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preosteoblast cells
Bone 71 (2015) 137–144
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Original Full Length Article
STAT-6 mediates TRAIL induced RANK ligand expression in stromal/preosteoblast cells Kumaran Sundaram a, Yuvaraj Sambandam a, Sundaravadivel Balasubramanian b, Balakrishnan Pillai a, Christina Voelkel-Johnson c, William L. Ries d, Sakamuri V. Reddy a,⁎ a
Charles P. Darby Children's Research Institute, Medical University of South Carolina, Charleston, SC, USA Department of Radiation Oncology, Medical University of South Carolina, Charleston, SC, USA Dept. of Microbiology & Immunology, Medical University of South Carolina, Charleston, SC, USA d College of Dental Medicine, Medical University of South Carolina, Charleston, SC, USA b c
a r t i c l e
i n f o
Article history: Received 24 July 2014 Revised 14 October 2014 Accepted 23 October 2014 Available online 30 October 2014 Edited by: Hong-Hee Kim Keywords: TRAIL RANK ligand STAT-6 Stromal/preosteoblasts
a b s t r a c t Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis, and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein, we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/ preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly, TRAIL treatment induced RANKL mRNA expression in these cells. In addition, TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition, siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus, our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Receptor activator of NF-κB ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells which induces osteoclast precursor cell differentiation to form multinucleated bone resorbing osteoclasts [1]. Elevated levels of RANKL expression have been associated with several skeletal diseases such as Paget's disease of bone and multiple myeloma [2,3]. A variety of cytokines and growth factors such as 1,25-(OH)2 vitamin D, parathyroid hormone (PTH), interleukin 1β (IL-1β), IL-11, and prostaglandin E2 (PGE2) induce RANKL expression in marrow stromal/preosteoblast cells [4]. Evidence suggests that MKP-1 is essential for vitamin D stimulated RANKL expression in bone marrow stromal cells [5]. Recently, protein phosphatase 2 A has been shown to regulate RANKL expression
⁎ Corresponding author at: Charles P. Darby Children's Research Institute, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA. Fax: +1 843 792 7927. E-mail address: [email protected] (S.V. Reddy).
http://dx.doi.org/10.1016/j.bone.2014.10.016 8756-3282/© 2013 Elsevier Inc. All rights reserved.
in mouse osteoblast cells [6]. Furthermore, lipopolysaccharide treatment increased the levels of RANKL expression through activation of toll-like receptors in primary murine osteoblasts [7]. It has also been shown that PTH stimulates RANKL expression through the cAMP/ protein kinase A/CREB cascade. Mouse RANKL expression is upregulated by vitamin D and PTH; however these transcriptional regulatory regions are far upstream to the transcriptional start site [8,9]. Runx2/Cbfa1 can enhance RANKL expression by increasing the ratio of immature to mature osteoblasts [10]. Also, DNA methylation has been shown to play an important role in modulation of RANKL and osteoprotegerin (OPG) expression in human bone [11]. We previously identified NFATc3 as a downstream target of CXCL13 signaling pathway to stimulate RANKL expression in oral squamous cell carcinoma cells [12]. Similarly, increased levels of extracellular calcium induced RANKL expression through activation of NFATc3 in mouse osteoblast cells [13]. Fibroblast growth factor (FGF)-2 induces RANKL production through COX-2mediated prostaglandin synthesis and by suppressing OPG expression [14]. We recently demonstrated that CREB (cAMP response element-binding) is a down-stream effector of CXCL5 signaling which enhances the RANKL expression in Paget's disease [15].
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Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/ Apo2/TNSF10) is a member of the TNF superfamily. TRAIL induces apoptosis in a variety of tumor cells but not in normal cells [16]. TRAIL is expressed on the surface of natural killer cells, lymphocytes, macrophages and dendritic cells [17]. TRAIL expressed either as a type II transmembrane protein or as a soluble protein is also detectable in different biological fluids under physiological conditions [18]. In humans, TRAIL can interact with four different TRAIL receptors. Two of these receptors are death receptors DR4 and DR5 which contain a death domain motif and promote apoptosis in tumor cells. In contrast, DcR1 and DcR2 receptors lack a death domain [19]. In addition, TRAIL can also bind with OPG, a decoy receptor of RANKL. Recently, it has been shown that TRAIL induces osteoclast differentiation under physiological and pathological conditions [20]. Furthermore, TRAIL increased osteoclast formation through a TNF receptor associated factor-6 (TRAF-6) dependent signaling pathway [21]. Multiple myeloma cancer cells produce high levels of TRAIL and have a positive correlation with osteolytic markers which suggested that TRAIL may play an important regulatory role in excess bone resorption in these patients [22]. However, TRAIL induced expression of RANKL in bone marrow stromal/preosteoblast cells is unknown. In the present study, we delineated the molecular mechanism underlying the TRAIL induced RANKL expression in these cells. 2. Materials and methods 2.1. Reagents Cell culture and DNA transfection reagents were purchased from Invitrogen (Carlsbad, CA). Recombinant human soluble TRAIL was purchased from Enzo Life Sciences (Farmingdale, NY). Anti-RANKL antibody was obtained from R&D systems Inc. (Minneapolis, MN). AntiSTAT-6 and anti-p-STAT-6 antibodies were purchased from Cell Signaling (Danvers, MA). Anti-TRAIL, anti-IκB, anti-p-IκB, anti-β-actin antibodies and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Super signal enhanced chemiluminescence (ECL) reagent was obtained from Amersham Bioscience (Piscataway, NJ) and nitrocellulose membranes were purchased from Millipore (Bedford, MA). pNF-κB-Luc reporter plasmid was obtained from Stratagene (La Jolla, CA) and a luciferase reporter assay system was obtained from Promega (Madison, WI). Chromatin Immunoprecipitation (ChIP) assay kit was purchased from Upstate (Temecula, CA). 2.2. Quantitative real-time RT-PCR Total RNA was isolated from the human bone marrow derived stromal/preosteoblast (SAKA-T) cells [23] stimulated with and without TRAIL (0–100 ng/ml) for 48 h, using RNAzol (Molecular Research Center, Inc., Cincinnati, OH). A reverse transcription reaction was performed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) at 25 °C for 5 min, 42 °C for 30 min followed by 85 °C for 5 min. The quantitative real-time PCR was performed using IQ™ SYBR Green Supermix in an iCycler and the primers specific for RANKL, OPG and GAPDH as described earlier [24]. Thermal cycling parameters were 94 °C for 3 min, followed by 40 cycles of amplifications at 94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min, and 72 °C for 5 min as the final elongation step. To quantify absolute copy number of mRNA expression in each sample, a standard curve for each mRNA was generated using a fivefold dilution of control RNA sample. The levels of mRNA expression were calculated as a ratio to GAPDH amplification. 2.3. Cell proliferation assay Human bone marrow derived stromal/preosteoblast cell line, SAKA-T was established by immortalization with SV40 virus large T-antigen as described previously [23,25]. SAKA-T cells were further transformed by telomerase expression enhancing the growth [26]. Cells were maintained
in Modified Eagle Medium (MEM) containing 10% fetal bovine serum (FBS) and supplemented with L-glutamine, penicillin and streptomycin. Cells were incubated at 37 °C in 5% CO2. SAKA-T cells were seeded in 96 well plates (1 × 104 cells/well) and stimulated with TRAIL (100 ng/ml) for 24 and 48 h. Cells were incubated with 10% (20 μl) Alamar Blue reagent (Invitrogen, cat#DAL1100) for 4 h and incubated at 37 °C and the fluorescence intensity was measured at 540 nm excitation and 594 nm emissions. Wells containing complete growth media alone with 20 μl of Alamar Blue were averaged and subtracted from all experimental readings [27]. 2.4. Western blot analysis SAKA-T or primary human bone marrow stromal cells (Stem cell Technologies Inc., Vancouver, cat# 70022) were seeded (5 × 105 cells/ well) in 6-well plates and supplemented with α-MEM containing 10% FBS. A day after seeding, cells were transfected with expression plasmids encoding TRAIL for 48 h. Cells were lysed in a cell lysis buffer and protein content of the samples was measured using the BCA protein assay reagent. Protein (20 μg) samples were then subjected to SDSPAGE using 12% Tris–HCl gels and blot transferred on to a nitrocellulose membrane, immunoblotted with anti-RANKL, anti-p-STAT-6, antiSTAT-6, anti-p-IκB, anti-IκB, anti-p-c-Jun and anti-β-actin antibodies. The bands were detected using the enhanced chemiluminescence detection system and quantified by densitometry analysis using the NIH ImageJ Program. 2.5. TRAIL expression vector construct The full-length of TRAIL cDNA was generated by RT-PCR using RNA isolated from normal human lymphocytes, inserted into the TA cloning system and verified by sequence analysis. TRAIL cDNA was subsequently cloned into the pIRES-EGFP vector as described [28]. 2.6. c-Jun N-terminal kinase (JNK) activity assay SAKA-T cells were cultured in the presence or absence of TRAIL (100 ng/ml) for different time points (0–3 h) and collected in ice-cold phosphate buffered saline (PBS), centrifuged at 3000 rpm for 5 min. Cells were resuspended in a cell lysis buffer with a protease inhibitor followed by brief sonication. Cell lysates were cleared by centrifugation for 5 min at 12,000 g. JNK activity was measured using a solid-phase GST-cJun (1–89 amino acids) fusion protein. Briefly, c-Jun kinase was coprecipitated with its substrate conjugated to glutathione-S-sepharose beads at 4 °C overnight. The precipitates were washed twice with cell lysis buffer followed by a kinase buffer (25 mM Tris (pH 7.5), 5 mM βglycerophosphate, 1 mM sodium vanadate, 2 mM dithiothreitol (DTT), 10 mM MgCl2) and resuspended in 50 μl of kinase buffer. The reaction was carried out at 30 °C for 30 min in the presence of 100 μM of ATP and stopped by addition of SDS-loading buffer. Proteins were separated by SDS-PAGE (15%) and blot transferred onto a nitrocellulose membrane. JNK activity was measured by using anti-p-c-Jun antibody. 2.7. Luciferase reporter gene assay SAKA-T cells transfected with NF-κB-Luc or hRANKL-pSTAT-Luc reporter plasmid construct [24] were co-transfected with siRNA (20 nM) against STAT-6 or scrambled control siRNA by lipofectamine. Cells were cultured in the presence or absence of TRAIL (100 ng/ml) for 48 h. Total cell lysates (20 μl) were mixed with 100 μl of the luciferase assay reagent and luminescence was measured for 10 s of integrated time using Sirius luminometer. β-Galactosidase activity was measured according to manufacturer's protocol (Promega). In brief, 100 μl of cell lysates was mixed with 100 μl of 2× assay buffer containing the substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) and incubated at 37 °C for 30 min. The mixture was added with 1 M sodium carbonate
