FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2150573669> ?p ?o ?g. }

• W2150573669 endingPage "4186" @default.
• W2150573669 startingPage "4175" @default.
• W2150573669 abstract "Article7 September 2006free access Secretion of L-glutamate from osteoclasts through transcytosis Riyo Morimoto Riyo Morimoto Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Shunsuke Uehara Shunsuke Uehara Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Shouki Yatsushiro Shouki Yatsushiro Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Narinobu Juge Narinobu Juge Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Zhaolin Hua Zhaolin Hua Departments of Neurology and Physiology, Graduate Programs in Neuroscience and Cell Biology, University of California San Francisco School of Medicine, CA, USA Search for more papers by this author Shigenori Senoh Shigenori Senoh Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Noriko Echigo Noriko Echigo Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Mitsuko Hayashi Mitsuko Hayashi Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Toshihide Mizoguchi Toshihide Mizoguchi Institute for Oral Science, Matsumoto Dental University, Shiojiri, Japan Search for more papers by this author Tadashi Ninomiya Tadashi Ninomiya Institute for Oral Science, Matsumoto Dental University, Shiojiri, Japan Search for more papers by this author Nobuyuki Udagawa Nobuyuki Udagawa Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan Search for more papers by this author Hiroshi Omote Hiroshi Omote Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Akitsugu Yamamoto Akitsugu Yamamoto Department of Cell Biology, Nagahama Institute of Bioscience and Technology, Nagahama, Japan Search for more papers by this author Robert H Edwards Robert H Edwards Departments of Neurology and Physiology, Graduate Programs in Neuroscience and Cell Biology, University of California San Francisco School of Medicine, CA, USA Search for more papers by this author Yoshinori Moriyama Corresponding Author Yoshinori Moriyama Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Riyo Morimoto Riyo Morimoto Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Shunsuke Uehara Shunsuke Uehara Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Shouki Yatsushiro Shouki Yatsushiro Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Narinobu Juge Narinobu Juge Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Zhaolin Hua Zhaolin Hua Departments of Neurology and Physiology, Graduate Programs in Neuroscience and Cell Biology, University of California San Francisco School of Medicine, CA, USA Search for more papers by this author Shigenori Senoh Shigenori Senoh Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Noriko Echigo Noriko Echigo Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Mitsuko Hayashi Mitsuko Hayashi Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Toshihide Mizoguchi Toshihide Mizoguchi Institute for Oral Science, Matsumoto Dental University, Shiojiri, Japan Search for more papers by this author Tadashi Ninomiya Tadashi Ninomiya Institute for Oral Science, Matsumoto Dental University, Shiojiri, Japan Search for more papers by this author Nobuyuki Udagawa Nobuyuki Udagawa Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan Search for more papers by this author Hiroshi Omote Hiroshi Omote Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Akitsugu Yamamoto Akitsugu Yamamoto Department of Cell Biology, Nagahama Institute of Bioscience and Technology, Nagahama, Japan Search for more papers by this author Robert H Edwards Robert H Edwards Departments of Neurology and Physiology, Graduate Programs in Neuroscience and Cell Biology, University of California San Francisco School of Medicine, CA, USA Search for more papers by this author Yoshinori Moriyama Corresponding Author Yoshinori Moriyama Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Search for more papers by this author Author Information Riyo Morimoto1,‡, Shunsuke Uehara1,‡, Shouki Yatsushiro1, Narinobu Juge1, Zhaolin Hua2, Shigenori Senoh1, Noriko Echigo1, Mitsuko Hayashi1, Toshihide Mizoguchi3, Tadashi Ninomiya3, Nobuyuki Udagawa4, Hiroshi Omote1, Akitsugu Yamamoto5, Robert H Edwards2 and Yoshinori Moriyama 1 1Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan 2Departments of Neurology and Physiology, Graduate Programs in Neuroscience and Cell Biology, University of California San Francisco School of Medicine, CA, USA 