p38 pathway which remains to be determined. The two important pathways that are associated with cell survival are the ERK 1/2 kinase and Akt, the downstream effector protein of PI 3-kinase. Several groups have reported in different cell types that CXCR7 regulates many important biological processes like cell survival, cell adhesion, and tumor development in animal models (Burns et al., 2006; Mazzinghi et al., 2008; Meijer et al., 2008; Raggo et al., 2005). In a recent study, Meijer et al. (2008) reported that CXCR7 is able to initiate powerful proliferation and migration-inducing signals, but the response apparently differs between cell types (Meijer et al., 2008). Both ERK and Akt are established to play a role in cell survival. Till now, it is not clear whether the function of CXCR7 is alternative or exclusive to that of CXCR4 in the human placental cells. The important finding of our study is that CXCR7, independently signals through CXCL12 in the human choriocarcinoma JAR cells as the entire experiments were performed by blocking the CXCR4 receptor using the specific monolclonal blocking antibody, anti-CXCR4 antibody 12G5. Anti-CXCR4 antibody 12G5 is well established to be a blocking antibody for CXCR4. Besides not inducing any immune response, it binds specifically to the CXCR4 receptor with a high binding affinity and functionally blocks
the CXCR4 receptors (Castellone et al., 2004; Ottaiano et al., 2005; Mazzinghi et al., 2008; Taichman et al., 2002; Weisel et al., 2009; Zheng et al., 1999). Our findings reveal that CXCL12 binds and signals through the CXCR7 in the human choriocarcinoma JAR cells and may provide useful insights in understanding the role of CXCL12-mediated signaling in the human choriocarcinoma cells and other cell types where the expression of CXCR4 is low or absent. The precise role of CXCR7 in the growth and development of the human placenta needs to be assessed by performing functional assays in addition to the CXCL12–CXCR7 signaling pathway in the human placental cytotrophoblasts and syncytiotrophobasts at different gestational stages. Further, this study also opens up the question about the previously established exclusive role of CXCR4 in organogenesis and survival. Keeping in view the recent findings of interaction between CXCR7 and CXCR4, it could be hypothesized that CXCR7 may assist in some of the functions of CXCR4 at some stages of the development.
CONCLUSION Our study demonstrates the expression of CXCR7 receptor and the CXCL12-mediated signaling through the CXCR7 receptor in the human choriocarcinoma JAR cells. CXCL12–CXCR7 interaction activated the ERK and Akt pathways in these cells (Figure 7). Inhibition of the ERK and Akt pathways using specific inhibitors (Wortmanin & PD98509) led to the activation of the p38 pathway. To conclude, CXCL12–CXCR7 interaction independently activates ERK and Akt signaling pathways in human choriocarcinoma JAR cells.
Figure 7. CXCL12–CXCR7 signaling pathways in human choriocarcinoma JAR cells.
ERK AND AKT PATHWAYS IN HUMAN CHORIOCARCINOMA CELLS ACKNOWLEDGMENTS We are thankful to Mr. Rajiv Kumar, Department of Pathology, All India Institute of Medical Sciences, New Delhi and Dr Neera Nath, Scientist, Department of Biochemistry, All India Institute of Medical Sciences, New Delhi for providing us the technical assistance in this research work. Declcaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES Aiuti A, Webb IJ, Bleul C, Springer T and Gutierrez-Ramos JC (1997). The chemokine CXCL12 is a chemoattractant for human CD34 þ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34 þ progenitors to peripheral blood. J Exp Med. 185: 111–120. Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, Moepps B, Arenzana-Seisdedos F, Thelen M, Bachelerie F (2005). The chemokine SDF.-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J Biol Chem. 280:35760–35766. Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti A, Springer TA (1996). A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med. 184: 1101–1109. Bleul CC, Schultze JL, Springer TA (1998). B lymphocyte chemotaxis regulated in association with microanatomic localization, differentiation state, and B cell receptor engagement. J Exp Med. 187: 753–762. Bromley SK, Mempel TR, Luster AD (2008). Orchestrating the orchestrators: chemokines in control of T cell traffic. Nat Immunol. 