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Modulation of osteoblast differentiation and bone mass by 5-HT2A receptor signaling in mice
European Journal of Pharmacology 762 (2015) 150–157
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Modulation of osteoblast differentiation and bone mass by 5-HT2A receptor signaling in mice Kenjiro Tanaka a, Takao Hirai a,n, Yukiko Ishibashi b,c, Nobuo Izumo d, Akifumi Togari a,n a
Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Aichi, Japan Department of Biochemistry, Faculty of Pharmaceutical Science, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-Ku, Yokohama, Kanagawa 245-0066, Japan c Division of Development Higher Brain Functions (University of Fukui), United Graduate School of Child Development, Osaka University, Kanazawa University and Hamamatsu University School of Medicine, Chiba University and University of Fukui, Fukui, Japan d Department of Clinical Pharmacology, Faculty of Pharmaceutical Science, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama, Kanagawa 245-0066, Japan b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 February 2015 Received in revised form 13 May 2015 Accepted 13 May 2015 Available online 27 May 2015
Recent studies reported that serotonin (5-hydroxytryptamine, 5-HT) may be an endogenous paracrine and/or autocrine factor that is used for intercellular communication in bone cells and between multiple organs regulating bone homeostasis. In the present study, we showed that the administration of MDL11939, a selective 5-HT2A receptor antagonist, reduced bone mass in mice. The loss of bone mass in MDL11939-treated mice was associated with impaired bone formation in vivo, as demonstrated by the lower expression of osterix (Osx) and osteocalcin than that in vehicle-treated mice. On the other hand, no significant differences were observed in osteoclast numbers between MDL11939- and vehicle-treated mice. The pharmacological blockade of 5-HT2A receptor signaling significantly decreased alkaline phosphatase activity in osteoblastic cells. In addition, the knockdown of the 5-HT2A receptor by a siRNA treatment decreased Osx, but not Runx2 gene expression in MC3T3-E1 cells. These results suggest that 5-HT2A receptor signaling mediated bone mass by regulating osteoblast differentiation. & 2015 Elsevier B.V. All rights reserved.
Keywords: 5-HT2A receptor Osteoblast differentiation Osterix Bone mass Bone formation Serotonin
1. Introduction Bone is a metabolically active organ that undergoes continuous remodeling throughout life. Bone remodeling is regulated by systemic hormones and paracrine/autocrine factors. Recent studies indicated that serotonin (5-hydroxytryptamine, 5-HT) may be an endogenous paracrine and/or autocrine factor that is used for intercellular communication in bone cells (Westbroek et al., 2001; Bliziotes et al., 2001) and between organs regulating bone homeostasis (Yadav et al., 2008; Zofková and Matucha, 2014). 5-HT is a biogenic amine that mediates the transmission of a wide variety of neuronal and non-neuronal information and also plays important roles in peripheral tissues (Barnes and Sharp, Abbreviations: 5-HT, 5-hydroxytryptamine; ALP, alkaline phosphatase; BFR, formation rate; BMD, bone mineral density; BMP, bone morphogenetic protein; BV/ TV, bone volume per total volume; DIC, day in culture; IGF, insulin-like growth factor; OC, Osteocalcin; Oc.N/BS, Osteoclast number/bone surface; Oc.S/BS, osteoclast surface/bone surface; Osx, Osterix; Runx2, Runt-related transcription factor 2; Tb.Th, trabecular thickness; TPH1, tryptophan hydroxylase 1; TRAP, tartrate-resistant acid phosphatase n Corresponding authors. Fax: þ 81 52 752 5988 E-mail addresses: [email protected] (T. Hirai), [email protected] (A. Togari). http://dx.doi.org/10.1016/j.ejphar.2015.05.048 0014-2999/& 2015 Elsevier B.V. All rights reserved.
1999; Gershon and Tack, 2007; McLean et al., 2007; Ramage and Villalón, 2008) as well as the central nervous system (CNS). The actions of 5-HT are known to be mediated through interactions with membrane-bound receptors that have been categorized into seven families and include at least 21 subtypes (Barnes and Sharp, 1999). All 5-HT receptor families, excluding the 5-HT3 receptor, which forms a ligand-gated ion channel, belong to the G proteincoupled receptor superfamily. Among them, 5-HT2A is associated with the contraction of vascular smooth muscle, platelet aggregation, thrombus formation, and coronary artery spasms. Previous studies identified a 5-HT-induced functional regulatory mechanism that was regulated by a 5-HT inactivatory system for cellular proliferation, differentiation, and maturation in osteoblasts in vitro as well as in the CNS, and enteric nervous system, and cardiovascular system (Westbroek et al., 2001; Bliziotes et al., 2001; Hirai et al., 2009). We also reported the expression patterns of mRNA for 5-HT2A and 5-HT2B receptors in osteoblasts, and suggested that the respective proteins may represent local primary targets for 5-HT signal transduction (Hirai et al., 2009). However, the actions of 5-HT in cells with osteoblastic lineages are not yet fully understood. In the present study, we determined whether 5-HT2A receptor signaling played a role in modulating bone physiology.
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2. Materials and methods 2.1. Materials MDL11939 (( 7)-α-phenyl-1-(2-phenylethyl)-4-piperidine methanol) hydrochloride was purchased from Sigma-Aldrich. All chemicals used were of analytical grade. 2.2. Animals and experimental procedures Sixty-seven male, seven-week-old C57BL/6J mice (Japan SLC Inc., Hamamatsu, Japan) were randomized by weight, assigned to groups, and acclimated to their cages for 1 week prior to the experiment. They were treated in accordance with the Guidelines for Animal Experiments at the School of Dentistry, Aichi-Gakuin University. Food and water were available ad libitum. The mice in each group were housed together under automatically controlled conditions of temperature (23 71 °C) and humidity (50 710%). 2.3. Administration MDL11939, a 5-HT2A receptor antagonist, was dissolved at a concentration of 25 mg/mL in 100% ethanol. In the first series of experiment, we studied the effect of MDL11939 on bone mass and TRAP activity in mice. Eight-week-old mice were used in this study. Mice were divided into three groups and given MDL11939 (0.3 mg/kg/day, n ¼6), MDL11939 (3 mg/kg/day, n ¼9), or vehicle (1.2% ethanol, n ¼9) by oral gavage once daily/7d/week for 2 weeks at a regular time. The second or third series of experiment was performed to evaluate the effect of MDL11939 on bone formation and gene expression. Eight-week-old mice were treated with MDL11939 at 3 mg/kg/day (n ¼11) or vehicle (1.2% ethanol, n ¼10 or 11) by oral gavage once daily/7d/week for 2 weeks at a regular time. Body weight was measured weekly and the MDL11939 dose was adjusted accordingly. 2.4. Microcomputed tomographic analysis The distal region of the right femur was subjected to a threedimensional m-computed tomography (mCT) analysis using an R-mCT mCT scanner (RIGAKU, Tokyo, Japan). Scanning was initiated 1.0 mm above the distal femoral growth plate, and a total of 75 consecutive 20-mm-thick sections were analyzed, encompassing a length of 1.5 mm of the secondary spongiosa. The measured volume of interest in the femur was obtained by selecting the cancellous bone (separate from the cortical shaft) using contour areas that were drawn semiautomatically (Kondo and Togari, 2011). TRI/ 3D-BON (Ratoc, Tokyo, Japan) software was used to analyze cancellous parameters: bone volume/total volume (BV/TV, %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm). 2.5. Bone histomorphometry In the dynamic histomorphometric analysis, all mice were injected intraperitoneally with calcein (10 mg/g) at 4 and 2 days before death. At the end of the experiments, the right femur of each mouse was dissected and fixed in 70% ethanol. Five-micrometer-thick sagittal sections were made as undecalcified sections. To assess the bone formation rate (BFR), metaphyseal cancellous bone in the femur was used to obtain the bone fraction in a rectangular area of 0.34 mm2 (0.5 0.67 mm), with its closest and furthest edges being 0.3 and 0.8 mm distal to the growth plate, respectively. At least two sections from independent animal for each group were subjected to the analysis. Regarding decalcified sections, the left tibiae of mice were dissected, fixed in 4%