3Institute for Oral Science, Matsumoto Dental University, Shiojiri, Japan 4Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan 5Department of Cell Biology, Nagahama Institute of Bioscience and Technology, Nagahama, Japan ‡These authors contributed equally to this work *Corresponding author. Department of Membrane Biochemistry, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan. Tel.: +81 86 251 7933/7934; Fax: +81 86 251 7935; E-mail: [email protected] The EMBO Journal (2006)25:4175-4186https://doi.org/10.1038/sj.emboj.7601317 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1−/− knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1−/− mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis. Introduction Bone resorption is an essential component of bone remodeling during the development, growth and turnover of bone. An imbalance contributes to the pathogenesis as well as the etiology of metabolic diseases such as osteoporosis and osteopetrosis (Eriksen, 1986; Rodan and Martin, 2000; Boyle et al, 2003). Osteoclasts are multinuclear cells originating from monocyte/macrophage lineage cells and are primarily responsible for bone resorption (Baron et al, 1994; Blair, 1998; Väänänen et al, 2000). During the resorption process, osteoclasts are highly polarized, form tightly sealed compartments (i.e., resorption lacunae) on the bone surface and secrete protons, chloride anions and proteases through the ruffled border, resulting in the degradation of the bone matrix. To transport a large amount of degradation products, osteoclasts develop an inwardly polarized membrane traffic called transcytosis: inorganic and organic degradation products and cathepsin K are taken up at the ruffled border through endocytosis, packaged into transcytotic vesicles, transported along microtubules to the basolateral plasma membrane and then secreted through exocytosis (Nesbitt and Horton, 1997; Salo et al, 1997; Mulari et al, 2003). The overall secretory processes are believed to be highly organized and coordinated with other bone functions, although the mechanisms and regulation of transcytosis remain poorly understood. L-Glutamate, an excitatory neurotransmitter of the central nervous system, acts as an intercellular messenger in peripheral non-neuronal cells, in which L-glutamate is stored in secretory vesicles and secreted through regulated exocytosis and, thus, enables intercellular chemical transmission in a paracrine or autocrine manner (Moriyama and Yamamoto, 2004). Although there is evidence for L-glutamate signaling in bone, its physiological significance has not been established (Serre et al, 1999; Bhangu et al, 2001; Hinoi et al, 2001; Skerry and Taylor, 2001; Mason, 2004). Bone cells express the GLAST-type Na+-dependent plasma membrane transporter, which terminates L-glutamate signals (Skerry and Taylor, 2001; Mason, 2004). Bone cells also express various functional glutamate receptors, and their stimulation affects the functions of osteoblasts and osteoclasts (Skerry and Taylor, 2001; Mason, 2004). However, it is still unresolved where, when and how L-glutamate appears as an intercellular messenger in bone. Although L-glutamate has been suggested to originate from L-glutamate-containing nerve terminals (Serre et al, 1999) or osteoblasts (Bhangu et al, 2001; Hinoi et al, 2002), secretion of L-glutamate from nerve terminals has not been detected. Furthermore, the mode of secretion of L-glutamate from osteoblasts differs from that of the exocytosis of L-glutamate in CNS or endocrine cells. Vesicular glutamate transporter (VGLUT) plays an essential role in L-glutamate signaling through vesicular storage of L-glutamate (Fremeau et al, 2004a;Moriyama and Yamamoto, 2004). There are three isoforms of VGLUT: VGLUT1, VGLUT2 and VGLUT3. VGLUT is a potential marker for the sites of L-glutamate signaling and can help reveal details of the mode of glutamate signaling in the body (Moriyama and Yamamoto, 2004). In the present study, we examined the expression and localization of VGLUT in bone tissue to determine how L-glutamate becomes an intercellular messenger. We found an unexpected link between L-glutamate signaling and bone resorption. We show that (1) mature osteoclasts possess VGLUT1-containing transcytotic vesicles and thus secrete L-glutamate and bone degradation products through transcytosis, (2) this glutamate signaling is involved in autoregulation of transcytosis and (3) impairment of the glutamate