9: 970–980. Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ, Wei K, McMaster BE, Wright K, Howard MC, Schall TJ (2006). A novel chemokine receptor for CXCL12and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med. 203: 2201–2213. Castellone MD, Guarino V, De Falco V, Carlomagno F, Basolo F, Faviana P, Kruhoffer M, Orntoft T, Russell JP, Rothstein JL, Fusco A, Santoro M, Melillo RM (2004). Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas. Oncogene. 23: 5958–5967. Corcione A, Ottonello L, Tortolina G, Facchetti P, Airoldi I, Guglielmino R, Dadati P, Truini M, Sozzani S, Dallegri F, Pistoia V (2000). Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells. J Natl Cancer Inst. 92: 628–635. Cross MJ, Stewart A, Hodgkin MN, Kerr DJ, Wakelam MJ (1995). Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem. 270: 25352–25355. D’Apuzzo M, Rolink A, Loetscher M, Hoxie JA, Clark-Lewis I, Melchers F, Baggiolini M, Moser B (1997). The chemokine
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RESEARCH ARTICLE
CXCL12–CXCR7 Signaling Activates ERK and Akt Pathways in Human Choriocarcinoma Cells Vishwas Tripathi1, Romsha Kumar1, Amit K. Dinda2, Jagdeep Kaur3, and Kalpana Luthra1 1
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India, 2Department of Pathology, All India Institute of Medical Sciences, New Delhi, India, and 3Department of Biotechnology, Panjab University, Chandigarh, India
Abstract CXCL12 acts as a physiological ligand for the chemokine receptor CXCR7. Chemokine receptor expression by human trophoblast and other placental cells have important implications for understanding the regulation of placental growth and development. We had previously reported the differential expression of CXCR7 in different stages of the human placenta suggesting its possible role in regulation of placental growth and development. In this study, we determined the expression of CXCR7 in human choriocarcinoma JAR cells at the mRNA level and protein level and the downstream signaling pathway mediated by CXCL12–CXCR7 interaction. We observed that binding of CXCL12 to CXCR7 activates the ERK and Akt cell-survival pathways in JAR cells. Inhibition of the ERK and Akt pathways using specific inhibitors (Wortmanin & PD98509) led to the activation of the p38 pathway. Our findings suggest a possible role of CXCR7 in activating the cell survival pathways ERK and Akt in human choriocarcinoma JAR cells. Keywords: RDC1, CXCR7, SDF-1, human placental cells, chemokine receptor, Chemokines
INTRODUCTION
apoptosis, and tumorigenesis. CXCL12 is a potent chemotactic factor for T cells (Kantele et al., 2000), monocytes (Bleul et al., 1996), B cells (D’Apuzzo et al., 1997; Bleul et al., 1998; Corcione et al., 2000), dendritic cells (Sozzani et al., 1997), mast cells (Lin et al., 2000), eosinophils (Nagase et al., 2001), and CD34þ hematopoietic progenitors (Aiuti et al., 1997; Mohle et al., 1998; Wang et al., 1998). CXCL12 regulates homing of hematopoietic stem cells to the bone marrow (Kawabata et al., 1999; Naiyer et al., 1999), megakaryocyte transepithelial migration (Hamada et al., 1998; Wang et al., 1998), platelet aggregation, and differentiation of early B cells, megakaryocytic-, and erythroid lineages (Nagasawa et al., 1996; Ma et al., 1998; Kowalska et al., 1999; Majka et al., 2000). CXCL12 binds to its cognate receptor CXCR4 and thus regulates the trafficking of normal and malignant cells. For many years, it was believed that CXCR4 was the only receptor for CXCL12. It has been recently observed that CXCL12 also binds to another transmembrane receptor called CXCR7 and this binding is very specific with high affinity (Balabanian et al., 2005). The affinity of CXCL12 to CXCR7 is approximately ten fold higher than the affinity of CXCL12 to CXCR4 (Balabanian et al., 2005; Burns et al., 2006). CXCR7, previously known as RDC1, is a chemokine receptor and is expressed in a number of mammalian tissues (Thelen & Thelen, 2008). Based on its binding to inflammatory and homing chemokines IFN-inducible T cell chemo-attractant (CXCL11) and CXCL12, CXCR7 is now classified as a deorphanized G-protein-coupled receptor (Balabanian et al., 2005; Burns et al., 2006).
