ORIGINAL RESEARCH ARTICLE
Journal of
Beclin-1 Is Required for RANKL-Induced Osteoclast Differentiation
Cellular Physiology
YEON-HO CHUNG,1,2 YOUNGSAENG JANG,1,2 BONGKUN CHOI,1,2 DA-HYUN SONG,1,2 EUN-JIN LEE,1,2 SANG-MIN KIM,1,2 YOUNGSUP SONG,1,2 SANG-WOOK KANG,1,2 SEUNG-YONG YOON,2 AND EUN-JU CHANG1,2* 1
Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea
2
Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Seoul, Korea
Beclin-1 plays a critical role in autophagy; however, it also contributes to other biological processes in a non-autophagic manner. Although studies have examined the non-autophagic role of autophagy proteins in the secretory function of osteoclasts (OC), the role of Beclin-1 is unclear. Here, we examined the role of Beclin-1 in OC differentiation, and found that mouse bone marrow macrophages (BMMs) showed increased expression of Beclin-1 upon RANKL stimulation in a p38- and NF-kappa B-dependent manner. During OC differentiation, Beclin1 localized to the mitochondria, where it was involved in the production of mitochondrial intracellular reactive oxygen species. Knockdown of Beclin-1 in RANKL-primed BMMs led to a significant reduction in RANKL-dependent osteoclastogenesis, which was accompanied by reduced NFATc1 induction. Furthermore, knockdown of Beclin-1 inhibited RANKL-mediated activation of JNK and p38, both of which act downstream of reactive oxygen species, resulting in the suppression of NFATc1 induction. Finally, overexpression of constitutively active NFATc1 rescued the phenotype induced by Beclin-1 knockdown, indicating that Beclin-1 mediates RANKL-induced osteoclastogenesis by regulating NFATc1 expression. These findings show that Beclin-1 plays a non-autophagic role in RANKL-induced osteoclastogenesis by inducing the production of reactive oxygen species and NFATc1. J. Cell. Physiol. 229: 1963–1971, 2014. © 2014 Wiley Periodicals, Inc.
Osteoclasts (OC), which are derived from hematopoietic monocyte/macrophage lineage cells, i.e., OC precursors, play a critical role in bone homeostasis by degrading and adsorbing old bone (Tanaka et al., 1993; Suda et al., 1999; Teitelbaum, 2000). The differentiation of OC precursor cells into OC is supported by two key factors: macrophage colonystimulating factor (M-CSF) and receptor activator of nuclear factor kB (NF-kB) ligand (RANKL). M-CSF binds to a receptor (c-fms) on OC and is required for OC survival and proliferation (Tanaka et al., 1993), while receptor activator of nuclear factor kB (NF-kB) ligand (RANKL) is a cytokine essential for osteoclastogenesis and OC survival (Suda et al., 1999; Teitelbaum, 2000). In OC precursor cells, M-CSF is responsible for the expression of receptor activator of nuclear factor-kB (NF-kB) (RANK) and that enables OC precursor cells to respond to RANKL at the early phase of OC differentiation. The RANKL–RANK interaction activates signaling pathways that induce the expression and activation of master transcription factors, including NF-kB, c-Fos, AP-1, and nuclear factor of activated T cells (NFAT) c1, which then drive osteoclastogenesis (Lee and Kim, 2003). Upon RANKL binding to RANK, TNFR-associated factor 6 (TRAF6) is recruited to the RANK intracellular domain. The TAB2 signaling complex then activates TGF-b-activated kinase 1 (TAK1), a MAPK kinase kinase, which in turn activates the inhibitory kappa B kinase (IKK) complex leading to the activation of the NF-kB pathway (Ninomiya-Tsuji et al., 1999; Mizukami et al., 2002). Also, RANK-mediated activation of extracellular signalregulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK induces NFATc1 expression (Takayanagi et al., 2002; Lee and Kim, 2003; Ikeda et al., 2004; Huang et al., 2006a; Huang et al., 2006b). In addition to activating MAPKs and the NF-kB pathway, RANKL also induces the production of reactive oxygen species (ROS) (also via JNK and p38), thereby playing a role in OC differentiation (Rhee, 1999; Lee et al., 2005). Finally, NFATc1 plays a critical role in OC © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .
differentiation by inducing the expression of osteoclast-specific genes, including osteoclast-associated receptor (OSCAR), cathepsin k, calcitonin receptor, and tartrate-resistant acid phosphatase (TRAP) (Takayanagi et al., 2002; Asagiri and Takayanagi, 2007). Beclin-1, the mammalian homolog of yeast Atg6, is an essential component of the class III phosphatidylinositol 3-kinase complex (PI3KC3), which plays a role in autophagy (Klionsky et al., 2003). Beclin-1 participates in autophagosome formation, a process involving the degradation of damaged intracellular organelles and protein aggregates, and in the recycling of macromolecules through fusion with lysosomes (Klionsky et al., 2003; Yang and Klionsky, 2010). However,
Conflict of Interest: None Contract grant sponsor: Korea Government (MSIP); Contract grant number: 2008-0062286. Contract grant sponsor: Republic of Korea and the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea; Contract grant number: 13-524. Contract grant sponsor: Ministry of Education, Science and Technology; Contract grant number: NRF-2013R1A1A3010455. *Correspondence to: Dr. Eun-Ju Chang, Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea. E-mail: [email protected] Manuscript Received: 26 December 2013 Manuscript Accepted: 11 April 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 15 April 2014. DOI: 10.1002/jcp.24646
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Beclin-1 also has non-autophagic functions in other cellular processes, including endocytic trafficking (Thoresen et al., 2010), phagocytosis (Sanjuan et al., 2007), and cytokinesis (Sagona et al., 2010); it even plays a role in pollen germination (Fujiki et al., 2007). Given that other autophagy proteins such as ATG5, ATG7, ATG4B, and LC3 regulate the secretory functions of OC (DeSelm et al., 2011; Chung et al., 2012), it is plausible that Beclin-1 plays a distinct role in these cells. However, its potential role and possible underlying mechanisms have not been addressed. Here, we show that RANKL up-regulated the expression of Beclin-1 via the p38 and JNK signaling pathways. Beclin-1 within OC mitochondria induced RANKL-mediated ROS production. This in turn increased NFATc1 expression, leading to OC differentiation and cathepsin K secretion. Taken together, these findings provide evidence of a non-autophagic role for Beclin-1 via RANKLinduced osteoclastogenesis. Materials and methods Reagents and antibodies RANKL and M-CSF were obtained from Pepro Tech Inc (Rocky Hill, NJ). The tartrate-resistant acid phosphatase (TRAP) Assay Kit, the Cathepsin K Activity Assay Kit, PD98059, SB203580 (SB), SP60025, Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), and the antimycin A and b-actin antibodies (Ab) were purchased from Sigma-Aldrich (St Louis, MO). Cell counting kit (CCK-8) was obtained from Dojindo (Kumamoto, Japan). RNAiMAX, Lipofectamine 2000, propidium iodide (PI), Alexa Fluor 488-conjugated phalloidin and DAPI were purchased from Invitrogen (Grand Island, NY). Antibodies against Beclin-1, p-ERK, ERK, p-JNK, JNK, p-p38, p38, p-ATF, p-TAK1, TAK1, p-MKK3/6, MKK3/6, and IkBa were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against NFATc1, cathepsin K, and GFP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-TOMM20 Ab and quinazoline (QZ) were purchased from Merck Millipore (Darmatadt, Germany). Donkey fluoresceinconjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG were obtained from Jackson Immunoresearch (West Grove, PA). Protease and phosphatase inhibitor cocktails were purchased from Thermo Pierce (Rockford, IL). Culture of bone marrow macrophages and osteoclast differentiation Bone marrow (BM) cells were isolated by flushing the marrow space in femurs and tibiae isolated from 6-week-old female ICR mice as described by Chang et al. (2008a). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Sciences in Seoul, Korea. Isolated cells were cultured for 12 h in a-minimal essential medium (a-MEM; Hyclone) supplemented with 10% FBS (Hyclone, Logan, UT) and 1% penicillin–streptomycin (Gibco, New York, NY). Non-adherent cells were collected after 12 h and cultured in petri dishes (Green Cross, Suwon, Korea) in the presence of M-CSF (30 ng/ml) for further 3 days. Adherent cells were considered to be bone marrowderived macrophages (BMMs) and were used as osteoclast (OC) precursor cells. BMM were cultured for 4 days with M-CSF (30 ng/ml) and RANKL (100 ng/ml) to induce differentiation into OC. OC formation was determined by TRAP staining according to the manufacturer’s instructions. The number of TRAP-positive multinucleated cells (MNCs; containing more than three nuclei) was counted under a light microscope. These cells were termed OC. Immunoblot analysis and cathepsin K activity assay Pre-fusion OCs were transfected with control- or Beclin-1-specific small interfering (si)RNAs for 48 h and then stimulated by culture in fresh osteoclastogenic media for 2 h. Culture media were collected JOURNAL OF CELLULAR PHYSIOLOGY