signaling stimulates bone resorption and may contribute to the development of osteoporosis. Results VGLUT1 was expressed in osteoclasts RT–PCR analysis demonstrated that osteoclasts derived from bone marrow cells contained mRNA encoding VGLUT1 and that other types of bone cells including primary osteoblasts, stroma cells and clonal bone cells did not express this mRNA (Figure 1A). Neither VGLUT2 nor VGLUT3 was expressed in any of the bone cells analyzed (Figure 1B). Northern blot analysis revealed mRNA of VGLUT1 in mature osteoclasts but not in undifferentiated cells (Figure 1C). Upon stimulation with RANKL, a receptor activator of the nuclear factor κB ligand, murine macrophage RAW264.7 cells can differentiate into osteoclasts (Toyomura et al, 2003) that then express the VGLUT1 gene (Figure 1A and C). These results indicate that the VGLUT1 gene was specifically expressed in osteoclasts during differentiation and that expression of the VGLUT2 and VGLUT3 genes in bone cells was negligible or below the detection limit of our assay. Figure 1.Inducible expression of VGLUT1 in osteoclasts. (A) RT–PCR analysis of brain, osteoblasts, MC3T3-E1 (clonal osteoblasts), MC3T3-G2/PA6 (clonal stroma cell line from calvariae), ST2 (clonal stroma cell line from bone marrow), C3H10T1/2 (clonal fibroblast cell line), osteoclasts, RAW264.7 macrophages and RAW264.7 cells treated with RANKL. The arrowhead indicates the VGLUT1 transcript. (B) VGLUT2 and VGLUT3 genes were not detectable in bone cells. Results of RT–PCR analysis of total cellular RNA are shown. Expression of G3PDH gene is also shown as a control. (C) Northern blotting revealing expression of the VGLUT1 gene in mature osteoclasts and RAW264.7 cells treated with RANKL. The G3PDH transcript, as a loading control, is also shown (lower panel). (D) Western blotting reveals the presence of VGLUT1 in RAW264.7 cells treated with RANKL. The presence of V-ATPase subunit A on the same blot is also shown. (E) RAW264.7 cells were cultured in the presence of RANKL for the indicated incubation periods (days) and the expression of VGLUT1 during osteoclastogenesis was observed by immunohistochemistry. Negative control with control IgG is also shown in insets. Bar=10 μm. (F) Osteoclasts (OC) in the femora of VGLUT1+/+ (wild type) mice visualized by TRAP staining (red) contain VGLUT1, which was visualized by the horseradish peroxidase-diaminobenzidine (HRP-DAB) method (charcoal). No VGLUT1 immunoreactivity was seen in osteoclasts from VGLUT1−/− mice. Bar=10 μm. Download figure Download PowerPoint Immunoblotting with VGLUT1 antibodies revealed that an immunoreactive polypeptide with an apparent molecular mass similar to that of VGLUT1 (∼62 kDa) appeared in RAW264.7 cells upon treatment of RANKL, whereas expression of the housekeeping vacuolar H+-ATPase (V-ATPase) subunit was the same before and after differentiation (Figure 1D). Inducible expression of VGLUT1 immunoreactivity in RAW264.7 cells treated with RANKL was confirmed by immunohistochemistry: VGLUT1 immunoreactivity appeared 3 days after induction and reached a steady-state level after 7 days (Figure 1E). The presence of VGLUT1 immunoreactivity in tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was confirmed in the femurs of VGLUT1+/+ (wild type) mice but not in those of VGLUT1−/− mice (Figure 1F). Essentially the same results were obtained in osteoclasts prepared from VGLUT1+/+ (wild type) mice but not in those of VGLUT1−/− mice (Supplementary Figure S1). Overall, these results demonstrate that VGLUT1 appeared in osteoclasts during osteoclastogenesis. VGLUT1 was associated with transcytotic vesicles To identify VGLUT1-containing organelles, we performed immunohistochemical analyses. After culturing on bone, an actin ring was observed, indicating the site of bone digestion (Figure 2A). The VGLUT1 immunoreactivity exhibited a punctated distribution throughout the cells and was especially abundant in the basolateral region, but less in the ruffled border region (Figure 2A and B). VGLUT1 was roughly co-localized with microtubules but not with actin (Figure 2A and B). VGLUT1 did not seem to be co-localized with Lamp2, TGN38, GM130 or transferrin receptor (TfR), which are markers for lysosomes, the Golgi apparatus, endosomes and recycling vesicles, respectively (Supplementary Figure S2), but rather was partially co-localized with lysobisphosphatidic acid, a phospholipid abundant in late endosomes (Figure 2C), and