Chemokines and their receptors have been implicated as pivotal players in many physiological and pathological conditions, but the expression and signaling pathways mediated by the interaction of chemokine and chemokine receptors at the materno–fetal interface is poorly understood. Chemokine receptor expression by human trophoblast and other placental cells have important implications for understanding the regulation of placental growth, development, and their role in materno–fetal HIV transmission. Chemokines belong to a superfamily of chemoattracting, cytokine-like proteins that bind to chemokine receptors and activate a number of distinct intracellular signaling pathways leading to functional responses such as chemotaxis, secretion, and transcriptional activation. (Thelen, 2001) Over 50 chemokines have been identified, and divided into four families (CXC, CX3C, CC, and C) on the basis of the positions of four conserved cysteine residues (Bromley et al., 2008). The chemokine CXCL12 (also called stromal cellderived factor-1, SDF-1) belongs to the family of CXC chemokines. It regulates many essential biological processes, including cardiac and neuronal development, stem cell motility, neovascularization, angiogenesis, Received 1 October 2013; accepted 12 December 2013. Address correspondence to Kalpana Luthra, Department of Biochemistry, All India Institute of Medical Sciences, Room No-3002, Ansari Nagar East, Gautam Nagar, New Delhi, Delhi 110029, India. E-mail: kalpanaluthra@ gmail.com
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Recent reports suggest that CXCR7 does not mediate a typical chemokine receptor response and is probably involved in CXCL12-mediated cell growth and survival (Thelen & Thelen, 2008). CXCR7 has been suggested to mediate CXCR4-like functions in subsets of leukocytes (Balabanian et al., 2005). Like CXCR4, CXCR7 is highly conserved between human and mouse (Heesen et al., 1998; Shimizu et al., 2000) and CXCR7/ mice die rapidly after birth (Sierro et al., 2007). In our previous study, we had assessed the expression of CXCR4 in the human placenta at different stages and observed its lower expression in the term as compared with the early placenta (Kumar et al., 2004). Having observed the expression of CXCR7 for the first time in the human placenta in a recently conducted study (Tripathi et al., 2009), we undertook to study the CXCL12 signaling mediated via CXCR7 in the human choriocarcinoma JAR cells.
MATERIALS AND METHODS Cell culture The human choriocarcinoma cell line JAR was obtained from National centre for cell sciences, India. Cells were plated in six-well plates at a density of 1.0–1.5 107 cells/ dish in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Rockville, MD,USA) containing 25 mM glucose, 20% fetal bovine serum, 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate; and were cultured at 37°C in 5% CO2. RNA isolation and RT-PCR To observe the expression of CXCR7 at the mRNA level in JAR cells, RT–PCR was performed after isolating total RNA from JAR cells by using the phenol–chloroform method. RNA was quantitated and checked for purity by determining the absorbance ratio at 260 and 280 nm. Two hundred nanogram of RNA was reverse transcribed in a total volume of 20 μl containing DTT (10 mM), dNTPs (1 mM each), random hexamers (5 mM; Promega, USA), RNasin (40 U/μl; Promega, USA) and 5X RT buffer with the addition of 200 U Superscript II Reverse Transcriptase (Promega, USA) for 2 h at 42°C. PCR was performed using 2.5 μl of the resultant cDNA in a 25 μl reaction volume containing 1X PCR buffer, 1.5 mM MgCl2, 2.5 mM dNTPs each, 1.25 U of Taq DNA polymerase (GIBCO BRL, USA) and 10 pmoles of CXCR7-specific forward and reverse primers, amplified at 67°C annealing temperature using following primer sequence (Microsynth, Switzerland) CXCR7: forward CTACA 3¢ CXCR7: reverse ACT 3¢
5¢
GGCTATGACACGCACTG
5¢
TGGTTGTGCTGCACGAG
