The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway

Toxicology and Applied Pharmacology 280 (2014) 502–510

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Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap

The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway Haitao Yu a, Yuxuan Du a, Xulong Zhang a, Ying Sun a, Shentao Li a, Yunpeng Dou a, Zhanguo Li b, Huihui Yuan a,⁎, Wenming Zhao a,⁎⁎ a b

Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China Department of Rheumatology & Immunology, Clinical Immunology Center, Peking University People's Hospital, No. 11 Xizhimen South Street, Beijing 100044, PR China

a r t i c l e

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Article history: Received 7 May 2014 Revised 18 August 2014 Accepted 22 August 2014 Available online 3 September 2014 Keywords: Aryl hydrocarbon receptor (Ahr) Rheumatoid arthritis (RA) Osteoblasts Proliferation Differentiation ERK signaling pathway

a b s t r a c t Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5 days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice. © 2014 Elsevier Inc. All rights reserved.

Introduction Rheumatoid arthritis (RA) is a prototypical systemic autoimmune disease that is characterized by chronic synovitis, and destruction of cartilage and bone. Bone formation by osteoblasts is impaired at erosion sites, which associates with the functional disability of patients with RA

Abbreviations: Ahr, aryl hydrocarbon receptor; AIP, Ahr-interacting protein; ALP, alkaline phosphatase; ARNT, aryl hydrocarbon receptor nuclear translocator; BMD, bone mineral density; CFA, complete Freund's adjuvant; CH-223191, 2-methyl-2H-pyrazole-3carboxylic acid; C II, bovine type II collagen; CIA, collagen-induced arthritis; DAB, 3,3′ diaminobenzidine; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; ECL, enhanced chemiluminescence; EDTA, ethylenediaminetetraacetic acid; ERK, extracellular receptoractivated kinase; FBS, fetal bovine serum; HE, hematoxylin and eosin; HSP, heat shock protein; IFA, incomplete Freund's adjuvant; IL, interleukin; pNPP, p-nitrophenyl phosphate; PVDF, polyvinylidene fluoride; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; RA, rheumatoid arthritis; TBST, Tris-Buffered Saline with Tween-20; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XRE, xenobiotic responsive element. ⁎ Corresponding author. Fax: +86 10 83911825. ⁎⁎ Corresponding author. Fax: +86 10 83911824. E-mail addresses: [email protected] (H. Yuan), [email protected] (W. Zhao).

http://dx.doi.org/10.1016/j.taap.2014.08.025 0041-008X/© 2014 Elsevier Inc. All rights reserved.

(Walsh and Gravallese, 2010; Schett and Gravallese, 2012). Inflammatory cells, cytokines, chemokines and other factors present at the local microenvironment aggravate the bone destruction in RA. The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor and it belongs to the Per-Arnt-Sim superfamily of proteins (Fukunaga et al., 1995). This ancient protein has been studied as a receptor for environmental contaminants and as a mediator of chemical toxicity within the last three decades. Interestingly, recent studies demonstrate that the Ahr has gained more attention in diseases of the immune system (Veldhoen et al., 2008; Nakahama et al., 2011; Monteleone et al., 2013), such as RA. Increasing Ahr activity in the synovial tissue aggravated the inflammatory response and exacerbated RA (Kobayashi et al., 2008). Ahr activation contributes to Th17 differentiation (Veldhoen et al., 2008, 2009; van Voorhis et al., 2013) and disturbs the balance of Th1/Th2 cell toward Th1 (Negishi et al., 2005; Hwang et al., 2014). In contrast, collagenimmunized Ahr knockout mice showed ameliorated synovitis due to decreased production of Th17 cells and proinflammatory cytokines, such as interleukin (IL)-1β and IL-6 (Nakahama et al., 2011). Meanwhile, Merja Korkalainen also reported that the Ahr activation ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), disrupted the