cathepsin K (Figure 2D), both of which are associated with the transcytotic pathway after endocytosis (Vääräniemi et al, 2004). Furthermore, VGLUT1 immunoreactivity was, in part, co-localized with the internalized fluorescent bone degradation products (Figure 2E). These results suggest that VGLUT1 was associated with the vesicles involved in the transcytotic pathway, such as transcytotic vesicles. Figure 2.Immunohistochemical localization of VGLUT1 in bone-resorbing osteoclasts. Osteoclasts were grown on pieces of bone. Localization of VGLUT1 was examined by double-labeling immunofluorescence microscopy. The staining pairs were as follows: VGLUT1 and actin (A), VGLUT1 and tubulin (B), VGLUT1 and lysobisphosphatidic acid (6C4) (C), VGLUT1 and cathepsin K (D) and VGLUT1 and fluorescent bone degradation products (E). The immunological localization was observed under a confocal microscope. A horizontal view of a merged picture is also shown. Bar=10 μm. Download figure Download PowerPoint VGLUT1-containing vesicles were identified through double immunoelectron microscopy. Consistent with the immunohistochemical observations made above, there were at least three types of vesicles associated with immunogold particles specific for VGLUT1 and bone degradation products: many clear vesicles with an average diameter of ∼300 nm in which VGLUT1 was present but in which immunoreactivity for bone degradation products was not detected (Figure 3A); larger vesicles located near basolateral regions that contained many VGLUT1 immunogold particles and some particles for bone degradation products (Figure 3B); and larger vesicles located near the ruffled border membrane that contained many immunogold particles for bone degradation products and a few immunogold particles for VGLUT1 (Figure 3C). We concluded that a population of VGLUT1 was associated with the transcytotic vesicles that contain bone degradation products. The dynamics of these vesicles during transcytosis is discussed later. Figure 3.Double gold labeling immunoelectron microscopy of bone-resorbing osteoclasts. Arrows and arrowheads indicate VGLUT1 (10 nm in diameter) and bone degradation product (5 nm in diameter), respectively. (A) VGLUT1-containing small vesicles; (B) vesicles containing both VGLUT1 and bone degradation products localized near the basolateral region; (C) vesicles containing bone degradation products but little VGLUT1 localized near the ruffled border membrane. Bar=100 nm. Download figure Download PowerPoint VGLUT1 in osteoclasts was functional We then determined whether VGLUT1-containing vesicles accumulate L-glutamate. Generally, V-ATPase supplies the driving force for the vesicular storage of L-glutamate (Maycox et al, 1990; Moriyama and Yamamoto, 2004). We prepared membrane vesicles from RAW264.7 cells treated with or without RANKL. Upon the addition of ATP, a substantial amount of L-glutamate was taken up by vesicles isolated from RAW264.7 cells treated with RANKL and much less was taken up by those isolated from undifferentiated control RAW264.7 cells (Figure 4A). The ATP-dependent L-glutamate uptake was blocked by carbonyl cyanide-m-chlorophenylhydrazone (CCCP, a proton conductor), bafilomycin A1 (V-ATPase inhibitor; Bowman et al, 1988) and Evans blue (an inhibitor of VGLUT; Roseth et al, 1998), but not affected by 10 mM D,L-aspartate (Figure 4B). A low concentration of Cl− (4 mM) was required for L-glutamate uptake; in the absence of Cl−, the uptake decreased to background level (Figure 4B). The results were fully consistent with the properties of VGLUT-mediated L-glutamate transport (Moriyama and Yamamoto, 2004), and demonstrated VGLUT1-dependent vesicular storage of L-glutamate in bone-resorbing RAW264.7 cells treated with RANKL. Figure 4.VGLUT1-mediated vesicular storage of L-glutamate. (A) Time course of L-glutamate uptake by membrane vesicles isolated from RAW264.7 cells treated with or without RANKL determined in the presence or absence of ATP; n=3. (B) Effects of various compounds on the ATP-dependent L-glutamate uptake by membrane vesicles of RAW264.7 cells treated with RANKL for 1 week. Additions: bafilomycin A1, 1 μM; CCCP, 1 μM; Evans blue, 1 μM; D,L-aspartate, 10 mM; KCl, 100 mM. In some experiments, KCl was substituted with K-acetate or omitted from the assay medium. The results are shown as means±s.e.m., n=4. Download figure Download PowerPoint Osteoclasts secreted L-glutamate through transcytosis Do osteoclasts