Expression of CXCR7 gene in MCF-7 cells was used as a positive control (PC) and expression of β-actin as an internal control. Immunocytochemistry To determine the expression of CXCR7 at the protein level in JAR cells, immunocytochemistry experiments were performed. JAR cells were grown on cover slips and fixed using chilled methanol, followed by staining with polyclonal anti human CXCR7 antibody (OPA115173, Affinity Bioreagent, USA) at concentration of 1:50 for 4 h at 37°C. Slides were then washed with PBS (pH 7.4) and treated with R.T.U. Vecta stain kit, (Vector universal quick kit, Vector Laboratories, U.S.A.). Slides were visualized after staining with FITC-conjugated secondary antibody. Two controls were taken, primary antibody (anti-human CXCR7) substituted with normal saline and MCF-7 cells were taken as a PC23 using polyclonal anti-human CXCR7 primary antibody (OPA115173, Affinity Bioreagent, USA) and FITC-conjugated secondary antibody (Vector Laboratories, USA). Expression of CXCR7 in all the samples were scored by five independent observers (three pathologists and two biochemists) blindly by a semi-quantitative scoring method as – negative, faintly or weakly positive, moderately positive, and strongly positive. Flow cytometry To determine the surface expression of CXCR4 and CXCR7, JAR cells were serum starved (to synchronize the cells) for 24 h in DMEM medium without serum containing 2% penicillin and streptomycin. Cells (1X106) were incubated at 4°C for 1 h with nonspecific isotypematched controls, mouse human IgG2A (MAB0031) (R&D systems, USA), and rabbit polyclonals to human IgG (Abcam Inc., USA), and 20 μg/ml of each of the following: anti-human CXCR4 (12 G5, R&D Systems, USA) (Castellone et al., 2004; Ottaiano et al., 2005; Mazzinghi et al., 2008; Taichman et al., 2002; Weisel et al., 2009; Zheng et al., 1999) and anti-human CXCR7/RDC1 (Abcam Inc., USA) (Cat no-ab12870) antibodies. The mouse or rabbit primary antibodies were then detected by incubating the cells at 25°C for 45 min with the respective fluorescein isothiocyanate (FITC)conjugated goat (Fab¢) 2 anti-mouse or rabbit IgG (R&D systems, USA) secondary antibodies. The cells were washed twice with phosphate-buffered saline, re-suspended, and fixed in 1% (w/v) paraformaldehyde for analysis. Ten thousand cells from each sample were evaluated for fluorescence detection using FACScan (BD Biosciences, USA), and the data were analyzed with Diva software (BD Biosciences, USA). Each of the experiments were performed in triplicate and repeated thrice.
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Western blotting To elucidate the signaling pathways triggered by the CXCL12–CXCR7 interaction, JAR cells were cultured to confluence, washed, and then serum starved in DMEM for 24 h. One million cells were pre-incubated for 1 h at 4°C with 20 μg/ml of anti-CXCR4 blocking antibody (12G5 R&D systems, USA) to block the CXCR4 receptor. Thereafter the cells were stimulated by 200 nM CXCL12 (R&D system, USA) at different selected time points followed by lysis in ice-cold RIPA buffer. Protein concentration was determined in the cell lysate using BCA estimation kit (Banglore genei, India). The lysate was clarified by centrifugation at 14,000 rpm for 10 min. Normalized lysate (30 μg) was resuspended in loading buffer and electrophoresed in 10% polyacrylamide gel under reducing conditions and transferred to nitrocellulose membrane. Another set of human choriocarcinoma JAR cells were preincubated at 37°C for 1 h with, 100 nM of wortmannin and 10 μM of PD98509 (Calbiochem, San Diego, CA) before adding CXCL12. Wortmannin is a selective, cell-permeable, and irreversible inhibitor of PI 3-kinase (Vlahakis et al., 2002; Cross et al., 1995) and PD98509 is a selective and cell-permeable inhibitor of MAP kinase kinase (MEK) and acts by inhibiting the activation of MAP kinase and subsequent phosphorylation of MAP kinase substrates (Vlahakis et al., 2002; Means et al., 2000). The blots were probed with antibodies specific for ERK phosphorylation at Thr202 and Tyr204, Akt phosphorylation at Ser473, and p38 phosphorylation at Thr180/Tyr182 (Cell signaling, USA). Membranes were stripped with stripping buffer (314 μl of BME, 0.5 mM Tris pH 6.8) for 30 min at 37°C, washed thoroughly and then reblotted with antibodies to ERK2 (Santa Cruz CA), Akt, and p38, (Cell signaling, USA). The protein bands were visualized using the ECL detection system (Lumi Glo, Cell signaling, USA), after incubating the membrane with anti-biotin HRP-linked (1:1000), antiHRP-linked (1:2000) secondary antibody. All Western blotting experiments were performed in duplicate and repeated thrice. In all the experiments, the respective PC (Cell signaling, USA) and lysate from Control (Ctrl) without blocking the CXCR4 receptor were also loaded.