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differentiation of osteoblasts and osteoclasts (Korkalainen et al., 2009). Although high expression of the Ahr enhanced synovitis, its contribution to bone loss has not been completely elucidated in RA. Based on these findings, we hypothesized that the active Ahr that was localized in osteoblasts had an increased ability to exacerbate bone loss in arthritis. In the present study, we explored the pathological role of Ahr in the bone destruction of collagen-induced arthritis (CIA) mice. To this aim, we analyzed the expression levels of Ahr mRNA and protein in the bone tissue of hind paws that were derived from CIA mice. Further, we assessed the localization of Ahr at the subcellular level, and then examined the effects of Ahr on the proliferation and differentiation of osteoblasts using an Ahr agonist, TCDD. These data showed that the high expression levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, TCDD-activated Ahr inhibited expression of the osteoblast marker alkaline phosphatase (ALP) in a dosedependent manner. Moreover, Ahr activation of the extracellular receptor-activated kinase (ERK) signaling pathway inhibited osteoblast proliferation and differentiation. Taken together, these results demonstrated that increased Ahr activity suppressed the functions of osteoblasts through activation of the ERK signaling pathway, which enabled bone erosion in arthritis. Materials and methods Reagents and chemicals. Trizol reagent, TCDD, TaqMan probes and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Life Technologies (Carlsbad, CA, USA), Cambridge Isotope Laboratories (Cambridge, MA, USA), Applied Biosystems (Foster City, California, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively. 2-Methyl-2H-pyrazole-3-carboxylic acid (CH-223191) and ERK inhibitor U0126 were from Sigma-Aldrich (St. Louis, MO, USA), and gentamycin, fungizone and ascorbic acid were purchased from Amresco (Solon, Ohio, USA). The bovine type II collagen (C II), complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were from Chondrex (Redmond, WA, USA). Cell culture media, penicillin, streptomycin and fetal bovine serum (FBS) were obtained from Hyclone (Life Technologies, CA, USA). Antibodies were obtained from the following vendors: the anti-Histone H3, anti-total-ERK, anti-phospho-ERK, anti-β-actin, Cy3-conjugated anti-mouse and FITC-conjugated antirabbit antibodies were from Cell Signaling Technology (Beverly, MA, USA); the anti-Ahr antibody was from Abcam (Cambridge, MA, USA); the anti-osteocalcin antibody was from Novus (Minneapolis, MN, USA); and the horseradish peroxidase-conjugated immunoglobulin G secondary antibody was obtained from Proteintech Group (Chicago, IL, USA). The following kits and reagents were used: GoScript Reverse Transcription System, cell lysis buffer, nuclear and cytoplasmic protein extraction kits, BCA protein assay kit and enhanced chemiluminescence (ECL) were from Thermomax (Columbia, MA, USA); the TaqMan PCR Master Mix was from Applied Biosystems (Foster City, CA, USA); SYBR Green Master Mix was from TaKaRa (Dalian, China); the alkaline phosphatase (ALP) assay kit was from Beyotime Institute of Biotechnology (Shanghai, China); and the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) was from Promega (Madison, WI, USA). Animals. Seven-week-old male DBA/1 mice (weight, 18 ± 2 g) were purchased from Beijing HFK Bio-technology Co. Ltd and were maintained under specific pathogen-free conditions. All of the animal experiments were approved by the Capital Medical University Institutional Animal Care and Use Committee (Permit ID: SCXK-2013-X-2). CIA models and evaluation. The CIA mouse model was utilized as described previously (Brand et al., 2007). Briefly, male DBA/1 mice were immunized intradermally at the base of the tail with 100 μg of bovine C II emulsified with an equal volume of CFA. On day 21, the animals were given a booster injection composed of 100 μg of bovine C II

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dissolved in an equal volume of IFA. Mice that were immunized with the adjuvant alone were used as normal controls. The onset of arthritic disease was defined as the appearance of definitive signs of edema and erythema in the paw. Animals were monitored every 4 days following the booster immunization. The incidence of arthritis was recorded and the paw swelling was measured using a 0.2 μm sliding caliper. The following clinical scores were assigned to evaluate the disease: 0 = no sign of arthritis; 1 = swelling and/or redness of the paw or one digit; 2 = two affected joints; 3 = more than two affected joints; and 4 = severe arthritis of the entire paw and digits (Cuzzocrea et al., 2005). The paw and leg tissues were harvested from each mouse after it was killed on day 49. The bone tissues of the paws were used for hematoxylin and eosin (HE) staining, immunohistochemistry, immunofluorescence, western blotting and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and the legs were used for isolating primary osteoblasts.