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paraformaldehyde, and then decalcified in 20% EDTA for 2 weeks. Sagittal section (5-mm thick) were made as decalcified sections and stained with TRAP for the osteoclast analysis. Measurements were made within an area of 0.8 mm2 (1.0 mm 0.8 mm), with its closest and furthest edges being 2.0 and 3.0 mm distal to the growth plate of the proximal ends of the tibia, respectively. Osteoclast number/bone surface (Oc.N/BS) and osteoclast surface/ bone surface (Oc.S/BS) were evaluated by scoring TRAP-positive multinucleated cells attached to the bone surface (Kondo and Togari, 2011). At least two sections from independent animal for each group were subjected to the analysis. 2.6. MC3T3-E1 cell cultures MC3T3-E1 cells were purchased from the RIKEN Cell Bank. MC3T3-E1 cells were cultured in α-MEM (Life Technologies) containing 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere. These cells were grown in non-supplemented medium until confluent (the first day of a confluent culture was referred to as day 0). When cells reached confluency, medium supplemented with 10 mM β-glycerophosphate and 10 mg/ml ascorbic acid was used. Media were changed every 2–4 days (Hirai et al., 2014a). 2.7. Determination of alkaline phosphatase (ALP) activity Cells were cultured for 10 days in culture (DIC) in the presence of MDL11939 at a concentration range of 1–10 mM or vehicle (0.01% ethanol) in order to determine the activity of alkaline phosphatase (ALP). ALP activity was determined as described previously (Fujita et al., 2001). In brief, cells were solubilized with 0.1% Triton X-100, followed by the determination of ALP activity in lysates using p-nitrophenol phosphate as a substrate. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad). ALP staining was performed using a standard protocol. In brief, cultured cells were rinsed in PBS, fixed using 10% buffered neutral formalin, rinsed with PBS, and then overlaid with 1.5 ml of 0.15 mg/ml 5-bromo-4-chloro-3-indolylphosphate plus 0.3 mg/ml nitro blue tetrazolium chloride in 0.1 M Tris–HCl (pH 9.5), 0.01 N NaOH, and 0.05 M MgCl2, followed by incubation at room temperature for 2 h in the dark. 2.8. C3H/10T1/2 cells and siRNA nucleofection C3H/10T1/2 cells were purchased from the Japanese Collection of Research Bioresources (JCRB, Japan) Cell Bank. C3H/10T1/2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1% penicillin/streptomycin to 70% confluency, followed by transient transfection with either interfering RNA (siRNA) targeting the 5HT2A receptor or non-silencing RNA diluted in Opti-MEM using Lipofectamine RNAiMAX according to the manufacturer's protocol (Life Technologies) as previously described (Hirai et al., 2014b). The siRNA used to silence the HTR2Atargeted gene (siRNA-HTR2A) and negative control siRNA (siRNANegative) were obtained from CosmoBio (Kyoto, Japan). The sequences of siRNA used to silence the HTR2A gene were as follows: HTR2A sense 5′-GGAGUAAGUUGGUGACAUA-3′, HTR2A antisense 5′-UAUGUCACCAACUUACUCC-3′. siRNA-HTR2A and siRNA-Negative were used at final concentrations of 10 nM. The medium was then replaced with fresh medium. Cells were harvested after 48 h for total RNA extraction at the indicated time points. 2.9. RNA extraction and quantitative RT-PCR As in vivo experiments, total RNA was extracted from femur (cancellous bone). As in vitro experiments, total RNA samples were
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extracted from the cells. In both cases, total RNA was isolated with an RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. One microgram of RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit according to the protocol of the manufacturer (Qiagen). Gene expression was analyzed with the StepOne-Plus real time PCR system with Step One Software v2.0 (Applied Biosystems). Reactions were performed in 20-mL volumes using a QuantiTect SYBR Green PCR Kit (Qiagen). Cycling conditions were 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data are presented as relative mRNA levels calculated by the equation 2–Ct (Ct¼ Ct of target gene minus Ct of GAPDH). The following primers were used: GAPDH, forward 5′-TGGAGAAACCTGCCAAGTATG-3′, reverse 5′-GGAGACAACCTGGTCCTCAG-3′; HTR2A, forward 5′-AGGCTCTTTTGTGGCATTTTTC-3′, reverse 5′TGGGCACCACATTACAACAAAC-3′; Runx2, forward 5′-AGGCACAGACAGAAGCTTGATG-3′, reverse 5′-GCGATCAGAGAACAAACTAGGTTTAGA-3; OC, forward 5′-GACCGCCTACAAACGCATCT-3′, reverse 5′-GGGCAGCACAGGTCCTAAATAGT-3′; Osterix, forward 5′-CCTCTGCGGGACTCAACAAC, reverse 5′-AAAGGTCAGCGTATGGCTTCTT-3′.
2.10. Data analysis Data are expressed as means 7S.D. Statistical differences were determined by one-way ANOVA followed by the Tukey HSD for multiple comparisons. P o0.05 was considered statistically significant.
3. Results 3.1. Decreases in bone mass by the administration of a 5-HT2A receptor antagonist to mice To evaluate the functionality of the 5-HT2A receptor in bone, we performed CT-based bone densitometry in cancellous bones following the oral administration of the 5-HT2A receptor antagonist, MDL11939 for 2 weeks (Fig. 1A). As shown in Fig. 1B, the BV/TV of the distal end of the femur was significantly lower in MDL11939treated mice than in vehicle-treated mice. The oral administration of MDL11939 at a dosage that influenced BV/TV had no effect on
Fig. 1. Blockade of 5-HT2A receptor signaling impaired trabecular bone properties in mice. (A) mCT image of the femur from 10-wk-old male administered vehicle or MDL11939. (B–E) CT-based bone densitometry of the femur from 10-wk-old male administered vehicle (Bar: white) or MDL11939 (Bar: black). B: bone volume per tissue volume (BV/TV). C: trabecular thickness (Tb.Th). D: trabecular number (Tb.N). E: trabecular separation (Tb.Sp). Values are expressed as means 7S.D.; n¼ 6–9 mice per group. *Po 0.05 significantly different from vehicle-treated mice.
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body weight [Average body weight (g;7 S.D.); 23.2 71.1 in the vehicle group, 22.6 71.1 in the MDL11939 (3 mg/kg) group]. Moreover, the trabecular thickness (Tb.Th) of the femur was also significantly lower in MDL11939-treated mice than in vehicletreated mice, and was dose-dependent (Fig. 1C). No significant differences were observed in Tb.N, and Tb.Sp between mice orally administered MDL11939 at 0.3 and 3 mg/kg and vehicle (Fig. 1D and E). In addition, we assessed the cortical bone mineral density (BMD) following the oral administration of MDL11939 at 3 mg/kg for 2 weeks. The cortical BMD of the tibia was significantly lower in MDL11939-treated mice than in vehicle-treated mice (Supplemental Fig. 1A). MDL11939-treated mice showed lower bone estimated strength by diminished bone rigidity indexes, which reflected the loss of cortical bone. The minimum moment of inertia of cross-sectional areas, which represents flexural rigidity, was significantly reduced in MDL11939-treated mice (Supplemental Fig. 1B). Moreover, torsional rigidity, as indicated by the polar moment of inertia of cross-sectional areas, was also significantly diminished in MDL11939-treated mice (Supplemental Fig. 1C), and, when taken together, demonstrated that mice administered the 5-HT2A receptor antagonist had a lower bone mass, cortical osteopenia, and a lower bone estimated strength. 3.2. No alteration in TRAP activity with the administration of MDL11939 to mice To examine the mechanisms responsible for bone loss due to the MDL11939 treatment, we next performed a histomorphometric analysis of bone resorption parameters such as the bone surface covered by TRAP-positive osteoclasts, Oc.S/BS, and Oc.N/BS (Fig. 2A). No significant differences were observed in Oc.S/BS or Oc. N/BS between mice orally administered MDL11939 at 0.3 and
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3 mg/kg and vehicle, which suggested that the pharmacological blockade of 5-HT2A receptor signaling did not affect osteoclast activity (Fig. 2B and C). 3.3. Effects of MDL11939 on bone formation To examine the mechanisms responsible for bone loss due to the MDL11939 treatment, we performed a bone histomorphometric analysis on bone formation parameters by assessing calcein double-labeling in the distal femur after the systemic administration of MDL11939 for 2 weeks. This analysis provided an in vivo estimation of osteoblastic activity with respect to the accumulation of bone mass. Bone formation parameters such as mineral surface/bone surface (MS/BS), the bone mineral apposition rate (MAR), and bone formation rates (BFRs) were significantly lower in MDL11939-administrated mice than in vehicle-administrated mice (Fig. 3A). We then examined the expression of gene-related bone formation using a quantitative real time PCR analysis. We extracted total RNA from the distal end of the femur and analyzed the mRNA expression of Runx2, Osterix (Osx), and Osteocalcin (OC). Consistent with the decrease observed in bone formation, the mRNA expression of Runx2, Osx, and OC in bone was significantly decreased in MDL11939-administrated mice (Fig. 3B), indicating that the pharmacological blockade of 5-HT2A receptor signaling led to bone loss concomitant with decreased bone formation. 3.4. Blockade of 5-HT2A receptor signaling impaired osteoblast differentiation To evaluate the function of the 5-HT2 receptor in osteoblastic cells, we examined the effects of MDL11939 on cell differentiation in MC3T3-E1 cells. As shown in Fig. 4A, sustained exposure to
Fig. 2. Effects of MDL11939 on osteoclast activity in mice. Mice were treated with MDL11939 at 3 mg/kg/day or vehicle for 2 weeks, followed by the staining of osteoclasts with TRAP. (A) Representative image of TRAP staining of the decalcified sections at the distal ends of the femur from 10-wk-old male administered vehicle (upper panel) or MDL11939 (lower panel). Arrowheads show TRAP-positive cells. Scale bars ¼100 mm. (B) Oc.S/BS and (C) Oc.N/BS. Values are expressed as means 7 S.D.; n ¼5–9 mice per group. *P o0.05 significantly different from vehicle-treated mice.