and centrifuged, and the protease activity of the cathepsin K in the supernatants was measured using a Cathepsin K Activity Assay Kit according to the manufacturer’s protocol. The supernatants were boiled in 5 SDS sample buffer and the proteins were resolved in 12% SDS-PAGE gels. The cells were washed with ice-cold PBS and lysed in modified RIPA buffer [50 mM Tris/HCl (pH 7.4), 1% Nonidet P40, 0.25% sodium deoxycholate, and 150 mM NaCl] containing protease and phosphatase inhibitors. Cell lysates were centrifuged at 10,000 g for 15 min, the supernatants were collected, and the proteins resolved in 10–12% SDS-PAGE gels. Separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), which was then blocked for 1 h with 5% BAS (MP biomedicals, Auckland, New Zealand) solution in Tris-buffered saline solution containing 0.1% Tween 20. The membrane was then incubated overnight at 4 °C with the appropriate primary Ab, washed, and incubated for 1 h at room temperature with the HRPconjugated secondary Ab. Reactive proteins were visualized using a chemiluminescence system (Merck-Millipore, Darmatadt, Germany). Small interfering RNA (siRNA) transfection and retroviral gene transfer Pre-designed control siRNA (4390843) and siRNA targeting mouse BECN1 were obtained from Ambion (Life Technology, Carlsbad, CA). Two different sequences were used to target mouse BECN1, BECN1 #1 (s80166), and BECN1 #2 (s80167). BECN1 #2 showed the greatest degree of interference. Oligonucleotide siRNAs were transfected into mouse BMMs or BMMs pretreated with RANKL (RANKL-primed BMMs) for 6 h in RNAiMAX reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s protocol. After 4 h, the medium was replaced with fresh osteoclastogenic media (a-MEM in the presence of MCSF (30 ng/ml) and RANKL (100 ng/ml)). The cells were analyzed at 24 h post-transfection, and endogenous Beclin-1 was detected by immunoblotting. For OC differentiation, the transfected cells were differentiated into OCs in the osteoclastogenic media for 4 days, fixed, and stained for TRAP. The population of TRAPþ cells, percentage of TRAPþ multinuclear cells (MNCs) in total cells, and TRAPþ MNCs with actin ring in TRAPþ MNCs was quantified. In this condition, the siRNA transfection efficiency of BMMs cells was ~92% when we assayed using siRNA carrying GFP. To generate retroviral stocks, Plat E packaging cell lines were transiently transfected with pMX-EGFP or pMX-NFATc1-EGFP constitutively active (CA) mutant vectors (CA-NFATc1) using Lipofectamine 2000 (Invitrogen) (Porter and Clipstone, 2002). The viral supernatant was collected at 48 h post-transfection. BMMs or OCP were incubated with the viral supernatant for 8 h in the presence of polybrene (10 mg/ml; Sigma), as described previously (Chang et al., 2008b). The transduction efficiency of retroviral CA-NFATc1 in BMMs cells was ~78%. After the retroviral supernatants were removed, BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 days. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR RNA was isolated from cells using Trizol Reagent (Life Technology, Carlsbad, CA) and 2 mg of RNA was reverse-transcribed using Super-Script II reverse transcriptase (Life Technologies). The resulting cDNA was amplified by PCR using the following primers: Beclin-1 (BECN1), 50 -GGCCAATAAGGGTCTGA-30 (forward) and 50 -GCTGCACACAGTCCAGAAAA-30 (reverse); NFATc1 (Nfatc1), 50 -GGGTCAGTGTGACCGAAGAT-30 (forward) and 50 -GGAAGTCAG-AAGTGGGTGGA-30 (reverse); cathepsin K (Ctsk), 50 -AATACCTCCCTCTCGATCCTACA-30 (forward) and 50 -GGTTCTTGACTGGAGTAACGTA-30 (reverse); TRAP (Acp5), 50 -TCCTGGCTCAAAAAGCAGTT-30 (forward) and 50 ACATAGCCCACACCGTTCTC-30 (reverse); and GAPDH
ROLE OF BECLIN-1 IN OSTEOCLASTOGENESIS (Gapdh), 50 -ACCACAGTCCA TGCCATCAC-30 (forward) and 50 -TCCACCACCCTGTTGCTGTA-30 (reverse). The PCR conditions were as follows: denaturation at 94 °C for 30 s, followed by annealing at 55–60 °C for 30 s, and an extension step at 72 °C for 1 min. The number of cycles fell within the linear range of amplification (28–30 cycles; GAPDH required 23 cycles). Quantitative real-time PCR (qRT-PCR) was performed using Power SYBR Green 1-Step Kit and an ABI 7000 Real Time PCR System (Applied Biosystems, Carlsbad, CA) according to manufacturer’s instructions. The sequences of primers used were the same as described above. Cell viability assay Control- or Beclin-1-specific siRNA-transfected RANKL-primed BMMs were cultured in the presence of RANKL (100 ng/ml) and M-CSF (30 ng/ml) for24 h and thenincubated with 10% CCKsolution inculturemediumfor1 hat37 °C.Theopticaldensitywasmeasuredin a microtiter plate reader (Bio-Rad, Hercules, CA) at 450 nm. Confocal microscopy BMMs or OCs grown on glass cover slips were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature and then permeabilized with 0.1% Triton X-100. After blocking with 1% BSA in PBS for 1 h, the cells were incubated with a primary antibody overnight at 4 °C. The cells were then washed and stained with DAPI and FITC- or Cy3-conjugated secondary antibodies. F-actin was stained using Alexa Fluor 488-conjugated phalloidin. After mounting, the fluorochromes were detected using a confocal laser-scanning microscope (LSM 710, Carl Zeiss). Measurement of mitochondrial superoxide production To measure mitochondrial superoxide production, control- or Beclin-1-specific siRNA-transfected BMMs were loaded with 5 mM Mitosox in the presence of M-CSF (30 ng/ml) and RANKL (30 ng/ml) for 1 h in the dark. The cells were then washed with warm PBS and further processed for analysis in a FACSCalibur flow cytometer according to the manufacturer’s instructions (Becton Dickinson, San Jose, CA). Statistics All quantitative experiments were performed at least in triplicate, and the data are expressed as the mean SD. Data were analyzed using Graphpad Prism 5 software, and the statistical significance of the differences between groups was determined using Student’s t test. A P value < 0.05 was considered significant. Results RANKL induces Beclin-1 expression during osteoclastogenesis
BMMs were differentiated into OC by culture in the presence of M-CSF and RANKL for 4 days (Figure 1A). We found that the expression of Beclin-1 protein increased during differentiation (Figure 1B). Beclin-1 expression increased 4.5-fold in cells treated with RANKL and M-CSF for 72 h, but did not increase in cells treated with M-CSF alone (Figure 1C and D), demonstrating that Beclin-1 expression is induced by RANKL signaling. To identify the signals induced by RANKL, BMMs were treated with PD98059 (an ERK inhibitor), SB203580 (SB; a p38 inhibitor), SP600125 (a JNK inhibitor), or QZ (a NF-kB activation inhibitor) in the presence of RANKL and M-CSF for 24 h. SB and QZ inhibited significantly RANKL-induced Beclin1 transcription (Figure 1E). These results suggest that RANKL induces Beclin-1 expression in OC via the p38 and NF-kB signaling pathways. JOURNAL OF CELLULAR PHYSIOLOGY
Beclin-1 is localized in the mitochondria and is involved in mitochondrial ROS production
Because the functions of proteins are dependent upon their location within cells, we next examined the subcellular location of Beclin-1 during osteoclastogenesis. A previous study reported that Beclin-1 localizes in the mitochondria, ER, and trans-Golgi network (Pattingre et al., 2005; Shi and Kehrl, 2010). As expected, an apparent cytosolic distribution of Beclin-1 was observed in BMM, pOC, and OC (Figure 2A). In addition, we found that Beclin-1 mainly co-localized with TOMM20, which is located in the outer mitochondrial membrane, during OC differentiation (Figure 2A). This was confirmed using the MitoTracker dye (data not shown). OC contains large numbers of mitochondria. These organelles supply energy to the vacuolar ATPase proton pump, which uses ATP to secrete protons (Hþ) into the resorption space and generates the ROS involved in osteoclast development (Brown and Breton, 1996; Srinivasan et al., 2010). We used Mitosox Red to examine whether downregulating Beclin-1 affected RANKL-induced mitochondrial ROS production. The efficiency of knockdown by Beclin-1-specific siRNA was confirmed by western blotting (Figure 2B). Beclin-1 siRNAtransfected BMMs showed a 74% reduction in mitochondrial superoxide levels compared with control siRNA-transfected cells (Figure 2C). The addition of the membrane permeable superoxide dismutase mimetic, MnTBAP, significantly inhibited mitochondrial superoxide levels. Similar to these conditions, Beclin-1 siRNA-transfected BMMs showed a 28.8% reduction compared with the control. However, treatment with antimycin A stimulated mitochondrial superoxide production (levels were 1.94-fold higher than in the controls) (Figure 2D). These data indicate that Beclin-1 plays a role in RANKLdependent ROS production. Knockdown of Beclin-1 inhibits RANKL-induced osteoclast differentiation
RANKL-dependent ROS production is an osteoclastogenic signal mediator required for OC differentiation (Rhee, 1999; Lee et al., 2005). Given the requirement for Beclin-1 for ROS generation in OC, it is possible that Beclin-1 may support RANKL-mediated signaling and is involved in OC differentiation. To investigate this, we examined the effect of Beclin-1 knockdown on RANKL-induced OC differentiation. siRNAmediated downregulation of Beclin-1 expression (Figure 3A) had no effect on the viability of RANKL-primed BMMs (Figure 3B). Interestingly, we observed a significant reduction in the number of TRAP-positive MNCs that were differentiated from Beclin-1-silenced BMMs (Figure 3C). Also, compared with that in control-siRNA-transfected cells, the expression of OC differentiation markers such as cathepsin K (Ctsk), NFATc1 (Nfatc1), and TRAP (Acp5) mRNA was downregulated in Beclin-1-siRNA-transfected cells (Figure 3D and E). Subsequently, the intracellular expression, secretion, and activity of cathepsin K were also reduced by Beclin-1 knockdown (Figure 3F and G), emphasizing the positive role of Beclin-1 in RANKLinduced OC differentiation. RANKL-mediated activation of JNK and p38 requires Beclin-1
To gain insight into the molecular mechanisms by which Beclin1 promotes osteoclastogenesis, we examined the effects of Beclin-1 knockdown on the early signaling pathways activated by M-CSF or RANKL. Increased activation of ERK, JNK, p38, and ATF2 were observed at 5 and 15 min after M-CSF treatment; these increases were not abrogated by knocking down Beclin-1 (Figure 4A). On the other hand, activation of the
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Fig. 1. RANKL-dependent induction of Beclin-1 during osteoclastogenesis. (A) Bone marrow-derived machrophages (BMMs) were cultured for 4 days with M-CSF (30 ng/ml) and RANKL (100 ng/ml) to induce differentiation into mature osteoclasts (OCs). After culture for 2 days (d2), cells were classed as pre-fusion osteoclasts (pOC). (B) Whole cell lysates were obtained from cultured cells at the indicated times and analyzed by western blotting with Abs specific for Beclin-1 or b-actin (loading control). (C) BMMs were treated with M-CSF plus RANKL or with M-CSF alone for the indicated times and Beclin-1 protein expression was examined by western blotting. (D) Changes in the amount of Beclin-1 protein relative to b-actin are shown. (E) BMMs were cultured without M-CSF and RANKL for 4 h (S) and then treated with M-CSF and RANKL in the presence of DMSO (Veh) PD98059 (PD, 10 mM), SB203580 (SB, 10 mM), SP600125 (SP, 10 mM), or quinazoline (QZ, 10 mM). After 24 h, the cells were harvested and subjected to quantitative real-time PCR (E) to examine Beclin-1 (BECN1) mRNA expression. Data are expressed as the mean SD. **P < 0.05, **P < 0.005, ***P < 0.0005 versus Veh.