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to stop the reaction and the color intensity was measured at 420 nM. The transfection efficiency was normalized by the β-galactosidase activity co-expressed in these cells. 2.8. Transcription factor array screening We used real-time RT-PCR based human transcription factor array screening for identification of TRAIL regulated transcription factors in SAKA-T cells stimulated with and without TRAIL (100 ng/ml) for 24 h. Total RNA was isolated from these cells using RNAzol reagent and reverse transcription reaction (RT) was performed. Real-time PCR was performed using RT qPCR Master Mix (RT2 Profiler PCR Array System (PAHS-075ZA) Qiagen Inc., Valentia, CA) in a 96-well plate to quantify the expression levels of 84 transcription factors. Thermal cycling parameters were 95 °C for 10 min, followed by 40 cycles of amplifications at 95 °C for 15 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min as the final elongation step. Relative levels of mRNA expression were normalized with housekeeping gene expression (GAPDH, HPRT and β-actin). Data analysis was performed in triplicates using the web portal http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php. 2.9. Chromatin immunoprecipitation (ChIP) SAKA-T cells were treated with or without TRAIL (100 ng/ml) for 24 h and cross-linked with 1% final concentration of formaldehyde at 37 °C for 10 min before harvest. Soluble chromatin was prepared by sonication to get an average DNA length of 200–1000 bp. The sheared soluble chromatin was precleared with blocked Protein G agarose, and 10% of the precleared chromatin was set aside as input control. Immunoprecipitation was carried out with 5 μg of anti-p-STAT-6 antibody overnight at 4 °C. Immune complexes were pulled down using Protein G agarose, washed and eluted twice with elution buffer (0.1 M NaHCO3, 1% SDS) and cross-linking reversed in 200 mM NaCl at 65 °C overnight with 20 μg RNase A. DNA was purified following proteinase K treatment with a PCR purification kit (Qiagen, MD). Then, PCR was performed using the gene specific primers for STAT binding region, 5′TTTACAGCAATGAGCAGACCT-3′ (sense) and 5′-CAGGATGCATGGGATT ACCT-3′ (antisense) DNA samples or input DNA fractions were analyzed by 35 cycles of PCR (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s). PCR products were subjected to electrophoresis using 2% agarose gels and visualized by ethidium bromide. The percentage of chromatinimmune complex precipitate DNA relative to input was calculated and shown as mean ± SE for three independent experiments. 3.0. Confocal microscopy analysis
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3. Results 3.1. TRAIL receptor expression in human bone marrow stromal/preosteoblast cells TRAIL interacts with receptors TRAILR1 (DR4), TRAILR2 (DR5), TRAILR3 (DcR1), and TRAILR4 (DcR2) [29]. It has been shown that osteoblast cells express TRAIL receptors [30]; however, TRAIL regulation of its receptor expression in human bone marrow derived stromal/ preosteoblast cells is unclear. Therefore, we examined the TRAIL modulation of its receptor expression in SAKA-T cell line. Cells were stimulated with and without TRAIL (100 ng/ml) for 48 h and total RNA isolated was subjected to real-time RT-PCR analysis for TRAIL receptor mRNA expression. As shown in Fig. 1A, TRAIL stimulation increased DcR1 (4.3-fold) and DR5 (3.5-fold) mRNA expression compared to unstimulated cells. However, no changes were observed in DcR2 and DR4 expression in these cells. Since OPG acts as a soluble TRAIL receptor [31], we also examined the TRAIL regulation of OPG mRNA expression in these cells. We identified that TRAIL significantly decreased OPG mRNA expression (Fig. 1B). Studies indicated that osteoblast cells express TRAIL receptors (DR4 and 5), however TRAIL stimulation does not induce apoptosis [32]. Therefore, we further tested the effect of TRAIL on proliferation of SAKA-T cells as described in Materials and methods. As shown in Fig. 1C, TRAIL treatment had no effect on SAKA-T cell proliferation. These results suggest that TRAIL differentially regulates receptor expression in human bone marrow stromal/preosteoblast cells. 3.2. TRAIL regulation of RANKL expression Recent evidence suggests that TRAIL directly promotes osteoclast differentiation/bone resorption activity [21] and modulates osteoblast differentiation [41]. However, TRAIL regulation of RANKL expression in human bone marrow stromal/preosteoblast cells is unknown. Therefore, SAKA-T cells were stimulated with TRAIL (100 ng/ml) for a variable period (0–48 h) and total RNA isolated from these cells was subjected to real-time RT-PCR analysis for RANKL mRNA expression. Interestingly, TRAIL significantly increased (5-fold) RANKL mRNA expression (Fig. 2A). Further, TRAIL stimulation significantly increased RANKL mRNA expression in a dose-dependent manner (Fig. 2B). In addition, total cell lysates obtained from SAKA-T cells transfected with the TRAIL expression vector [28] were subjected to western blot analysis for RANKL expression. As shown in Fig. 2C, TRAIL over-expression demonstrated a significant increase in RANKL expression in these cells. These results suggest that TRAIL induces RANKL expression in bone marrow stromal/preosteoblast cells. 3.3. TRAIL signaling in stromal/preosteoblast cells
4
SAKA-T cells were seeded (1 × 10 /well) onto 22 mm coverslips in 6-well plates. Cells were treated with and without TRAIL (100 ng/ml) for 1 h and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Then, cells were permeabilized with 0.1% Triton X-100 for 5 min and blocked for 1 h with PBS containing 10% donkey serum. Cells were incubated with primary antibody against p-STAT-6 (1:100 dilution) in PBS and incubated overnight at 4 °C. After extensive washing with PBS, cells were incubated with Alexa 568-conjugated antirabbit IgG. Nuclear staining was performed with DAPI and localization of p-STAT-6 was visualized by IX81 confocal microscope (Olympus, IX81; 60 ×). Z-stack images were obtained at 0.5 μm thickness stepsize and images were then rotated along the X-axis and Y-axis using the Fluoview 1000 software. 3.1. Statistical analysis Results are presented as mean ± SE for three independent experiments and were compared by Student's t-test. Values were considered significantly different for p b 0.05.
TRAIL can induce NF-κB activation for normal cell survival [33]. Therefore, we next examined the effect of TRAIL on NF-κB activation in stromal/preosteoblast cells. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for 0–3 h. Western blot analysis of total cell lysates demonstrated that TRAIL significantly increased the levels of p-IκB expression. However, there was no change in total IκB expression in these cells (Fig. 3A). We further examined the NF-κB reporter activity in these cells. NF-κB-Luc reporter plasmid was transiently transfected into SAKA-T cells using lipofectamine. Cells were cultured in the presence or absence of TRAIL (100 ng/ml) for 48 h. Total cell lysates obtained from these cells were analyzed for luciferase activity. TRAIL treatment increased (4.5-fold) NF-κB reporter activity compared to unstimulated cells (Fig. 3B). It has also been shown that JNK modulates RANKL expression in stromal/preosteoblast cells [34]. We therefore tested TRAIL regulation of JNK activity in these cells. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for variable time-points (0–3 h) and total cell lysates obtained were assayed for JNK activity as described in Materials and methods. As shown in Fig. 3C, TRAIL
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Fig. 1. Effect of TRAIL on receptor expression and proliferation of human bone marrow derived stromal/preosteoblast cells. (A) TRAIL regulation of receptor expression. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for 48 h and total RNA isolated was subjected to real-time RT-PCR analysis for DR4, DR5, DcR1 and DcR2, and (B) OPG mRNA expression. The absolute levels of mRNA expression in the samples were normalized by GAPDH mRNA expression. (C) TRAIL does not alter proliferation of SAKA-T cells. Cells were treated with TRAIL (100 ng/ml) for 0–48 h and proliferation [fluorescence intensity as arbitrary units (AU)] was assayed as described. Data represent triplicate studies with mean ± SE (*p b 0.05).
treatment increased (5-fold) JNK activity over basal levels of expression in these cells in a time-dependent manner. 3.4. Participation of STAT-6 in TRAIL induction of RANKL expression To further identify TRAIL regulated transcription factors which modulate RANKL expression, we used real-time RT-PCR based human transcription factor array screening of 84 transcription factors. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for 24 h and total RNA
isolated was used as a template for identification of transcription factors and data obtained were analyzed by web portal as described in Materials and methods. We thus identified a 3.2- and 3.0-fold increase in STAT-6 and myogenic differentiated 1 (MYOD1) mRNA expression in TRAIL stimulated SAKA-T cells, respectively (Fig. 4A). STAT transcription factors have been implicated in regulating gene expression in stromal/preosteoblast cells. Therefore, we examined the TRAIL modulation of STAT-6 activation in these cells. Western blot analysis of total cell lysates obtained from the primary human bone marrow stromal cells
Fig. 2. TRAIL induces RANKL expression in SAKA-T cells. (A). TRAIL induces RANKL mRNA expression. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for indicated time-points and cells were stimulated with different concentrations of TRAIL (0–100 ng/ml) for 48 h (B) and total RNA isolated was subjected to real-time RT-PCR analysis for RANKL mRNA expression. The absolute levels of RANKL mRNA expression in the samples were normalized by GAPDH mRNA expression. Data represent triplicate studies with mean ± SE (*p b 0.05). (C). Overexpression of TRAIL increases RANKL levels. SAKA-T cells were transfected with empty vector (EV) or TRAIL expression vector. After 48 h, total cell lysates were subjected to western blot analysis for TRAIL and RANKL expression. β-Actin expression served as control.