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Fig. 3. Effects of the systemic administration of MDL11939 on bone formation. (A) An analysis of the mineralized surface/bone surface (MS/BS), mineral apposition rates (MAR), and bone formation rate (BFR) in the cancellous bone compartment of the distal femur metaphysis from 10-wk-old male administered vehicle (Bar: white) or MDL11939 (Bar: black). n ¼11 mice per group. Values are expressed as means 7 S.D. *Po 0.05, significantly different from vehicle-treated mice. (B) Total RNA was isolated from the distal region of the right femur in 10-week-old male mice administered vehicle (Bar: white) or MDL11939 (Bar: black), followed by the determination of Runx2, Osx, and OC mRNA levels by real time qRT-PCR using specific primers. Values are expressed as means 7S.D.; n ¼10–11 mice per group. *Po 0.05, significantly different from vehicle-treated mice.
Fig. 4. Blockade of 5-HT2A receptor signaling impaired osteoblast differentiation. (A) Alkaline phosphatase activity in MC3T3-E1 cells cultured with MDL11939. Cells were cultured in α-MEM in either the presence or absence of MDL11939 at a concentration range of 1–10 mM for 10 DIC, followed by determination of the activity of alkaline phosphatase. Values are the mean 7 S.D. from four independent cultures. *Po 0.05, significantly different from each control value obtained in the absence of MDL11939. (B and C) Real time PCR analyses of osteoblast-related genes. Osteoblasts were cultured for 10 DIC with MDL11939, and then subjected to real time PCR. Data are expressed as the mean7 S.D. after normalization by GAPDH expression from three or four different experiments. *P o0.05, significantly different from each control value obtained in the absence of MDL11939. Figures are representative of data from three independent experiments.
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MDL11939 significantly decreased ALP activity in MC3T3-E1 cells in a concentration-dependent manner. Moreover, primary mouse osteoblasts were cultured for 13 DIC in the presence of ketanserin, a 5-HT2A specific antagonist, at a concentration range of 3–10 mM in order to determine the activity of ALP. The results obtained showed that sustained exposure to ketanserin significantly prevented increases in ALP activity in osteoblasts cultured for 13 DIC in a concentration-dependent manner (Supplemental Fig. 2A). To better characterize these mechanisms, the effects of the 5-HT2A selective antagonist, sarpogrelate were also determined (Supplemental Fig. 2B). Osteoblasts were cultured for 13 DIC in the sustained presence of sarpogrelate at 10 mM. All these 5-HT2A receptor antagonists were invariably effective in significantly inhibiting the activity of ALP in osteoblasts cultured for 13 DIC. Sustained exposure to sarpogrelate at 30 mM inhibited the activity of ALP by 450%, which was similar to that induced by ketanserin at 10 mM (Supplemental Fig. 2). The results of the real time PCR analysis revealed that the expression of Osx was significantly lower in osteoblasts cultured for 10 DIC in the presence of 10 mM MDL11939 than in cells cultured in the absence of MDL11939 (Fig. 4B). Furthermore, sustained exposure to MDL11939 at 1–10 mM decreased the expression of OC in osteoblasts cultured for 10 DIC (Fig. 4B). To further evaluate the potential role of 5-HT2A receptor signaling in osteogenesis, we attempted to elucidate the gene expression of Osx in C3H10T1/2 cells transfected with small interfering RNA (siRNA) for the knockdown of 5-HT2A receptor. Cells were transfected with siRNA for HTR2A, followed by the determination of HTR2A levels using real time qRT-PCR. The results obtained demonstrated that, after transfection, HTR2A levels were significantly decreased in C3H10T1/2 cells transfected with HTR2A siRNA (Fig. 5A). A significant decrease in Osx expression was observed in C3H10T1/2 cells under these conditions (Fig. 5B). No significant differences were observed in Runx2 gene expression between
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siRNA-HTR2A and siRNA-Negative treated cells (Fig. 5C). These results indicated that 5-HT2A receptor signaling regulated osteoblast differentiation through Osx gene expression.
4. Discussion We herein demonstrated that 5-HT2A receptor signaling regulated osteoblast differentiation and bone mineral density in mice. Although several previous studies reported the functional expression of the 5-HT receptor in osteoblasts, no direct evidence for the role of 5-HT2A receptor signaling in the mechanisms underlying cellular differentiation in osteoblasts currently exists in the literature. To the best of our knowledge, this is the first study to show that 5-HT2A receptor signaling in osteoblasts plays a physiologically significant role in bone metabolism. Several studies previously suggested that 5-HT receptor signaling regulated cellular function in osteoblasts, osteocytes, and osteoclasts (Yadav et al., 2008; Hirai et al., 2010; Bliziotes et al., 2006; Chabbi-Achengli et al., 2012). 5-HT2B receptor expression during osteoblast differentiation in vitro was shown to be increased in primary cultured mouse osteoblasts (Hirai et al., 2009), and the ablation of the 5-HT2B receptor in female mice led to osteopenia due to impaired bone formation (Collet et al., 2008). In addition, 5-HT2B receptor signaling in osteoblasts was found to regulate cellular proliferation, differentiation, and functions (Baudry et al., 2010; Locker et al., 2006; Li et al., 2013). Previous studies e confirmed the pattern of expression for the 5-HT receptor in osteoblastic cells (Hirai et al., 2009). A reverse transcription polymerase chain reaction analysis revealed the expression of mRNA for the receptors of 5-HT1B, 5-HT1D, 5-HT2A, and 5-HT2B in primary cultured mouse calvarial osteoblasts. An immunohistochemical analysis clearly demonstrated that the 5-HT2A
Fig. 5. Silencing HTR2A down-regulated the expression of osterix in C3H/10T1/2 cells. C3H/10T1/2 cells were treated with HTR2A siRNA (siRNA-HTR2A) or non-silencing RNA (siRNA-Negative), followed by further cultivation for 48 h and the subsequent determination of (A) HTR2A, (B) Osx, and (C) Runx2 levels by real time qRT-PCR. Relative mRNA expression was normalized to GAPDH. Each value represents the means 7 S.D. of four independent determinations. *Po 0.05, significantly different from value treated with siRNA-Negative. Figures are representative of data from three independent experiments.
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receptor was expressed in both osteoblasts and osteoclasts in adult mouse bone sections (data not shown). In the present study, we showed that the oral administration of MDL11939, a selective and high affinity 5-HT2A receptor antagonist, significantly decreased bone mass and led to skeletal fragility, as reflected by the decreased expression of Osx and OC (Figs. 1 and 3), which suggested that 5-HT plays a significant role in modulating bone physiology, at least through 5-HT2A receptor signaling. Additionally, the administration of MDL11939 led to a reduction in bone formation without effective osteoclast activity (Fig. 3). Therefore, we speculated that 5-HT2A receptor signaling may regulate bone formation and cellular differentiation as well as the 5-HT2B receptor signaling in osteoblasts. Previous studies suggested that the 5-HT2A receptor was expressed in osteoblasts (Westbroek et al.,2001; Hirai et al., 2009) and regulated cellular proliferation in MC3T3-E1 cells (Hirai et al., 2010). In this study, the pharmacological blockade of 5-HT2A receptor signaling significantly decreased cellular differentiation in MC3T3-E1 cells and primary cultured osteoblasts (Supplemental Fig. 2). Furthermore, knockdown of the 5-HT2A receptor by a siRNA treatment also decreased Osx gene expression in C3H10T1/2 cells. Osx is a key transcription factor that is required for osteoblast differentiation and bone formation (Nakashima et al., 2002). The absence of osteoblast differentiation in Osx mutant embryos was shown to lead to small bones that did not express osteoblastspecific marker genes (Zhou et al., 2010). After birth, the absence Osx in mice causes multiple skeletal phenotypes including the lack of new bone formation, absence of the resorption of mineralized cartilage, and defects in osteocyte maturation and function (Sinha and Zhou 2013). Several studies reported the expression of osteoblast-related genes was controlled by the bone morphogenetic protein 2 (BMP2) and insulin-like growth factor 1 (IGF1) signaling pathways in osteoblast differentiation. The expression of Osx, which is activated by the BMP2/IGF1 signaling pathway, was also suggested to involve the mitogen-activated protein kinase (MAPK) pathway rather than BMP2/Smad/Runx2 signaling (Celil and Campbell, 2005; Celil et al., 2005). Three distinctive MAPK pathways have been identified to date; the extracellular regulated kinases (ERK1/2, also known as p42/44 MAPK), p38 MAPK, and JNK/ SAPK pathways. These are known to modulate the phosphorylation and, hence, activation status of transcription factors and link transmembrane signaling with gene-induction events in the nucleus (Sugden and Clerk, 1997). The activation of ERK provides a mitogenic and differentiating signal in osteoblasts (Lai et al., 2001). ERK also plays critical roles in osteoblast differentiation and skeletal development (Ge et al., 2007). We previously demonstrated that ERK activity was markedly increased by 5-HT2A receptor signaling in MC3T3-E1 cells, indicating that activation of the MAPK/ ERK pathway played an important role in cellular function regulated by 5-HT2A receptor signaling in osteoblastic cells (Hirai et al., 2010). Thus, these results indicated that 5-HT2A receptor signaling participated in osteoblast differentiation, at least through the regulation of Osx, but not Runx2 expression. 5-HT appears to play dual roles in the mechanisms underlying the maintenance of homeostasis as an autocrine and/or paracrine mediator in bone development. A question in this analysis is the origin of the 5-HT ligand that binds 5-HT2A receptors in osteoblasts. One possibility is that bone cells themselves could produce 5HT, in which case the effects from the ligand would be autocrine and/or paracrine mediator in bone cells. Previous studies showed that the expression of the initial and rate-limiting enzyme in 5HT synthesis, tryptophan hydroxylase 1 (TPH1) in osteoblastic and osteocytic cells (Gustafsson et al., 2006; Bliziotes et al., 2006). Using real-time RT-PCR, we also observed that TPH1 mRNA is expressed in MC3T3-E1 cells (Data not shown). These findings indicate that osteoblasts are capable of synthesizing 5HT, and thus
can produce paracrine/autocrine effects on bone cells. In this study, the negative effects of antagonists for 5-HT2A receptor on osteoblastic activities favor the idea that these antagonists would prevent the cellular differentiation through blockade of the 5-HT2A receptor signaling under sustained stimulation by 5HT. We analyzed the concentration of 5-HT in the culture medium of primary cultured osteoblasts by the HPLC procedure as described previously (Hirai et al., 2009). The concentration of 5-HT in the culture medium (α-MEM þ10% FBS) was approximately 0.3 mM. Therefore, the 5-HT2A receptor expressed in cultured osteoblasts would be under stimulation by 5-HT present in culture medium. Furthermore, 5-HT secreted by osteoclast precursors has more recently been shown to be crucially involved in osteoclast differentiation and acted on neighboring osteoblasts through a paracrine system (Chabbi-Achengli et al., 2012). The endogenous agonist 5-HT may be supplied to osteoblasts by neighboring osteoclasts, by osteoblasts themselves, and by blood circulation. Therefore, 5-HT2A receptor signaling in osteoblasts may regulate bone mass through an autocrine and/or paracrine system in local areas in bone. However, we cannot deny the possibility that effects of MDL11939 might be under the regulation of CNS-mediated skeletal effects, rather than an autocrine and/or paracrine system. Recent study showed that brainstem-derived 5-HT favors bone mass accrual following its binding to 5-HT2C receptors on ventromedial hypothalamic neuron (Yadav et al., 2009). Further studies are necessary to investigate the contribution of 5-HT2A receptor in these processes. Taken together, these results suggest that the 5-HT2A receptor is expressed in bone to regulate bone mass. An evaluation of the functionality of the 5-HT2A receptor in bone may provide an important insight for future discoveries and the development of innovative drugs for the treatment of various bone diseases in humans.