secrete L-glutamate? As the exocytosis of L-glutamate is usually driven by membrane depolarization, we measured the L-glutamate present in the medium after depolarization of osteoclasts with KCl. KCl at 50 mM depolarizes bone-resorbing osteoclasts through a voltage-gated K+ channel (Kajiya et al, 2003). We found that KCl stimulated the secretion of L-glutamate from RAW264.7 cells treated with RANKL and to a lesser extent from undifferentiated control cells (Figure 5A). The KCl-evoked L-glutamate secretion was dependent on extracellular Ca2+ and temperature: depletion of Ca2+ by treatment with EGTA-AM inhibited the secretion by 80% and low temperature (24°C) abolished the secretion (Figure 5B). Consistent with its effect on the vesicular storage of L-glutamate (Figure 4), bafilomycin A1 decreased L-glutamate secretion (Figure 5B). These properties are similar to those reported for the regulated exocytosis of L-glutamate by neurons and endocrine cells (Moriyama and Yamamoto, 2004). Figure 5.Regulated secretion of L-glutamate and fluorescent bone degradation products from RAW264.7 cells treated with RANKL. (A) RAW264.7 cells treated with RANKL or RAW264.7 cells (2.0 × 105 cells/dish) were stimulated with 50 mM KCl. The L-glutamate released was measured. (B) L-Glutamate secretion after 20 min is shown. In some experiments, cells were treated for 2 h with 1 μM bafilomycin A1, 50 μM EGTA-AM or 10 μM nocodazole, and then stimulated with KCl. (C) Fluorescent bone degradation products in the medium under the conditions in panel B were assessed fluorometrically. The results are means±s.e.m., n=4. Asterisks indicate statistically significant numbers (*P<0.01, **P<0.001). ATP stimulates the secretion of L-glutamate (D) and fluorescent bone degradation products (E) from RAW264.7 cells treated with RANKL. The assay was started by the addition of 1 mM ATP. In some experiments, 0.1 mM PPADS was also included. The results are means±s.e.m., n=4. Asterisks indicate statistically significant numbers (*P<0.01, **P<0.001). Download figure Download PowerPoint As a population of VGLUT1 is associated with transcytotic vesicles, secretion of L-glutamate may accompany, at least in part, the secretion of bone degradation products. As expected, KCl stimulated secretion of fluorescent bone degradation products (Figure 5C). The secretion also required extracellular Ca2+ and was sensitive to temperature and bafilomycin A1 (Figure 5C). Bafilomycin A1 is also known to inhibit transcytosis (Palokangas et al, 1997; Stenbeck and Horton, 2004). Furthermore, nocodazole, a reagent that dissociates microtubules and, through this, inhibits transcytosis (Stenbeck and Horton, 2004), inhibited the secretion of both L-glutamate and bone degradation products to a similar extent (Figure 5B and C). These results support the idea that L-glutamate was co-secreted with bone degradation products through transcytosis. It is important to ask what kinds of stimuli trigger transcytosis under physiological conditions. We found that ATP at 1 mM stimulated the secretion of both L-glutamate (Figure 5D) and bone degradation products (Figure 5E), and was blocked by pyridoxalphosphate-6-azophenyl-2′-4′-disulfonic acid (PPADS), a selective antagonist of ionotropic purinergic receptors (Ralevic and Burnstock, 1998). As ATP is secreted from osteoblasts upon mechanical stimulation and depolarizes osteoclasts so as to increase the cytosolic concentration of Ca2+ via ionotropic purinergic receptors (Naemsch et al, 2001; Romanello et al, 2001), the results suggest that ATP was one of the physiological stimuli for secretion of L-glutamate and bone degradation products. L-Glutamate secretion from osteoclasts from VGLUT1−/− mice We then assessed L-glutamate secretion from osteoclasts prepared from VGLUT1−/− mice. The number and morphology of osteoclasts from VGLUT1+/− heterozygotes and VGLUT1−/− homozygotes were apparently comparable to those of wild-type osteoclasts. As shown in Figure 6A and B, KCl stimulated the secretion of L-glutamate in osteoclasts from wild-type and heterozygotic mice by a factor of ∼2. The KCl-evoked secretion of L-glutamate was not observed in the case of osteoclasts from VGLUT1−/− homozygotes (Figure 6C). Essentially the same results were obtained when the L-glutamate secretion was evoked by ATP (Figure 6D). In contrast to the L-glutamate secretion, KCl-mediated secretion of bone degradation products was not altered in osteoclasts of