TDAG8 activation inhibits osteoclastic bone resorption - The FASEB ...

The FASEB Journal article fj.13-233106. Published online November 12, 2013.

The FASEB Journal • Research Communication

TDAG8 activation inhibits osteoclastic bone resorption Hisako Hikiji,*,1 Daisuke Endo,† Kyoji Horie,‡ Takeshi Harayama,* Noriyuki Akahoshi,§ Hidemitsu Igarashi,§ Yasuyuki Kihara,* Keisuke Yanagida,* Junji Takeda,‡ Takehiko Koji,† Takao Shimizu,* and Satoshi Ishii*,§,2 *Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; †Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; ‡Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; and §Department of Immunology, Graduate School of Medicine, Akita University, Akita, Japan Although the roles of acids in bone metabolism are well characterized, the function of proton-sensing receptors in bone metabolism remains to be explored. In this study, we evaluated the role of proton-sensing receptor T-cell death-associated gene 8 (TDAG8) in osteoclastic activity during bone loss after ovariectomy. Through observations of bone mineral content, we found that pathological bone resorption was significantly exacerbated in mice homozygous for a gene trap mutation in the Tdag8 gene. Furthermore, osteoclasts from the homozygous mutant mice resorbed calcium in vitro more than the osteoclasts from the heterozygous mice did. Impaired osteoclast formation under acidic conditions was ameliorated in cultures of bone marrow cells by Tdag8 gene mutation. Extracellular acidification changed the cell morphology of osteoclasts via the TDAG8-Rho signaling pathway. These results suggest that the enhancement of TDAG8 function represents a new strategy for preventing bone resorption diseases, such as osteoporosis.—Hikiji, H., Endo, D., Horie, K., Harayama, T., Akahoshi, N., Igarashi, H., Kihara, Y., Yanagida, K., Takeda, J., Koji, T., Shimizu, T., Satoshi, I. TDAG8 activation inhibits osteoclastic bone resorption. FASEB J. 28, 000 – 000 (2014). www.fasebj.org ABSTRACT

Key Words: proton-sensing receptor 䡠 GPR65 䡠 Rho 䡠 cell morphology 䡠 T-cell death-associated gene 8 Bone remodeling is essential for the optimal control of calcium homeostasis and is performed by the concerted actions of osteoclasts and osteoblasts (1). Osteoclasts are multinucleated cells that are unique in their ability to degrade bone, and their activities are regu-

Abbreviations: BMC/TV, bone mineral content per tissue volume; BV/TV, bone volume per tissue volume; cAMP, cyclic adenosine monophosphate; GFP, green fluorescent protein; gt, gene trap; GTP, guanosine triphosphate; HRP, horseradish peroxidase; M-CSF, macrophage colony-stimulating factor; ␮CT, microcomputed tomography; OGR1, ovarian cancer G-protein-coupled receptor 1; RANKL, receptor activator of NF-␬B ligand; TDAG8, T-cell death-associated gene 8; SSC, standard saline citrate; TRAP, tartrate-resistant acid phosphatase; WT, wild-type

lated by hormones such as parathyroid hormone, vitamin D3, and calcitonin (1). Furthermore, there are paracrine/autocrine factors that affect osteoclastic activities, such as platelet-activating factor (2) and leukotriene B4 (3). Bone resorption is initiated by the secretion of protons through vacuolar-type ATPase and the passive transport of chloride through a chloride channel (1). To generate protons, carbonic anhydrase converts CO2 and H2O into protons and HCO3⫺. Osteoclast-mediated extracellular acidification enhances bone resorption, which results in the formation of resorbing compartments (that is, lacunae) on the bone surfaces (4). Although osteoclasts create the local extracellular acidic milieu around themselves, it is still unknown how the cells detect and respond to this extracellular acidity. There are several families of channels and receptors that are activated by extracellular acidic pH. The former includes acid-sensing ion channels (5), transient receptor potential cation channels (6), and hyperpolarization-activated cation channels (7). The latter involves acid-sensing G-protein-coupled receptors (8), such as the ovarian cancer G-protein-coupled receptor 1 (OGR1; also known as GPR68; ref. 9); GPR4 (9); G2A (also known as GPR132; ref. 10); and T-cell deathassociated gene 8 (TDAG8; also known as GPR65; refs. 11, 12). In humans, TDAG8 mRNA is highly expressed in peripheral blood leukocytes, lymph nodes, and the spleen (13). Previously, we showed that human TDAG8 stimulates cyclic adenosine monophosphate (cAMP) formation, activates Rho, and induces stress fiber formation in response to extracellular acidification in stably transfected cells (11). We also revealed that TDAG8 mediates the extracellular acidification-induced inhibition of proinflammatory cytokine production in mouse macrophages (14). In addition, we 1 Current address: Department of Oral Functional Management, School of Oral Health Sciences, Kyushu Dental University, Fukuoka 803-8580, Japan. 2 Correspondence: Department of Immunology, Graduate School of Medicine, Akita University, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan. E-mail: [email protected] doi: 10.1096/fj.13-233106 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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recently demonstrated that TDAG8 promotes tumor cell growth and survival in vivo and in vitro, strongly suggesting that TDAG8 serves as an extracellular proton sensor responsible for the adaptation of the tumor to the acidic milieu (15). Ovariectomy, a model of postmenopausal osteoporosis, causes bone resorption by an acute decrease in serum estrogen levels (16). Estrogen deficiency results in a bone-remodeling imbalance in which bone resorption exceeds bone formation (1). Because osteoclasts are the major cells involved in bone loss (17), they play an important role in bone resorption of ovariectomized mice. In the present study, we found an inhibitory role for TDAG8 in osteoclastic bone resorption through the analysis of ovariectomized mice homozygous for a gene trap (gt) mutation in the Tdag8 gene (Tdag8gt/gt) (18). Our in vitro data consistently demonstrated that the Tdag8 gene mutation resulted in an increase in osteoclastic activity, suggesting that TDAG8 in osteoclasts counteracts the increased bone resorption in osteoporosis by sensing the extracellular acidic milieu.