JNK and p38 signaling pathways was inhibited in Beclin-1silenced BMMs treated with RANKL (Figure 4B). NFATc1, a pivotal transcription factor during osteoclastogenesis (Takayanagi et al., 2002), is induced by RANKL via the JNK and p38 MAPK signaling pathways (Ikeda et al., 2004; Huang et al., 2006a). Thus, we next examined the expression of NFATc1 protein in BMMs transfected with Beclin-1 siRNA or control siRNA in the presence of RANKL. NFATc1 expression was significantly increased by RANKL, but it was markedly inhibited by Beclin-1 knockdown (Figure 4C). This suggests that Beclin-1 mediates RANKL-dependent NFATc1 induction in a JNK- and p38 MAPK-dependent manner. Overexpression of active NFATc1 restores osteoclastogenesis in Beclin-1-knockdown BMMs
To confirm that NFATc1 plays a critical role in Beclin-1 function during osteoclastogenesis, BMMs or RANKL-induced OCPs were infected with a retrovirus harboring a constitutively active form of NFATc1 (NFATc1 CA) (Figure 5A). Transduction of NFATc1-CA, but not of control retroviruses, rescued the RANKL-induced TRAP-positive OC formation that was suppressed by Beclin-1 siRNA (Figure 5B), JOURNAL OF CELLULAR PHYSIOLOGY
and the number of TRAP-positive MNCs increased significantly (Figure 5C). These results clearly suggest that Belcin-1 plays a critical role in RANKL-induced NFATc1 expression, thereby contributing to osteoclastogenesis. Discussion
A number of studies report non-autophagic roles for autophagy proteins, including ATG5, ATG7, and LC3, in OCs (DeSelm et al., 2011; Chung et al., 2012). Deletion of ATG5 or ATG7 from cells of the macrophage lineage has no effect on OC differentiation; however, it does impair the secretion of lysosomal contents and bone resorption (DeSelm et al., 2011). The present study showed that the Beclin-1 plays an important role in RANKL-induced OC differentiation, which is distinct from the functions of other autophagy-related proteins in OC. We found that the Beclin-1 expression was up-regulated by M-CSF plus RANKL, but not by M-CSF alone, during osteoclastogenesis, and that this up-regulation was dependent upon NF-kB and p38 activation (Figure 1). The NF-kB pathway is crucial for RANKL-mediated OC development (Iotsova et al., 1997). Upon stimulation with RANKL, activated IkB kinase (IKK) induces the phosphorylation of serine 536 within
ROLE OF BECLIN-1 IN OSTEOCLASTOGENESIS
the p65 transactivation domain, thereby promoting translocation of p50/p65 to the nucleus (Iotsova et al., 1997). Thus, it is likely that these signals are responsible for the binding of p65/ RelA to the kB site within the BECN1 promoter in response to RANKL, thereby up-regulating BECN1 mRNA and protein expression (Copetti et al., 2009). The localization of Beclin-1 in the mitochondria of OC might be related to its role in ROS production (Figure 2). Intracellular mitochondrial ROS are generated via the TRAF6, Rac1, and NADPH oxidase (Nox) 1 pathway (Rhee, 1999). Beclin-1 contains two TRAF6-binding motifs, which are responsible for binding to activated TRAF6 (Shi and Kehrl, 2010). Since TRAF6 is a pivotal adaptor molecule for both RANK and intracellular
ROS production, we assumed that Beclin-1 might be associated with TRAF6. However, we did not detect any interaction between endogenous TRAF6 and Beclin-1 in BMMs or OC upon RANKL stimulation (data not shown). On the other hand, Beclin-1 was initially identified as a Bcl-2-interacting myosin like coiled-coil protein, which interacts with Bcl-2 through Bcl-2 homology domain 3 (BH3) (Liang et al., 1998). A recent study reported that Bcl-2 regulates intracellular redox status in cancer cells by physically interacting with COX Va (the Va subunit of cytochrome c oxidase; complex IV) (Chen and Pervaiz, 2010). Accordingly, we suggest that Beclin-1 might be involved in regulating mitochondrial ROS generation by interacting with Bcl-2. However, further studies are needed to
Fig. 2. Beclin-1 localization and its role in osteoclast mitochondria. (A) BMMs, pOCs (1d), OCs (3d), or mature OCs (4d) were fixed; immunostained for Beclin-1 (green), TOMM20 (red), and F-actin (blue, pseudocolor); and then stained with the nuclear dye, DAPI (cyan, pseudocolor). Right panels of mOC image show low magnification image of mOC and contain actin ring structure in the box. Scale bars: 12.5 mm and 50 mm (low magnification image of mOC). (B) BMMs were transfected with a control siRNA or with two Beclin-1-specific siRNAs for 24 h. Knockdown of Beclin-1 protein expression was examined by western blotting. (C) Transfected BMMs were pretreated with antimycin A (20 mM) or MnTBAP (200 mM) for 30 min, stimulated with RANKL (400 ng/ml) for 1 h, and then stained with Mitosox 5 mM for 30 min. Mitochondrial-derived superoxide generation was measured by flow cytometry. For each histogram, the y axis shows the number of cells and the x axis shows the FL2 channel (Mitosox Red). (D) The intensity of Mitosox Red fluorescence was compared with that in control siRNAtransfected cells. Data are expressed as the mean SD. **P < 0.005 versus control.
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Fig. 3. Role of Beclin-1 in RANKL-induced osteoclastogenesis. (A–G) BMMs were primed with RANKL for 6 h and then transfected with control siRNA or Beclin-1-specific siRNA. Transfected RANKL-primed BMMs were cultured with M-CSF and RANKL for 24 h, and Beclin-1 protein expression (A) and cell viability (B) were analyzed by western blotting and a CCK-8 assay, respectively. Transfected RANKL-primed BMMs were differentiated into OCs in osteoclastogenic medium. Cells were then fixed and stained with TRAP solution and TRAP-positive multinucleated OCs (MNCs) with different number of nuclei were counted in five different categories; 1 to 2 nuclei, 3–10 nuclei, 11–20 nuclei, 21–50 nuclei, and those with greater than 50 nuclei. Mature TRAP-positive MNCs were photographed under a light microscope (magnification 100). Scale bars: 200 mm (C). Transfected BMMs were cultured in the presence of M-CSF and RANKL for the indicated times, and mRNA levels including BECN1 (Beclin-1), Ctsk (cathepsin K), Nfatc1 (NFATc1), Acp5 (TRAP), and Gapdh (GAPDH) were analyzed by semiquantitative RT-PCR (D) and quantitative real-time PCR (E). Cells were differentiated into pOCs by incubation in osteoclastogenic medium for 2 days. The osteoclastogenic medium was then replaced with fresh medium containing RANKL (100 ng/ml) and M-CSF (30 ng/ml). Supernatants and cell lysates were collected at the indicated times and analyzed by western blotting to measure Beclin-1 and cathepsin K expression (F). Cathepsin K activity in the supernatant was evaluated using a cathepsin k activity assay (G). Data are expressed as the mean SD. **P < 0.05, **P < 0.005, ***P < 0.0005 versus control.