Figure 1. Expression of CXCR7 at the RNA level in JAR cells (Lane 1 showing expression of β-actin, (172 bp), Lanes 2 and 3 showing expression of CXCR7 (100 bp) in JAR and MCF-7 cells, respectively.
the expression of CXCR7 in MCF-7 cells (a PC, Figure 1, Lane 3). Expression of CXCR7 in JAR cells was evaluated at the protein level by immunocytochemistry and flow cytometry using specific polyclonal anti-human CXCR7 antibody (OPA-15173, Affinity Bioreagent, USA). A distinct surface expression of CXCR7 protein was observed in JAR cells on immunocytochemical staining (Figure 2B). Treatment of the JAR human choriocarcinoma cells with different concentrations of the anti-CXCR4 blocking monoclonal antibody 12G5, demonstrated maximal expression of CXCR4 receptors at 20 μg/ml of the mAb (Figure 3A). Thereafter, the expression of CXCR7/RDC1 was determined (by flow cytometry) using anti-CXCR7 antibody after treating cells with the anti-CXCR4 (12G5) blocking antibody (20 μg/ml) to completely block the
RESULTS JAR cells expressed CXCR7 both at the mRNA (RT-PCR) and at the protein level (by immunocytochemistry and flowcytometry). The expression of CXCR7 was confirmed, both at the mRNA and protein level in MCF-7 cells (known to express CXCR7 (Burns et al., 2006), which served as a PC. The expression of mRNA after RT-PCR was obtained in JAR cells. A 100 base pair amplified product of CXCR7 was obtained and the results were reproducible. A representative gel photograph reveals the expression of CXCR7 mRNA in JAR cells (Figure 1, Lane 2) and
Figure 2. (A) Negative control (without primary antibody). (B) Surface expression of CXCR7 protein in JAR cells (using FITC staining). (C) PC (MCF-7 cells showing expression of RDC1).
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Figure 3. (A) Showing expression of CXCR4 receptors in JAR cells (for identification of dosage of anti-CXCR4 antibody required to block the receptor). (B) Showing expression of CXCR7 protein on JAR cells by flow cytometric analysis.
CXCR4 receptors expressed on human choriocarcinoma JAR cells. More than 75% cells were found to express CXCR7 (Figure 3B). Anti-CXCR4 mAb 12G5, at a concentration of 20 μg/ml, was then used as a blocking antibody in further experiments to study the signaling via CXCR7 after blocking the CXCR4 receptors.