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Fig. 3. TRAIL enhances NF-κB and JNK activity. (A). TRAIL activation of IκB. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for indicated time-points. Total cell lysates were subjected to western blot analysis for IκB and p-IκB expression. (B). TRAIL enhances NF-κB-Luc reporter gene activity. SAKA-T cells were transfected with EV or NF-κB-Luc reporter plasmids and stimulated with and without TRAIL (100 ng/ml) for 48 h. Total cell lysates prepared were assayed for luciferase activity (RLU, relative light units). The transfection efficiency was normalized by β-galactosidase activity co-expressed in these cells. Values are expressed as mean ± SE for triplicate studies (*p b 0.05). (C). TRAIL increases JNK activity. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for indicated time-points (0–3 h) and total cell lysates were analyzed to measure JNK as described in Materials and methods. β-Actin expression served as control.
stimulated with TRAIL showed an increase (10-fold) in the levels of p-STAT-6 expression (Fig. 4B). Furthermore, confocal microscopy analysis of SAKA-T cells treated with TRAIL revealed nuclear localization of p-STAT-6 (Fig. 4C-i). Z-stack images further confirm the nuclear localization of p-STAT-6 in these cells (Fig. 4C-ii). We previously identified STAT binding element (ATTTGGGGAA) at far upstream in the hRANKL promoter region [24]. Long-range enhancer elements have also been shown to modulate RANKL gene expression [35]. Therefore, we next examined p-STAT-6 binding to the hRANKL gene promoter region by ChIP assay using anti-p-STAT-6 antibody as described. Chromatin immune complexes obtained from TRAIL stimulated SAKA-T cells were analyzed by PCR using hRANKL genespecific primers for the STAT-binding region [24]. As shown in Fig. 5A, TRAIL treatment significantly increased p-STAT-6 binding to the hRANKL promoter region. We detected no binding in immunoprecipitate obtained with a control IgG. Next, we examined the potential role of STAT-6 to modulate RANKL gene promoter activity in response to TRAIL stimulation. SAKA-T cells were transiently cotransfected with hRANKL-pSTAT reporter plasmid with siRNA against STAT-6 or control siRNA and cultured in the presence and absence of TRAIL for 48 h. siRNA suppression of STAT-6 inhibits TRAIL stimulated hRANKL-pSTAT luciferase reporter gene activity. In contrast, no change in gene promoter activity was observed in empty vector (EV) and control siRNA transfected cells (Fig. 5B). Transfection efficiency was normalized by β-galactosidase activity co-expressed in these cells. These results suggest that STAT-6 is a down-stream effector of TRAIL signaling to induce RANKL expression in human bone marrow stromal/preosteoblast cells. 4. Discussion Studies have identified that several cytokines regulate RANKL expression in stromal/osteoblast lineage cells and immune responsive T-cells which play an important role in osteoclast development and bone resorption activity under normal and pathologic conditions [36,37]. Herein, we have demonstrated that TRAIL induces RANKL expression in human bone marrow stromal/preosteoblast cells suggesting that TRAIL can enhance osteoclast differentiation through elevated levels of RANKL expression in the bone microenvironment.
Furthermore, TRAIL deficient mice showed normal bone phenotype [38]. Therefore, TRAIL induction of RANKL expression may have a pathologic role with respect to osteoclast development and bone destruction. TRAIL promotes cell survival, migration, and proliferation in a variety of normal cells [39]. In contrast, TRAIL induces cell death in many cancer cells [40]. Human osteoblasts have been shown to express TRAIL-R and are resistant to TRAIL mediated apoptosis [30]. It has been reported that the osteoblast sensitivity to TRAIL was due to the upregulation of DR5 and down-regulation of DcR2 [41]. However, this study demonstrated that TRAIL induced the expression of DR5 and DcR1 receptors with no change in proliferation of human bone marrow stromal/preosteoblast cells. OPG can serve as a soluble decoy receptor for both RANKL and TRAIL [42]. Therefore, TRAIL inhibition of OPG and induction of RANKL expression in bone marrow stromal/preosteoblast cells may enhance osteoclast formation and bone resorption activity in pathologic conditions. Consistently, it has been shown that OPG deficient mice lead to severe alveolar bone loss [43]. Also, TRAIL has been shown to block the inhibitory effect of OPG on RANKL induced osteoclast formation [44]. Elevated levels of TRAIL have been associated with periodontitis [45]. These studies suggested that OPG may inhibit alveolar bone loss induced by RANKL and TRAIL in periodontitis conditions. Furthermore, TRAIL has been shown to induce apoptosis in a variety of cancer cell types which includes multiple myeloma [46]. Also, breast cancer cells have been shown to express RANKL, TRAIL and its receptors. Therefore, our results suggest that TRAIL induced RANKL expression may have a functional role in tumor progression and bone metastasis [47]. Our transcription factor array screening identified TRAIL upregulation of STAT-6 and MyoD1 transcription factor expression. It has been shown that MyoD promotes osteoblast differentiation [48]. However, we did not identify a putative binding motif for MyoD in the hRANKL promoter region. This study identifies a functional role for TRAIL activation of STAT-6 to induce RANKL expression in bone marrow stromal/preosteoblast cells. Previously, it has been shown that STAT-3 activation in stromal/osteoblastic cells is required for induction of the RANKL expression by interleukin-1 (IL-1) but not 1,25-dihydroxyvitamin D3 or parathyroid hormone [49]. Recently, calcineurin inhibitor, tacrolimus has been shown to affect the RANKL expression in IL-6 stimulated
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Fig. 4. TRAIL up-regulation of STAT-6 transcription factor expression. (A) Identification of STAT-6 transcription factor up-regulation by TRAIL. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for 24 h. Total RNA isolated was subjected to RT2-Profiler array screening of 84 human transcription factors array by real-time RT-PCR in triplicate studies as described in Materials and methods. (B). TRAIL induces p-STAT-6 expression in normal human bone marrow derived primary stromal/preosteoblast cells. Cells were stimulated with and without TRAIL (100 ng/ml) for indicated time-points (0–3 h) and total cell lysates were subjected to western blot analysis for STAT-6 and p-STAT-6 expression. (C-i). Confocal microscopy analysis for pSTAT-6 nuclear localization. SAKA-T cells were stimulated with and without TRAIL (100 ng/ml) for 1 h and immunostained for p-STAT-6. Nuclear staining was performed by DAPI and the merged image demonstrates nuclear localization of p-STAT-6 in TRAIL stimulated cells. (C-ii) Z-stack images of p-STAT-6 nuclear localization along x-axis and y-axis.
fibroblast-like synoviocytes through STAT-3 [50]. IL-6 in combination with IL-6 soluble receptor and oncostatin M has been shown to induce RANKL expression in stromal cells via STAT-3 [51]. We previously showed that STAT-1 is a downstream effector of FGF-2 signaling to stimulate RANKL expression [24]. Therefore, STAT family members show differential cytokine response and functional significance with respect to RANKL expression. This study demonstrated that TRAIL induced p-STAT-6 expression and ChIP assay revealed that TRAIL stimulation increased p-STAT-6 binding to the human RANKL promoter region suggesting STAT-6 participation in TRAIL control of RANKL gene expression. Inhibition of hRANKL-STAT promoter activity through siRNA
suppression further confirms the specificity for STAT-6 participation in TRAIL induction of RANKL gene expression. However, we did not observe STAT6 participation in TRAIL down-regulation of OPG (data not shown). STAT-6 and NF-kB have been shown to bind cooperatively to a DNA probe containing both sites and synergize activation of transcription by IL-4 [52]. We further demonstrated that TRAIL enhances NF-kB and JNK activity. hRANKL promoter has been shown to contain STAT and NF-kB responsive elements [53,54]. The activation and interaction between STAT3 and NF-κB signaling pathways collaboratively linked inflammation to cancer [55]. Therefore, it is possible that cooperative mechanisms among STAT-6 with other transcription factors may exist
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Fig. 5. STAT-6 mediates TRAIL increased hRANKL promoter activity. (A). ChIP assay for p-STAT-6 binding to the hRANKL gene promoter region. (i) SAKA-T cells were stimulated with and without TRAIL (100 ng/ml) for 24 h and a ChIP assay was performed using antip-STAT-6 antibody as described in Materials and methods. (ii) Chromatin immunoprecipitate obtained in the ChIP assay was quantified by real-time PCR. (B). STAT-6 regulates hRANKL gene promoter activity. SAKA-T cells were transfected with hRANKL-pSTAT with or without STAT-6 siRNA and control scrambled siRNA in the presence and absence of TRAIL (100 ng/ml) for 48 h. Cells transfected with EV served as control. Total cell lysates obtained from these cells were assayed for luciferase activity and normalized with βgalactosidase activity co-expressed in these cells. Data represent triplicate studies with a mean ± SE (*p b 0.05).
in TRAIL up-regulation of RANKL gene transcription. Further, JNK activity has been shown to be required for STAT activation [56,57]. We previously showed that a JNK inhibitor abrogates the CXCL13 stimulated RANKL expression in oral cancer cells [12]. Therefore, TRAIL induction of JNK activation may play a role in STAT6 activation to enhance RANKL expression. Thus, our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in human bone marrow stromal/preosteoblast cells.
Acknowledgments We thank the confocal microscopy core facility in the Division of Cardiology at the Medical University of South Carolina, Charleston, SC.
References [1] Takahashi N, Udagawa N, Suda T. A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 1999;256:449–55. [2] Menaa C, Reddy SV, Kurihara N, Maeda H, Anderson D, Cundy T, et al. Enhanced RANK ligand expression and responsivity of bone marrow cells in Paget's disease of bone. J Clin Invest 2000;105:1833–8. [3] Roodman GD, Dougall WC. RANK ligand as a therapeutic target for bone metastases and multiple myeloma. Cancer Treat Rev 2008;34:92–101. [4] Horwood NJ, Elliott J, Martin TJ, Gillespie MT. Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. Endocrinology 1998;139:4743–6.