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explore whether Beclin-1 regulates ROS production in OC precursor cells in response to RANKL. Although the relationship between ROS and NFATc1 expression identified in this study is obscure, some reports suggest that RANKL-dependent ROS production induces the transcriptional activity of NFATc1 (Huang et al., 2001; Ke et al., 2006; Han et al., 2007; Srinivasan et al., 2010). Taken
together, these studies suggest that ROS mediate the activation of MAPKs (e.g., JNK and p38) in OC precursor cells (Lee et al., 2005), and that this is associated with NFATc1 induction. Our findings show that knockdown of Beclin-1 inhibited RANKL-mediated mitochondrial ROS production (Figure 2C and D) and JNK and p38 MAPK activation (Figure 4), resulting in the failure of NFATc1 induction (Figure 4).
Fig. 4. Beclin-1 knockdown inhibits RANKL-mediated JNK and p38 activation. (A–C) BMMs were transfected with control- or Beclin-1specific siRNA and serum-starved for 2 h before stimulation with 30 ng/ml M-CSF (A) and 400 ng/ml RANKL (B) for the indicated times. Lysates were analyzed by western blotting with antibodies against molecules involved in the MAPK and NF-kB signaling pathways. To examine NFATc1 expression, transfected BMMs were stimulated with RANKL (100 ng/ml) in the presence of M-CSF (30 ng/ml) for the indicated times and then immunoblotted with antibodies against NFATc1 and Beclin-1 (C).
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Fig. 5. Effect of constitutively active NFATc1 on Beclin-1 knockdown-induced inhibition of osteoclastogenesis. (A) BMMs or RANKL-primed BMMs were infected with retroviruses harboring a control vector (pMX) or with a constitutively active NFATc1 (NFATc1 CA) construct. Overexpression of NFATc1 CA was confirmed by western blotting at 24 h post-infection. (B) BMMs or RANKL-primed BMMs were transfected with control- or Beclin-1-specific siRNAs for 4 h, and then infected with control or NFATc1-CA retroviruses. Cells were then cultured in the presence of M-CSF and RANKL for 4 days, fixed, and stained for TRAP (magnification 100). Scale bars: 200 mm. (C) The number of mature TRAP-positive MNCs was counted under a light microscope. Data are expressed as the mean SD. **P < 0.05, **P < 0.005, *** P < 0.0005 versus pMX.
Taken together, the results of the present study suggest that Beclin-1 may act as a molecular link that mediates mitochondrial ROS and NFATc1 expression, thereby contributing to RANKL-induced OC differentiation. Thus, Beclin-1 appears to play a non-autophagic role during osteoclastogenesis. Acknowledgments
This work was supported by a National Research Foundation of Korea (NRF) MRC grant funded by the Korea Government (MSIP) (2008-0062286), Republic of Korea and the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea (13-524). This project was supported in part by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (NRF2013R1A1A3010455). Literature Cited Asagiri M, Takayanagi H. 2007. The molecular understanding of osteoclast differentiation. Bone 40:251–264. Brown D, Breton S. 1996. Mitochondria-rich, proton-secreting epithelial cells. J Exp Biol 199:2345–2358. Chang EJ, Ha J, Huang H, Kim HJ, Woo JH, Lee Y, Lee ZH, Kim JH, Kim HH. 2008a. The JNKdependent CaMK pathway restrains the reversion of committed cells during osteoclast differentiation. J Cell Sci 121:2555–2564.
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Chang EJ, Ha J, Oerlemans F, Lee YJ, Lee SW, Ryu J, Kim HJ, Lee Y, Kim HM, Choi JY, Kim JY, Shin CS, Pak YK, Tanaka S, Wieringa B, Lee ZH, Kim HH. 2008b. Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption. Nat Med 14:966–972. Chen ZX, Pervaiz S. 2010. Involvement of cytochrome c oxidase subunits Va and Vb in the regulation of cancer cell metabolism by Bcl-2. Cell Death Differ 17:408–420. Chung YH, Yoon SY, Choi B, Sohn DH, Yoon KH, Kim WJ, Kim DH, Chang EJ. 2012. Microtubule-associated protein light chain 3 regulates Cdc42-dependent actin ring formation in osteoclast. Int J Biochem Cell Biol 44:989–997. Copetti T, Bertoli C, Dalla E, Demarchi F, Schneider C. 2009. P65/RelA modulates BECN1 transcription and autophagy. Mol Cell Biol 29:2594–2608. DeSelm CJ, Miller BC, Zou W, Beatty WL, van Meel E, Takahata Y, Klumperman J, Tooze SA, Teitelbaum SL, Virgin HW. 2011. Autophagy proteins regulate the secretory component of osteoclastic bone resorption (vol 21, pg 966, 2011). Dev Cell 21:1179. Fujiki Y, Yoshimoto K, Ohsumi Y. 2007. An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination. Plant Physiol 143:1132–1139. Han KY, Yang D, Chang EJ, Lee Y, Huang H, Sung SH, Lee ZH, Kim YC, Kim HH. 2007. Inhibition of osteoclast differentiation and bone resorption by sauchinone. Biochem Pharmacol 74:911–923. Huang C, Li J, Costa M, Zhang Z, Leonard SS, Castranova V, Vallyathan V, Ju G, Shi X. 2001. Hydrogen peroxide mediates activation of nuclear factor of activated T cells (NFAT) by nickel subsulfide. Cancer Res 61:8051–8057. Huang H, Chang EJ, Ryu J, Lee ZH, Lee Y, Kim HH. 2006. Induction of c-Fos and NFATc1 during RANKL-stimulated osteoclast differentiation is mediated by the p38 signaling pathway. Biochem Biophys Res Commun 351:99–105. Huang H, Ryu J, Ha J, Chang EJ, Kim HJ, Kim HM, Kitamura T, Lee ZH, Kim HH. 2006. Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NFkappaB transactivation by RANKL. Cell Death Differ 13:1879–1891. Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, Hata K, Yamashita K, Hiraga T, Watanabe T, Kukita T, Yoshioka K, Rao A, Yoneda T. 2004. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Invest 114:475–484. Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, Bravo R. 1997. Osteopetrosis in mice lacking NF-kappa B1 and NF-kappa B2. Nat Med 3:1285–1289. Ke Q, Li J, Ding J, Ding M, Wang L, Liu B, Costa M, Huang C. 2006. Essential role of ROSmediated NFAT activation in TNF-alpha induction by crystalline silica exposure. Am J Physiol Lung Cell Mol Physiol 291:L257–L264.
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Klionsky DJ, Cregg JM, Dunn WA, Jr., Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y. 2003. A unified nomenclature for yeast autophagyrelated genes. Dev Cell 5:539–545. Lee NK, Choi YG, Baik JY, Han SY, Jeong DW, Bae YS, Kim N, Lee SY. 2005. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood 106:852– 859. Lee ZH, Kim HH. 2003. Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts. Biochem Bioph Res Co 305:211–214. Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G, Herman B, Levine B. 1998. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol 72:8586–8596. Mizukami J, Takaesu G, Akatsuka H, Sakurai H, Ninomiya-Tsuji J, Matsumoto K, Sakurai N. 2002. Receptor activator of NF-kappa B ligand (RANKL) activates TAK1 mitogenactivated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6. Mol Cell Biol 22:992–1000. Ninomiya-Tsuji J, Kishimoto K, Hiyama A, Inoue J, Cao ZD, Matsumoto K. 1999. The kinase TAK1 can activate the NIK-I kappa B as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 398:252–256. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B. 2005. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122:927–939. Porter CM, Clipstone NA. 2002. Sustained NFAT signaling promotes a Th1-like pattern of gene expression in primary murine CD4þ T cells. J Immunol (Baltimore, MD: 1950) 168:4936–4945. Rhee SG. 1999. Redox signaling: hydrogen peroxide as intracellular messenger. Exp Mol Med 31:53–59.
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Sagona AP, Nezis IP, Pedersen NM, Liestol K, Poulton J, Rusten TE, Skotheim RI, Raiborg C, Stenmark H. 2010. PtdIns(3)P controls cytokinesis through KIF13A-mediated recruitment of FYVE-CENT to the midbody. Nat Cell Biol 12:362–371. Sanjuan MA, Dillon CP, Tait SWG, Moshiach S, Dorsey F, Connell S, Komatsu M, Tanaka K, Cleveland JL, Withoff S, Green DR. 2007. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450:1253–1257. Shi CS, Kehrl JH. 2010. TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy. Sci Signal 3:ra42. Srinivasan S, Koenigstein A, Joseph J, Sun L, Kalyanaraman B, Zaidi M, Avadhani NG. 2010. Role of mitochondrial reactive oxygen species in osteoclast differentiation. Ann NY Acad Sci 1192:245–252. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. 1999. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J, Wagner EF, Mak TW, Kodama T, Taniguchi T. 2002. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 3:889–901. Tanaka S, Takahashi N, Udagawa N, Tamura T, Akatsu T, Stanley ER, Kurokawa T, Suda T. 1993. Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors. J Clin Invest 91:257–263. Teitelbaum SL. 2000. Bone resorption by osteoclasts. Science 289:1504–1508. Thoresen SB, Pedersen NM, Liestol K, Stenmark H. 2010. A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic. Exp Cell Res 316:3368–3378. Yang Z, Klionsky DJ. 2010. Eaten alive: a history of macroautophagy. Nat Cell Biol 12:814–822.