Cell culture and treatment. Primary osteoblasts were isolated from murine long bones and cultured as previously described (Bakker and Klein-Nulend, 2012) with general modification. The long bone was cut into 1–2 mm2 pieces, which were then placed onto 75 cm2 flasks with Dulbecco's Modified Eagle Medium/Low Glucose (DMEM/Low Glucose) supplemented with 100 U/ml penicillin, 50 μg/ml streptomycin sulfate, 50 μg/ml gentamycin, 1.25 μg/ml fungizone, 100 μg/ml ascorbic acid, and 10% FBS, and grown at 37 °C in a humidified atmosphere of 5% CO2. At 80% confluency, the primary osteoblasts were digested with a 0.25% trypsin solution at 37 °C for 10 min. Subsequently, the primary osteoblasts were seeded onto 24-well plates filled with climb piece (8 × 104 cells/well) for identification (see online Supplementary data, Fig. S1) and detection of Ahr with immunofluorescence, and the expression of Ahr in 6-well plates (4.5 × 105 cells/well) was evaluated after 5 days. Furthermore, the primary osteoblasts were treated with variable concentrations of TCDD (0.1, 1.0 and 10 nM) and/or CH223191 (10 μM) to measure the effect on the mRNA expression levels of ALP and Cyp1a1 and on the rate of proliferation after 3 and 5 day time points. The MC3T3-E1 cell line was purchased from the Chinese Academy of Medical Sciences and Peking Union Medical College and cultured in αMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere of 5% CO2. At 80% confluency, the cells were digested with 0.25% trypsin and placed onto 6-well plates at a density of 1 × 105 cells/well with αMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate. The medium was changed every 2–3 days. The cells were then seeded onto 96-well plates at a density of 3 × 103 cells/well with 100 μl of medium containing TCDD (10 nM) and/or U0126 (10 μM), and the number of viable cells was measured at 3 days or 5 days. Then, to measure the expression of ALP and cytochromes Cyp1a1 and the activity of ALP, cells (1 × 105 cells/well) were seeded onto 6-well plates and treated with TCDD (0.1, 1.0 and 10 nM) and/or CH-223191 (10 μM) or U0126 (10 μM) for 15, 30, 45, 60, and 120 min, or 5 days.

Histology. The paws were fixed with 4% neutral formalin and decalcified in 20% ethylenediaminetetraacetic acid (EDTA) for 4 weeks. The sections were hydrated, embedded in paraffin, and cut into 4 μm sections for HE staining, immunohistochemistry and immunofluorescence. The severity of the inflammatory cell infiltration in the HE sections was scored using a semi-quantitative scale as previously described (Stolina et al., 2009). Immunohistochemistry was performed to detect immunoreaction of the Ahr using an anti-Ahr antibody (1:100). The positive signals were detected with 3,3′ diaminobenzidine (DAB), and then Mayer's hematoxylin was used to counterstain the slides. The scores for the immunoreactivity of Ahr were classified as previously described (Nonaka et al., 2012).