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[5] Griffin III AC, Kern MJ, Kirkwood KL. MKP-1 is essential for canonical vitamin Dinduced signaling through nuclear import and regulates RANKL expression and function. Mol Endocrinol 2012;26:1682–93. [6] Okamura H, Yang D, Yoshida K, Haneji T. Protein phosphatase 2A Calpha is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts. FEBS Lett 2013;587:48–53. [7] Kikuchi T, Matsuguchi T, Tsuboi N, Mitani A, Tanaka S, Matsuoka M, et al. Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via Toll-like receptors. J Immunol 2001;166:3574–9. [8] Fu Q, Manolagas SC, O'Brien CA. Parathyroid hormone controls receptor activator of NF-kappaB ligand gene expression via a distant transcriptional enhancer. Mol Cell Biol 2006;26:6453–68. [9] Kim S, Yamazaki M, Zella LA, Shevde NK, Pike JW. Activation of receptor activator of NF-kappaB ligand gene expression by 1,25-dihydroxyvitamin D3 is mediated through multiple long-range enhancers. Mol Cell Biol 2006;26:6469–86. [10] O'Brien CA, Kern B, Gubrij I, Karsenty G, Manolagas SC. Cbfa1 does not regulate RANKL gene activity in stromal/osteoblastic cells. Bone 2002;30:453–62. [11] Delgado-Calle J, Sanudo C, Fernandez AF, Garcia-Renedo R, Fraga MF, Riancho JA. Role of DNA methylation in the regulation of the RANKL-OPG system in human bone. Epigenetics 2012;7:83–91. [12] Yuvaraj S, Griffin AC, Sundaram K, Kirkwood KL, Norris JS, Reddy SV. A novel function of CXCL13 to stimulate RANK ligand expression in oral squamous cell carcinoma cells. Mol Cancer Res 2009;7:1399–407. [13] Lee HL, Bae OY, Baek KH, Kwon A, Hwang HR, Qadir AS, et al. High extracellular calcium-induced NFATc3 regulates the expression of receptor activator of NFkappaB ligand in osteoblasts. Bone 2011;49:242–9. [14] Nakagawa N, Yasuda H, Yano K, Mochizuki S, Kobayashi N, Fujimoto H, et al. Basic fibroblast growth factor induces osteoclast formation by reciprocally regulating the production of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in mouse osteoblastic cells. Biochem Biophys Res Commun 1999;265:158–63. [15] Sundaram K, Rao DS, Ries WL, Reddy SV. CXCL5 stimulation of RANK ligand expression in Paget's disease of bone. Lab Invest 2013;93:472–9. [16] Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673–82. [17] Almasan A, Ashkenazi A. Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy. Cytokine Growth Factor Rev 2003;14:337–48. [18] Davanzo R, Zauli G, Monasta L, Vecchi Brumatti L, Abate MV, Ventura G, et al. Human colostrum and breast milk contain high levels of TNF-related apoptosis-inducing ligand (TRAIL). J Hum Lact 2013;29:23–5. [19] Picarda G, Trichet V, Teletchea S, Heymann D, Redini F. TRAIL receptor signaling and therapeutic option in bone tumors: the trap of the bone microenvironment. Am J Cancer Res 2012;2:45–64. [20] Brunetti G, Oranger A, Mori G, Sardone F, Pignataro P, Coricciati M, et al. TRAIL effect on osteoclast formation in physiological and pathological conditions. Front Biosci (Elite Ed) 2011;3:1154–61. [21] Yen ML, Hsu PN, Liao HJ, Lee BH, Tsai HF. TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. PLoS One 2012;7:e38048. [22] Kawano Y, Ueno S, Abe M, Kikukawa Y, Yuki H, Iyama K, et al. TRAIL produced from multiple myeloma cells is associated with osteolytic markers. Oncol Rep 2012;27: 39–44. [23] Roccisana JL, Kawanabe N, Kajiya H, Koide M, Roodman GD, Reddy SV. Functional role for heat shock factors in the transcriptional regulation of human RANK ligand gene expression in stromal/osteoblast cells. J Biol Chem 2004;279:10500–7. [24] Sundaram K, Senn J, Yuvaraj S, Rao DS, Reddy SV. FGF-2 stimulation of RANK ligand expression in Paget's disease of bone. Mol Endocrinol 2009;23:1445–54. [25] Darimont C, Avanti O, Tromvoukis Y, Vautravers-Leone P, Kurihara N, Roodman GD, et al. SV40 T antigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiated phenotype. Cell Growth Differ 2002;13:59–67. [26] Takahashi S, Reddy SV, Dallas M, Devlin R, Chou JY, Roodman GD. Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation. Endocrinology 1995;136:1441–9. [27] Stathem M, Marimuthu S, O'Neal J, Rathmell JC, Chesney JA, Beverly LJ, et al. Glucose availability and glycolytic metabolism dictate glycosphingolipid levels. J Cell Biochem 2014 (E-pub ahead of print). [28] Voelkel-Johnson C, King DL, Norris JS. Resistance of prostate cancer cells to soluble TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) can be overcome by doxorubicin or adenoviral delivery of full-length TRAIL. Cancer Gene Ther 2002;9:164–72. [29] Petak I, Douglas L, Tillman DM, Vernes R, Houghton JA. Pediatric rhabdomyosarcoma cell lines are resistant to Fas-induced apoptosis and highly sensitive to TRAILinduced apoptosis. Clin Cancer Res 2000;6:4119–27. [30] Atkins GJ, Bouralexis S, Evdokiou A, Hay S, Labrinidis A, Zannettino AC, et al. Human osteoblasts are resistant to Apo2L/TRAIL-mediated apoptosis. Bone 2002;31:448–56. [31] Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 1998;273:14363–7. [32] Bu R, Borysenko CW, Li Y, Cao L, Sabokbar A, Blair HC. Expression and function of TNF-family proteins and receptors in human osteoblasts. Bone 2003;33:760–70. [33] Zauli G, Sancilio S, Cataldi A, Sabatini N, Bosco D, Di Pietro R. PI-3K/Akt and NFkappaB/IkappaBalpha pathways are activated in Jurkat T cells in response to TRAIL treatment. J Cell Physiol 2005;202:900–11. [34] Okahashi N, Inaba H, Nakagawa I, Yamamura T, Kuboniwa M, Nakayama K, et al. Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway. Infect Immun 2004; 72:1706–14.
144
K. Sundaram et al. / Bone 71 (2015) 137–144
[35] Kim S, Yamazaki M, Zella LA, Meyer MB, Fretz JA, Shevde NK, et al. Multiple enhancer regions located at significant distances upstream of the transcriptional start site mediate RANKL gene expression in response to 1,25-dihydroxyvitamin D3. J Steroid Biochem Mol Biol 2007;103:430–4. [36] Xiong J, Onal M, Jilka RL, Weinstein RS, Manolagas SC, O'Brien CA. Matrix-embedded cells control osteoclast formation. Nat Med 2011;17:1235–41. [37] Fumoto T, Takeshita S, Ito M, Ikeda K. Physiological functions of osteoblast lineage and T cell-derived RANKL in bone homeostasis. J Bone Miner Res 2014;29:830–42. [38] Sedger LM, Glaccum MB, Schuh JC, Kanaly ST, Williamson E, Kayagaki N, et al. Characterization of the in vivo function of TNF-alpha-related apoptosis-inducing ligand, TRAIL/Apo2L, using TRAIL/Apo2L gene-deficient mice. Eur J Immunol 2002;32: 2246–54. [39] Di Pietro R, Zauli G. Emerging non-apoptotic functions of tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)/Apo2L. J Cell Physiol 2004;201:331–40. [40] Wang S, El-Deiry WS. TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 2003;22:8628–33. [41] Brunetti G, Oranger A, Carbone C, Mori G, Sardone FR, Mori C, et al. Osteoblasts display different responsiveness to TRAIL-induced apoptosis during their differentiation process. Cell Biochem Biophys 2013;67:1127–36. [42] Shipman CM, Croucher PI. Osteoprotegerin is a soluble decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand and can function as a paracrine survival factor for human myeloma cells. Cancer Res 2003;63:912–6. [43] Koide M, Kobayashi Y, Ninomiya T, Nakamura M, Yasuda H, Arai Y, et al. Osteoprotegerin-deficient male mice as a model for severe alveolar bone loss: comparison with RANKL-overexpressing transgenic male mice. Endocrinology 2013; 154:773–82. [44] Zauli G, Rimondi E, Nicolin V, Melloni E, Celeghini C, Secchiero P. TNF-related apoptosis-inducing ligand (TRAIL) blocks osteoclastic differentiation induced by RANKL plus M-CSF. Blood 2004;104:2044–50. [45] Lucas H, Bartold PM, Dharmapatni AA, Holding CA, Haynes DR. Inhibition of apoptosis in periodontitis. J Dent Res 2010;89:29–33. [46] Secchiero P, Zauli G. Tumor-necrosis-factor-related apoptosis-inducing ligand and the regulation of hematopoiesis. Curr Opin Hematol 2008;15:42–8.
[47] Labovsky V, Vallone VB, Martinez LM, Otaegui J, Chasseing NA. Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines. Cancer Cell Int 2012;12:29. [48] Hewitt J, Lu X, Gilbert L, Nanes MS. The muscle transcription factor MyoD promotes osteoblast differentiation by stimulation of the Osterix promoter. Endocrinology 2008;149:3698–707. [49] O'Brien CA, Gubrij I, Lin SC, Saylors RL, Manolagas SC. STAT3 activation in stromal/ osteoblastic cells is required for induction of the receptor activator of NF-kappaB ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D3 or parathyroid hormone. J Biol Chem 1999;274:19301–8. [50] Choe JY, Park KY, Park SH, Lee SI, Kim SK. Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6R-mediated RANKL expression through JAK2–STAT3– SOCS3 signaling pathway in fibroblast-like synoviocytes. Arthritis Res Ther 2013; 15:R26. [51] Bishop KA, Meyer MB, Pike JW. A novel distal enhancer mediates cytokine induction of mouse RANKl gene expression. Mol Endocrinol 2009;23:2095–110. [52] Shen CH, Stavnezer J. Interaction of stat6 and NF-kappaB: direct association and synergistic activation of interleukin-4-induced transcription. Mol Cell Biol 1998;18: 3395–404. [53] Bishop KA, Coy HM, Nerenz RD, Meyer MB, Pike JW. Mouse Rankl expression is regulated in T cells by c-Fos through a cluster of distal regulatory enhancers designated the T cell control region. J Biol Chem 2011;286:20880–91. [54] Srivastava S, Matsuda M, Hou Z, Bailey JP, Kitazawa R, Herbst MP, et al. Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells. J Biol Chem 2003;278:46171–8. [55] Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein Cell 2013;4:176–85. [56] Decker T, Kovarik P. Serine phosphorylation of STATs. Oncogene 2000;19:2628–37. [57] Kim JH, Lee SC, Ro J, Kang HS, Kim HS, Yoon S. Jnk signaling pathway-mediated regulation of Stat3 activation is linked to the development of doxorubicin resistance in cancer cell lines. Biochem Pharmacol 2010;79:373–80.