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Beclin-1 Is Required for RANKL-Induced Osteoclast Differentiation
Cellular Physiology
YEON-HO CHUNG,1,2 YOUNGSAENG JANG,1,2 BONGKUN CHOI,1,2 DA-HYUN SONG,1,2 EUN-JIN LEE,1,2 SANG-MIN KIM,1,2 YOUNGSUP SONG,1,2 SANG-WOOK KANG,1,2 SEUNG-YONG YOON,2 AND EUN-JU CHANG1,2* 1
Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea
2
Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Seoul, Korea
Beclin-1 plays a critical role in autophagy; however, it also contributes to other biological processes in a non-autophagic manner. Although studies have examined the non-autophagic role of autophagy proteins in the secretory function of osteoclasts (OC), the role of Beclin-1 is unclear. Here, we examined the role of Beclin-1 in OC differentiation, and found that mouse bone marrow macrophages (BMMs) showed increased expression of Beclin-1 upon RANKL stimulation in a p38- and NF-kappa B-dependent manner. During OC differentiation, Beclin1 localized to the mitochondria, where it was involved in the production of mitochondrial intracellular reactive oxygen species. Knockdown of Beclin-1 in RANKL-primed BMMs led to a significant reduction in RANKL-dependent osteoclastogenesis, which was accompanied by reduced NFATc1 induction. Furthermore, knockdown of Beclin-1 inhibited RANKL-mediated activation of JNK and p38, both of which act downstream of reactive oxygen species, resulting in the suppression of NFATc1 induction. Finally, overexpression of constitutively active NFATc1 rescued the phenotype induced by Beclin-1 knockdown, indicating that Beclin-1 mediates RANKL-induced osteoclastogenesis by regulating NFATc1 expression. These findings show that Beclin-1 plays a non-autophagic role in RANKL-induced osteoclastogenesis by inducing the production of reactive oxygen species and NFATc1. J. Cell. Physiol. 229: 1963–1971, 2014. © 2014 Wiley Periodicals, Inc.
Osteoclasts (OC), which are derived from hematopoietic monocyte/macrophage lineage cells, i.e., OC precursors, play a critical role in bone homeostasis by degrading and adsorbing old bone (Tanaka et al., 1993; Suda et al., 1999; Teitelbaum, 2000). The differentiation of OC precursor cells into OC is supported by two key factors: macrophage colonystimulating factor (M-CSF) and receptor activator of nuclear factor kB (NF-kB) ligand (RANKL). M-CSF binds to a receptor (c-fms) on OC and is required for OC survival and proliferation (Tanaka et al., 1993), while receptor activator of nuclear factor kB (NF-kB) ligand (RANKL) is a cytokine essential for osteoclastogenesis and OC survival (Suda et al., 1999; Teitelbaum, 2000). In OC precursor cells, M-CSF is responsible for the expression of receptor activator of nuclear factor-kB (NF-kB) (RANK) and that enables OC precursor cells to respond to RANKL at the early phase of OC differentiation. The RANKL–RANK interaction activates signaling pathways that induce the expression and activation of master transcription factors, including NF-kB, c-Fos, AP-1, and nuclear factor of activated T cells (NFAT) c1, which then drive osteoclastogenesis (Lee and Kim, 2003). Upon RANKL binding to RANK, TNFR-associated factor 6 (TRAF6) is recruited to the RANK intracellular domain. The TAB2 signaling complex then activates TGF-b-activated kinase 1 (TAK1), a MAPK kinase kinase, which in turn activates the inhibitory kappa B kinase (IKK) complex leading to the activation of the NF-kB pathway (Ninomiya-Tsuji et al., 1999; Mizukami et al., 2002). Also, RANK-mediated activation of extracellular signalregulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK induces NFATc1 expression (Takayanagi et al., 2002; Lee and Kim, 2003; Ikeda et al., 2004; Huang et al., 2006a; Huang et al., 2006b). In addition to activating MAPKs and the NF-kB pathway, RANKL also induces the production of reactive oxygen species (ROS) (also via JNK and p38), thereby playing a role in OC differentiation (Rhee, 1999; Lee et al., 2005). Finally, NFATc1 plays a critical role in OC © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .
differentiation by inducing the expression of osteoclast-specific genes, including osteoclast-associated receptor (OSCAR), cathepsin k, calcitonin receptor, and tartrate-resistant acid phosphatase (TRAP) (Takayanagi et al., 2002; Asagiri and Takayanagi, 2007). Beclin-1, the mammalian homolog of yeast Atg6, is an essential component of the class III phosphatidylinositol 3-kinase complex (PI3KC3), which plays a role in autophagy (Klionsky et al., 2003). Beclin-1 participates in autophagosome formation, a process involving the degradation of damaged intracellular organelles and protein aggregates, and in the recycling of macromolecules through fusion with lysosomes (Klionsky et al., 2003; Yang and Klionsky, 2010). However,
Conflict of Interest: None Contract grant sponsor: Korea Government (MSIP); Contract grant number: 2008-0062286. Contract grant sponsor: Republic of Korea and the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea; Contract grant number: 13-524. Contract grant sponsor: Ministry of Education, Science and Technology; Contract grant number: NRF-2013R1A1A3010455. *Correspondence to: Dr. Eun-Ju Chang, Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea. E-mail: [email protected] Manuscript Received: 26 December 2013 Manuscript Accepted: 11 April 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 15 April 2014. DOI: 10.1002/jcp.24646
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Beclin-1 also has non-autophagic functions in other cellular processes, including endocytic trafficking (Thoresen et al., 2010), phagocytosis (Sanjuan et al., 2007), and cytokinesis (Sagona et al., 2010); it even plays a role in pollen germination (Fujiki et al., 2007). Given that other autophagy proteins such as ATG5, ATG7, ATG4B, and LC3 regulate the secretory functions of OC (DeSelm et al., 2011; Chung et al., 2012), it is plausible that Beclin-1 plays a distinct role in these cells. However, its potential role and possible underlying mechanisms have not been addressed. Here, we show that RANKL up-regulated the expression of Beclin-1 via the p38 and JNK signaling pathways. Beclin-1 within OC mitochondria induced RANKL-mediated ROS production. This in turn increased NFATc1 expression, leading to OC differentiation and cathepsin K secretion. Taken together, these findings provide evidence of a non-autophagic role for Beclin-1 via RANKLinduced osteoclastogenesis. Materials and methods Reagents and antibodies RANKL and M-CSF were obtained from Pepro Tech Inc (Rocky Hill, NJ). The tartrate-resistant acid phosphatase (TRAP) Assay Kit, the Cathepsin K Activity Assay Kit, PD98059, SB203580 (SB), SP60025, Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), and the antimycin A and b-actin antibodies (Ab) were purchased from Sigma-Aldrich (St Louis, MO). Cell counting kit (CCK-8) was obtained from Dojindo (Kumamoto, Japan). RNAiMAX, Lipofectamine 2000, propidium iodide (PI), Alexa Fluor 488-conjugated phalloidin and DAPI were purchased from Invitrogen (Grand Island, NY). Antibodies against Beclin-1, p-ERK, ERK, p-JNK, JNK, p-p38, p38, p-ATF, p-TAK1, TAK1, p-MKK3/6, MKK3/6, and IkBa were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against NFATc1, cathepsin K, and GFP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-TOMM20 Ab and quinazoline (QZ) were purchased from Merck Millipore (Darmatadt, Germany). Donkey fluoresceinconjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG were obtained from Jackson Immunoresearch (West Grove, PA). Protease and phosphatase inhibitor cocktails were purchased from Thermo Pierce (Rockford, IL). Culture of bone marrow macrophages and osteoclast differentiation Bone marrow (BM) cells were isolated by flushing the marrow space in femurs and tibiae isolated from 6-week-old female ICR mice as described by Chang et al. (2008a). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Sciences in Seoul, Korea. Isolated cells were cultured for 12 h in a-minimal essential medium (a-MEM; Hyclone) supplemented with 10% FBS (Hyclone, Logan, UT) and 1% penicillin–streptomycin (Gibco, New York, NY). Non-adherent cells were collected after 12 h and cultured in petri dishes (Green Cross, Suwon, Korea) in the presence of M-CSF (30 ng/ml) for further 3 days. Adherent cells were considered to be bone marrowderived macrophages (BMMs) and were used as osteoclast (OC) precursor cells. BMM were cultured for 4 days with M-CSF (30 ng/ml) and RANKL (100 ng/ml) to induce differentiation into OC. OC formation was determined by TRAP staining according to the manufacturer’s instructions. The number of TRAP-positive multinucleated cells (MNCs; containing more than three nuclei) was counted under a light microscope. These cells were termed OC. Immunoblot analysis and cathepsin K activity assay Pre-fusion OCs were transfected with control- or Beclin-1-specific small interfering (si)RNAs for 48 h and then stimulated by culture in fresh osteoclastogenic media for 2 h. Culture media were collected JOURNAL OF CELLULAR PHYSIOLOGY