MATERIALS AND METHODS Mice All animal procedures were performed in accordance with the guidelines for animal research at The University of Tokyo and were approved by The University of Tokyo Ethics Committee for Animal Experiments. The Tdag8 mutant (TM88) mouse line was generated by the gene trap strategy (18). The Sleeping Beauty transposon was inserted into the genome of a male mouse germ-line cell on a mixed (BDF1 and ICR) genetic background. The insertion site mapped to intron 1 of the Tdag8 gene. The Sleeping Beauty transposon contains a splicing acceptor sequence, internal ribosome entry site, ␤-galactosidase gene, polyadenylation signal, CAG promoter, green fluorescent protein (GFP) gene, and splicing donor sequence (18). Therefore, this insertion mutated the Tdag8 gene by forcing exon 1 at the 5= end of the insertion site to splice with the splice acceptor of the Sleeping Beauty transposon and by preventing it from splicing to exon 2 at the 3= end of the insertion site, thereby preventing the production of mature Tdag8 mRNA (18). Tdag8⫹/gt mice were backcrossed for 9 generations with C57BL/6N mice. Tdag8gt/gt mice were then obtained by mating with Tdag8⫹/gt mice. The genotypes of the offspring were then determined (14). Tdag8⫹/gt and/or wild-type (WT) littermates were used as the controls. The mice were given access to a standard laboratory diet and water ad libitum. Ovariectomy Tdag8gt/gt, Tdag8⫹/gt, and WT 10-wk-old female mice, under anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight; Somnopentyl; Kyoritsu, Tokyo, Japan), underwent either bilateral ovariectomy or a sham procedure in which the bilateral ovaries were exteriorized but not removed. The mice were euthanized 4 wk after the surgical procedure. The body weights of the Tdag8gt/gt female mice (ovariectomized group, 22.1⫾1.4 g, n⫽11; sham-operation group, 21.7⫾1.5 g, n⫽12) were indistinguishable from those of the WT female mice (ovariectomized group, 22.1⫾ 1.1 g, n⫽11; sham-operation group, 21.3⫾1.3 g, n⫽9) and the 2