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For dual-labeled immunofluorescence staining, the hind paw sections were deparaffinized and blocked with goat serum and then incubated with an anti-Ahr antibody (1:100) and an anti-osteocalcin antibody (1:50) overnight at 4 °C. After washing with PBS, the sections were next incubated with a Cy3-conjugated anti-mouse secondary antibody (1:100) and a FITC-conjugated anti-rabbit secondary antibody (1:100) for 90 min at room temperature. For immunofluorescence, primary osteoblasts were placed onto glass coverslips in 24-well plates at 5 × 104 cells/well. Ethyl alcohol (95%) was used to fix the osteoblasts for 20 min, and then Triton X-100 (0.1%) was used to permeate the osteoblasts for 20 min. Goat serum (10%) was added for 20 min, and the cells were then incubated with the anti-Ahr (1:1000) antibody overnight at 4 °C. A Cy3-conjugated secondary antibody (1:200) was incubated for 90 min. Nuclei were stained with DAPI (1:200) for 3 min. Images were acquired using a fluorescence microscope (Leica DM4000B, Leica Biosystems UK Ltd, Newcastle Upon Tyne, UK) and analyzed by Image-Pro Plus 6.0 software (MC, New York, USA). Protein extraction and western blotting analysis. The hind paw bone tissue was ground in liquid nitrogen, and MC3T3-E1 cells were washed with cold phosphate-buffered saline, and then the protein was extracted using cell lysis buffer according to the manufacturer's protocol. A nuclear and cytoplasmic protein extraction kit was used to extract the nuclear and cytoplasmic protein from primary osteoblasts according to the manufacturer's instructions. Briefly, proteins were resolved by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in Tris-Buffered Saline with Tween-20 (TBST), the membranes were incubated with antibodies against Ahr (1:100), ERK (1:1000), phospho-ERK (1:1000), Histone H3 (1:1000), or β-actin (1:1000) at 4 °C overnight. After washing, the membranes were incubated with HRP-conjugated IgG secondary antibodies for 45 min at room temperature. The western blotting signals were detected via the ECL-Plus western blotting system. The protein bands were visualized by ECL reagents using the Bio-Rad ChemiDoc XRS + system (Bio-Rad, USA). The band intensities were quantified using ImageJ 2.x. qRT-PCR. Total RNA was extracted with Trizol reagent according to the manufacturer's protocol. Complementary DNA (cDNA) synthesis was performed using the GoScript Reverse Transcription System. qRT-PCR was performed with an IQ-5 PCR Detection System (Bio-Rad, USA). Specifically designed TaqMan probes for Ahr (Mm00478932_m1) and GAPDH (Mm9999915_g1) were used to detect Ahr expression levels with the following conditions: denaturation for 10 min at 95 °C, and annealing for 15 s at 60 °C. Analysis of the ALP, Cyp1a1 and GAPDH gene expression levels was performed using the SYBR Green Master Mix, and the gene-specific primers that were used are listed in Table 1. The PCR reaction was initiated with an incubation step of 3 min at 95 °C to activate the AmpliTaq Gold DNA Polymerase. This was followed by 40 cycles of a denaturation step for 30 s at 95 °C and annealing for 15 s at 60.2 °C for ALP and GAPDH. Each primer was designed using melting curve analysis to confirm that primer-dimers were not formed in the reaction. The levels of Ahr transcript were normalized to those of GAPDH and reported as a fold change. The comparative CT method was used in this study. Cell proliferation assay. Detection of the proliferation of MC3T3-E1 cells and primary osteoblasts was carried out with the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) following a previously described method (Miron et al., 2013). After 3 or 5 days, 20 μl/well of the CellTiter 96 solution was added for 1 h at 37 °C in a humidified 5% CO2 atmosphere, and the absorbance was recorded at 490 nm using an ELISA plate reader. ALP enzyme activity analysis. The ALP-specific activity of the cells was measured following the manufacturer's instruction. The cells were

Table 1 Primer sequences for quantitative real-time RT-PCR (qRT-PCR). Gene ALP Cyp1a1 GAPDH

Primer sequence Sense Antisense Sense Antisense Sense Antisense

5′-GGACGGTGAACGGGAAAAT-3′ 5′-CTTCTCCACCGTGGGTCTCA-3′ 5′-GTCAGGACAGGAAGCTGGAC-3′ 5′-GAGGCTCCACGAGATAGCAG-3′ 5′-CTTTGTCAAGCTCATTTCCTGG-3′ 5′-TCTTGCTCAGTGTCCTTGC-3′