all three mice genotypes (Figure 6E). Figure 6.Both KCl- and ATP-dependent L-glutamate secretion were impaired in osteoclasts from VGLUT1−/− mice. The time course of KCl-dependent L-glutamate secretion from osteoclasts (2.0 × 105 cells/dish) prepared from bone marrow of wild-type VGLUT1+/+ (A), VGLUT1+/− (B) or VGLUT1−/− (C) was measured as in Figure 5 in the presence (open squares) or absence (closed squares) of 50 mM KCl. (D) In osteoclasts (2.0 × 105 cells/dish) prepared from wild-type and VGLUT1−/− mice, L-glutamate secretion were measured 20 min after stimulation with 1 mM ATP. (E) Secretion of bone degradation products from osteoclasts under the conditions in panels A–C was assessed fluorometrically after 20 min. The results are means±s.e.m., n=3. Download figure Download PowerPoint mGluR8-mediated autoregulation of transcytosis Subsequently, we investigated the role of the released L-glutamate. As L-glutamate signaling in peripheral tissues usually involves an autoregulatory mechanism by way of metabotropic receptors (Yamada et al, 1998; Uehara et al, 2004), we investigated whether mGluRs are expressed in osteoclasts, and if so, whether they regulate transcytosis. Among known mGluRs, RT–PCR indicated the presence of mRNAs of mGluR3, mGluR4 and mGluR8 in RAW264.7 cells treated with RANKL and the presence of mGluR3, mGluR5 and mGluR8 in osteoclasts (Figure 7A and Supplementary Figure S3). Immunoreactivity for mGluR8 (Figure 7B) but not mGluR3 and mGluR4 was detected by immunohistochemical analysis in RAW264.7 cells treated with RANKL (data not shown), suggesting expression of mGluR8 in mature osteoclasts. Figure 7.mGluR-mediated regulation of the secretion of L-glutamate and fluorescent bone degradation products from RAW264.7 cells treated with RANKL. (A) Expression of mGluR8 gene as revealed by RT–PCR. (B) Expression of mGluR8 as revealed by immunoblotting (upper panel) and immunohistochemistry (lower panel). Bar=10 μm. (C) RAW264.7 cells treated with RANKL (2.0 × 105 cells/dish) were incubated in the presence or absence of agonists or antagonists of glutamate receptors or DBcAMP as indicated and then stimulated with 50 mM KCl. The L-glutamate released after 20 min is shown. The results are means±s.e.m., n=3. Asterisks and marks indicate statistically significant numbers (**P<0.001 compared with control, #P<0.001 compared with +ACPT-I). (D) Fluorescent bone degradation products in the medium under the conditions in panel A were assessed fluorometrically. The results are means±s.e.m., n=3. Asterisks and marks indicate statistically significant numbers (*P<0.01 compared with control, #P<0.001 compared with +ACPT-I). (E) cAMP content in the osteoclast-like cells (2.0 × 105 cells/dish) under the conditions in panel A was measured. The results are means±s.e.m., n=3. Asterisks and marks indicate statistically significant numbers (*P<0.01 compared with control, #P<0.001 compared with +ACPT-I, †P<0.001, compared with +L-glutamate). Additions: L-glutamate, 0.5 mM; ACPT-I, 50 μM; CPPG, 100 μM; DBcAMP, 1 mM. Download figure Download PowerPoint It is important to ask whether mGluR8 regulates transcytosis. Upon the addition of (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I), a specific agonist of class III mGluRs (Acher et al, 1997), KCl-evoked secretions of both L-glutamate and bone degradation products decreased by about 90 and 87% of the control, respectively, and the inhibitions were blocked by the (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG), a specific antagonist (Toms et al, 1996), and dibutylylcAMP (DBcAMP), a non-hydrolyzable cAMP analogue (Figure 7C and D). Agonists for other mGluRs and NMDA receptor did not affect the secretions of L-glutamate (data not shown). mGlu" @default.
• W2150573669 created "2016-06-24" @default.
• W2150573669 creator A5004964979 @default.
• W2150573669 creator A5005943342 @default.
• W2150573669 creator A5013379195 @default.
• W2150573669 creator A5017377536 @default.
• W2150573669 creator A5022444323 @default.
• W2150573669 creator A5027616900 @default.
• W2150573669 creator A5027966918 @default.
• W2150573669 creator A5029372795 @default.
• W2150573669 creator A5030611509 @default.
• W2150573669 creator A5034236679 @default.
• W2150573669 creator A5034320377 @default.
• W2150573669 creator A5035126720 @default.
• W2150573669 creator A5070750668 @default.
• W2150573669 creator A5079870914 @default.
• W2150573669 creator A5086559857 @default.