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Tdag8⫹/gt female mice (ovariectomized group, 22.8⫾1.5 g, n⫽11; sham-operation group, 23.6⫾2.2 g, n⫽10). Analysis of bone phenotypes Mouse hindlimb bones were subjected to radiographic examination. The femurs were dissected and stored in 70% ethanol. Microcomputed tomography (␮CT; inspeXio SMX90CT; Shimadzu, Kyoto, Japan) was used to assess the bone mineral content and bone mass of the trabecular bone in the distal femoral metaphysis with a 12-␮m isotropic voxel size with 40 kV of tube voltage and 100 ␮A of tube current. Three-dimensional CT images were reconstituted and analyzed with a TRI system (Ratoc, Tokyo, Japan). Decalcification The procedure for decalcification is described elsewhere (19). Briefly, decalcifying solutions used in this experiment are Morse’s solution, containing 10% sodium citrate and 22.5% formic acid (20) and 10% EDTA (pH 7.4). The tissues, which had been fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 16 h at room temperature, were decalcified with Morse’s solution for 3 d or 10% EDTA for 7 d. After decalcification was complete, the tissues decalcified with Morse’s solution were embedded in paraffin by a routine procedure, sectioned into 5-␮m slices, and used for immunohistochemistry and in situ hybridization, and those decalcified with 10% EDTA were used for tartrate-resistant acid phosphatase (TRAP) staining. In situ hybridization A 41-base sequence corresponding to nt 495–535 of mouse Tdag8 cDNA (GenBank accession no. NM_008152) was selected. The sequence for the antisense probe was 5=CGAGGTGGCGCTGCTCTTCAATGCACATGCTGTTCATCGCC-3= and for the sense probe was 5=GGCGATGAACAGCATGTGCATTGAAGAGCAGCGCCACCTCG-3=. These oligo-DNAs were labeled at their 3= ends with digoxigenin-11-dUTP and terminal deoxynucleotidyl transferase, and in situ hybridization was performed (21). Briefly, the sections were deparaffinized, treated with 0.2 N HCl for 20 min and 20 ␮g/ml proteinase K at 37°C for 15 min. After postfixation with 4% paraformaldehyde in PBS, the sections were immersed in 2 mg/ml glycine in PBS for 30 min and kept in 40% deionized formamide in 4⫻ standard saline citrate (SSC; 1⫻ SSC⫽0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.0) until hybridization. Hybridization was performed for 15–17 h at 37°C with 1 ␮g/ml digoxigenin-labeled Tdag8 oligo-DNA probe in the hybridization medium. Then, the slides were washed twice with 2⫻ SSC/50% formamide at 37°C, followed by 2⫻ SSC at room temperature. The signals were detected by fluorescence immunohistochemistry with rhodamine-conjugated sheep polyclonal anti-digoxigenin antibody (Roche Diagnostics, Mannheim, Germany). To examine the level of hybridizable RNAs in the tissue sections, the 28S rRNA probe was used as a positive control (22) and signals were detected by enzyme immunohistochemistry with horseradish peroxidase (HRP)conjugated mouse monoclonal anti-T-T dimer antibody (Kyowa Medex, Tokyo, Japan). Immunohistochemistry for TDAG8 The sections were deparaffinized with toluene, rehydrated through a graded ethanol series, and immersed in 0.2%

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HIKIJI ET AL.

Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. After inhibition of endogenous peroxidase activity with 0.3% H2O2 in methanol for 15 min, the sections were preincubated with 500 ␮g/ml normal goat IgG and 1% BSA in PBS for 1 h, to block nonspecific binding of antibodies. Unless otherwise specified, all reactions were conducted at room temperature. Then, the sections were reacted with rabbit polyclonal antihuman GPR65 antibody (Wako, Osaka, Japan) for 16 h. After washing with 0.075% Brij 35 in PBS, they were reacted with HRP-conjugated goat anti-rabbit IgG for 1 h. After a wash in 0.075% Brij 35 in PBS, the HRP sites were visualized with DAB, Ni, Co, and H2O2, according to the method of Adams (23). As a negative control, normal rabbit IgG was used at the same concentration instead of the primary antibody. TRAP staining was performed with a kit according to the manufacturer’s protocol (Wako). Primary osteoclast culture Bone marrow was flushed from the femurs and tibiae of 6-8-wk-old male mice. Osteoclasts were differentiated from the bone marrow cells by stimulation with receptor activator of NF-␬B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) (24). Briefly, the bone marrow cells from 1 or 2 mice were cultured in ␣MEM (Life Technologies, Rockville, MD, USA) containing 10% FBS (SAFC Biosciences, Lenexa, KS, USA) with soluble RANKL (30 ng/ml; PeproTech, Rocky Hill, NJ) and M-CSF (50 ng/ml; R&D Systems, Minneapolis, MN, USA) for 5 d. The cells were then stained with 0.01% naphthol AS-MX phosphate (Sigma-Aldrich) in the presence of 100 mM l(⫹)-tartaric acid (pH 5.0; Wako), to detect TRAP activity. TRAP-positive cells with more than 3 nuclei were counted as viable osteoclasts.