5. Conclusion In the present study, we identified a novel regulatory component in the actions of 5-HT and showed, for the first time, that 5-HT2A receptor signaling played an important role in regulating bone mass.
Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research to A.T. (26462827) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2015.05. 048.
References Barnes, N.M., Sharp, T., 1999. A review of central 5-HT receptors and their function. Neuropharmacology 38, 1083–1152. Baudry, A., Bitard, J., Mouillet-Richard, S., Locker, M., Poliard, A., Launay, J.M., Kellermann, O., 2010. Serotonergic 5-HT(2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C. J. Biol. Chem. 285, 26066–26073. Bliziotes, M.M., Eshleman, A.J., Zhang, X.W., Wiren, K.M., 2001. Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake. Bone 29, 477–486. Bliziotes, M., Eshleman, A., Burt-Pichat, B., Zhang, X.W., Hashimoto, J., Wiren, K.,
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Chenu, P., 2006. Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells. Bone 39, 1313–1321. Celil, A.B., Campbell, P.G., 2005a. BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. J. Biol. Chem. 280, 31353–31359. Celil, A.B., Hollinger, J.O., Campbell, P.G., 2005b. Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling. J. Cell. Biochem. 95, 518–528. Chabbi-Achengli, Y., Coudert, A.E., Callebert, J., Geoffroy, V., Côté, F., Collet, C., de Vernejoul, M.C., 2012. Decreased osteoclastogenesis in serotonin-deficient mice. Proc. Natl. Acad. Sci. USA 109, 2567–2572. Collet, C., Schiltz, C., Geoffroy, V., Maroteaux, L., Launay, J.M., de Vernejoul, M.C., 2008. The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation. FASEB J. 22, 418–427. Fujita, T., Fukuyama, R., Izumo, N., Hirai, T., Meguro, T., Nakamuta, H., Koida, M., 2001. Transactivation of core binding factor alpha1 as a basic mechanism to trigger parathyroid hormone-induced osteogenesis. Jpn. J. Pharmacol. 86, 405–416. Ge, C., Xiao, G., Jiang, D., Franceschi, R.T., 2007. Critical role of the extracellular signal-regulated kinase-MAPK pathway in osteoblast differentiation and skeletal development. J. Cell Biol. 176, 709–718. Gershon, M.D., Tack, J., 2007. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 132, 397–414. Gustafsson, B.I., Thommesen, L., Stunes, A.K., Tommeras, K., Westbroek, I., Waldum, H.L., Slørdahl, K., Tamburstuen, M.V., Reseland, J.E., Syversen, U., 2006. Serotonin and fluoxetine modulate bone cell function in vitro. J. Cell. Biochem. 98, 139–151. Hirai, T., Tokumo, K., Tsuchiya, D., Nishio, H., 2009. Expression of mRNA for 5-HT2 receptors and proteins related to inactivation of 5-HT in mouse osteoblasts. J. Pharmacol. Sci. 109, 319–323. Hirai, T., Kaneshige, K., Kurosaki, T., Nishio, H., 2010. Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells. Biochem. Biophys. Res. Commun. 396, 278–282. Hirai, T., Tanaka, K., Togari, A., 2014a. ß-adrenergic receptor signaling regulates Ptgs2 by driving circadian gene expression in osteoblasts. J. Cell Sci. 127, 3711–3719. Hirai, T., Tanaka, K., Togari, A., 2014b. α1-adrenergic receptor signaling in osteoblasts regulates clock genes and bone morphogenetic protein 4 expression through up-regulation of the transcriptional factor nuclear factor IL-3 (Nfil3)/E4 promoter-binding protein 4 (E4BP4). J. Biol. Chem. 289, 17174–17183. Kondo, H., Togari, A., 2011. Continuous treatment with a low-dose ß-agonist reduces bone mass by increasing bone resorption without suppressing bone
157
formation. Calcif. Tissue Int. 88, 23–32. Lai, C.F., Chaudhary, L., Fausto, A., Halstead, L.R., Ory, D.S., Avioli, L.V., Cheng, S.L., 2001. Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells. J. Biol. Chem. 276, 14443–14450. Li, X., Ma, Y., Wu, X., Hao, Z., Yin, J., Shen, J., Li, X., Zhang, P., 2013. Wang H. Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT(2B) receptor and the ERK1/2 signalling pathway. Biochem. Biophys. Res. Commun. 441, 809–814. Locker, M., Bitard, J., Collet, C., Poliard, A., Mutel, V., Launay, J.M., Kellermann, O., 2006. Stepwise control of osteogenic differentiation by 5-HT(2B) receptor signaling: nitric oxide production and phospholipase A2 activation. Cell. Signal. 18, 628–639. McLean, P.G., Borman, R.A., Lee, K., 2007. 5-HT in the enteric nervous system: gut function and neuropharmacology. Trends Neurosci. 30, 9–13. Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J.M., Behringer, R.R., de Crombrugghe, B., 2002. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29. Ramage, A.G., Villalón, C.M., 2008. 5-hydroxytryptamine and cardiovascular regulation. Trends Pharmacol. Sci. 29, 472–481. Sinha, K.M., Zhou, X., 2013. Genetic and molecular control of osterix in skeletal formation. J. Cell. Biochem. 114, 975–984. Sugden, P.H., Clerk, A., 1997. Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors. Cell. Signal. 9, 337–351. Westbroek, I., van der Plas, A., de Rooij, K.E., Klein-Nulend, J., Nijweide, P.J., 2001. Expression of serotonin receptors in bone. J. Biol. Chem. 276, 28961–28968. Yadav, V.K., Ryu, J.H., Suda, N., Tanaka, K.F., Gingrich, J.A., Schütz, G., Glorieux, F.H., Chiang, C.Y., Zajac, J.D., Insogna, K.L., Mann, J.J., Hen, R., Ducy, P., Karsenty, G., 2008. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 135, 825–837. Yadav, V.K., Oury, F., Suda, N., Liu, Z.W., Gao, X.B., Confavreux, C., Klemenhagen, K.C., Tanaka, K.F., Gingrich, J.A., Guo, X.E., Tecott, L.H., Mann, J.J., Hen, R., Horvath, T.L., Karsenty, G., 2009. A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure. Cell 138, 976–989. Zhou, X., Zhang, Z., Feng, J.Q., Dusevich, V.M., Sinha, K., Zhang, H., Darnay, B.G., de Crombrugghe, B., 2010. Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice. Proc. Natl. Acad. Sci. USA 107, 12919–12924. Zofková, I., Matucha, P., 2014. New insights into the physiology of bone regulation: the role of neurohormones. Physiol. Res. 63, 421–427.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Modulation of osteoblast differentiation and bone mass by 5-HT2A receptor signaling in mice Kenjiro Tanaka a, Takao Hirai a,n, Yukiko Ishibashi b,c, Nobuo Izumo d, Akifumi Togari a,n a
Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Aichi, Japan Department of Biochemistry, Faculty of Pharmaceutical Science, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-Ku, Yokohama, Kanagawa 245-0066, Japan c Division of Development Higher Brain Functions (University of Fukui), United Graduate School of Child Development, Osaka University, Kanazawa University and Hamamatsu University School of Medicine, Chiba University and University of Fukui, Fukui, Japan d Department of Clinical Pharmacology, Faculty of Pharmaceutical Science, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama, Kanagawa 245-0066, Japan b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 February 2015 Received in revised form 13 May 2015 Accepted 13 May 2015 Available online 27 May 2015
Recent studies reported that serotonin (5-hydroxytryptamine, 5-HT) may be an endogenous paracrine and/or autocrine factor that is used for intercellular communication in bone cells and between multiple organs regulating bone homeostasis. In the present study, we showed that the administration of MDL11939, a selective 5-HT2A receptor antagonist, reduced bone mass in mice. The loss of bone mass in MDL11939-treated mice was associated with impaired bone formation in vivo, as demonstrated by the lower expression of osterix (Osx) and osteocalcin than that in vehicle-treated mice. On the other hand, no significant differences were observed in osteoclast numbers between MDL11939- and vehicle-treated mice. The pharmacological blockade of 5-HT2A receptor signaling significantly decreased alkaline phosphatase activity in osteoblastic cells. In addition, the knockdown of the 5-HT2A receptor by a siRNA treatment decreased Osx, but not Runx2 gene expression in MC3T3-E1 cells. These results suggest that 5-HT2A receptor signaling mediated bone mass by regulating osteoblast differentiation. & 2015 Elsevier B.V. All rights reserved.