washed with PBS and collected with a cell scraper. The cell supernatant was discarded after centrifugation at 20,000 ×g for 5 min. Lysis buffer (100 μl) was added to the cell pellets for 10 min to detect total protein and ALP activity. An ALP assay kit was used to determine ALP activity in the lysate by measuring p-nitrophenyl phosphate (pNPP) hydrolysis according to the manufacturer's protocol. The reaction was terminated after 30 min of incubation at 37 °C and the reaction mixture was measured at 405 nm. A standard curve was produced using a series of diluted standards with known concentrations. The ALP-specific activity was calculated using a standard curve. The total protein content was measured using a BCA protein assay kit. The results were expressed in millimoles of p-nitrophenol produced per milligram of protein. Radiography. The Micro-CT LCT-200 scanner (Aloka LaTheta Laboratory, Japan) was used for detecting the bone mineral density (BMD) after mice were anesthetized with chloral hydrate (10%) on day 49. Xray images were analyzed by reconstruction of 3D quantitative analyses using VGStudio MAX 2.0 software. Statistical analysis. Spearman's correlation analysis was used to examine the relationship between bone mineral density and changes in Ahr-related expression patterns. Data were analyzed using a statistical package (SPSS 11.5, Chicago, IL, USA). Unpaired t-tests (two-tailed) were used for statistical analysis of qRT-PCR, western blotting and ALP activity. A p value less than 0.05 was considered to be statistically significant. Results Upregulation of Ahr in the joints of CIA mice The relationship between Ahr expression and bone erosion was investigated in a well-established animal model of RA, the CIA mouse model. Following a booster immunization, mice developed progressive arthritis with an 83.3% incidence rate (Fig. 1A). In addition, the paw swelling and clinical scores both increased after the onset of arthritis in CIA mice in comparison with normal controls (Figs. 1B–C). The synovitis and associated destruction, evaluated by histology and radiography methods, corresponded with the signs and symptoms of arthritic mice (Figs. 1D–G). To determine the pathophysiologic role of Ahr in the bone destruction that occurs in arthritis, the expression of Ahr in arthritic bone tissues of the paw was measured. Significantly higher expression levels of Ahr were observed in arthritic joints compared to controls using an immunohistochemistry assay, especially in interphalangeal joint bone tissue (Figs. 2A–B). In addition, mRNA and protein expression levels of Ahr were consistently higher in CIA joint bone tissues than those from control mice (Figs. 2C–E). Furthermore, the mRNA expression levels of Ahr plotted against BMD showed a significantly negative correlation (r = −0.748, p = 0.005; Fig. 2F). Localization of increased Ahr expression in osteoblasts To identify whether increased Ahr expression is localized in osteoblasts, we studied the expression levels of Ahr and osteocalcin, an

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Fig. 1. CIA development in DBA/1 mice. Mice were injected intradermally with bovine type II collagen to induce arthritis (n = 6/group). The incidence of arthritis (A), paw swelling (B), and clinical score (C) were recorded until day 49. Representative joints on the day of euthanization were analyzed with HE staining (D, E), X-ray scanning (F) and BMD (G). Data are present as the mean ± SEM. *p b 0.05; **p b 0.01. Bar indicates 200 μm.

osteoblast-specific marker (Oyajobi et al., 1999), in the hind paws. The results showed that high expression of Ahr is primarily localized in osteoblasts in CIA mice (Fig. 3A). To further investigate the potential effect of Ahr in osteoblasts, primary osteoblasts were isolated and cultured. Immunofluorescent staining showed that levels of Ahr in perinuclear and nuclear were both higher in CIA osteoblasts than normal controls (Fig. 3B). Next, the fluorescence intensity of Ahr-specific staining was calculated, and this revealed a 12.5% augmented staining pattern in the nucleus of CIA osteoblasts (Fig. 3C). Consistent with these observations, qRT-PCR analysis showed that the mRNA levels of Ahr were significantly high in CIA osteoblasts compared to controls (Fig. 3D). In addition, western blotting analysis showed that the expression of Ahr was significantly higher in osteoblasts of CIA mice than in controls, particularly in the nucleus of osteoblasts (Figs. 3E–F).

Inhibition of osteoblast development with increasing Ahr activity To further test the potential effects of activated Ahr on osteoblast proliferation and differentiation, we introduced an Ahr agonist (TCDD) (Liu and Jefcoate, 2006) to cultured osteoblasts. Cyp1a1 is a standard marker of Ahr transcriptional activity (Chmill et al., 2010) and is often used to detect the activation status of Ahr (Kazantseva et al., 2012). As expected, Cyp1a1 expression was significantly high 5 days following treatment with various doses of TCDD in the preosteoblast MC3T3-E1 cell line and in primary osteoblasts (Figs. 4A–B). Activated Ahr significantly inhibited the proliferation capacity of preosteoblast MC3T3-E1 cells on days 3 and 5 (Fig. 4C). Compared with normal controls, the proliferation of CIA osteoblasts was significantly reduced by 6.7% and 8.3% with TCDD-treated on days 3 and 5, respectively (Fig. 4D).