and centrifuged, and the protease activity of the cathepsin K in the supernatants was measured using a Cathepsin K Activity Assay Kit according to the manufacturer’s protocol. The supernatants were boiled in 5 SDS sample buffer and the proteins were resolved in 12% SDS-PAGE gels. The cells were washed with ice-cold PBS and lysed in modified RIPA buffer [50 mM Tris/HCl (pH 7.4), 1% Nonidet P40, 0.25% sodium deoxycholate, and 150 mM NaCl] containing protease and phosphatase inhibitors. Cell lysates were centrifuged at 10,000 g for 15 min, the supernatants were collected, and the proteins resolved in 10–12% SDS-PAGE gels. Separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), which was then blocked for 1 h with 5% BAS (MP biomedicals, Auckland, New Zealand) solution in Tris-buffered saline solution containing 0.1% Tween 20. The membrane was then incubated overnight at 4 °C with the appropriate primary Ab, washed, and incubated for 1 h at room temperature with the HRPconjugated secondary Ab. Reactive proteins were visualized using a chemiluminescence system (Merck-Millipore, Darmatadt, Germany). Small interfering RNA (siRNA) transfection and retroviral gene transfer Pre-designed control siRNA (4390843) and siRNA targeting mouse BECN1 were obtained from Ambion (Life Technology, Carlsbad, CA). Two different sequences were used to target mouse BECN1, BECN1 #1 (s80166), and BECN1 #2 (s80167). BECN1 #2 showed the greatest degree of interference. Oligonucleotide siRNAs were transfected into mouse BMMs or BMMs pretreated with RANKL (RANKL-primed BMMs) for 6 h in RNAiMAX reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s protocol. After 4 h, the medium was replaced with fresh osteoclastogenic media (a-MEM in the presence of MCSF (30 ng/ml) and RANKL (100 ng/ml)). The cells were analyzed at 24 h post-transfection, and endogenous Beclin-1 was detected by immunoblotting. For OC differentiation, the transfected cells were differentiated into OCs in the osteoclastogenic media for 4 days, fixed, and stained for TRAP. The population of TRAPþ cells, percentage of TRAPþ multinuclear cells (MNCs) in total cells, and TRAPþ MNCs with actin ring in TRAPþ MNCs was quantified. In this condition, the siRNA transfection efficiency of BMMs cells was ~92% when we assayed using siRNA carrying GFP. To generate retroviral stocks, Plat E packaging cell lines were transiently transfected with pMX-EGFP or pMX-NFATc1-EGFP constitutively active (CA) mutant vectors (CA-NFATc1) using Lipofectamine 2000 (Invitrogen) (Porter and Clipstone, 2002). The viral supernatant was collected at 48 h post-transfection. BMMs or OCP were incubated with the viral supernatant for 8 h in the presence of polybrene (10 mg/ml; Sigma), as described previously (Chang et al., 2008b). The transduction efficiency of retroviral CA-NFATc1 in BMMs cells was ~78%. After the retroviral supernatants were removed, BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 days. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR RNA was isolated from cells using Trizol Reagent (Life Technology, Carlsbad, CA) and 2 mg of RNA was reverse-transcribed using Super-Script II reverse transcriptase (Life Technologies). The resulting cDNA was amplified by PCR using the following primers: Beclin-1 (BECN1), 50 -GGCCAATAAGGGTCTGA-30 (forward) and 50 -GCTGCACACAGTCCAGAAAA-30 (reverse); NFATc1 (Nfatc1), 50 -GGGTCAGTGTGACCGAAGAT-30 (forward) and 50 -GGAAGTCAG-AAGTGGGTGGA-30 (reverse); cathepsin K (Ctsk), 50 -AATACCTCCCTCTCGATCCTACA-30 (forward) and 50 -GGTTCTTGACTGGAGTAACGTA-30 (reverse); TRAP (Acp5), 50 -TCCTGGCTCAAAAAGCAGTT-30 (forward) and 50 ACATAGCCCACACCGTTCTC-30 (reverse); and GAPDH
ROLE OF BECLIN-1 IN OSTEOCLASTOGENESIS (Gapdh), 50 -ACCACAGTCCA TGCCATCAC-30 (forward) and 50 -TCCACCACCCTGTTGCTGTA-30 (reverse). The PCR conditions were as follows: denaturation at 94 °C for 30 s, followed by annealing at 55–60 °C for 30 s, and an extension step at 72 °C for 1 min. The number of cycles fell within the linear range of amplification (28–30 cycles; GAPDH required 23 cycles). Quantitative real-time PCR (qRT-PCR) was performed using Power SYBR Green 1-Step Kit and an ABI 7000 Real Time PCR System (Applied Biosystems, Carlsbad, CA) according to manufacturer’s instructions. The sequences of primers used were the same as described above. Cell viability assay Control- or Beclin-1-specific siRNA-transfected RANKL-primed BMMs were cultured in the presence of RANKL (100 ng/ml) and M-CSF (30 ng/ml) for24 h and thenincubated with 10% CCKsolution inculturemediumfor1 hat37 °C.Theopticaldensitywasmeasuredin a microtiter plate reader (Bio-Rad, Hercules, CA) at 450 nm. Confocal microscopy BMMs or OCs grown on glass cover slips were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature and then permeabilized with 0.1% Triton X-100. After blocking with 1% BSA in PBS for 1 h, the cells were incubated with a primary antibody overnight at 4 °C. The cells were then washed and stained with DAPI and FITC- or Cy3-conjugated secondary antibodies. F-actin was stained using Alexa Fluor 488-conjugated phalloidin. After mounting, the fluorochromes were detected using a confocal laser-scanning microscope (LSM 710, Carl Zeiss). Measurement of mitochondrial superoxide production To measure mitochondrial superoxide production, control- or Beclin-1-specific siRNA-transfected BMMs were loaded with 5 mM Mitosox in the presence of M-CSF (30 ng/ml) and RANKL (30 ng/ml) for 1 h in the dark. The cells were then washed with warm PBS and further processed for analysis in a FACSCalibur flow cytometer according to the manufacturer’s instructions (Becton Dickinson, San Jose, CA). Statistics All quantitative experiments were performed at least in triplicate, and the data are expressed as the mean SD. Data were analyzed using Graphpad Prism 5 software, and the statistical significance of the differences between groups was determined using Student’s t test. A P value < 0.05 was considered significant. Results RANKL induces Beclin-1 expression during osteoclastogenesis
BMMs were differentiated into OC by culture in the presence of M-CSF and RANKL for 4 days (Figure 1A). We found that the expression of Beclin-1 protein increased during differentiation (Figure 1B). Beclin-1 expression increased 4.5-fold in cells treated with RANKL and M-CSF for 72 h, but did not increase in cells treated with M-CSF alone (Figure 1C and D), demonstrating that Beclin-1 expression is induced by RANKL signaling. To identify the signals induced by RANKL, BMMs were treated with PD98059 (an ERK inhibitor), SB203580 (SB; a p38 inhibitor), SP600125 (a JNK inhibitor), or QZ (a NF-kB activation inhibitor) in the presence of RANKL and M-CSF for 24 h. SB and QZ inhibited significantly RANKL-induced Beclin1 transcription (Figure 1E). These results suggest that RANKL induces Beclin-1 expression in OC via the p38 and NF-kB signaling pathways. JOURNAL OF CELLULAR PHYSIOLOGY
Beclin-1 is localized in the mitochondria and is involved in mitochondrial ROS production
Because the functions of proteins are dependent upon their location within cells, we next examined the subcellular location of Beclin-1 during osteoclastogenesis. A previous study reported that Beclin-1 localizes in the mitochondria, ER, and trans-Golgi network (Pattingre et al., 2005; Shi and Kehrl, 2010). As expected, an apparent cytosolic distribution of Beclin-1 was observed in BMM, pOC, and OC (Figure 2A). In addition, we found that Beclin-1 mainly co-localized with TOMM20, which is located in the outer mitochondrial membrane, during OC differentiation (Figure 2A). This was confirmed using the MitoTracker dye (data not shown). OC contains large numbers of mitochondria. These organelles supply energy to the vacuolar ATPase proton pump, which uses ATP to secrete protons (Hþ) into the resorption space and generates the ROS involved in osteoclast development (Brown and Breton, 1996; Srinivasan et al., 2010). We used Mitosox Red to examine whether downregulating Beclin-1 affected RANKL-induced mitochondrial ROS production. The efficiency of knockdown by Beclin-1-specific siRNA was confirmed by western blotting (Figure 2B). Beclin-1 siRNAtransfected BMMs showed a 74% reduction in mitochondrial superoxide levels compared with control siRNA-transfected cells (Figure 2C). The addition of the membrane permeable superoxide dismutase mimetic, MnTBAP, significantly inhibited mitochondrial superoxide levels. Similar to these conditions, Beclin-1 siRNA-transfected BMMs showed a 28.8% reduction compared with the control. However, treatment with antimycin A stimulated mitochondrial superoxide production (levels were 1.94-fold higher than in the controls) (Figure 2D). These data indicate that Beclin-1 plays a role in RANKLdependent ROS production. Knockdown of Beclin-1 inhibits RANKL-induced osteoclast differentiation
RANKL-dependent ROS production is an osteoclastogenic signal mediator required for OC differentiation (Rhee, 1999; Lee et al., 2005). Given the requirement for Beclin-1 for ROS generation in OC, it is possible that Beclin-1 may support RANKL-mediated signaling and is involved in OC differentiation. To investigate this, we examined the effect of Beclin-1 knockdown on RANKL-induced OC differentiation. siRNAmediated downregulation of Beclin-1 expression (Figure 3A) had no effect on the viability of RANKL-primed BMMs (Figure 3B). Interestingly, we observed a significant reduction in the number of TRAP-positive MNCs that were differentiated from Beclin-1-silenced BMMs (Figure 3C). Also, compared with that in control-siRNA-transfected cells, the expression of OC differentiation markers such as cathepsin K (Ctsk), NFATc1 (Nfatc1), and TRAP (Acp5) mRNA was downregulated in Beclin-1-siRNA-transfected cells (Figure 3D and E). Subsequently, the intracellular expression, secretion, and activity of cathepsin K were also reduced by Beclin-1 knockdown (Figure 3F and G), emphasizing the positive role of Beclin-1 in RANKLinduced OC differentiation. RANKL-mediated activation of JNK and p38 requires Beclin-1
To gain insight into the molecular mechanisms by which Beclin1 promotes osteoclastogenesis, we examined the effects of Beclin-1 knockdown on the early signaling pathways activated by M-CSF or RANKL. Increased activation of ERK, JNK, p38, and ATF2 were observed at 5 and 15 min after M-CSF treatment; these increases were not abrogated by knocking down Beclin-1 (Figure 4A). On the other hand, activation of the
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Fig. 1. RANKL-dependent induction of Beclin-1 during osteoclastogenesis. (A) Bone marrow-derived machrophages (BMMs) were cultured for 4 days with M-CSF (30 ng/ml) and RANKL (100 ng/ml) to induce differentiation into mature osteoclasts (OCs). After culture for 2 days (d2), cells were classed as pre-fusion osteoclasts (pOC). (B) Whole cell lysates were obtained from cultured cells at the indicated times and analyzed by western blotting with Abs specific for Beclin-1 or b-actin (loading control). (C) BMMs were treated with M-CSF plus RANKL or with M-CSF alone for the indicated times and Beclin-1 protein expression was examined by western blotting. (D) Changes in the amount of Beclin-1 protein relative to b-actin are shown. (E) BMMs were cultured without M-CSF and RANKL for 4 h (S) and then treated with M-CSF and RANKL in the presence of DMSO (Veh) PD98059 (PD, 10 mM), SB203580 (SB, 10 mM), SP600125 (SP, 10 mM), or quinazoline (QZ, 10 mM). After 24 h, the cells were harvested and subjected to quantitative real-time PCR (E) to examine Beclin-1 (BECN1) mRNA expression. Data are expressed as the mean SD. **P < 0.05, **P < 0.005, ***P < 0.0005 versus Veh.