logic bone culture system; BD Biosciences, Franklin Lakes, NJ, USA). After removal of the cells with a bleach solution (6% NaOCl and 5.2% NaCl), the dishes were washed with water and photographed under a light microscope (BH-2; Olympus, Tokyo, Japan). The area of the calcium phosphateresorbed pits was measured with the image-processing application software ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA). Confocal microscopy Bone marrow cells were seeded onto 35-mm poly-d-lysinecoated, glass-bottomed dishes (Iwaki, Tokyo, Japan) in ␣MEM containing 10% FBS with soluble RANKL (30 ng/ml) and M-CSF (100 ng/ml). On d 5, the osteoclasts were fixed in PBS containing 3.7% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. For actin labeling, the osteoclasts were incubated with 0.03% rhodamine-phalloidin and 0.1% Triton X-100 in PBS for 40 min. The stained cells were observed with a confocal laser-scanning microscope (LSM510; Carl Zeiss, Oberkochen, Germany). Osteoclast survival assay Bone marrow cells were cultured in ␣MEM containing 10% FBS with soluble RANKL (30 ng/ml) and M-CSF (50 ng/ml) for 4 d. The pH of the medium was changed from 7.4 to 7.6, 7.4, 7.0, or 6.4 on d 4, and then the cells were cultured for an additional 18 h without soluble RANKL and M-CSF. To calculate the survival rate, the number of surviving osteoclasts was counted under a light microscope (BH-2; Olympus), before and after the additional 18 h incubation. ELISA for guanosine triphosphate (GTP)-bound Rho

RT-PCR analysis RNA was extracted from the primary osteoclasts cultured in the presence of RANKL (30 ng/ml) and M-CSF (50 ng/ml) for 5 d, with or without dexamethasone (100 nM) added for the final 24 h. cDNA was synthesized from 0.8 ␮g total RNA by oligo(dT) priming using Superscript II reverse transcriptase (Life Technologies). To quantify the mRNA levels of Tdag8 and ␤-actin, the resultant cDNA was amplified in duplicate by real-time PCR with the following primers: mouse Tdag8, 5=-AAGAGAAGCATCCCTCCAGAAAC-3= and 5=-GAATCTTACAGGAGATTGGAGATTG-3=; mouse Ogr1, 5=-ACCACACCATCCACCAGACAC-3= and 5=-AGCCAGAAGGGAAGTGAACAGA-3=; mouse G2a, 5=-CTCTATGGTGTTTCTGTGCCTGTCT3= and 5=-GCTCTGTGGGCAAGTGTGTCT-3=; mouse Gpr4, 5=-GCTGGTGTCTTCATCTCCTCTGT-3= and 5=-GGTCTGGGTTTGCTTTGTGG-3=; and mouse B-actin, 5=-GCTGTGCTATGTTGCTCTAGACTT-3= and 5=-AATTGAATGTAGTTTCATGGATGC-3=. The following protocol was used: 40 cycles of 95°C for 15 s, 60°C for 5 s, and 72°C for 5 s. Osteoclast formation assay Bone marrow cells were cultured in ␣MEM containing 10% FBS with soluble RANKL (30 ng/ml) and M-CSF (50 ng/ml) for 2 d. The pH of the medium was changed from 7.4 to 7.0 on d 2. The number of osteoclasts was counted on d 5. Calcium resorption assay Bone marrow cells were cultured in ␣MEM containing 10% FBS with soluble RANKL (30 ng/ml) and M-CSF (100 ng/ml) for 9 d on calcium phosphate-coated dishes (BioCoat Osteo-

Osteoclasts were serum starved for 15 min before treatment with a physiological salt solution (containing 130 mM NaCl, 0.9 mM NaH2PO4, 5.4 mM KCl, 0.8 mM MgSO4, 1.0 mM CaCl2, 25 mM glucose, and 0.1% BSA) buffered with HEPES/ EPPS/MES at pH 6.4 or 7.4 for 2 min (9). After cell lysis, 12.5 ␮g protein was subjected to ELISA for GTP-Rho with a G-LISA Rho Activation Assay Biochem kit (Cytoskeleton, Denver, CO, USA). Statistical analysis All values are expressed as means ⫾ sd. The means of multiple groups were compared by analysis of variance (Prism; GraphPad Software, La Jolla, CA, USA). The statistical significance of the differences was determined by Dunnett’s multiple comparison test. The mean values of 2 groups were compared using the unpaired 2-tailed t test (Prism). Values of P ⬍ 0.05 were considered statistically significant.

RESULTS Tdag8gt/gt mice are susceptible to bone loss induced by ovariectomy We constructed Tdag8gt/gt mice using the Sleeping Beauty transposon system (18). These mice contained a transposon insertion in the Tdag8 gene on a C57BL/6N genetic background. The mice grew apparently normally by gross appearance and were fertile. As shown in Materials and Methods, the mean body weight of the

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Figure 1. Radiographic analysis of the hindlimb. A–D) Morphometric analysis of the metaphyseal region of the femur from ovariectomized mice by ␮CT. A) Mineral content. B) BV/TV. C) Number. D) Separation. *P ⬍ 0.01, #P ⬍ 0.05 vs. ovariectomized Tdag8gt/gt mice (n⫽9 –12 animals from ⱖ5 independent experiments). E) Representative ␮CT images of the metaphyseal region of the femurs. Black and white arrowheads (bottom left panel) indicate cortical and trabecular bone, respectively. Note the highly porous inside of the bone in the ovariectomized Tdag8gt/gt mice. Scale bar ⫽ 2 mm. OVX, ovariectomy; sham, sham operation.