To monitor the inhibitory effect of Ahr in osteoblast differentiation, the mRNA levels of ALP, a marker of osteoblast differentiation (Orriss et al., 2012), were measured using qRT-PCR. ALP expression in TCDDtreated MC3T3-E1 cells declined in a dose-dependent manner on day 5 (data not shown). After TCDD-treatment (10 nM) for 5 days, ALP mRNA expression was reduced in MC3T3-E1 cells (65.9%) and in primary normal (41.2%) and CIA osteoblasts (53.3%) in comparison with their controls. To further confirm that the reduction of ALP upon treatment with TCDD is dependent on Ahr, we used an Ahr-specific antagonist, CH-223191. The results showed that the expression of ALP was reversed after CH-223191 treatment in both MC3T3-E1 cells and primary osteoblasts at day 5 (Figs. 4E–F). Activation of the ERK signaling pathway by Ahr Previous studies reported that the MAPK signaling pathway is essential for osteoblast proliferation and differentiation (Orriss et al., 2012; Greenblatt et al., 2013; Sharma et al., 2013). Therefore, we examined the effect of activated Ahr on the ERK/MAPK intracellular signaling pathway. Our data showed that activated Ahr induced the phosphorylation of ERK after 45 min of stimulation with TCDD in the MC3T3-E1 cell line, and the level of phosphorylation increased significantly at 5 days (Fig. 5A). To confirm whether the activation of the ERK signaling pathway was related with the high expression of Ahr, the Ahr inhibitor CH-223191 was used. The data showed that the Ahr inhibitor suppressed the ERK phosphorylation that was induced by Ahr (Figs. 5B–C). Subsequently, an ERK inhibitor (U0126) was used to further explore the role of ERK in suppressing osteoblast proliferation and differentiation. As expected, U0126 blocked the Ahr-induced ERK signaling pathway after 5 days of stimulation (Figs. 5D–E). Intriguingly, the ERK

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Fig. 2. Upregulation of Ahr in the joints of CIA mice. Mice were induced arthritis by bovine type II and euthanized on day 49. The interphalangeal joints were collected for measuring the expression of Ahr. The Ahr expression levels in interphalangeal joints was determined using immunohistochemistry (A–B), western blotting (C–D) and qRT-PCR (E) analysis on the day of euthanization. Spearman's correlation analysis (F) showed a negative correlation between the levels of the Ahr and BMD in bone tissues (r = −0.748, p = 0.005). The slides were stained with DAB as substrate. The brown dots stand for the intensity of Ahr expression. Data are present as the mean ± SEM. *p b 0.05. Bar indicates 100 μm and the red arrows indicate Ahrpositive staining.

inhibitor also recovered the proliferative capacity of MC3T3-E1 cells that were treated with the Ahr activator, TCDD, at day 5 (Fig. 5F). More importantly, compared to the Ahr activator group (TCDD treatment only), the protein expression levels of ALP in MC3T3-E1 cells were restored by increasing 2-fold at day 5 of ERK inhibition (Fig. 5G). Discussion Bone erosion is a central feature of RA and it is related to poor functional outcome (Schett and Gravallese, 2012). Although various factors are recognized to regulate these processes, the mechanisms involved in bone resorption have not been completely elucidated. In the present study, we reported that high expression levels of Ahr corresponded with bone erosion in arthritic mice. Moreover, we explained that high levels of Ahr corresponded with bone loss and inhibited osteoblast proliferation and differentiation through stimulation of the ERK signaling pathway. These findings support our hypothesis that activated Ahr localized in osteoblasts is involved in bone erosion in arthritis. Ahr is ubiquitously expressed in vertebrate cells and it mediates heterodimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a structurally related protein that is recognized by DNA (Kewley et al., 2004). In the absence of bound agonist, Ahr is present in the cytoplasm where it forms a complex with heat shock protein (HSP) 90, Ahr-interacting protein (AIP), and p23 (Stevens et al., 2009). Upon binding with its ligand, Ahr undergoes a conformational change, translocates to the nucleus, and dimerizes with ARNT. Within the nucleus, the Ahr/ARNT heterodimer binds to a specific sequence, designated as the xenobiotic responsive element (XRE), which causes