CXCL12 on interaction with CXCR7 in JAR cells (after blocking CXCR4 receptors) triggered phosphorylation of ERK1/2 and the extent of phosphorylation was maximal at 10 min on incubation of the cells with CXCL12 at different time periods followed by Western blotting (Figure 4A). In order to determine the activation of MAPK pathway in JAR cells, MAP kinase, the upstream regulator of ERK 1/2, was inhibited by incubating the cells with the inhibitor PD98509 before CXCL12 treatment. The specificity and efficacy of PD98509 as an inhibitor was checked by determining ERK 1/2 phosphorylation following CXCL12 treatment. CXCL12 triggered phosphorylation of ERK 1/2 was abrogated in the presence of MEK inhibitor (Figure 4B). Furthermore PD98509 did not affect the cell surface expression of CXCR4 and CXCR7 when analyzed by flow cytometry (P ns, data not shown). These results demonstrate the activation of the MAPK pathway (ERK 1/2) by CXCL12–CXCR7 interaction in JAR cells. Another important kinase is Akt which is a serine/ threonine Kinase. It is involved in mediating various physiological responses, such as inhibition of apoptosis and stimulation of cell proliferation. Further, to assess the involvement of Akt, Western blotting was performed after stimulating JAR cells (pretreated with anti-CXCR4 antibody 12G5) with CXCL12. The results of Western blotting showed that CXCL12, on interaction with CXCR7, triggered activation of AKT (Figure 5A). CXCL12-triggered phosphorylation of AKT was completely abrogated in the presence of wortmannin inhibitor (Figure 5B) which is a selective inhibitor of PI 3-kinase (Vlahakis et al., 2002; Cross et al., 1995). Pre-treating the cells with wortmannin before activation of CXCL12 did not alter the cell surface expression of CXCR7 (data not shown). The specificity of the wortmannin was demonstrated by its ability to inhibit Akt phosphorylation following CXCL12 treatment. To determine the kinetics of CXCL12 activation of Akt via the CXCR7 receptor, cells were incubated with CXCL12 for progressively longer duration (Figure 5A). Akt phosphorylation was maximal and peaked at 10 min after which it vanished slowly. To investigate whether CXCR7 receptors signals through the cell survival pathways similar to the CXCR4
Figure 4. CXCL12–CXCR7 interaction-mediated ERK1/2 phosphorylation in the JAR placental cells. (A) JAR cells pre-treated with anti-CXCR4 antibody followed by incubation with 200 nM CXCL12 for various time points (0, 5, 10, 15, and 20 min) lysed and blotted using anti-phospho ERK1/2 and anti-ERK2 antibodies. (B) JAR cells pre-treated with anti-CXCR4 antibody followed by incubation with 200 nM CXCL12, untreated or pre-incubated with ERK1/2 inhibitor PD98509. Membranes were blotted using anti-phospho ERK1/2 and anti-ERK2 antibodies. Lane 1, Untreated; Lane 2, Treated with CXCL12; Lane 3, PD98509 CXCL12.
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Figure 5. CXCL12–CXCR7 interaction-mediated Akt phosphorylation placental cells JAR. (A) JAR cells pre-treated with anti-CXCR4 antibody, were incubated with 200 nM CXCL12 for various time points, and then lysed and blotted using anti phospho Akt and anti-Akt antibodies. (B) JAR cells pre-treated with anti-CXCR4 antibody or pre-incubated with PI 3-kinase inhibitor Wortmannin, and then treated with 200 nM CXCL12. PC (Cat no-9273, Cell signaling, USA) was loaded in the gel to check for specificity. Membranes were blotted using anti-phospho Akt and anti-Akt antibodies. Lane 1, Pretreated with anti-CXCR4 antibody then treated with CXCL12; Lane 2, JAR cells treated with CXCL12 without blocking the CXCR4 receptor; Lane 3, PC (Cat no-9273, Cell signaling, USA); Lane 4, JAR cells pretreated with Wortmannin then treated with CXCL12.
receptor via the Akt-p38 balance in JAR placental cells, we determined the activation of phosphorylated p38 at different time periods (1–30 min), after treating the cells with CXCL12. We did not observe p38 phosphorylation (Figure 6A) suggesting that unlike CXCR4, CXCR7 on CXCL12 interaction (after blocking the CXCR4 receptors) does not activate the phosphorylation of p38. This unique feature of CXCR7 receptor that it does not activate p38, suggests its exclusive role in activating the ERK and Akt cell survival pathways. The p38 kinase is stimulated by different types of cellular stress such as UV radiation, heat shock, inflammatory cytokines including TNFa-1 and IL-1 etc. On investigation we observed that on blocking the cell survival pathways using Akt inhibitor wortmannin
and ERK inhibitor PD98509, CXCR7–CXCL12 interaction activates the p38 pathway (Figure 6B).