Tdag8gt/gt mice was indistinguishable from that of the WT and Tdag8⫹/gt mice, which served as the controls. In addition, the following parameters were within normal range in the sera of the Tdag8gt/gt mice, when compared with those of Tdag8⫹/gt mice: total protein, albumin, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, amylase, ␥-glutamyl transferase, total cholesterol, HDL-cholesterol, triglyceride, total bilirubin, and glucose (data not shown). We conducted ␮CT analysis of the third to the fifth lumbar vertebrae (L3–L5) to identify the basal bone phenotypes from naive mice. The results demonstrated that there was no significant difference in trabecular bone mineral density between the WT and Tdag8gt/gt mice (data not shown). We examined the role of TDAG8 in bone resorption of ovariectomized mice. ␮CT analysis was used to compare the trabecular bone mineral content per tissue volume (BMC/TV) of the metaphyseal region in the femurs of the ovariectomized female mice with that of the sham-operation mice. Tissue volume means the volume of the total bone tissue and includes the trabecular bone and bone marrow but not the cortical bone. There were no significant differences in BMC/TV values between the sham-operation Tdag8gt/gt, Tdag8⫹/gt, and WT mice. As expected, the BMC/TV values were significantly reduced by ovariectomy in Tdag8gt/gt mice as well as Tdag8⫹/gt mice and WT mice (Fig. 1A). Of note, the BMC/TV value of the ovariectomized Tdag8gt/gt mice was significantly less than that of the ovariectomized Tdag8⫹/gt and WT mice. The ␮CT analysis also revealed that the trabecular bone volume per tissue volume (BV/TV) in the me4

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taphyseal region of the femurs was significantly reduced in the ovariectomized Tdag8gt/gt mice, Tdag8⫹/gt mice, and WT mice, compared with the individual sham-operation mice (Fig. 1B). Similar to the BMC/TV value, the ovariectomized Tdag8gt/gt mice displayed a greater reduction in the femoral BV/TV than the ovariectomized Tdag8⫹/gt mice or WT mice. Two other parameters related to BV/TV, the trabecular number (the linear density of the trabecular bone; Fig. 1C) and trabecular separation (the distance between the edges of the trabecular bone; Fig. 1D), also indicated that the bone volume of the ovariectomized Tdag8gt/gt mice significantly decreased compared with that of the ovariectomized Tdag8⫹/gt or WT mice, which is consistent with the representative images shown in Fig. 1E. Taken together, Tdag8gt/gt mice developed more severe bone resorption than the Tdag8⫹/gt or WT mice did. Therefore, TDAG8 appears to protect bone from aberrant resorption after ovariectomy. TDAG8 is expressed in osteoclasts In situ hybridization with the labeled antisense probe showed that Tdag8 mRNA is located in the osteoclasts within bone tissues of sham-treated and ovariectomized mice (Fig. 2A, E). In the presence of an excess of unlabeled antisense probe, the hybridization signal was essentially abolished in the osteoclasts (Fig. 2B, F). Furthermore, the labeled sense probe gave no signal in the osteoclasts (Fig. 2C, G). The hybridization signals were also detected in the bone marrow cells other than the osteoclasts, consistent with a previous report (25). However, some signals in these bone marrow cells were not abolished by addition of an excess unlabeled probe (Fig. 2B, F). Similar signals were obtained, even with



HIKIJI ET AL.

Figure 2. In situ hybridization for Tdag8 mRNA in mouse bone tissues. Femoral bone sections from ovariectomized (A–D) and sham-operation (E–H) WT mice. In situ hybridization with the digoxigenin-labeled antisense probe (red) shows the expression of Tdag8 mRNA in osteoclasts of both sham-operation and ovariectomized mice (arrows, A, E). Negative controls with either the labeled antisense probe with a 50-fold excess of unlabeled antisense probe (arrows, B, F) or the labeled sense probe (arrows, C, G) were devoid of signal in osteoclasts. Some of the hybridization signals in bone marrow cells other than osteoclasts (arrowheads, A, E) may have been due to nonspecific staining, since similar signals were obtained in negative controls performed with the equivalent sections (arrowheads, B, C, F, G). To evaluate the levels of hybridizable RNAs in the tissue sections, the 28S rRNA complementary probe was used (D, H). Blue color represents nuclear staining with DAPI. Scale bar ⫽ 50 ␮m.