JNK and p38 signaling pathways was inhibited in Beclin-1silenced BMMs treated with RANKL (Figure 4B). NFATc1, a pivotal transcription factor during osteoclastogenesis (Takayanagi et al., 2002), is induced by RANKL via the JNK and p38 MAPK signaling pathways (Ikeda et al., 2004; Huang et al., 2006a). Thus, we next examined the expression of NFATc1 protein in BMMs transfected with Beclin-1 siRNA or control siRNA in the presence of RANKL. NFATc1 expression was significantly increased by RANKL, but it was markedly inhibited by Beclin-1 knockdown (Figure 4C). This suggests that Beclin-1 mediates RANKL-dependent NFATc1 induction in a JNK- and p38 MAPK-dependent manner. Overexpression of active NFATc1 restores osteoclastogenesis in Beclin-1-knockdown BMMs
To confirm that NFATc1 plays a critical role in Beclin-1 function during osteoclastogenesis, BMMs or RANKL-induced OCPs were infected with a retrovirus harboring a constitutively active form of NFATc1 (NFATc1 CA) (Figure 5A). Transduction of NFATc1-CA, but not of control retroviruses, rescued the RANKL-induced TRAP-positive OC formation that was suppressed by Beclin-1 siRNA (Figure 5B), JOURNAL OF CELLULAR PHYSIOLOGY
and the number of TRAP-positive MNCs increased significantly (Figure 5C). These results clearly suggest that Belcin-1 plays a critical role in RANKL-induced NFATc1 expression, thereby contributing to osteoclastogenesis. Discussion
A number of studies report non-autophagic roles for autophagy proteins, including ATG5, ATG7, and LC3, in OCs (DeSelm et al., 2011; Chung et al., 2012). Deletion of ATG5 or ATG7 from cells of the macrophage lineage has no effect on OC differentiation; however, it does impair the secretion of lysosomal contents and bone resorption (DeSelm et al., 2011). The present study showed that the Beclin-1 plays an important role in RANKL-induced OC differentiation, which is distinct from the functions of other autophagy-related proteins in OC. We found that the Beclin-1 expression was up-regulated by M-CSF plus RANKL, but not by M-CSF alone, during osteoclastogenesis, and that this up-regulation was dependent upon NF-kB and p38 activation (Figure 1). The NF-kB pathway is crucial for RANKL-mediated OC development (Iotsova et al., 1997). Upon stimulation with RANKL, activated IkB kinase (IKK) induces the phosphorylation of serine 536 within
ROLE OF BECLIN-1 IN OSTEOCLASTOGENESIS
the p65 transactivation domain, thereby promoting translocation of p50/p65 to the nucleus (Iotsova et al., 1997). Thus, it is likely that these signals are responsible for the binding of p65/ RelA to the kB site within the BECN1 promoter in response to RANKL, thereby up-regulating BECN1 mRNA and protein expression (Copetti et al., 2009). The localization of Beclin-1 in the mitochondria of OC might be related to its role in ROS production (Figure 2). Intracellular mitochondrial ROS are generated via the TRAF6, Rac1, and NADPH oxidase (Nox) 1 pathway (Rhee, 1999). Beclin-1 contains two TRAF6-binding motifs, which are responsible for binding to activated TRAF6 (Shi and Kehrl, 2010). Since TRAF6 is a pivotal adaptor molecule for both RANK and intracellular
ROS production, we assumed that Beclin-1 might be associated with TRAF6. However, we did not detect any interaction between endogenous TRAF6 and Beclin-1 in BMMs or OC upon RANKL stimulation (data not shown). On the other hand, Beclin-1 was initially identified as a Bcl-2-interacting myosin like coiled-coil protein, which interacts with Bcl-2 through Bcl-2 homology domain 3 (BH3) (Liang et al., 1998). A recent study reported that Bcl-2 regulates intracellular redox status in cancer cells by physically interacting with COX Va (the Va subunit of cytochrome c oxidase; complex IV) (Chen and Pervaiz, 2010). Accordingly, we suggest that Beclin-1 might be involved in regulating mitochondrial ROS generation by interacting with Bcl-2. However, further studies are needed to
Fig. 2. Beclin-1 localization and its role in osteoclast mitochondria. (A) BMMs, pOCs (1d), OCs (3d), or mature OCs (4d) were fixed; immunostained for Beclin-1 (green), TOMM20 (red), and F-actin (blue, pseudocolor); and then stained with the nuclear dye, DAPI (cyan, pseudocolor). Right panels of mOC image show low magnification image of mOC and contain actin ring structure in the box. Scale bars: 12.5 mm and 50 mm (low magnification image of mOC). (B) BMMs were transfected with a control siRNA or with two Beclin-1-specific siRNAs for 24 h. Knockdown of Beclin-1 protein expression was examined by western blotting. (C) Transfected BMMs were pretreated with antimycin A (20 mM) or MnTBAP (200 mM) for 30 min, stimulated with RANKL (400 ng/ml) for 1 h, and then stained with Mitosox 5 mM for 30 min. Mitochondrial-derived superoxide generation was measured by flow cytometry. For each histogram, the y axis shows the number of cells and the x axis shows the FL2 channel (Mitosox Red). (D) The intensity of Mitosox Red fluorescence was compared with that in control siRNAtransfected cells. Data are expressed as the mean SD. **P < 0.005 versus control.
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Fig. 3. Role of Beclin-1 in RANKL-induced osteoclastogenesis. (A–G) BMMs were primed with RANKL for 6 h and then transfected with control siRNA or Beclin-1-specific siRNA. Transfected RANKL-primed BMMs were cultured with M-CSF and RANKL for 24 h, and Beclin-1 protein expression (A) and cell viability (B) were analyzed by western blotting and a CCK-8 assay, respectively. Transfected RANKL-primed BMMs were differentiated into OCs in osteoclastogenic medium. Cells were then fixed and stained with TRAP solution and TRAP-positive multinucleated OCs (MNCs) with different number of nuclei were counted in five different categories; 1 to 2 nuclei, 3–10 nuclei, 11–20 nuclei, 21–50 nuclei, and those with greater than 50 nuclei. Mature TRAP-positive MNCs were photographed under a light microscope (magnification 100). Scale bars: 200 mm (C). Transfected BMMs were cultured in the presence of M-CSF and RANKL for the indicated times, and mRNA levels including BECN1 (Beclin-1), Ctsk (cathepsin K), Nfatc1 (NFATc1), Acp5 (TRAP), and Gapdh (GAPDH) were analyzed by semiquantitative RT-PCR (D) and quantitative real-time PCR (E). Cells were differentiated into pOCs by incubation in osteoclastogenic medium for 2 days. The osteoclastogenic medium was then replaced with fresh medium containing RANKL (100 ng/ml) and M-CSF (30 ng/ml). Supernatants and cell lysates were collected at the indicated times and analyzed by western blotting to measure Beclin-1 and cathepsin K expression (F). Cathepsin K activity in the supernatant was evaluated using a cathepsin k activity assay (G). Data are expressed as the mean SD. **P < 0.05, **P < 0.005, ***P < 0.0005 versus control.