a variety of biological effects (Reyes et al., 1992) including toxicity, evolution and immunology. Several reports revealed that Ahr agonists cause bone-specific developmental defects (Miettinen et al., 2005; Korkalainen et al., 2009). Moreover, in a female transgenic mouse that expressed a constitutively active Ahr, the levels of ALP were decreased and bone formation was deteriorated (Wejheden et al., 2010). These results demonstrated the key roles of Ahr in bone development. RA is a systemic autoimmune disease characterized with inflammatory cells infiltration and chronic bone erosion. Ahr plays critical roles in the development of RA (Nguyen et al., 2013). Ahr deficiency in T cells suppresses the development of collagen-induced arthritis by increasing Th17 and decreasing Th1 cell populations in lymph nodes from CIA mice (Nakahama et al., 2011). Contradictorily, others reported that Ahr ligands (TCDD, ITE) reduced Th1 type cytokine production (Kerkvliet et al., 1996; Nugent et al., 2013). In addition, several groups have shown that Ahr activation contributes to Th17 differentiation (Kimura et al., 2008; Veldhoen et al., 2008, 2009; van Voorhis et al., 2013). Many researchers agreed with the explanation that T cell disorders in RA patients induced the expression of RANKL (a noble osteoclast factor) in osteoblasts, leading the bone destruction in RA (Kong et al., 1999; Anandarajah, 2009). Osteoblasts and osteoclasts are major cells that occur in bone tissue, and an imbalance in osteoblasts and osteoclasts leads to bone destruction. Osteoclasts are derived from hemopoietic progenitors of the monocyte–macrophage lineage (Park et al., 2014). In addition, the development of arthritis was not suppressed in macrophages of Ahr knock-out mice (LysM-Cre Ahrflox/flox) (Nakahama et al., 2011). Moreover, other study also showed that Ahr activation treatment with TCDD

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Fig. 3. The expression of Ahr in osteoblasts. The hind paws of normal (up) and CIA mice (down) were double-stained (A) with antibodies specific for the Ahr (red) and the osteoblast marker osteocalcin (OC; green). Subcellular localization of the Ahr in primary osteoblasts derived from normal mice (up) and CIA mice (down) was detected by immunofluorescent staining (B). The fluorescence intensity in nuclei (C) was calculated using ImagePro Plus 6.0 software. The qRT-PCR (D) analysis of mRNA expression levels of the Ahr in primary osteoblasts from CIA and normal mice. Western blot (E) analysis detected the expression of the Ahr in the cytoplasm and nucleus of primary osteoblasts derived from CIA and normal mice. The intensity (F) was calculated using ImageJ 2.x. DAPI (blue) was used for counterstaining nuclei. N = nucleus, C = cytoplasm. Arrows show double-positive cells. Data are represented as the mean ± SEM. *p b 0.05; **p b 0.01; ***p b 0.001.

did not cause osteoclastic bone resorption (Ilvesaro et al., 2005). Therefore, we postulated that Ahr in hematopoietic progenitors and osteoclasts did not play a key role in bone destruction in RA development. In this study, the level of Ahr in osteoblasts was measured and high Ahr expression was detected in joint bone tissues of an arthritic model that displayed bone erosion locally and osteoporosis systemically and was negatively correlated with BMD. These results suggested that greater expression of Ahr might contribute to bone erosion in RA.

Immunofluorescence analysis demonstrated that enhanced Ahr translocation into the osteoblast nuclear compartment activated its signaling pathway and mediated its biological functions. The precise regulation of osteoblast proliferation and differentiation is necessary for skeletal development (Saito et al., 2013). Previous reports show that the differentiation of osteoblasts is reduced by Ahr when exposed to an Ahrspecific ligand (Naruse et al., 2002; Ryan et al., 2007; Korkalainen et al., 2009). Our studies also revealed that active Ahr inhibited

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Fig. 4. Inhibition of osteoblast development by increased Ahr activity. The Ahr agonist TCDD was used to treat the pre-osteoblast cell line MC3T3-E1 and the primary osteoblasts derived from normal and CIA mice for 5 days, and this resulted in a dose-dependent increase in the mRNA expression levels of Cyp1a1, a marker of Ahr activation. The pre-osteoblast cell line MC3T3-E1 and the primary osteoblasts derived from CIA mice and normal mice were treated with 10 nM TCDD, an agonist of Ahr, which was dissolved in 0.1% DMSO. The relative ratio of cell proliferation was measured by the MTS assay (C–D) on days 3 and 5. Cells were treated with 10 nM TCDD for 5 days with or without pretreatment using the Ahr-specific antagonist CH-223191 (10 μM) for 1 h and 0.1% DMSO alone as a control. ALP was analyzed by qRT-PCR (E–F) in the pre-osteoblast cell line MC3T3-E1 and in primary osteoblasts from normal and CIA mice. Data are represented by the mean ± SEM (n = 4). *p b 0.05, **/##p b 0.01, ***/###p b 0.001, versus negative control.