the labeled sense probe (Fig. 2C, G). Thus, these signals seem to be due to the autofluorescence of bone marrow cells. Such nonspecific signals appeared consistently in the bone marrow cells of the Tdag8gt/gt mice, but not in the osteoclasts (Supplemental Fig. S1). We next examined the localization of TDAG8 protein in bone tissues by immunostaining (Fig. 3). In line with the data of in situ hybridization (Fig. 2), TDAG8 was detected in the osteoclasts and bone marrow cells of the WT mice. Both plasma membrane and intracellular TDAG8 were detectable in the osteoclasts (Fig. 3B). Some plasma membrane regions facing lacunae were positive for TDAG8. The intracellular TDAG8 may

correspond to molecules that were synthesized de novo and/or internalized from plasma membrane after activation (11). Little or no signal was observed in the bone tissues from the Tdag8gt/gt mice (Fig. 3E). Given that the local acidic milieu is created only by osteoclasts, it is reasonable to assume that TDAG8 is activated by protons on the cell surface of osteoclasts in an autocrine manner. Thus, we focused on osteoclasts in the following studies. To elucidate the role of the Tdag8 gene in osteoclastic activity, we investigated the expression of Tdag8 mRNA in the primary osteoclasts. As shown in Fig. 4A, Tdag8 mRNA expression was detected in the primary osteoclasts that were differen-

Figure 3. Immunostaining for TDAG8 in mouse bone tissues. A–F) Cross sections of mouse femurs stained with hematoxylin and eosin (H&E; A, D), an anti-TDAG8 antibody (anti-TDAG8; B, E), or nonimmune IgG (rabbit IgG; C, F). Top panels (A–C) and bottom panels (D–F) are from WT and Tdag8gt/gt mice, respectively. TDAG8 protein was detected in the osteoclasts (arrowheads) and other bone marrow cells of WT mice (B), but not of Tdag8gt/gt mice (E). G) representative image of the TRAP staining of the osteoclasts. Scale bar ⫽ 10 ␮m. TDAG8 ACTIVATION AMELIORATES BONE LOSS Downloaded from www.fasebj.org to IP 185.107.94.33. The FASEB Journal Vol., No. , pp:, November, 2017

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ent from that in Tdag8⫹/gt mice at pH 7.4, Tdag8gt/gt osteoclasts resorbed more calcium than Tdag8⫹/gt osteoclasts in vitro (Fig. 5C, D). This result indicates that the calcium-resorbing activity of individual Tdag8gt/gt osteoclasts was enhanced compared with Tdag8⫹/gt actin ring was deformed to severalosteoclasts in the medium, even at normal pH. We also observed that the survival rate of the osteoclasts increased under acidic conditions. The survival rate of the Tdag8gt/gt osteoclasts under acidic conditions was similar to that of the Tdag8⫹/gt osteoclasts (Fig. 5E), suggesting that the increased survival of osteoclasts under acidic conditions is independent of TDAG8. TDAG8 changes the morphology of osteoclasts

Figure 4. Expression of proton-sensing G-protein-coupled receptors in primary osteoclasts. A) Quantitative PCR analysis of Tdag8 mRNA expression in osteoclasts differentiated from bone marrow cells in the presence of RANKL (30 ng/ml) and M-CSF (50 ng/ml) for 5 d, with or without dexamethasone (Dex; 100 nM) for the final 24 h. Similar results were obtained in 3 independent experiments. N.D., not detected. *P ⬍ 0.05 vs. WT osteoclasts without drug treatment (n⫽3, independent cultures). B) Quantitative PCR analysis of the mRNA expression of Tdag8, Ogr1, G2a, and Gpr4 in the primary osteoclasts (n⫽6, independent cultures).

tiated from the bone marrow cells in the presence of the RANKL and M-CSF. In the thymus, glucocorticoid (dexamethasone) has been shown to increase the expression of the Tdag8 gene (18, 26, 27), and we consistently observed that dexamethasone enhanced the expression of Tdag8 mRNA in the osteoclast cultures (Fig. 4A). Among the 3 other proton-sensing G-protein-coupled receptors tested, both Ogr1 and G2a mRNAs were expressed in the primary osteoclasts, whereas Gpr4 mRNA was not detected (Fig. 4B). TDAG8 deficiency enhances osteoclast formation and osteoclastic calcium resorption Under the more acidic culture conditions at pH 7.0 compared with the physiological culture conditions at pH 7.4, osteoclast formation was inhibited in the bone marrow cell cultures derived from Tdag8⫹/gt mice (Fig. 5A). Furthermore, the resulting Tdag8⫹/gt osteoclasts were rather small (Fig. 5B). However, the observed inhibition was ameliorated in the Tdag8gt/gt osteoclasts (Fig. 5A), which were larger than the Tdag8⫹/gt osteoclasts at acidic pH (Fig. 5B). These results show that Tdag8gt/gt osteoclast formation was resistant to acidic pH. Although osteoclast formation in Tdag8gt/gt mice was not significantly differ6