• W2150573669 date "2006-09-07" @default.
• W2150573669 modified "2023-10-14" @default.
• W2150573669 title "Secretion of L-glutamate from osteoclasts through transcytosis" @default.
• W2150573669 cites W1586341181 @default.
• W2150573669 cites W1931173751 @default.
• W2150573669 cites W1968556089 @default.
• W2150573669 cites W1970143992 @default.
• W2150573669 cites W1971817436 @default.
• W2150573669 cites W1972183007 @default.
• W2150573669 cites W1974688989 @default.
• W2150573669 cites W1976961529 @default.
• W2150573669 cites W1981919734 @default.
• W2150573669 cites W1984206132 @default.
• W2150573669 cites W2006950202 @default.
• W2150573669 cites W2014577644 @default.
• W2150573669 cites W2015839100 @default.
• W2150573669 cites W2016444704 @default.
• W2150573669 cites W2021016137 @default.
• W2150573669 cites W2022079801 @default.
• W2150573669 cites W2028930552 @default.
• W2150573669 cites W2035501441 @default.
• W2150573669 cites W2037718442 @default.
• W2150573669 cites W2043651158 @default.
• W2150573669 cites W2046019785 @default.
• W2150573669 cites W2050123488 @default.
• W2150573669 cites W2053168080 @default.
• W2150573669 cites W2058774752 @default.
• W2150573669 cites W2062712606 @default.
• W2150573669 cites W2064230636 @default.
• W2150573669 cites W2071667853 @default.
• W2150573669 cites W2074830850 @default.
• W2150573669 cites W2077386452 @default.
• W2150573669 cites W2077497516 @default.
• W2150573669 cites W2078409020 @default.
• W2150573669 cites W2092494777 @default.
• W2150573669 cites W2093252878 @default.
• W2150573669 cites W2093353836 @default.
• W2150573669 cites W2093719268 @default.
• W2150573669 cites W2099757747 @default.
• W2150573669 cites W2101156303 @default.
• W2150573669 cites W2104523951 @default.
• W2150573669 cites W2116730131 @default.
• W2150573669 cites W2117445941 @default.
• W2150573669 cites W2118676056 @default.
• W2150573669 cites W2128520952 @default.
• W2150573669 cites W2169061872 @default.
• W2150573669 cites W2269603020 @default.
• W2150573669 cites W2342591351 @default.
• W2150573669 cites W2413218385 @default.
• W2150573669 cites W2951764089 @default.
• W2150573669 cites W4235771167 @default.
• W2150573669 doi "https://doi.org/10.1038/sj.emboj.7601317" @default.
• W2150573669 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1570443" @default.
• W2150573669 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16957773" @default.
• W2150573669 hasPublicationYear "2006" @default.
• W2150573669 type Work @default.
• W2150573669 sameAs 2150573669 @default.
• W2150573669 citedByCount "81" @default.
• W2150573669 countsByYear W21505736692012 @default.
• W2150573669 countsByYear W21505736692013 @default.
• W2150573669 countsByYear W21505736692014 @default.
• W2150573669 countsByYear W21505736692015 @default.
• W2150573669 countsByYear W21505736692016 @default.
• W2150573669 countsByYear W21505736692017 @default.
• W2150573669 countsByYear W21505736692020 @default.
• W2150573669 countsByYear W21505736692021 @default.
• W2150573669 countsByYear W21505736692022 @default.
• W2150573669 countsByYear W21505736692023 @default.
• W2150573669 crossrefType "journal-article" @default.
• W2150573669 hasAuthorship W2150573669A5004964979 @default.
• W2150573669 hasAuthorship W2150573669A5005943342 @default.
• W2150573669 hasAuthorship W2150573669A5013379195 @default.
• W2150573669 hasAuthorship W2150573669A5017377536 @default.
• W2150573669 hasAuthorship W2150573669A5022444323 @default.
• W2150573669 hasAuthorship W2150573669A5027616900 @default.
• W2150573669 hasAuthorship W2150573669A5027966918 @default.
• W2150573669 hasAuthorship W2150573669A5029372795 @default.
• W2150573669 hasAuthorship W2150573669A5030611509 @default.
• W2150573669 hasAuthorship W2150573669A5034236679 @default.
• W2150573669 hasAuthorship W2150573669A5034320377 @default.
• W2150573669 hasAuthorship W2150573669A5035126720 @default.

• next