DISCUSSION The present study was undertaken to determine the downstream signaling of CXCL12 mediated via CXCR7 in the human choriocarcinoma JAR cells JAR. Our study reveals that CXCL12–CXCR7 interaction activates ERK and Akt pathways in the human choriocarcinoma JAR cells. CXCR7, also known as RDC1, is a recently deorphanized chemokine receptor which shares the ligand
Figure 6. CXCL12–CXCR7 interaction does not activate the p38 phosphorylation. On blocking the survival pathways by pre incubating the cells with the Akt inhibitor wortmannin and ERK inhibitor PD98509, CXCR7–CXCL12 interaction activated the phosphorylation of p38. JAR cells, incubated with 200 nM CXCL12 for various time points, and then lysed and blotted using anti-phospho p38 and anti-p38 antibodies. PC (Cat no-9213) was loaded in the gel to check for specificity. Membranes were blotted using anti-phospho p38 and anti-p38 antibodies. (A) Lane 1, Untreated; Lane 2, without blocking the CXCR4 receptor CXCL12 for 2 min; Lane 3, PC; Lane 4, Treated with anti CXCR4 CXCL12 for 2 min; Lane 5, Treated with anti CXCR4 CXCL12 for 5 min; Lane 6, Treated with anti CXCR4 CXCL12 for 10 min; Lane 7, Treated with anti CXCR4 CXCL12 for 15 min. (B) JAR cells were untreated or preincubated with Wortmannin (100 nM), PD98509 (10 μM) and without blocking the CXCR4 receptor followed by stimulation with 200 nM CXCL12.Western blotting was done using specific antibodies. Lane 1, Treated with anti CXCR4 CXCL12 (without any inhibitor); Lane 2, Treated with anti CXCR4 Wortmannin CXCL12; Lane 3, Treated with anti CXCR4 PD98509 CXCL12; Lane 4, Cells without blocking the CXCR4 receptor CXCL12.
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CXCL12 with another chemokine receptor CXCR4 and plays a crucial role in regulating immunity, angiogenesis, stem cell trafficking, and mediating organ-specific metastases of cancer (Xueqing et al., 2010). In our previous study, we reported for the first time that CXCR7 is differentially expressed and its expression increases at the term stage of the human placenta unlike the CXCR4 (Tripathi et al., 2009) The interaction between CXCR4 and CXCL12 was previously thought to be exclusive as CXCR4 was the only known receptor of CXCL12. However, Balabanian et al. (2005) observed that CXCL12 binds and signals through CXCR7 expressed on the surface of primary T cells and participates in lymphocyte motility. This breakthrough opened a new question about the role of CXCL12-mediated signaling pathways in those cells where CXCR4 is less expressed. The precise role of CXCR7 in the human placenta needs to be elucidated. We therefore undertook to study CXCL12 signaling mediated via CXCR7 in placental cells (JAR). CXCL12–CXCR7 interaction led to activation of the ERK and Akt pathways in JAR cells. Further, the inhibition of Akt and ERK using specific inhibitors (wortmannin & PD98509) abrogated the phosphorylation of ERK and Akt. Moreover, our study revealed that the CXCL12–CXCR7 interaction did not activate p38 pathway and therefore did not favor cell death. An interesting observation was the activation of the p38 pathway after blocking the ERK and Akt pathways using pathway-specific inhibitors. One probable reason could be that the Akt and/or ERK pathway/s may have a negative regulatory effect on the p38 pathway which remains to be determined. The two important pathways that are associated with cell survival are the ERK 1/2 kinase and Akt, the downstream effector protein of PI 3-kinase. Several groups have reported in different cell types that CXCR7 regulates many important biological processes like cell survival, cell adhesion, and tumor development in animal models (Burns et al., 2006; Mazzinghi et al., 2008; Meijer et al., 2008; Raggo et al., 2005). In a recent study, Meijer et al. (2008) reported that CXCR7 is able to initiate powerful proliferation and migration-inducing signals, but the response apparently differs between cell types (Meijer et al., 2008). Both ERK and Akt are established to play a role in cell survival. Till now, it is not clear whether the function of CXCR7 is alternative or exclusive to that of CXCR4 in the human placental cells. The important finding of our study is that CXCR7, independently signals through CXCL12 in the human choriocarcinoma JAR cells as the entire experiments were performed by blocking the CXCR4 receptor using the specific monolclonal blocking antibody, anti-CXCR4 antibody 12G5. Anti-CXCR4 antibody 12G5 is well established to be a blocking antibody for CXCR4. Besides not inducing any immune response, it binds specifically to the CXCR4 receptor with a high binding affinity and functionally blocks
the CXCR4 receptors (Castellone et al., 2004; Ottaiano et al., 2005; Mazzinghi et al., 2008; Taichman et al., 2002; Weisel et al., 2009; Zheng et al., 1999). Our findings reveal that CXCL12 binds and signals through the CXCR7 in the human choriocarcinoma JAR cells and may provide useful insights in understanding the role of CXCL12-mediated signaling in the human choriocarcinoma cells and other cell types where the expression of CXCR4 is low or absent. The precise role of CXCR7 in the growth and development of the human placenta needs to be assessed by performing functional assays in addition to the CXCL12–CXCR7 signaling pathway in the human placental cytotrophoblasts and syncytiotrophobasts at different gestational stages. Further, this study also opens up the question about the previously established exclusive role of CXCR4 in organogenesis and survival. Keeping in view the recent findings of interaction between CXCR7 and CXCR4, it could be hypothesized that CXCR7 may assist in some of the functions of CXCR4 at some stages of the development.