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Original Full Length Article
STAT-6 mediates TRAIL induced RANK ligand expression in stromal/preosteoblast cells Kumaran Sundaram a, Yuvaraj Sambandam a, Sundaravadivel Balasubramanian b, Balakrishnan Pillai a, Christina Voelkel-Johnson c, William L. Ries d, Sakamuri V. Reddy a,⁎ a
Charles P. Darby Children's Research Institute, Medical University of South Carolina, Charleston, SC, USA Department of Radiation Oncology, Medical University of South Carolina, Charleston, SC, USA Dept. of Microbiology & Immunology, Medical University of South Carolina, Charleston, SC, USA d College of Dental Medicine, Medical University of South Carolina, Charleston, SC, USA b c
a r t i c l e
i n f o
Article history: Received 24 July 2014 Revised 14 October 2014 Accepted 23 October 2014 Available online 30 October 2014 Edited by: Hong-Hee Kim Keywords: TRAIL RANK ligand STAT-6 Stromal/preosteoblasts
a b s t r a c t Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis, and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein, we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/ preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly, TRAIL treatment induced RANKL mRNA expression in these cells. In addition, TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition, siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus, our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Receptor activator of NF-κB ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells which induces osteoclast precursor cell differentiation to form multinucleated bone resorbing osteoclasts [1]. Elevated levels of RANKL expression have been associated with several skeletal diseases such as Paget's disease of bone and multiple myeloma [2,3]. A variety of cytokines and growth factors such as 1,25-(OH)2 vitamin D, parathyroid hormone (PTH), interleukin 1β (IL-1β), IL-11, and prostaglandin E2 (PGE2) induce RANKL expression in marrow stromal/preosteoblast cells [4]. Evidence suggests that MKP-1 is essential for vitamin D stimulated RANKL expression in bone marrow stromal cells [5]. Recently, protein phosphatase 2 A has been shown to regulate RANKL expression
⁎ Corresponding author at: Charles P. Darby Children's Research Institute, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA. Fax: +1 843 792 7927. E-mail address: [email protected] (S.V. Reddy).
http://dx.doi.org/10.1016/j.bone.2014.10.016 8756-3282/© 2013 Elsevier Inc. All rights reserved.
in mouse osteoblast cells [6]. Furthermore, lipopolysaccharide treatment increased the levels of RANKL expression through activation of toll-like receptors in primary murine osteoblasts [7]. It has also been shown that PTH stimulates RANKL expression through the cAMP/ protein kinase A/CREB cascade. Mouse RANKL expression is upregulated by vitamin D and PTH; however these transcriptional regulatory regions are far upstream to the transcriptional start site [8,9]. Runx2/Cbfa1 can enhance RANKL expression by increasing the ratio of immature to mature osteoblasts [10]. Also, DNA methylation has been shown to play an important role in modulation of RANKL and osteoprotegerin (OPG) expression in human bone [11]. We previously identified NFATc3 as a downstream target of CXCL13 signaling pathway to stimulate RANKL expression in oral squamous cell carcinoma cells [12]. Similarly, increased levels of extracellular calcium induced RANKL expression through activation of NFATc3 in mouse osteoblast cells [13]. Fibroblast growth factor (FGF)-2 induces RANKL production through COX-2mediated prostaglandin synthesis and by suppressing OPG expression [14]. We recently demonstrated that CREB (cAMP response element-binding) is a down-stream effector of CXCL5 signaling which enhances the RANKL expression in Paget's disease [15].
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Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/ Apo2/TNSF10) is a member of the TNF superfamily. TRAIL induces apoptosis in a variety of tumor cells but not in normal cells [16]. TRAIL is expressed on the surface of natural killer cells, lymphocytes, macrophages and dendritic cells [17]. TRAIL expressed either as a type II transmembrane protein or as a soluble protein is also detectable in different biological fluids under physiological conditions [18]. In humans, TRAIL can interact with four different TRAIL receptors. Two of these receptors are death receptors DR4 and DR5 which contain a death domain motif and promote apoptosis in tumor cells. In contrast, DcR1 and DcR2 receptors lack a death domain [19]. In addition, TRAIL can also bind with OPG, a decoy receptor of RANKL. Recently, it has been shown that TRAIL induces osteoclast differentiation under physiological and pathological conditions [20]. Furthermore, TRAIL increased osteoclast formation through a TNF receptor associated factor-6 (TRAF-6) dependent signaling pathway [21]. Multiple myeloma cancer cells produce high levels of TRAIL and have a positive correlation with osteolytic markers which suggested that TRAIL may play an important regulatory role in excess bone resorption in these patients [22]. However, TRAIL induced expression of RANKL in bone marrow stromal/preosteoblast cells is unknown. In the present study, we delineated the molecular mechanism underlying the TRAIL induced RANKL expression in these cells. 2. Materials and methods 2.1. Reagents Cell culture and DNA transfection reagents were purchased from Invitrogen (Carlsbad, CA). Recombinant human soluble TRAIL was purchased from Enzo Life Sciences (Farmingdale, NY). Anti-RANKL antibody was obtained from R&D systems Inc. (Minneapolis, MN). AntiSTAT-6 and anti-p-STAT-6 antibodies were purchased from Cell Signaling (Danvers, MA). Anti-TRAIL, anti-IκB, anti-p-IκB, anti-β-actin antibodies and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Super signal enhanced chemiluminescence (ECL) reagent was obtained from Amersham Bioscience (Piscataway, NJ) and nitrocellulose membranes were purchased from Millipore (Bedford, MA). pNF-κB-Luc reporter plasmid was obtained from Stratagene (La Jolla, CA) and a luciferase reporter assay system was obtained from Promega (Madison, WI). Chromatin Immunoprecipitation (ChIP) assay kit was purchased from Upstate (Temecula, CA). 2.2. Quantitative real-time RT-PCR Total RNA was isolated from the human bone marrow derived stromal/preosteoblast (SAKA-T) cells [23] stimulated with and without TRAIL (0–100 ng/ml) for 48 h, using RNAzol (Molecular Research Center, Inc., Cincinnati, OH). A reverse transcription reaction was performed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) at 25 °C for 5 min, 42 °C for 30 min followed by 85 °C for 5 min. The quantitative real-time PCR was performed using IQ™ SYBR Green Supermix in an iCycler and the primers specific for RANKL, OPG and GAPDH as described earlier [24]. Thermal cycling parameters were 94 °C for 3 min, followed by 40 cycles of amplifications at 94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min, and 72 °C for 5 min as the final elongation step. To quantify absolute copy number of mRNA expression in each sample, a standard curve for each mRNA was generated using a fivefold dilution of control RNA sample. The levels of mRNA expression were calculated as a ratio to GAPDH amplification. 2.3. Cell proliferation assay Human bone marrow derived stromal/preosteoblast cell line, SAKA-T was established by immortalization with SV40 virus large T-antigen as described previously [23,25]. SAKA-T cells were further transformed by telomerase expression enhancing the growth [26]. Cells were maintained
in Modified Eagle Medium (MEM) containing 10% fetal bovine serum (FBS) and supplemented with L-glutamine, penicillin and streptomycin. Cells were incubated at 37 °C in 5% CO2. SAKA-T cells were seeded in 96 well plates (1 × 104 cells/well) and stimulated with TRAIL (100 ng/ml) for 24 and 48 h. Cells were incubated with 10% (20 μl) Alamar Blue reagent (Invitrogen, cat#DAL1100) for 4 h and incubated at 37 °C and the fluorescence intensity was measured at 540 nm excitation and 594 nm emissions. Wells containing complete growth media alone with 20 μl of Alamar Blue were averaged and subtracted from all experimental readings [27]. 2.4. Western blot analysis SAKA-T or primary human bone marrow stromal cells (Stem cell Technologies Inc., Vancouver, cat# 70022) were seeded (5 × 105 cells/ well) in 6-well plates and supplemented with α-MEM containing 10% FBS. A day after seeding, cells were transfected with expression plasmids encoding TRAIL for 48 h. Cells were lysed in a cell lysis buffer and protein content of the samples was measured using the BCA protein assay reagent. Protein (20 μg) samples were then subjected to SDSPAGE using 12% Tris–HCl gels and blot transferred on to a nitrocellulose membrane, immunoblotted with anti-RANKL, anti-p-STAT-6, antiSTAT-6, anti-p-IκB, anti-IκB, anti-p-c-Jun and anti-β-actin antibodies. The bands were detected using the enhanced chemiluminescence detection system and quantified by densitometry analysis using the NIH ImageJ Program. 2.5. TRAIL expression vector construct The full-length of TRAIL cDNA was generated by RT-PCR using RNA isolated from normal human lymphocytes, inserted into the TA cloning system and verified by sequence analysis. TRAIL cDNA was subsequently cloned into the pIRES-EGFP vector as described [28]. 2.6. c-Jun N-terminal kinase (JNK) activity assay SAKA-T cells were cultured in the presence or absence of TRAIL (100 ng/ml) for different time points (0–3 h) and collected in ice-cold phosphate buffered saline (PBS), centrifuged at 3000 rpm for 5 min. Cells were resuspended in a cell lysis buffer with a protease inhibitor followed by brief sonication. Cell lysates were cleared by centrifugation for 5 min at 12,000 g. JNK activity was measured using a solid-phase GST-cJun (1–89 amino acids) fusion protein. Briefly, c-Jun kinase was coprecipitated with its substrate conjugated to glutathione-S-sepharose beads at 4 °C overnight. The precipitates were washed twice with cell lysis buffer followed by a kinase buffer (25 mM Tris (pH 7.5), 5 mM βglycerophosphate, 1 mM sodium vanadate, 2 mM dithiothreitol (DTT), 10 mM MgCl2) and resuspended in 50 μl of kinase buffer. The reaction was carried out at 30 °C for 30 min in the presence of 100 μM of ATP and stopped by addition of SDS-loading buffer. Proteins were separated by SDS-PAGE (15%) and blot transferred onto a nitrocellulose membrane. JNK activity was measured by using anti-p-c-Jun antibody. 2.7. Luciferase reporter gene assay SAKA-T cells transfected with NF-κB-Luc or hRANKL-pSTAT-Luc reporter plasmid construct [24] were co-transfected with siRNA (20 nM) against STAT-6 or scrambled control siRNA by lipofectamine. Cells were cultured in the presence or absence of TRAIL (100 ng/ml) for 48 h. Total cell lysates (20 μl) were mixed with 100 μl of the luciferase assay reagent and luminescence was measured for 10 s of integrated time using Sirius luminometer. β-Galactosidase activity was measured according to manufacturer's protocol (Promega). In brief, 100 μl of cell lysates was mixed with 100 μl of 2× assay buffer containing the substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) and incubated at 37 °C for 30 min. The mixture was added with 1 M sodium carbonate