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The regulation of morphological changes in osteoclasts is highly related to their functions (17, 28). Because the Tdag8⫹/gt and Tdag8gt/gt osteoclasts expressed GFP under the control of the constitutive CAG promoter (29), we were able to visualize the cells under a laser-scanning confocal microscope without staining (Fig. 6A–F, green). The actin was stained with rhodamine-phalloidin. The “actin ring” of osteoclasts is the actin-rich peripheral sealing zone characteristic of mature osteoclasts (Fig. 6A, B, red; ref. 30). After 40 min of stimulation with an acidic buffer at pH 6.4, the Tdag8⫹/gt osteoclasts looked abnormally shrunken (Fig. 6C, green), and the actin ring was deformed to several small, open circles (Fig. 6C, red). In contrast, the Tdag8gt/gt osteowas enhanced compared with clasts did not alter their morphology at pH 6.4 (Fig. 6D, green), although the deformation of the actin ring and the formation of the small, open circles did occur (Fig. 6D, red). Rho has been implicated in the modulation of the F-actin cytoskeleton and cell shape (31) and in the formation of stress fibers in osteoclasts (32). Y-27632 is a cell-permeable and selective inhibitor of Rho-associated protein kinases (33). The acidic pH-induced retraction of osteoclasts was suppressed by Y-27632 (Fig. 6E, green), whereas the deformation of the actin ring was not inhibited (Fig. 6E, red). On the acid treatment, the level of active GTP-bound Rho was significantly increased in the Tdag8⫹/gt osteoclasts but not in the Tdag8gt/gt osteoclasts (Fig. 6G). These results indicate that acidic pH stimulated the actin stress fiber-related retraction of osteoclasts through the TDAG8-Rho signaling pathway, although the deformation of the actin ring was independent of this pathway. An increase in cAMP accumulation by acidic pH was not observed in the osteoclasts, probably because the resulting cAMP level was too low to detect under our experimental conditions (11). DISCUSSION This study is the first to demonstrate the inhibitory effects of TDAG8 on bone resorption induced by ovariectomy. An increased ability of osteoclasts to resorb calcium in vitro probably accounts for the enhanced bone resorption in ovariectomized Tdag8gt/gt mice. TDAG8 in osteoclasts is likely to counteract the enhanced bone resorption in osteoporosis by sensing the extracellular acidic milieu generated by the osteoclasts themselves.



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Figure 5. Role of TDAG8 in the formation, calcium resorption activity, and survival rate of osteoclasts. A) Osteoclast formation from bone marrow cells. *P ⬍ 0.001 vs. Tdag8⫹/gt osteoclasts cultured at pH 7.0 (n⫽6 wells). B) TRAPstained images of the primary osteoclasts. The number of Tdag8⫹/gt osteoclasts was lower than that of the Tdag8gt/gt osteoclasts at pH 7.0. Scale bar ⫽ 500 ␮m. C) Calcium resorption by the primary osteoclasts. Osteoclasts were cultured on calcium phosphate–coated dishes at pH 7.4. *P ⫽ 0.0075 vs. Tdag8⫹/gt osteoclasts (n⫽3 wells). D) Representative images of calcium resorption by primary osteoclasts. Scale bar ⫽ 1 mm. E) Increased survival rate of the osteoclasts under acidic conditions was independent of TDAG8 (n⫽6 wells). Similar results were obtained in 3 independent experiments (A, C, E). *P ⬍ 0.05 vs. Tdag8⫹/gt osteoclasts cultured at pH 7.6 (n⫽6 wells).

We found the phenotypes of Tdag8gt/gt mice only after ovariectomy, that is, under estrogen-deficient conditions. Estrogen deficiency leads to increased osteoclast number and activity, probably because of decreasing osteoprotegerin expression and also the increase in RANKL expression in osteoblasts (34). In addition, the RANKL-activated osteoclasts are reported to be rich in vacuolar-type ATPase, a key molecule for extracellular acidification (35, 36), which is necessary for bone resorption. Thus, it is plausible that the intensely activated TDAG8 under estrogen-deficient conditions becomes involved in the regulation of osteoclastic activity in vivo. In our in vitro culture experiments, osteoclasts were differentiated from bone marrow cells in the medium containing RANKL as well as M-CSF. Thus, we assume that the differentiated osteoclasts have already been in a state of activation, replicating the estrogen-deficient conditions in vivo. Given the accelerated formation and enhanced calcium resorption of the Tdag8gt/gt osteoclasts in vitro, the proton-sensing signaling through TDAG8 appears to negatively regulate the formation and function of osteoclasts in the pathological state. It is interesting that the Tdag8gt/gt osteoclasts showed enhanced calcium resorption, even under in a normal pH condition. This observation strongly suggests that TDAG8 actually senses the local acidic milieu beneath osteoclasts, leading to the regulation of osteoclastic calcium resorption under in vitro culture conditions. There are 4 proton-sensing G-protein-coupled receptors: OGR1 (9), GPR4 (9), G2A (10), and TDAG8 (11, 12). We detected mRNA expression of all these genes,