CONCLUSION Our study demonstrates the expression of CXCR7 receptor and the CXCL12-mediated signaling through the CXCR7 receptor in the human choriocarcinoma JAR cells. CXCL12–CXCR7 interaction activated the ERK and Akt pathways in these cells (Figure 7). Inhibition of the ERK and Akt pathways using specific inhibitors (Wortmanin & PD98509) led to the activation of the p38 pathway. To conclude, CXCL12–CXCR7 interaction independently activates ERK and Akt signaling pathways in human choriocarcinoma JAR cells.
Figure 7. CXCL12–CXCR7 signaling pathways in human choriocarcinoma JAR cells.
ERK AND AKT PATHWAYS IN HUMAN CHORIOCARCINOMA CELLS ACKNOWLEDGMENTS We are thankful to Mr. Rajiv Kumar, Department of Pathology, All India Institute of Medical Sciences, New Delhi and Dr Neera Nath, Scientist, Department of Biochemistry, All India Institute of Medical Sciences, New Delhi for providing us the technical assistance in this research work. Declcaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES Aiuti A, Webb IJ, Bleul C, Springer T and Gutierrez-Ramos JC (1997). The chemokine CXCL12 is a chemoattractant for human CD34 þ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34 þ progenitors to peripheral blood. J Exp Med. 185: 111–120. Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, Moepps B, Arenzana-Seisdedos F, Thelen M, Bachelerie F (2005). The chemokine SDF.-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J Biol Chem. 280:35760–35766. Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti A, Springer TA (1996). A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med. 184: 1101–1109. Bleul CC, Schultze JL, Springer TA (1998). B lymphocyte chemotaxis regulated in association with microanatomic localization, differentiation state, and B cell receptor engagement. J Exp Med. 187: 753–762. Bromley SK, Mempel TR, Luster AD (2008). Orchestrating the orchestrators: chemokines in control of T cell traffic. Nat Immunol. 9: 970–980. Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ, Wei K, McMaster BE, Wright K, Howard MC, Schall TJ (2006). A novel chemokine receptor for CXCL12and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med. 203: 2201–2213. Castellone MD, Guarino V, De Falco V, Carlomagno F, Basolo F, Faviana P, Kruhoffer M, Orntoft T, Russell JP, Rothstein JL, Fusco A, Santoro M, Melillo RM (2004). Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas. Oncogene. 23: 5958–5967. Corcione A, Ottonello L, Tortolina G, Facchetti P, Airoldi I, Guglielmino R, Dadati P, Truini M, Sozzani S, Dallegri F, Pistoia V (2000). Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells. J Natl Cancer Inst. 92: 628–635. Cross MJ, Stewart A, Hodgkin MN, Kerr DJ, Wakelam MJ (1995). Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem. 270: 25352–25355. D’Apuzzo M, Rolink A, Loetscher M, Hoxie JA, Clark-Lewis I, Melchers F, Baggiolini M, Moser B (1997). The chemokine
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