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to stop the reaction and the color intensity was measured at 420 nM. The transfection efficiency was normalized by the β-galactosidase activity co-expressed in these cells. 2.8. Transcription factor array screening We used real-time RT-PCR based human transcription factor array screening for identification of TRAIL regulated transcription factors in SAKA-T cells stimulated with and without TRAIL (100 ng/ml) for 24 h. Total RNA was isolated from these cells using RNAzol reagent and reverse transcription reaction (RT) was performed. Real-time PCR was performed using RT qPCR Master Mix (RT2 Profiler PCR Array System (PAHS-075ZA) Qiagen Inc., Valentia, CA) in a 96-well plate to quantify the expression levels of 84 transcription factors. Thermal cycling parameters were 95 °C for 10 min, followed by 40 cycles of amplifications at 95 °C for 15 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min as the final elongation step. Relative levels of mRNA expression were normalized with housekeeping gene expression (GAPDH, HPRT and β-actin). Data analysis was performed in triplicates using the web portal http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php. 2.9. Chromatin immunoprecipitation (ChIP) SAKA-T cells were treated with or without TRAIL (100 ng/ml) for 24 h and cross-linked with 1% final concentration of formaldehyde at 37 °C for 10 min before harvest. Soluble chromatin was prepared by sonication to get an average DNA length of 200–1000 bp. The sheared soluble chromatin was precleared with blocked Protein G agarose, and 10% of the precleared chromatin was set aside as input control. Immunoprecipitation was carried out with 5 μg of anti-p-STAT-6 antibody overnight at 4 °C. Immune complexes were pulled down using Protein G agarose, washed and eluted twice with elution buffer (0.1 M NaHCO3, 1% SDS) and cross-linking reversed in 200 mM NaCl at 65 °C overnight with 20 μg RNase A. DNA was purified following proteinase K treatment with a PCR purification kit (Qiagen, MD). Then, PCR was performed using the gene specific primers for STAT binding region, 5′TTTACAGCAATGAGCAGACCT-3′ (sense) and 5′-CAGGATGCATGGGATT ACCT-3′ (antisense) DNA samples or input DNA fractions were analyzed by 35 cycles of PCR (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s). PCR products were subjected to electrophoresis using 2% agarose gels and visualized by ethidium bromide. The percentage of chromatinimmune complex precipitate DNA relative to input was calculated and shown as mean ± SE for three independent experiments. 3.0. Confocal microscopy analysis
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3. Results 3.1. TRAIL receptor expression in human bone marrow stromal/preosteoblast cells TRAIL interacts with receptors TRAILR1 (DR4), TRAILR2 (DR5), TRAILR3 (DcR1), and TRAILR4 (DcR2) [29]. It has been shown that osteoblast cells express TRAIL receptors [30]; however, TRAIL regulation of its receptor expression in human bone marrow derived stromal/ preosteoblast cells is unclear. Therefore, we examined the TRAIL modulation of its receptor expression in SAKA-T cell line. Cells were stimulated with and without TRAIL (100 ng/ml) for 48 h and total RNA isolated was subjected to real-time RT-PCR analysis for TRAIL receptor mRNA expression. As shown in Fig. 1A, TRAIL stimulation increased DcR1 (4.3-fold) and DR5 (3.5-fold) mRNA expression compared to unstimulated cells. However, no changes were observed in DcR2 and DR4 expression in these cells. Since OPG acts as a soluble TRAIL receptor [31], we also examined the TRAIL regulation of OPG mRNA expression in these cells. We identified that TRAIL significantly decreased OPG mRNA expression (Fig. 1B). Studies indicated that osteoblast cells express TRAIL receptors (DR4 and 5), however TRAIL stimulation does not induce apoptosis [32]. Therefore, we further tested the effect of TRAIL on proliferation of SAKA-T cells as described in Materials and methods. As shown in Fig. 1C, TRAIL treatment had no effect on SAKA-T cell proliferation. These results suggest that TRAIL differentially regulates receptor expression in human bone marrow stromal/preosteoblast cells. 3.2. TRAIL regulation of RANKL expression Recent evidence suggests that TRAIL directly promotes osteoclast differentiation/bone resorption activity [21] and modulates osteoblast differentiation [41]. However, TRAIL regulation of RANKL expression in human bone marrow stromal/preosteoblast cells is unknown. Therefore, SAKA-T cells were stimulated with TRAIL (100 ng/ml) for a variable period (0–48 h) and total RNA isolated from these cells was subjected to real-time RT-PCR analysis for RANKL mRNA expression. Interestingly, TRAIL significantly increased (5-fold) RANKL mRNA expression (Fig. 2A). Further, TRAIL stimulation significantly increased RANKL mRNA expression in a dose-dependent manner (Fig. 2B). In addition, total cell lysates obtained from SAKA-T cells transfected with the TRAIL expression vector [28] were subjected to western blot analysis for RANKL expression. As shown in Fig. 2C, TRAIL over-expression demonstrated a significant increase in RANKL expression in these cells. These results suggest that TRAIL induces RANKL expression in bone marrow stromal/preosteoblast cells. 3.3. TRAIL signaling in stromal/preosteoblast cells
4
SAKA-T cells were seeded (1 × 10 /well) onto 22 mm coverslips in 6-well plates. Cells were treated with and without TRAIL (100 ng/ml) for 1 h and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Then, cells were permeabilized with 0.1% Triton X-100 for 5 min and blocked for 1 h with PBS containing 10% donkey serum. Cells were incubated with primary antibody against p-STAT-6 (1:100 dilution) in PBS and incubated overnight at 4 °C. After extensive washing with PBS, cells were incubated with Alexa 568-conjugated antirabbit IgG. Nuclear staining was performed with DAPI and localization of p-STAT-6 was visualized by IX81 confocal microscope (Olympus, IX81; 60 ×). Z-stack images were obtained at 0.5 μm thickness stepsize and images were then rotated along the X-axis and Y-axis using the Fluoview 1000 software. 3.1. Statistical analysis Results are presented as mean ± SE for three independent experiments and were compared by Student's t-test. Values were considered significantly different for p b 0.05.
TRAIL can induce NF-κB activation for normal cell survival [33]. Therefore, we next examined the effect of TRAIL on NF-κB activation in stromal/preosteoblast cells. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for 0–3 h. Western blot analysis of total cell lysates demonstrated that TRAIL significantly increased the levels of p-IκB expression. However, there was no change in total IκB expression in these cells (Fig. 3A). We further examined the NF-κB reporter activity in these cells. NF-κB-Luc reporter plasmid was transiently transfected into SAKA-T cells using lipofectamine. Cells were cultured in the presence or absence of TRAIL (100 ng/ml) for 48 h. Total cell lysates obtained from these cells were analyzed for luciferase activity. TRAIL treatment increased (4.5-fold) NF-κB reporter activity compared to unstimulated cells (Fig. 3B). It has also been shown that JNK modulates RANKL expression in stromal/preosteoblast cells [34]. We therefore tested TRAIL regulation of JNK activity in these cells. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for variable time-points (0–3 h) and total cell lysates obtained were assayed for JNK activity as described in Materials and methods. As shown in Fig. 3C, TRAIL
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Fig. 1. Effect of TRAIL on receptor expression and proliferation of human bone marrow derived stromal/preosteoblast cells. (A) TRAIL regulation of receptor expression. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for 48 h and total RNA isolated was subjected to real-time RT-PCR analysis for DR4, DR5, DcR1 and DcR2, and (B) OPG mRNA expression. The absolute levels of mRNA expression in the samples were normalized by GAPDH mRNA expression. (C) TRAIL does not alter proliferation of SAKA-T cells. Cells were treated with TRAIL (100 ng/ml) for 0–48 h and proliferation [fluorescence intensity as arbitrary units (AU)] was assayed as described. Data represent triplicate studies with mean ± SE (*p b 0.05).
treatment increased (5-fold) JNK activity over basal levels of expression in these cells in a time-dependent manner. 3.4. Participation of STAT-6 in TRAIL induction of RANKL expression To further identify TRAIL regulated transcription factors which modulate RANKL expression, we used real-time RT-PCR based human transcription factor array screening of 84 transcription factors. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for 24 h and total RNA
isolated was used as a template for identification of transcription factors and data obtained were analyzed by web portal as described in Materials and methods. We thus identified a 3.2- and 3.0-fold increase in STAT-6 and myogenic differentiated 1 (MYOD1) mRNA expression in TRAIL stimulated SAKA-T cells, respectively (Fig. 4A). STAT transcription factors have been implicated in regulating gene expression in stromal/preosteoblast cells. Therefore, we examined the TRAIL modulation of STAT-6 activation in these cells. Western blot analysis of total cell lysates obtained from the primary human bone marrow stromal cells
Fig. 2. TRAIL induces RANKL expression in SAKA-T cells. (A). TRAIL induces RANKL mRNA expression. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for indicated time-points and cells were stimulated with different concentrations of TRAIL (0–100 ng/ml) for 48 h (B) and total RNA isolated was subjected to real-time RT-PCR analysis for RANKL mRNA expression. The absolute levels of RANKL mRNA expression in the samples were normalized by GAPDH mRNA expression. Data represent triplicate studies with mean ± SE (*p b 0.05). (C). Overexpression of TRAIL increases RANKL levels. SAKA-T cells were transfected with empty vector (EV) or TRAIL expression vector. After 48 h, total cell lysates were subjected to western blot analysis for TRAIL and RANKL expression. β-Actin expression served as control.