Keywords: 5-HT2A receptor Osteoblast differentiation Osterix Bone mass Bone formation Serotonin
1. Introduction Bone is a metabolically active organ that undergoes continuous remodeling throughout life. Bone remodeling is regulated by systemic hormones and paracrine/autocrine factors. Recent studies indicated that serotonin (5-hydroxytryptamine, 5-HT) may be an endogenous paracrine and/or autocrine factor that is used for intercellular communication in bone cells (Westbroek et al., 2001; Bliziotes et al., 2001) and between organs regulating bone homeostasis (Yadav et al., 2008; Zofková and Matucha, 2014). 5-HT is a biogenic amine that mediates the transmission of a wide variety of neuronal and non-neuronal information and also plays important roles in peripheral tissues (Barnes and Sharp, Abbreviations: 5-HT, 5-hydroxytryptamine; ALP, alkaline phosphatase; BFR, formation rate; BMD, bone mineral density; BMP, bone morphogenetic protein; BV/ TV, bone volume per total volume; DIC, day in culture; IGF, insulin-like growth factor; OC, Osteocalcin; Oc.N/BS, Osteoclast number/bone surface; Oc.S/BS, osteoclast surface/bone surface; Osx, Osterix; Runx2, Runt-related transcription factor 2; Tb.Th, trabecular thickness; TPH1, tryptophan hydroxylase 1; TRAP, tartrate-resistant acid phosphatase n Corresponding authors. Fax: þ 81 52 752 5988 E-mail addresses: [email protected] (T. Hirai), [email protected] (A. Togari). http://dx.doi.org/10.1016/j.ejphar.2015.05.048 0014-2999/& 2015 Elsevier B.V. All rights reserved.
1999; Gershon and Tack, 2007; McLean et al., 2007; Ramage and Villalón, 2008) as well as the central nervous system (CNS). The actions of 5-HT are known to be mediated through interactions with membrane-bound receptors that have been categorized into seven families and include at least 21 subtypes (Barnes and Sharp, 1999). All 5-HT receptor families, excluding the 5-HT3 receptor, which forms a ligand-gated ion channel, belong to the G proteincoupled receptor superfamily. Among them, 5-HT2A is associated with the contraction of vascular smooth muscle, platelet aggregation, thrombus formation, and coronary artery spasms. Previous studies identified a 5-HT-induced functional regulatory mechanism that was regulated by a 5-HT inactivatory system for cellular proliferation, differentiation, and maturation in osteoblasts in vitro as well as in the CNS, and enteric nervous system, and cardiovascular system (Westbroek et al., 2001; Bliziotes et al., 2001; Hirai et al., 2009). We also reported the expression patterns of mRNA for 5-HT2A and 5-HT2B receptors in osteoblasts, and suggested that the respective proteins may represent local primary targets for 5-HT signal transduction (Hirai et al., 2009). However, the actions of 5-HT in cells with osteoblastic lineages are not yet fully understood. In the present study, we determined whether 5-HT2A receptor signaling played a role in modulating bone physiology.
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2. Materials and methods 2.1. Materials MDL11939 (( 7)-α-phenyl-1-(2-phenylethyl)-4-piperidine methanol) hydrochloride was purchased from Sigma-Aldrich. All chemicals used were of analytical grade. 2.2. Animals and experimental procedures Sixty-seven male, seven-week-old C57BL/6J mice (Japan SLC Inc., Hamamatsu, Japan) were randomized by weight, assigned to groups, and acclimated to their cages for 1 week prior to the experiment. They were treated in accordance with the Guidelines for Animal Experiments at the School of Dentistry, Aichi-Gakuin University. Food and water were available ad libitum. The mice in each group were housed together under automatically controlled conditions of temperature (23 71 °C) and humidity (50 710%). 2.3. Administration MDL11939, a 5-HT2A receptor antagonist, was dissolved at a concentration of 25 mg/mL in 100% ethanol. In the first series of experiment, we studied the effect of MDL11939 on bone mass and TRAP activity in mice. Eight-week-old mice were used in this study. Mice were divided into three groups and given MDL11939 (0.3 mg/kg/day, n ¼6), MDL11939 (3 mg/kg/day, n ¼9), or vehicle (1.2% ethanol, n ¼9) by oral gavage once daily/7d/week for 2 weeks at a regular time. The second or third series of experiment was performed to evaluate the effect of MDL11939 on bone formation and gene expression. Eight-week-old mice were treated with MDL11939 at 3 mg/kg/day (n ¼11) or vehicle (1.2% ethanol, n ¼10 or 11) by oral gavage once daily/7d/week for 2 weeks at a regular time. Body weight was measured weekly and the MDL11939 dose was adjusted accordingly. 2.4. Microcomputed tomographic analysis The distal region of the right femur was subjected to a threedimensional m-computed tomography (mCT) analysis using an R-mCT mCT scanner (RIGAKU, Tokyo, Japan). Scanning was initiated 1.0 mm above the distal femoral growth plate, and a total of 75 consecutive 20-mm-thick sections were analyzed, encompassing a length of 1.5 mm of the secondary spongiosa. The measured volume of interest in the femur was obtained by selecting the cancellous bone (separate from the cortical shaft) using contour areas that were drawn semiautomatically (Kondo and Togari, 2011). TRI/ 3D-BON (Ratoc, Tokyo, Japan) software was used to analyze cancellous parameters: bone volume/total volume (BV/TV, %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm). 2.5. Bone histomorphometry In the dynamic histomorphometric analysis, all mice were injected intraperitoneally with calcein (10 mg/g) at 4 and 2 days before death. At the end of the experiments, the right femur of each mouse was dissected and fixed in 70% ethanol. Five-micrometer-thick sagittal sections were made as undecalcified sections. To assess the bone formation rate (BFR), metaphyseal cancellous bone in the femur was used to obtain the bone fraction in a rectangular area of 0.34 mm2 (0.5 0.67 mm), with its closest and furthest edges being 0.3 and 0.8 mm distal to the growth plate, respectively. At least two sections from independent animal for each group were subjected to the analysis. Regarding decalcified sections, the left tibiae of mice were dissected, fixed in 4%
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paraformaldehyde, and then decalcified in 20% EDTA for 2 weeks. Sagittal section (5-mm thick) were made as decalcified sections and stained with TRAP for the osteoclast analysis. Measurements were made within an area of 0.8 mm2 (1.0 mm 0.8 mm), with its closest and furthest edges being 2.0 and 3.0 mm distal to the growth plate of the proximal ends of the tibia, respectively. Osteoclast number/bone surface (Oc.N/BS) and osteoclast surface/ bone surface (Oc.S/BS) were evaluated by scoring TRAP-positive multinucleated cells attached to the bone surface (Kondo and Togari, 2011). At least two sections from independent animal for each group were subjected to the analysis. 2.6. MC3T3-E1 cell cultures MC3T3-E1 cells were purchased from the RIKEN Cell Bank. MC3T3-E1 cells were cultured in α-MEM (Life Technologies) containing 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere. These cells were grown in non-supplemented medium until confluent (the first day of a confluent culture was referred to as day 0). When cells reached confluency, medium supplemented with 10 mM β-glycerophosphate and 10 mg/ml ascorbic acid was used. Media were changed every 2–4 days (Hirai et al., 2014a). 2.7. Determination of alkaline phosphatase (ALP) activity Cells were cultured for 10 days in culture (DIC) in the presence of MDL11939 at a concentration range of 1–10 mM or vehicle (0.01% ethanol) in order to determine the activity of alkaline phosphatase (ALP). ALP activity was determined as described previously (Fujita et al., 2001). In brief, cells were solubilized with 0.1% Triton X-100, followed by the determination of ALP activity in lysates using p-nitrophenol phosphate as a substrate. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad). ALP staining was performed using a standard protocol. In brief, cultured cells were rinsed in PBS, fixed using 10% buffered neutral formalin, rinsed with PBS, and then overlaid with 1.5 ml of 0.15 mg/ml 5-bromo-4-chloro-3-indolylphosphate plus 0.3 mg/ml nitro blue tetrazolium chloride in 0.1 M Tris–HCl (pH 9.5), 0.01 N NaOH, and 0.05 M MgCl2, followed by incubation at room temperature for 2 h in the dark. 2.8. C3H/10T1/2 cells and siRNA nucleofection C3H/10T1/2 cells were purchased from the Japanese Collection of Research Bioresources (JCRB, Japan) Cell Bank. C3H/10T1/2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1% penicillin/streptomycin to 70% confluency, followed by transient transfection with either interfering RNA (siRNA) targeting the 5HT2A receptor or non-silencing RNA diluted in Opti-MEM using Lipofectamine RNAiMAX according to the manufacturer's protocol (Life Technologies) as previously described (Hirai et al., 2014b). The siRNA used to silence the HTR2Atargeted gene (siRNA-HTR2A) and negative control siRNA (siRNANegative) were obtained from CosmoBio (Kyoto, Japan). The sequences of siRNA used to silence the HTR2A gene were as follows: HTR2A sense 5′-GGAGUAAGUUGGUGACAUA-3′, HTR2A antisense 5′-UAUGUCACCAACUUACUCC-3′. siRNA-HTR2A and siRNA-Negative were used at final concentrations of 10 nM. The medium was then replaced with fresh medium. Cells were harvested after 48 h for total RNA extraction at the indicated time points. 2.9. RNA extraction and quantitative RT-PCR As in vivo experiments, total RNA was extracted from femur (cancellous bone). As in vitro experiments, total RNA samples were
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extracted from the cells. In both cases, total RNA was isolated with an RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. One microgram of RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit according to the protocol of the manufacturer (Qiagen). Gene expression was analyzed with the StepOne-Plus real time PCR system with Step One Software v2.0 (Applied Biosystems). Reactions were performed in 20-mL volumes using a QuantiTect SYBR Green PCR Kit (Qiagen). Cycling conditions were 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data are presented as relative mRNA levels calculated by the equation 2–Ct (Ct¼ Ct of target gene minus Ct of GAPDH). The following primers were used: GAPDH, forward 5′-TGGAGAAACCTGCCAAGTATG-3′, reverse 5′-GGAGACAACCTGGTCCTCAG-3′; HTR2A, forward 5′-AGGCTCTTTTGTGGCATTTTTC-3′, reverse 5′TGGGCACCACATTACAACAAAC-3′; Runx2, forward 5′-AGGCACAGACAGAAGCTTGATG-3′, reverse 5′-GCGATCAGAGAACAAACTAGGTTTAGA-3; OC, forward 5′-GACCGCCTACAAACGCATCT-3′, reverse 5′-GGGCAGCACAGGTCCTAAATAGT-3′; Osterix, forward 5′-CCTCTGCGGGACTCAACAAC, reverse 5′-AAAGGTCAGCGTATGGCTTCTT-3′.
2.10. Data analysis Data are expressed as means 7S.D. Statistical differences were determined by one-way ANOVA followed by the Tukey HSD for multiple comparisons. P o0.05 was considered statistically significant.
3. Results 3.1. Decreases in bone mass by the administration of a 5-HT2A receptor antagonist to mice To evaluate the functionality of the 5-HT2A receptor in bone, we performed CT-based bone densitometry in cancellous bones following the oral administration of the 5-HT2A receptor antagonist, MDL11939 for 2 weeks (Fig. 1A). As shown in Fig. 1B, the BV/TV of the distal end of the femur was significantly lower in MDL11939treated mice than in vehicle-treated mice. The oral administration of MDL11939 at a dosage that influenced BV/TV had no effect on
Fig. 1. Blockade of 5-HT2A receptor signaling impaired trabecular bone properties in mice. (A) mCT image of the femur from 10-wk-old male administered vehicle or MDL11939. (B–E) CT-based bone densitometry of the femur from 10-wk-old male administered vehicle (Bar: white) or MDL11939 (Bar: black). B: bone volume per tissue volume (BV/TV). C: trabecular thickness (Tb.Th). D: trabecular number (Tb.N). E: trabecular separation (Tb.Sp). Values are expressed as means 7S.D.; n¼ 6–9 mice per group. *Po 0.05 significantly different from vehicle-treated mice.
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body weight [Average body weight (g;7 S.D.); 23.2 71.1 in the vehicle group, 22.6 71.1 in the MDL11939 (3 mg/kg) group]. Moreover, the trabecular thickness (Tb.Th) of the femur was also significantly lower in MDL11939-treated mice than in vehicletreated mice, and was dose-dependent (Fig. 1C). No significant differences were observed in Tb.N, and Tb.Sp between mice orally administered MDL11939 at 0.3 and 3 mg/kg and vehicle (Fig. 1D and E). In addition, we assessed the cortical bone mineral density (BMD) following the oral administration of MDL11939 at 3 mg/kg for 2 weeks. The cortical BMD of the tibia was significantly lower in MDL11939-treated mice than in vehicle-treated mice (Supplemental Fig. 1A). MDL11939-treated mice showed lower bone estimated strength by diminished bone rigidity indexes, which reflected the loss of cortical bone. The minimum moment of inertia of cross-sectional areas, which represents flexural rigidity, was significantly reduced in MDL11939-treated mice (Supplemental Fig. 1B). Moreover, torsional rigidity, as indicated by the polar moment of inertia of cross-sectional areas, was also significantly diminished in MDL11939-treated mice (Supplemental Fig. 1C), and, when taken together, demonstrated that mice administered the 5-HT2A receptor antagonist had a lower bone mass, cortical osteopenia, and a lower bone estimated strength. 3.2. No alteration in TRAP activity with the administration of MDL11939 to mice To examine the mechanisms responsible for bone loss due to the MDL11939 treatment, we next performed a histomorphometric analysis of bone resorption parameters such as the bone surface covered by TRAP-positive osteoclasts, Oc.S/BS, and Oc.N/BS (Fig. 2A). No significant differences were observed in Oc.S/BS or Oc. N/BS between mice orally administered MDL11939 at 0.3 and
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3 mg/kg and vehicle, which suggested that the pharmacological blockade of 5-HT2A receptor signaling did not affect osteoclast activity (Fig. 2B and C). 3.3. Effects of MDL11939 on bone formation To examine the mechanisms responsible for bone loss due to the MDL11939 treatment, we performed a bone histomorphometric analysis on bone formation parameters by assessing calcein double-labeling in the distal femur after the systemic administration of MDL11939 for 2 weeks. This analysis provided an in vivo estimation of osteoblastic activity with respect to the accumulation of bone mass. Bone formation parameters such as mineral surface/bone surface (MS/BS), the bone mineral apposition rate (MAR), and bone formation rates (BFRs) were significantly lower in MDL11939-administrated mice than in vehicle-administrated mice (Fig. 3A). We then examined the expression of gene-related bone formation using a quantitative real time PCR analysis. We extracted total RNA from the distal end of the femur and analyzed the mRNA expression of Runx2, Osterix (Osx), and Osteocalcin (OC). Consistent with the decrease observed in bone formation, the mRNA expression of Runx2, Osx, and OC in bone was significantly decreased in MDL11939-administrated mice (Fig. 3B), indicating that the pharmacological blockade of 5-HT2A receptor signaling led to bone loss concomitant with decreased bone formation. 3.4. Blockade of 5-HT2A receptor signaling impaired osteoblast differentiation To evaluate the function of the 5-HT2 receptor in osteoblastic cells, we examined the effects of MDL11939 on cell differentiation in MC3T3-E1 cells. As shown in Fig. 4A, sustained exposure to
Fig. 2. Effects of MDL11939 on osteoclast activity in mice. Mice were treated with MDL11939 at 3 mg/kg/day or vehicle for 2 weeks, followed by the staining of osteoclasts with TRAP. (A) Representative image of TRAP staining of the decalcified sections at the distal ends of the femur from 10-wk-old male administered vehicle (upper panel) or MDL11939 (lower panel). Arrowheads show TRAP-positive cells. Scale bars ¼100 mm. (B) Oc.S/BS and (C) Oc.N/BS. Values are expressed as means 7 S.D.; n ¼5–9 mice per group. *P o0.05 significantly different from vehicle-treated mice.