except Gpr4, in primary mouse osteoclasts. Among these proton sensors, OGR1 has been considered to enhance osteoclast formation and bone resorption (37–39). Consistent with the report of Ludwig et al. (9), activation of OGR1 in RAW 264.7 osteoclast-like cells was shown to mediate calcium influx by acid stimulation (38). Meanwhile, acid-stimulated TDAG8 elicited cAMP production, Rho activation, and stress fiber formation (11, 12). Thus, there may be a balance between these two proton-sensing G protein-coupled receptors to maintain normal osteoclast formation and function under acidic conditions. We observed that the Tdag8gt/gt osteoclast cultures responded to acid stimulation, resulting in an increased survival rate and the formation of small, open circles in connection with the actin ring deformation. Although detailed mechanisms remain to be clarified, it is plausible that proton-sensing proteins other than TDAG8 are involved in these responses. Notably, the osteoclasts expressed mRNA of Ogr1 and G2a in addition to Tdag8, as shown in Fig. 4B. The levels of ovariectomy-induced bone resorption appeared small in our current data. This effect may be partly due to the genetic background of our mice (i.e., C57BL/6 mice). The effect of ovariectomy on trabecular bone reportedly varied among inbred mouse strains. Bouxsein et al. (40) showed that C57BL/6 mice had significant bone loss but less than that of BALB/c mice after ovariectomy. However, given that many studies using ovariectomized knockout mice have revealed osteoclast dysfunction under the C57BL/6 genetic background (34,


7

Figure 6. Morphologic changes in osteoclasts mediated through the TDAG8Rho signaling pathway. A–F) Images of rhodaminephalloidin staining of primary osteoclasts. The Tdag8 ⫹ / g t osteoclasts, which originally had a round shape (A), were markedly shrunken 40 min after the pH was decreased to 6.4 (C), whereas the Tdag8gt/gt osteoclasts were unchanged (B, D). The acidic pH-induced retraction was suppressed by Y-27632 in Tdag8⫹/gt osteoclasts (E), whereas this compound had little or no effect on Tdag8gt/gt osteoclasts (F). In parallel, the actin ring in the Tdag8⫹/gt and Tdag8gt/gt osteoclasts was comparably deformed to several small, open circles after 40 min in acidic conditions (at pH 6.4). Similar results were obtained in 3 independent experiments. A =–F =) Schematic drawings of the cells (indicated by asterisks in A–F, respectively) depict the cell morphologies. Green, cytoplasmic GFP; red, rhodamine-labeled actin. G) Rho activation in the acid-treated osteoclasts. ELISA for Rho in an active GTP-bound state was performed in triplicate. Absorbance ratio (acid-treated cells to control cells) of Tdag8⫹/gt mice was compared with that of Tdag8gt/gt mice. Scale bar ⫽ 50 ␮m. *P ⫽ 0.002 vs. Tdag8⫹/gt osteoclasts (n⫽3 independent experiments).

41– 43), this commonly used mouse strain probably provided us a practical model for studying the pathogenesis of postmenopausal osteoporosis. In summary, we propose that TDAG8 functions in inhibiting bone resorption; this mechanism is possibly responsible for maintaining bone homeostasis. Many therapeutic agents are currently being investigated to prevent bone resorptive disease (44). Our findings suggest that chemical compounds with agonist and/or potentiator activity at TDAG8 can act as therapeutic agents for bone resorptive diseases such as osteoporosis. The authors thank K. Ohori, C. Kanokoda, and S. Kondo for technical assistance and all the members of their laboratory (Department of Biochemistry and Molecular Biology, University of Tokyo) for support and valuable suggestions. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.S., S.I., and H.H.), a Health and Labor Sciences research grant for the research on allergic disease and immunology from the Ministry of Health, Labor, and Welfare of Japan (to S.I.), and a grant to the Respiratory Failure Research Group from the Ministry of Health, Labor, and Welfare of Japan (to S.I.). The authors declare no conflicts of interest.

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TDAG8 activation inhibits osteoclastic bone resorption Hisako Hikiji, Daisuke Endo, Kyoji Horie, et al. FASEB J published online November 12, 2013 Access the most recent version at doi:10.1096/fj.13-233106

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