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Fig. 3. TRAIL enhances NF-κB and JNK activity. (A). TRAIL activation of IκB. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for indicated time-points. Total cell lysates were subjected to western blot analysis for IκB and p-IκB expression. (B). TRAIL enhances NF-κB-Luc reporter gene activity. SAKA-T cells were transfected with EV or NF-κB-Luc reporter plasmids and stimulated with and without TRAIL (100 ng/ml) for 48 h. Total cell lysates prepared were assayed for luciferase activity (RLU, relative light units). The transfection efficiency was normalized by β-galactosidase activity co-expressed in these cells. Values are expressed as mean ± SE for triplicate studies (*p b 0.05). (C). TRAIL increases JNK activity. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for indicated time-points (0–3 h) and total cell lysates were analyzed to measure JNK as described in Materials and methods. β-Actin expression served as control.
stimulated with TRAIL showed an increase (10-fold) in the levels of p-STAT-6 expression (Fig. 4B). Furthermore, confocal microscopy analysis of SAKA-T cells treated with TRAIL revealed nuclear localization of p-STAT-6 (Fig. 4C-i). Z-stack images further confirm the nuclear localization of p-STAT-6 in these cells (Fig. 4C-ii). We previously identified STAT binding element (ATTTGGGGAA) at far upstream in the hRANKL promoter region [24]. Long-range enhancer elements have also been shown to modulate RANKL gene expression [35]. Therefore, we next examined p-STAT-6 binding to the hRANKL gene promoter region by ChIP assay using anti-p-STAT-6 antibody as described. Chromatin immune complexes obtained from TRAIL stimulated SAKA-T cells were analyzed by PCR using hRANKL genespecific primers for the STAT-binding region [24]. As shown in Fig. 5A, TRAIL treatment significantly increased p-STAT-6 binding to the hRANKL promoter region. We detected no binding in immunoprecipitate obtained with a control IgG. Next, we examined the potential role of STAT-6 to modulate RANKL gene promoter activity in response to TRAIL stimulation. SAKA-T cells were transiently cotransfected with hRANKL-pSTAT reporter plasmid with siRNA against STAT-6 or control siRNA and cultured in the presence and absence of TRAIL for 48 h. siRNA suppression of STAT-6 inhibits TRAIL stimulated hRANKL-pSTAT luciferase reporter gene activity. In contrast, no change in gene promoter activity was observed in empty vector (EV) and control siRNA transfected cells (Fig. 5B). Transfection efficiency was normalized by β-galactosidase activity co-expressed in these cells. These results suggest that STAT-6 is a down-stream effector of TRAIL signaling to induce RANKL expression in human bone marrow stromal/preosteoblast cells. 4. Discussion Studies have identified that several cytokines regulate RANKL expression in stromal/osteoblast lineage cells and immune responsive T-cells which play an important role in osteoclast development and bone resorption activity under normal and pathologic conditions [36,37]. Herein, we have demonstrated that TRAIL induces RANKL expression in human bone marrow stromal/preosteoblast cells suggesting that TRAIL can enhance osteoclast differentiation through elevated levels of RANKL expression in the bone microenvironment.
Furthermore, TRAIL deficient mice showed normal bone phenotype [38]. Therefore, TRAIL induction of RANKL expression may have a pathologic role with respect to osteoclast development and bone destruction. TRAIL promotes cell survival, migration, and proliferation in a variety of normal cells [39]. In contrast, TRAIL induces cell death in many cancer cells [40]. Human osteoblasts have been shown to express TRAIL-R and are resistant to TRAIL mediated apoptosis [30]. It has been reported that the osteoblast sensitivity to TRAIL was due to the upregulation of DR5 and down-regulation of DcR2 [41]. However, this study demonstrated that TRAIL induced the expression of DR5 and DcR1 receptors with no change in proliferation of human bone marrow stromal/preosteoblast cells. OPG can serve as a soluble decoy receptor for both RANKL and TRAIL [42]. Therefore, TRAIL inhibition of OPG and induction of RANKL expression in bone marrow stromal/preosteoblast cells may enhance osteoclast formation and bone resorption activity in pathologic conditions. Consistently, it has been shown that OPG deficient mice lead to severe alveolar bone loss [43]. Also, TRAIL has been shown to block the inhibitory effect of OPG on RANKL induced osteoclast formation [44]. Elevated levels of TRAIL have been associated with periodontitis [45]. These studies suggested that OPG may inhibit alveolar bone loss induced by RANKL and TRAIL in periodontitis conditions. Furthermore, TRAIL has been shown to induce apoptosis in a variety of cancer cell types which includes multiple myeloma [46]. Also, breast cancer cells have been shown to express RANKL, TRAIL and its receptors. Therefore, our results suggest that TRAIL induced RANKL expression may have a functional role in tumor progression and bone metastasis [47]. Our transcription factor array screening identified TRAIL upregulation of STAT-6 and MyoD1 transcription factor expression. It has been shown that MyoD promotes osteoblast differentiation [48]. However, we did not identify a putative binding motif for MyoD in the hRANKL promoter region. This study identifies a functional role for TRAIL activation of STAT-6 to induce RANKL expression in bone marrow stromal/preosteoblast cells. Previously, it has been shown that STAT-3 activation in stromal/osteoblastic cells is required for induction of the RANKL expression by interleukin-1 (IL-1) but not 1,25-dihydroxyvitamin D3 or parathyroid hormone [49]. Recently, calcineurin inhibitor, tacrolimus has been shown to affect the RANKL expression in IL-6 stimulated
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Fig. 4. TRAIL up-regulation of STAT-6 transcription factor expression. (A) Identification of STAT-6 transcription factor up-regulation by TRAIL. SAKA-T cells were stimulated with TRAIL (100 ng/ml) for 24 h. Total RNA isolated was subjected to RT2-Profiler array screening of 84 human transcription factors array by real-time RT-PCR in triplicate studies as described in Materials and methods. (B). TRAIL induces p-STAT-6 expression in normal human bone marrow derived primary stromal/preosteoblast cells. Cells were stimulated with and without TRAIL (100 ng/ml) for indicated time-points (0–3 h) and total cell lysates were subjected to western blot analysis for STAT-6 and p-STAT-6 expression. (C-i). Confocal microscopy analysis for pSTAT-6 nuclear localization. SAKA-T cells were stimulated with and without TRAIL (100 ng/ml) for 1 h and immunostained for p-STAT-6. Nuclear staining was performed by DAPI and the merged image demonstrates nuclear localization of p-STAT-6 in TRAIL stimulated cells. (C-ii) Z-stack images of p-STAT-6 nuclear localization along x-axis and y-axis.
fibroblast-like synoviocytes through STAT-3 [50]. IL-6 in combination with IL-6 soluble receptor and oncostatin M has been shown to induce RANKL expression in stromal cells via STAT-3 [51]. We previously showed that STAT-1 is a downstream effector of FGF-2 signaling to stimulate RANKL expression [24]. Therefore, STAT family members show differential cytokine response and functional significance with respect to RANKL expression. This study demonstrated that TRAIL induced p-STAT-6 expression and ChIP assay revealed that TRAIL stimulation increased p-STAT-6 binding to the human RANKL promoter region suggesting STAT-6 participation in TRAIL control of RANKL gene expression. Inhibition of hRANKL-STAT promoter activity through siRNA
suppression further confirms the specificity for STAT-6 participation in TRAIL induction of RANKL gene expression. However, we did not observe STAT6 participation in TRAIL down-regulation of OPG (data not shown). STAT-6 and NF-kB have been shown to bind cooperatively to a DNA probe containing both sites and synergize activation of transcription by IL-4 [52]. We further demonstrated that TRAIL enhances NF-kB and JNK activity. hRANKL promoter has been shown to contain STAT and NF-kB responsive elements [53,54]. The activation and interaction between STAT3 and NF-κB signaling pathways collaboratively linked inflammation to cancer [55]. Therefore, it is possible that cooperative mechanisms among STAT-6 with other transcription factors may exist
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Fig. 5. STAT-6 mediates TRAIL increased hRANKL promoter activity. (A). ChIP assay for p-STAT-6 binding to the hRANKL gene promoter region. (i) SAKA-T cells were stimulated with and without TRAIL (100 ng/ml) for 24 h and a ChIP assay was performed using antip-STAT-6 antibody as described in Materials and methods. (ii) Chromatin immunoprecipitate obtained in the ChIP assay was quantified by real-time PCR. (B). STAT-6 regulates hRANKL gene promoter activity. SAKA-T cells were transfected with hRANKL-pSTAT with or without STAT-6 siRNA and control scrambled siRNA in the presence and absence of TRAIL (100 ng/ml) for 48 h. Cells transfected with EV served as control. Total cell lysates obtained from these cells were assayed for luciferase activity and normalized with βgalactosidase activity co-expressed in these cells. Data represent triplicate studies with a mean ± SE (*p b 0.05).
in TRAIL up-regulation of RANKL gene transcription. Further, JNK activity has been shown to be required for STAT activation [56,57]. We previously showed that a JNK inhibitor abrogates the CXCL13 stimulated RANKL expression in oral cancer cells [12]. Therefore, TRAIL induction of JNK activation may play a role in STAT6 activation to enhance RANKL expression. Thus, our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in human bone marrow stromal/preosteoblast cells.
Acknowledgments We thank the confocal microscopy core facility in the Division of Cardiology at the Medical University of South Carolina, Charleston, SC.
References [1] Takahashi N, Udagawa N, Suda T. A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 1999;256:449–55. [2] Menaa C, Reddy SV, Kurihara N, Maeda H, Anderson D, Cundy T, et al. Enhanced RANK ligand expression and responsivity of bone marrow cells in Paget's disease of bone. J Clin Invest 2000;105:1833–8. [3] Roodman GD, Dougall WC. RANK ligand as a therapeutic target for bone metastases and multiple myeloma. Cancer Treat Rev 2008;34:92–101. [4] Horwood NJ, Elliott J, Martin TJ, Gillespie MT. Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. Endocrinology 1998;139:4743–6.