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explore whether Beclin-1 regulates ROS production in OC precursor cells in response to RANKL. Although the relationship between ROS and NFATc1 expression identified in this study is obscure, some reports suggest that RANKL-dependent ROS production induces the transcriptional activity of NFATc1 (Huang et al., 2001; Ke et al., 2006; Han et al., 2007; Srinivasan et al., 2010). Taken
together, these studies suggest that ROS mediate the activation of MAPKs (e.g., JNK and p38) in OC precursor cells (Lee et al., 2005), and that this is associated with NFATc1 induction. Our findings show that knockdown of Beclin-1 inhibited RANKL-mediated mitochondrial ROS production (Figure 2C and D) and JNK and p38 MAPK activation (Figure 4), resulting in the failure of NFATc1 induction (Figure 4).
Fig. 4. Beclin-1 knockdown inhibits RANKL-mediated JNK and p38 activation. (A–C) BMMs were transfected with control- or Beclin-1specific siRNA and serum-starved for 2 h before stimulation with 30 ng/ml M-CSF (A) and 400 ng/ml RANKL (B) for the indicated times. Lysates were analyzed by western blotting with antibodies against molecules involved in the MAPK and NF-kB signaling pathways. To examine NFATc1 expression, transfected BMMs were stimulated with RANKL (100 ng/ml) in the presence of M-CSF (30 ng/ml) for the indicated times and then immunoblotted with antibodies against NFATc1 and Beclin-1 (C).
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Fig. 5. Effect of constitutively active NFATc1 on Beclin-1 knockdown-induced inhibition of osteoclastogenesis. (A) BMMs or RANKL-primed BMMs were infected with retroviruses harboring a control vector (pMX) or with a constitutively active NFATc1 (NFATc1 CA) construct. Overexpression of NFATc1 CA was confirmed by western blotting at 24 h post-infection. (B) BMMs or RANKL-primed BMMs were transfected with control- or Beclin-1-specific siRNAs for 4 h, and then infected with control or NFATc1-CA retroviruses. Cells were then cultured in the presence of M-CSF and RANKL for 4 days, fixed, and stained for TRAP (magnification 100). Scale bars: 200 mm. (C) The number of mature TRAP-positive MNCs was counted under a light microscope. Data are expressed as the mean SD. **P < 0.05, **P < 0.005, *** P < 0.0005 versus pMX.
Taken together, the results of the present study suggest that Beclin-1 may act as a molecular link that mediates mitochondrial ROS and NFATc1 expression, thereby contributing to RANKL-induced OC differentiation. Thus, Beclin-1 appears to play a non-autophagic role during osteoclastogenesis. Acknowledgments
This work was supported by a National Research Foundation of Korea (NRF) MRC grant funded by the Korea Government (MSIP) (2008-0062286), Republic of Korea and the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea (13-524). This project was supported in part by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (NRF2013R1A1A3010455). Literature Cited Asagiri M, Takayanagi H. 2007. The molecular understanding of osteoclast differentiation. Bone 40:251–264. Brown D, Breton S. 1996. Mitochondria-rich, proton-secreting epithelial cells. J Exp Biol 199:2345–2358. Chang EJ, Ha J, Huang H, Kim HJ, Woo JH, Lee Y, Lee ZH, Kim JH, Kim HH. 2008a. The JNKdependent CaMK pathway restrains the reversion of committed cells during osteoclast differentiation. J Cell Sci 121:2555–2564.
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Chang EJ, Ha J, Oerlemans F, Lee YJ, Lee SW, Ryu J, Kim HJ, Lee Y, Kim HM, Choi JY, Kim JY, Shin CS, Pak YK, Tanaka S, Wieringa B, Lee ZH, Kim HH. 2008b. Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption. Nat Med 14:966–972. Chen ZX, Pervaiz S. 2010. Involvement of cytochrome c oxidase subunits Va and Vb in the regulation of cancer cell metabolism by Bcl-2. Cell Death Differ 17:408–420. Chung YH, Yoon SY, Choi B, Sohn DH, Yoon KH, Kim WJ, Kim DH, Chang EJ. 2012. Microtubule-associated protein light chain 3 regulates Cdc42-dependent actin ring formation in osteoclast. Int J Biochem Cell Biol 44:989–997. Copetti T, Bertoli C, Dalla E, Demarchi F, Schneider C. 2009. P65/RelA modulates BECN1 transcription and autophagy. Mol Cell Biol 29:2594–2608. DeSelm CJ, Miller BC, Zou W, Beatty WL, van Meel E, Takahata Y, Klumperman J, Tooze SA, Teitelbaum SL, Virgin HW. 2011. Autophagy proteins regulate the secretory component of osteoclastic bone resorption (vol 21, pg 966, 2011). Dev Cell 21:1179. Fujiki Y, Yoshimoto K, Ohsumi Y. 2007. An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination. Plant Physiol 143:1132–1139. Han KY, Yang D, Chang EJ, Lee Y, Huang H, Sung SH, Lee ZH, Kim YC, Kim HH. 2007. Inhibition of osteoclast differentiation and bone resorption by sauchinone. Biochem Pharmacol 74:911–923. Huang C, Li J, Costa M, Zhang Z, Leonard SS, Castranova V, Vallyathan V, Ju G, Shi X. 2001. Hydrogen peroxide mediates activation of nuclear factor of activated T cells (NFAT) by nickel subsulfide. Cancer Res 61:8051–8057. Huang H, Chang EJ, Ryu J, Lee ZH, Lee Y, Kim HH. 2006. Induction of c-Fos and NFATc1 during RANKL-stimulated osteoclast differentiation is mediated by the p38 signaling pathway. Biochem Biophys Res Commun 351:99–105. Huang H, Ryu J, Ha J, Chang EJ, Kim HJ, Kim HM, Kitamura T, Lee ZH, Kim HH. 2006. Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NFkappaB transactivation by RANKL. Cell Death Differ 13:1879–1891. Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, Hata K, Yamashita K, Hiraga T, Watanabe T, Kukita T, Yoshioka K, Rao A, Yoneda T. 2004. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Invest 114:475–484. Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, Bravo R. 1997. Osteopetrosis in mice lacking NF-kappa B1 and NF-kappa B2. Nat Med 3:1285–1289. Ke Q, Li J, Ding J, Ding M, Wang L, Liu B, Costa M, Huang C. 2006. Essential role of ROSmediated NFAT activation in TNF-alpha induction by crystalline silica exposure. Am J Physiol Lung Cell Mol Physiol 291:L257–L264.
ROLE OF BECLIN-1 IN OSTEOCLASTOGENESIS
Klionsky DJ, Cregg JM, Dunn WA, Jr., Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y. 2003. A unified nomenclature for yeast autophagyrelated genes. Dev Cell 5:539–545. Lee NK, Choi YG, Baik JY, Han SY, Jeong DW, Bae YS, Kim N, Lee SY. 2005. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood 106:852– 859. Lee ZH, Kim HH. 2003. Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts. Biochem Bioph Res Co 305:211–214. Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G, Herman B, Levine B. 1998. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol 72:8586–8596. Mizukami J, Takaesu G, Akatsuka H, Sakurai H, Ninomiya-Tsuji J, Matsumoto K, Sakurai N. 2002. Receptor activator of NF-kappa B ligand (RANKL) activates TAK1 mitogenactivated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6. Mol Cell Biol 22:992–1000. Ninomiya-Tsuji J, Kishimoto K, Hiyama A, Inoue J, Cao ZD, Matsumoto K. 1999. The kinase TAK1 can activate the NIK-I kappa B as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 398:252–256. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B. 2005. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122:927–939. Porter CM, Clipstone NA. 2002. Sustained NFAT signaling promotes a Th1-like pattern of gene expression in primary murine CD4þ T cells. J Immunol (Baltimore, MD: 1950) 168:4936–4945. Rhee SG. 1999. Redox signaling: hydrogen peroxide as intracellular messenger. Exp Mol Med 31:53–59.
JOURNAL OF CELLULAR PHYSIOLOGY
Sagona AP, Nezis IP, Pedersen NM, Liestol K, Poulton J, Rusten TE, Skotheim RI, Raiborg C, Stenmark H. 2010. PtdIns(3)P controls cytokinesis through KIF13A-mediated recruitment of FYVE-CENT to the midbody. Nat Cell Biol 12:362–371. Sanjuan MA, Dillon CP, Tait SWG, Moshiach S, Dorsey F, Connell S, Komatsu M, Tanaka K, Cleveland JL, Withoff S, Green DR. 2007. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450:1253–1257. Shi CS, Kehrl JH. 2010. TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy. Sci Signal 3:ra42. Srinivasan S, Koenigstein A, Joseph J, Sun L, Kalyanaraman B, Zaidi M, Avadhani NG. 2010. Role of mitochondrial reactive oxygen species in osteoclast differentiation. Ann NY Acad Sci 1192:245–252. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. 1999. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J, Wagner EF, Mak TW, Kodama T, Taniguchi T. 2002. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 3:889–901. Tanaka S, Takahashi N, Udagawa N, Tamura T, Akatsu T, Stanley ER, Kurokawa T, Suda T. 1993. Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors. J Clin Invest 91:257–263. Teitelbaum SL. 2000. Bone resorption by osteoclasts. Science 289:1504–1508. Thoresen SB, Pedersen NM, Liestol K, Stenmark H. 2010. A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic. Exp Cell Res 316:3368–3378. Yang Z, Klionsky DJ. 2010. Eaten alive: a history of macroautophagy. Nat Cell Biol 12:814–822.
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