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Fig. 3. Effects of the systemic administration of MDL11939 on bone formation. (A) An analysis of the mineralized surface/bone surface (MS/BS), mineral apposition rates (MAR), and bone formation rate (BFR) in the cancellous bone compartment of the distal femur metaphysis from 10-wk-old male administered vehicle (Bar: white) or MDL11939 (Bar: black). n ¼11 mice per group. Values are expressed as means 7 S.D. *Po 0.05, significantly different from vehicle-treated mice. (B) Total RNA was isolated from the distal region of the right femur in 10-week-old male mice administered vehicle (Bar: white) or MDL11939 (Bar: black), followed by the determination of Runx2, Osx, and OC mRNA levels by real time qRT-PCR using specific primers. Values are expressed as means 7S.D.; n ¼10–11 mice per group. *Po 0.05, significantly different from vehicle-treated mice.
Fig. 4. Blockade of 5-HT2A receptor signaling impaired osteoblast differentiation. (A) Alkaline phosphatase activity in MC3T3-E1 cells cultured with MDL11939. Cells were cultured in α-MEM in either the presence or absence of MDL11939 at a concentration range of 1–10 mM for 10 DIC, followed by determination of the activity of alkaline phosphatase. Values are the mean 7 S.D. from four independent cultures. *Po 0.05, significantly different from each control value obtained in the absence of MDL11939. (B and C) Real time PCR analyses of osteoblast-related genes. Osteoblasts were cultured for 10 DIC with MDL11939, and then subjected to real time PCR. Data are expressed as the mean7 S.D. after normalization by GAPDH expression from three or four different experiments. *P o0.05, significantly different from each control value obtained in the absence of MDL11939. Figures are representative of data from three independent experiments.
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MDL11939 significantly decreased ALP activity in MC3T3-E1 cells in a concentration-dependent manner. Moreover, primary mouse osteoblasts were cultured for 13 DIC in the presence of ketanserin, a 5-HT2A specific antagonist, at a concentration range of 3–10 mM in order to determine the activity of ALP. The results obtained showed that sustained exposure to ketanserin significantly prevented increases in ALP activity in osteoblasts cultured for 13 DIC in a concentration-dependent manner (Supplemental Fig. 2A). To better characterize these mechanisms, the effects of the 5-HT2A selective antagonist, sarpogrelate were also determined (Supplemental Fig. 2B). Osteoblasts were cultured for 13 DIC in the sustained presence of sarpogrelate at 10 mM. All these 5-HT2A receptor antagonists were invariably effective in significantly inhibiting the activity of ALP in osteoblasts cultured for 13 DIC. Sustained exposure to sarpogrelate at 30 mM inhibited the activity of ALP by 450%, which was similar to that induced by ketanserin at 10 mM (Supplemental Fig. 2). The results of the real time PCR analysis revealed that the expression of Osx was significantly lower in osteoblasts cultured for 10 DIC in the presence of 10 mM MDL11939 than in cells cultured in the absence of MDL11939 (Fig. 4B). Furthermore, sustained exposure to MDL11939 at 1–10 mM decreased the expression of OC in osteoblasts cultured for 10 DIC (Fig. 4B). To further evaluate the potential role of 5-HT2A receptor signaling in osteogenesis, we attempted to elucidate the gene expression of Osx in C3H10T1/2 cells transfected with small interfering RNA (siRNA) for the knockdown of 5-HT2A receptor. Cells were transfected with siRNA for HTR2A, followed by the determination of HTR2A levels using real time qRT-PCR. The results obtained demonstrated that, after transfection, HTR2A levels were significantly decreased in C3H10T1/2 cells transfected with HTR2A siRNA (Fig. 5A). A significant decrease in Osx expression was observed in C3H10T1/2 cells under these conditions (Fig. 5B). No significant differences were observed in Runx2 gene expression between
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siRNA-HTR2A and siRNA-Negative treated cells (Fig. 5C). These results indicated that 5-HT2A receptor signaling regulated osteoblast differentiation through Osx gene expression.
4. Discussion We herein demonstrated that 5-HT2A receptor signaling regulated osteoblast differentiation and bone mineral density in mice. Although several previous studies reported the functional expression of the 5-HT receptor in osteoblasts, no direct evidence for the role of 5-HT2A receptor signaling in the mechanisms underlying cellular differentiation in osteoblasts currently exists in the literature. To the best of our knowledge, this is the first study to show that 5-HT2A receptor signaling in osteoblasts plays a physiologically significant role in bone metabolism. Several studies previously suggested that 5-HT receptor signaling regulated cellular function in osteoblasts, osteocytes, and osteoclasts (Yadav et al., 2008; Hirai et al., 2010; Bliziotes et al., 2006; Chabbi-Achengli et al., 2012). 5-HT2B receptor expression during osteoblast differentiation in vitro was shown to be increased in primary cultured mouse osteoblasts (Hirai et al., 2009), and the ablation of the 5-HT2B receptor in female mice led to osteopenia due to impaired bone formation (Collet et al., 2008). In addition, 5-HT2B receptor signaling in osteoblasts was found to regulate cellular proliferation, differentiation, and functions (Baudry et al., 2010; Locker et al., 2006; Li et al., 2013). Previous studies e confirmed the pattern of expression for the 5-HT receptor in osteoblastic cells (Hirai et al., 2009). A reverse transcription polymerase chain reaction analysis revealed the expression of mRNA for the receptors of 5-HT1B, 5-HT1D, 5-HT2A, and 5-HT2B in primary cultured mouse calvarial osteoblasts. An immunohistochemical analysis clearly demonstrated that the 5-HT2A
Fig. 5. Silencing HTR2A down-regulated the expression of osterix in C3H/10T1/2 cells. C3H/10T1/2 cells were treated with HTR2A siRNA (siRNA-HTR2A) or non-silencing RNA (siRNA-Negative), followed by further cultivation for 48 h and the subsequent determination of (A) HTR2A, (B) Osx, and (C) Runx2 levels by real time qRT-PCR. Relative mRNA expression was normalized to GAPDH. Each value represents the means 7 S.D. of four independent determinations. *Po 0.05, significantly different from value treated with siRNA-Negative. Figures are representative of data from three independent experiments.
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receptor was expressed in both osteoblasts and osteoclasts in adult mouse bone sections (data not shown). In the present study, we showed that the oral administration of MDL11939, a selective and high affinity 5-HT2A receptor antagonist, significantly decreased bone mass and led to skeletal fragility, as reflected by the decreased expression of Osx and OC (Figs. 1 and 3), which suggested that 5-HT plays a significant role in modulating bone physiology, at least through 5-HT2A receptor signaling. Additionally, the administration of MDL11939 led to a reduction in bone formation without effective osteoclast activity (Fig. 3). Therefore, we speculated that 5-HT2A receptor signaling may regulate bone formation and cellular differentiation as well as the 5-HT2B receptor signaling in osteoblasts. Previous studies suggested that the 5-HT2A receptor was expressed in osteoblasts (Westbroek et al.,2001; Hirai et al., 2009) and regulated cellular proliferation in MC3T3-E1 cells (Hirai et al., 2010). In this study, the pharmacological blockade of 5-HT2A receptor signaling significantly decreased cellular differentiation in MC3T3-E1 cells and primary cultured osteoblasts (Supplemental Fig. 2). Furthermore, knockdown of the 5-HT2A receptor by a siRNA treatment also decreased Osx gene expression in C3H10T1/2 cells. Osx is a key transcription factor that is required for osteoblast differentiation and bone formation (Nakashima et al., 2002). The absence of osteoblast differentiation in Osx mutant embryos was shown to lead to small bones that did not express osteoblastspecific marker genes (Zhou et al., 2010). After birth, the absence Osx in mice causes multiple skeletal phenotypes including the lack of new bone formation, absence of the resorption of mineralized cartilage, and defects in osteocyte maturation and function (Sinha and Zhou 2013). Several studies reported the expression of osteoblast-related genes was controlled by the bone morphogenetic protein 2 (BMP2) and insulin-like growth factor 1 (IGF1) signaling pathways in osteoblast differentiation. The expression of Osx, which is activated by the BMP2/IGF1 signaling pathway, was also suggested to involve the mitogen-activated protein kinase (MAPK) pathway rather than BMP2/Smad/Runx2 signaling (Celil and Campbell, 2005; Celil et al., 2005). Three distinctive MAPK pathways have been identified to date; the extracellular regulated kinases (ERK1/2, also known as p42/44 MAPK), p38 MAPK, and JNK/ SAPK pathways. These are known to modulate the phosphorylation and, hence, activation status of transcription factors and link transmembrane signaling with gene-induction events in the nucleus (Sugden and Clerk, 1997). The activation of ERK provides a mitogenic and differentiating signal in osteoblasts (Lai et al., 2001). ERK also plays critical roles in osteoblast differentiation and skeletal development (Ge et al., 2007). We previously demonstrated that ERK activity was markedly increased by 5-HT2A receptor signaling in MC3T3-E1 cells, indicating that activation of the MAPK/ ERK pathway played an important role in cellular function regulated by 5-HT2A receptor signaling in osteoblastic cells (Hirai et al., 2010). Thus, these results indicated that 5-HT2A receptor signaling participated in osteoblast differentiation, at least through the regulation of Osx, but not Runx2 expression. 5-HT appears to play dual roles in the mechanisms underlying the maintenance of homeostasis as an autocrine and/or paracrine mediator in bone development. A question in this analysis is the origin of the 5-HT ligand that binds 5-HT2A receptors in osteoblasts. One possibility is that bone cells themselves could produce 5HT, in which case the effects from the ligand would be autocrine and/or paracrine mediator in bone cells. Previous studies showed that the expression of the initial and rate-limiting enzyme in 5HT synthesis, tryptophan hydroxylase 1 (TPH1) in osteoblastic and osteocytic cells (Gustafsson et al., 2006; Bliziotes et al., 2006). Using real-time RT-PCR, we also observed that TPH1 mRNA is expressed in MC3T3-E1 cells (Data not shown). These findings indicate that osteoblasts are capable of synthesizing 5HT, and thus
can produce paracrine/autocrine effects on bone cells. In this study, the negative effects of antagonists for 5-HT2A receptor on osteoblastic activities favor the idea that these antagonists would prevent the cellular differentiation through blockade of the 5-HT2A receptor signaling under sustained stimulation by 5HT. We analyzed the concentration of 5-HT in the culture medium of primary cultured osteoblasts by the HPLC procedure as described previously (Hirai et al., 2009). The concentration of 5-HT in the culture medium (α-MEM þ10% FBS) was approximately 0.3 mM. Therefore, the 5-HT2A receptor expressed in cultured osteoblasts would be under stimulation by 5-HT present in culture medium. Furthermore, 5-HT secreted by osteoclast precursors has more recently been shown to be crucially involved in osteoclast differentiation and acted on neighboring osteoblasts through a paracrine system (Chabbi-Achengli et al., 2012). The endogenous agonist 5-HT may be supplied to osteoblasts by neighboring osteoclasts, by osteoblasts themselves, and by blood circulation. Therefore, 5-HT2A receptor signaling in osteoblasts may regulate bone mass through an autocrine and/or paracrine system in local areas in bone. However, we cannot deny the possibility that effects of MDL11939 might be under the regulation of CNS-mediated skeletal effects, rather than an autocrine and/or paracrine system. Recent study showed that brainstem-derived 5-HT favors bone mass accrual following its binding to 5-HT2C receptors on ventromedial hypothalamic neuron (Yadav et al., 2009). Further studies are necessary to investigate the contribution of 5-HT2A receptor in these processes. Taken together, these results suggest that the 5-HT2A receptor is expressed in bone to regulate bone mass. An evaluation of the functionality of the 5-HT2A receptor in bone may provide an important insight for future discoveries and the development of innovative drugs for the treatment of various bone diseases in humans.