143
[5] Griffin III AC, Kern MJ, Kirkwood KL. MKP-1 is essential for canonical vitamin Dinduced signaling through nuclear import and regulates RANKL expression and function. Mol Endocrinol 2012;26:1682–93. [6] Okamura H, Yang D, Yoshida K, Haneji T. Protein phosphatase 2A Calpha is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts. FEBS Lett 2013;587:48–53. [7] Kikuchi T, Matsuguchi T, Tsuboi N, Mitani A, Tanaka S, Matsuoka M, et al. Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via Toll-like receptors. J Immunol 2001;166:3574–9. [8] Fu Q, Manolagas SC, O'Brien CA. Parathyroid hormone controls receptor activator of NF-kappaB ligand gene expression via a distant transcriptional enhancer. Mol Cell Biol 2006;26:6453–68. [9] Kim S, Yamazaki M, Zella LA, Shevde NK, Pike JW. Activation of receptor activator of NF-kappaB ligand gene expression by 1,25-dihydroxyvitamin D3 is mediated through multiple long-range enhancers. Mol Cell Biol 2006;26:6469–86. [10] O'Brien CA, Kern B, Gubrij I, Karsenty G, Manolagas SC. Cbfa1 does not regulate RANKL gene activity in stromal/osteoblastic cells. Bone 2002;30:453–62. [11] Delgado-Calle J, Sanudo C, Fernandez AF, Garcia-Renedo R, Fraga MF, Riancho JA. Role of DNA methylation in the regulation of the RANKL-OPG system in human bone. Epigenetics 2012;7:83–91. [12] Yuvaraj S, Griffin AC, Sundaram K, Kirkwood KL, Norris JS, Reddy SV. A novel function of CXCL13 to stimulate RANK ligand expression in oral squamous cell carcinoma cells. Mol Cancer Res 2009;7:1399–407. [13] Lee HL, Bae OY, Baek KH, Kwon A, Hwang HR, Qadir AS, et al. High extracellular calcium-induced NFATc3 regulates the expression of receptor activator of NFkappaB ligand in osteoblasts. Bone 2011;49:242–9. [14] Nakagawa N, Yasuda H, Yano K, Mochizuki S, Kobayashi N, Fujimoto H, et al. Basic fibroblast growth factor induces osteoclast formation by reciprocally regulating the production of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in mouse osteoblastic cells. Biochem Biophys Res Commun 1999;265:158–63. [15] Sundaram K, Rao DS, Ries WL, Reddy SV. CXCL5 stimulation of RANK ligand expression in Paget's disease of bone. Lab Invest 2013;93:472–9. [16] Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673–82. [17] Almasan A, Ashkenazi A. Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy. Cytokine Growth Factor Rev 2003;14:337–48. [18] Davanzo R, Zauli G, Monasta L, Vecchi Brumatti L, Abate MV, Ventura G, et al. Human colostrum and breast milk contain high levels of TNF-related apoptosis-inducing ligand (TRAIL). J Hum Lact 2013;29:23–5. [19] Picarda G, Trichet V, Teletchea S, Heymann D, Redini F. TRAIL receptor signaling and therapeutic option in bone tumors: the trap of the bone microenvironment. Am J Cancer Res 2012;2:45–64. [20] Brunetti G, Oranger A, Mori G, Sardone F, Pignataro P, Coricciati M, et al. TRAIL effect on osteoclast formation in physiological and pathological conditions. Front Biosci (Elite Ed) 2011;3:1154–61. [21] Yen ML, Hsu PN, Liao HJ, Lee BH, Tsai HF. TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. PLoS One 2012;7:e38048. [22] Kawano Y, Ueno S, Abe M, Kikukawa Y, Yuki H, Iyama K, et al. TRAIL produced from multiple myeloma cells is associated with osteolytic markers. Oncol Rep 2012;27: 39–44. [23] Roccisana JL, Kawanabe N, Kajiya H, Koide M, Roodman GD, Reddy SV. Functional role for heat shock factors in the transcriptional regulation of human RANK ligand gene expression in stromal/osteoblast cells. J Biol Chem 2004;279:10500–7. [24] Sundaram K, Senn J, Yuvaraj S, Rao DS, Reddy SV. FGF-2 stimulation of RANK ligand expression in Paget's disease of bone. Mol Endocrinol 2009;23:1445–54. [25] Darimont C, Avanti O, Tromvoukis Y, Vautravers-Leone P, Kurihara N, Roodman GD, et al. SV40 T antigen and telomerase are required to obtain immortalized human adult bone cells without loss of the differentiated phenotype. Cell Growth Differ 2002;13:59–67. [26] Takahashi S, Reddy SV, Dallas M, Devlin R, Chou JY, Roodman GD. Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation. Endocrinology 1995;136:1441–9. [27] Stathem M, Marimuthu S, O'Neal J, Rathmell JC, Chesney JA, Beverly LJ, et al. Glucose availability and glycolytic metabolism dictate glycosphingolipid levels. J Cell Biochem 2014 (E-pub ahead of print). [28] Voelkel-Johnson C, King DL, Norris JS. Resistance of prostate cancer cells to soluble TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) can be overcome by doxorubicin or adenoviral delivery of full-length TRAIL. Cancer Gene Ther 2002;9:164–72. [29] Petak I, Douglas L, Tillman DM, Vernes R, Houghton JA. Pediatric rhabdomyosarcoma cell lines are resistant to Fas-induced apoptosis and highly sensitive to TRAILinduced apoptosis. Clin Cancer Res 2000;6:4119–27. [30] Atkins GJ, Bouralexis S, Evdokiou A, Hay S, Labrinidis A, Zannettino AC, et al. Human osteoblasts are resistant to Apo2L/TRAIL-mediated apoptosis. Bone 2002;31:448–56. [31] Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 1998;273:14363–7. [32] Bu R, Borysenko CW, Li Y, Cao L, Sabokbar A, Blair HC. Expression and function of TNF-family proteins and receptors in human osteoblasts. Bone 2003;33:760–70. [33] Zauli G, Sancilio S, Cataldi A, Sabatini N, Bosco D, Di Pietro R. PI-3K/Akt and NFkappaB/IkappaBalpha pathways are activated in Jurkat T cells in response to TRAIL treatment. J Cell Physiol 2005;202:900–11. [34] Okahashi N, Inaba H, Nakagawa I, Yamamura T, Kuboniwa M, Nakayama K, et al. Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway. Infect Immun 2004; 72:1706–14.
144
K. Sundaram et al. / Bone 71 (2015) 137–144
[35] Kim S, Yamazaki M, Zella LA, Meyer MB, Fretz JA, Shevde NK, et al. Multiple enhancer regions located at significant distances upstream of the transcriptional start site mediate RANKL gene expression in response to 1,25-dihydroxyvitamin D3. J Steroid Biochem Mol Biol 2007;103:430–4. [36] Xiong J, Onal M, Jilka RL, Weinstein RS, Manolagas SC, O'Brien CA. Matrix-embedded cells control osteoclast formation. Nat Med 2011;17:1235–41. [37] Fumoto T, Takeshita S, Ito M, Ikeda K. Physiological functions of osteoblast lineage and T cell-derived RANKL in bone homeostasis. J Bone Miner Res 2014;29:830–42. [38] Sedger LM, Glaccum MB, Schuh JC, Kanaly ST, Williamson E, Kayagaki N, et al. Characterization of the in vivo function of TNF-alpha-related apoptosis-inducing ligand, TRAIL/Apo2L, using TRAIL/Apo2L gene-deficient mice. Eur J Immunol 2002;32: 2246–54. [39] Di Pietro R, Zauli G. Emerging non-apoptotic functions of tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)/Apo2L. J Cell Physiol 2004;201:331–40. [40] Wang S, El-Deiry WS. TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 2003;22:8628–33. [41] Brunetti G, Oranger A, Carbone C, Mori G, Sardone FR, Mori C, et al. Osteoblasts display different responsiveness to TRAIL-induced apoptosis during their differentiation process. Cell Biochem Biophys 2013;67:1127–36. [42] Shipman CM, Croucher PI. Osteoprotegerin is a soluble decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand and can function as a paracrine survival factor for human myeloma cells. Cancer Res 2003;63:912–6. [43] Koide M, Kobayashi Y, Ninomiya T, Nakamura M, Yasuda H, Arai Y, et al. Osteoprotegerin-deficient male mice as a model for severe alveolar bone loss: comparison with RANKL-overexpressing transgenic male mice. Endocrinology 2013; 154:773–82. [44] Zauli G, Rimondi E, Nicolin V, Melloni E, Celeghini C, Secchiero P. TNF-related apoptosis-inducing ligand (TRAIL) blocks osteoclastic differentiation induced by RANKL plus M-CSF. Blood 2004;104:2044–50. [45] Lucas H, Bartold PM, Dharmapatni AA, Holding CA, Haynes DR. Inhibition of apoptosis in periodontitis. J Dent Res 2010;89:29–33. [46] Secchiero P, Zauli G. Tumor-necrosis-factor-related apoptosis-inducing ligand and the regulation of hematopoiesis. Curr Opin Hematol 2008;15:42–8.
[47] Labovsky V, Vallone VB, Martinez LM, Otaegui J, Chasseing NA. Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines. Cancer Cell Int 2012;12:29. [48] Hewitt J, Lu X, Gilbert L, Nanes MS. The muscle transcription factor MyoD promotes osteoblast differentiation by stimulation of the Osterix promoter. Endocrinology 2008;149:3698–707. [49] O'Brien CA, Gubrij I, Lin SC, Saylors RL, Manolagas SC. STAT3 activation in stromal/ osteoblastic cells is required for induction of the receptor activator of NF-kappaB ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D3 or parathyroid hormone. J Biol Chem 1999;274:19301–8. [50] Choe JY, Park KY, Park SH, Lee SI, Kim SK. Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6R-mediated RANKL expression through JAK2–STAT3– SOCS3 signaling pathway in fibroblast-like synoviocytes. Arthritis Res Ther 2013; 15:R26. [51] Bishop KA, Meyer MB, Pike JW. A novel distal enhancer mediates cytokine induction of mouse RANKl gene expression. Mol Endocrinol 2009;23:2095–110. [52] Shen CH, Stavnezer J. Interaction of stat6 and NF-kappaB: direct association and synergistic activation of interleukin-4-induced transcription. Mol Cell Biol 1998;18: 3395–404. [53] Bishop KA, Coy HM, Nerenz RD, Meyer MB, Pike JW. Mouse Rankl expression is regulated in T cells by c-Fos through a cluster of distal regulatory enhancers designated the T cell control region. J Biol Chem 2011;286:20880–91. [54] Srivastava S, Matsuda M, Hou Z, Bailey JP, Kitazawa R, Herbst MP, et al. Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells. J Biol Chem 2003;278:46171–8. [55] Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein Cell 2013;4:176–85. [56] Decker T, Kovarik P. Serine phosphorylation of STATs. Oncogene 2000;19:2628–37. [57] Kim JH, Lee SC, Ro J, Kang HS, Kim HS, Yoon S. Jnk signaling pathway-mediated regulation of Stat3 activation is linked to the development of doxorubicin resistance in cancer cell lines. Biochem Pharmacol 2010;79:373–80.