osteoblast differentiation, exacerbating bone loss in RA. Compared to control, the expression of ALP was reduced in CIA osteoblasts when TCDD (10 nM) was added for 5 days (Fig. 4F). Because of the higher levels of Ahr that localized in CIA osteoblasts compared to control, its activation will inhibit osteoblast differentiation significantly. Activated Ahr attenuated the proliferative capacity of osteoblasts and this further supported the role of Ahr in arthritic bone loss, which substantiated previous studies. As we know, Ahr acts as a cyto-ubiquitin ligase to regulate selective protein degradation. It has been reported that activation of the Ahr by its ligands regulates cellular development and differentiation, such as the generation of regulatory T cells (Treg) and the balance of Th1/Th2, through binding of their translocators (Funatake et al., 2005; Negishi et al., 2005; Kimura et al., 2008). ERK/MAPK is serine/threonine kinase pathways that play critical roles in the control of cell proliferation and differentiation (Cargnello and Roux, 2011; Greenblatt et al., 2013). Moreover, Tan et al. found that TCDD-stimulated MAPKs were critical for the induction of Ahrdependent gene transcription and Cyp1a1 expression (Tan et al.,

2002). This knowledge prompted us to determine the effect of Ahr on the ERK1/2 signaling pathways in osteoblasts. Interestingly, our studies showed that active Ahr significantly enhanced ERK phosphorylation levels in the MC3T3-E1 cell line, which is in accordance with a previous study in macrophages (Cheon et al., 2007). Our results suggest that the activation of the ERK/MAPK signaling pathway involves the pathological changes of osteoblasts upon Ahr activation. The inhibition of proliferation and differentiation was reversed in MC3T3-E1 cells that were pretreated with an ERK inhibitor (U0126), which further suggests that the ERK/MAPK signaling pathway, a main mechanism for Ahr activation, suppressed osteoblast proliferation and differentiation. Although the exact mechanisms of ERK/MAPK activity in the Ahr-induced osteoblast pathological changes are not known, our data demonstrate that it is necessary to suppress osteoblast proliferation and differentiation, corresponding with other researches (Liao et al., 2008; Feng et al., 2012). Conversely, some other reports demonstrated that ERK activation promoted osteoblast proliferation and/or differentiation (Lou et al., 2000; Lai et al., 2001; Ge et al., 2007; Hu

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Fig. 5. Activation of the ERK signaling pathway by increased Ahr activity. Activated Ahr by 10 nM TCDD stimulated MC3T3-E1 cells for 15 min, 30 min, 45 min, 60 min, 120 min and 5 days. The expression of p-ERK1/2 and ERK (A) was detected by western blot analysis. The MC3T3-E1 cells were treated with 10 nM TCDD for 5 days with or without the Ahr-specific antagonist CH-223191 (10 μM) for 1 h, and then the expression of p-ERK1/2 and ERK (B) was assessed by western blotting. The MC3T3-E1 cells were pretreated with or without an ERK inhibitor (10 μM, U0126) for 1 h before treatment with 10 nM TCDD for 5 days. The expression levels of p-ERK1/2 and ERK (D) were detected by western blot analysis. The intensity (C, E) was calculated using ImageJ 2.x. The percentage of proliferation cells (F) was measured using the MTS assay. The ALP activity (G) was assessed by the ALP assay. Data are represented as the mean ± SEM. *p b 0.05, **p b 0.01, ***p b 0.001.

et al., 2013). Therefore, more studies are needed to elucidate the role of ERK signal pathway in osteoblast proliferation and differentiation. Furthermore, although we found that high expression levels of Ahr in osteoblasts stimulated the ERK signaling pathway, which corresponded with bone erosion in arthritis mice, the precise translational mechanisms of the Ahr/ARNT complex in the cytoplasm of osteoblasts need to be further studied. To summarize, we confirm that active Ahr suppresses osteoblast proliferation and differentiation via activation of the ERK signaling pathway, which in turn enables bone erosion in CIA mice. This indicates that the Ahr may be a potential drug target to prevent bone erosion in RA.

Conflict of interest statement The authors have no conflicts of interest or financial disclosures.

Acknowledgments This study was supported by the State Key Development Program for Basic Research of China (Grant No. 2010CB529106) and the National Nature Science Foundations of China (Grant Nos. 31400767 and 31370936).

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