5. Conclusion In the present study, we identified a novel regulatory component in the actions of 5-HT and showed, for the first time, that 5-HT2A receptor signaling played an important role in regulating bone mass.
Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research to A.T. (26462827) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2015.05. 048.
References Barnes, N.M., Sharp, T., 1999. A review of central 5-HT receptors and their function. Neuropharmacology 38, 1083–1152. Baudry, A., Bitard, J., Mouillet-Richard, S., Locker, M., Poliard, A., Launay, J.M., Kellermann, O., 2010. Serotonergic 5-HT(2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C. J. Biol. Chem. 285, 26066–26073. Bliziotes, M.M., Eshleman, A.J., Zhang, X.W., Wiren, K.M., 2001. Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake. Bone 29, 477–486. Bliziotes, M., Eshleman, A., Burt-Pichat, B., Zhang, X.W., Hashimoto, J., Wiren, K.,
K. Tanaka et al. / European Journal of Pharmacology 762 (2015) 150–157
Chenu, P., 2006. Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells. Bone 39, 1313–1321. Celil, A.B., Campbell, P.G., 2005a. BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. J. Biol. Chem. 280, 31353–31359. Celil, A.B., Hollinger, J.O., Campbell, P.G., 2005b. Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling. J. Cell. Biochem. 95, 518–528. Chabbi-Achengli, Y., Coudert, A.E., Callebert, J., Geoffroy, V., Côté, F., Collet, C., de Vernejoul, M.C., 2012. Decreased osteoclastogenesis in serotonin-deficient mice. Proc. Natl. Acad. Sci. USA 109, 2567–2572. Collet, C., Schiltz, C., Geoffroy, V., Maroteaux, L., Launay, J.M., de Vernejoul, M.C., 2008. The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation. FASEB J. 22, 418–427. Fujita, T., Fukuyama, R., Izumo, N., Hirai, T., Meguro, T., Nakamuta, H., Koida, M., 2001. Transactivation of core binding factor alpha1 as a basic mechanism to trigger parathyroid hormone-induced osteogenesis. Jpn. J. Pharmacol. 86, 405–416. Ge, C., Xiao, G., Jiang, D., Franceschi, R.T., 2007. Critical role of the extracellular signal-regulated kinase-MAPK pathway in osteoblast differentiation and skeletal development. J. Cell Biol. 176, 709–718. Gershon, M.D., Tack, J., 2007. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 132, 397–414. Gustafsson, B.I., Thommesen, L., Stunes, A.K., Tommeras, K., Westbroek, I., Waldum, H.L., Slørdahl, K., Tamburstuen, M.V., Reseland, J.E., Syversen, U., 2006. Serotonin and fluoxetine modulate bone cell function in vitro. J. Cell. Biochem. 98, 139–151. Hirai, T., Tokumo, K., Tsuchiya, D., Nishio, H., 2009. Expression of mRNA for 5-HT2 receptors and proteins related to inactivation of 5-HT in mouse osteoblasts. J. Pharmacol. Sci. 109, 319–323. Hirai, T., Kaneshige, K., Kurosaki, T., Nishio, H., 2010. Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells. Biochem. Biophys. Res. Commun. 396, 278–282. Hirai, T., Tanaka, K., Togari, A., 2014a. ß-adrenergic receptor signaling regulates Ptgs2 by driving circadian gene expression in osteoblasts. J. Cell Sci. 127, 3711–3719. Hirai, T., Tanaka, K., Togari, A., 2014b. α1-adrenergic receptor signaling in osteoblasts regulates clock genes and bone morphogenetic protein 4 expression through up-regulation of the transcriptional factor nuclear factor IL-3 (Nfil3)/E4 promoter-binding protein 4 (E4BP4). J. Biol. Chem. 289, 17174–17183. Kondo, H., Togari, A., 2011. Continuous treatment with a low-dose ß-agonist reduces bone mass by increasing bone resorption without suppressing bone
157
formation. Calcif. Tissue Int. 88, 23–32. Lai, C.F., Chaudhary, L., Fausto, A., Halstead, L.R., Ory, D.S., Avioli, L.V., Cheng, S.L., 2001. Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells. J. Biol. Chem. 276, 14443–14450. Li, X., Ma, Y., Wu, X., Hao, Z., Yin, J., Shen, J., Li, X., Zhang, P., 2013. Wang H. Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT(2B) receptor and the ERK1/2 signalling pathway. Biochem. Biophys. Res. Commun. 441, 809–814. Locker, M., Bitard, J., Collet, C., Poliard, A., Mutel, V., Launay, J.M., Kellermann, O., 2006. Stepwise control of osteogenic differentiation by 5-HT(2B) receptor signaling: nitric oxide production and phospholipase A2 activation. Cell. Signal. 18, 628–639. McLean, P.G., Borman, R.A., Lee, K., 2007. 5-HT in the enteric nervous system: gut function and neuropharmacology. Trends Neurosci. 30, 9–13. Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J.M., Behringer, R.R., de Crombrugghe, B., 2002. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29. Ramage, A.G., Villalón, C.M., 2008. 5-hydroxytryptamine and cardiovascular regulation. Trends Pharmacol. Sci. 29, 472–481. Sinha, K.M., Zhou, X., 2013. Genetic and molecular control of osterix in skeletal formation. J. Cell. Biochem. 114, 975–984. Sugden, P.H., Clerk, A., 1997. Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors. Cell. Signal. 9, 337–351. Westbroek, I., van der Plas, A., de Rooij, K.E., Klein-Nulend, J., Nijweide, P.J., 2001. Expression of serotonin receptors in bone. J. Biol. Chem. 276, 28961–28968. Yadav, V.K., Ryu, J.H., Suda, N., Tanaka, K.F., Gingrich, J.A., Schütz, G., Glorieux, F.H., Chiang, C.Y., Zajac, J.D., Insogna, K.L., Mann, J.J., Hen, R., Ducy, P., Karsenty, G., 2008. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 135, 825–837. Yadav, V.K., Oury, F., Suda, N., Liu, Z.W., Gao, X.B., Confavreux, C., Klemenhagen, K.C., Tanaka, K.F., Gingrich, J.A., Guo, X.E., Tecott, L.H., Mann, J.J., Hen, R., Horvath, T.L., Karsenty, G., 2009. A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure. Cell 138, 976–989. Zhou, X., Zhang, Z., Feng, J.Q., Dusevich, V.M., Sinha, K., Zhang, H., Darnay, B.G., de Crombrugghe, B., 2010. Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice. Proc. Natl. Acad. Sci. USA 107, 12919–12924. Zofková, I., Matucha, P., 2014. New insights into the physiology of bone regulation: the role of neurohormones. Physiol. Res. 63, 421–427.