Signaling Cascade /Nfatc1 Β Through Regulating the GSK3 Akt Induces

Akt Induces Osteoclast Differentiation through Regulating the GSK3 β/NFATc1 Signaling Cascade

This information is current as Jang Bae Moon, Jung Ha Kim, Kabsun Kim, Bang Ung of September 25, 2021. Youn, Aeran Ko, Soo Young Lee and Nacksung Kim J Immunol 2012; 188:163-169; Prepublished online 30 November 2011; doi: 10.4049/jimmunol.1101254

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Akt Induces Osteoclast Differentiation through Regulating the GSK3b/NFATc1 Signaling Cascade

Jang Bae Moon,* Jung Ha Kim,* Kabsun Kim,* Bang Ung Youn,* Aeran Ko,* Soo Young Lee,† and Nacksung Kim*

SHIP is an SH2-containing inositol-5-phosphatase expressed in hematopoietic cells. It hydrolyzes the PI3K product PI(3,4,5)P3 and blunts the PI3K-initiated signaling pathway. Although the PI3K/Akt pathway has been shown to be important for osteoclastogenesis, the molecular events involved in osteoclast differentiation have not been revealed. We demonstrate that Akt induces osteoclast differentiation through regulating the GSK3b/NFATc1 signaling cascade. Inhibition of the PI3K by LY294002 reduces formation of osteoclasts and attenuates the expression of NFATc1, but not that of c-Fos. Conversely, overexpression of Akt in bone marrow-derived macrophages (BMMs) strongly induced NFATc1 expression without affecting c-Fos expression, suggesting that

PI3K/Akt-mediated NFATc1 induction is independent of c-Fos during RANKL-induced osteoclastogenesis. In addition, we found Downloaded from that overexpression of Akt enhances formation of an inactive form of GSK3b (phospho-GSK3b) and nuclear localization of NFATc1, and that overexpression of a constitutively active form of GSK3b attenuates osteoclast formation through downregulation of NFATc1. Furthermore, BMMs from SHIP knockout mice show the increased expression levels of phospho-Akt and phospho-GSK3b, as well as the enhanced osteoclastogenesis, compared with wild type. However, overexpression of a constitutively active form of GSK3b attenuates RANKL-induced osteoclast differentiation from SHIP-deﬁcient BMMs. Our data suggest that the PI3K/Akt/GSK3b/NFATc1 signaling axis plays an important role in RANKL-induced osteoclastogenesis. The Journal of http://www.jimmunol.org/ Immunology, 2012, 188: 163–169.

one homeostasis is tightly regulated by bone-resorbing NFATc1, osteoclast-associated receptor (OSCAR), and tartrate- activity of osteoclasts and bone-forming activity of os- resistant acid phosphatase (TRAP) (3, 6). Among the induced B teoblasts. Osteoclasts, which are multinucleated cells genes, NFATc1 in particular acts as a key modulator of osteo- specialized in bone resorption, are differentiated from monocyte- clastogenesis. Ectopic expression of NFATc1 in the precursors macrophage lineage cells of hematopoietic origin. Their differ- efficiently induces differentiation into functional osteoclasts even entiation is triggered by two critical factors supplied by osteoblasts, in the absence of RANKL (7). Moreover, NFATc1-deficient em- k by guest on September 25, 2021 M-CSF and receptor activator of NF- B ligand (RANKL) (1–3). bryonic stem cells fail to differentiate into osteoclasts in response The essential role of these osteotropic factors in osteoclast for- to RANKL (8), suggesting indispensable role of NFATc1 in osteo- mation is clearly demonstrated in genetic studies in which op/op clastogenesis. mouse, which lacks M-CSF, shows osteopetrotic phenotype be- Phosphoinositide 3-kinase (PI3K) is a lipid kinase that phos- cause of the defective osteoclast formation (4). RANKL-deficient phorylates phosphoinositides at the 39-OH position of the inositol mice also display a prominent osteopetrotic phenotype because of the complete absence of osteoclasts (5). ring. D3-phosphoinositides generated by PI3K recruit PH-domain- Upon binding of RANKL to its receptor RANK, downstream containing proteins such as Akt and mediates various cellular signaling pathways are activated including NF-kB, JNK, p38 functions, including mitogenesis, survival, motility, and differen- MAPK, extracellular signal-related kinase, and Akt to induce the tiation. However, the signaling pathways mediated by PI3K can be expression of osteoclastogenesis-related genes such as c-Fos, blunted by a phosphatase, such as phosphatase and tensin homolog (PTEN) and SHIP. SHIP is a Src homology (SH) 2-containing *National Research Laboratory for Regulation of Bone Metabolism and Disease, inositol-5-phosphatase widely expressed in hematopoietic cells. Medical Research Center for Gene Regulation, Research Institute of Medical Scien- SHIP specifically hydrolyzes the 59-phosphate group from phos- ces, Chonnam National University Medical School, Gwangju 501-746, Korea; and †Department of Bioinspired Science and the Department of Life Science, Ewha phatidylinositol-3,4,5-triphosphate (PIP3), the major product of Womans University, Seoul 120-750, Korea PI3K and negatively regulates PI3K activity (9). Whereas PI3K Received for publication May 2, 2011. Accepted for publication October 27, 2011. initiates and regulates the signals promoting osteoclast precursors 2 2 This work was supported by a Korea Science and Engineering Foundation National survival and differentiation, SHIP / mice show increased num- Research Laboratory Program grant funded by the Korean government (R0A-2007- 000-20025-0) and a grant from the Korea Science and Engineering Foundation bers of osteoclast precursors and enhanced osteoclastogenesis, re- through the Medical Research Center for Gene Regulation at Chonnam National sulting in severe osteoporosis (10). These data suggest that PI3K is University (R13-2002-013-03001-0). involved in osteoclast differentiation. Address correspondence and reprint requests to Prof. Nacksung Kim, Department of PI3K and phospholipid dependent activation of the serine/ Pharmacology, Chonnam National University Medical School, Hak-Dong 5, Dong- Ku, Gwangju 501-746, Korea. E-mail address: [email protected] threonine kinase Akt (also known as PKB) mediate the anti- The online version of this article contains supplemental material. apoptotic function in a variety of cell types (11). PI3K/Akt sig- Abbreviations used in this article: KO, knockout; MNC, mononuclear cell; PTEN, naling pathway also has been shown to regulate osteoclasts phosphatase and tensin homolog; RANKL, receptor activator of NF-kB ligand; survival and differentiation (12, 13). However, the precise mech- TRAP, tartrate-resistant acid phosphatase; WT, wild type. anism by which Akt regulates the differentiation of osteoclasts Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 remains unknown. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101254 164 ROLE OF THE Akt/GSK3b/NFATc1 AXIS DURING OSTEOCLASTOGENESIS

Upon stimulation by insulin or growth factors, Akt phosphory- Lamin B1 (Santa Cruz). HRP-conjugated secondary Abs (Amersham lates glycogen synthase kinase-3b (GSK3b) at serine residue and Biosciences) were probed and developed with ECL solution (Millipore). subsequently inhibits the kinase activity of GSK3b,suggestingthat Signals were detected and analyzed by LAS3000 luminescent image an- alyzer (Fuji Photo Film). GSK3b is one substrate for Akt (14). It has been shown that GSK3b can modulate the activity of NFATc transcription factors Transfection and reporter assay (15, 16). NFAT family members are regulated primarily at the level For transfection of reporter plasmids, 293T cells were plated into 24-well of their subcellular localization (17). GSK3b directly phosphor- plates (2 3 104 cells per well) 24 h prior to transfection. Plasmid DNA was ylates NFATc1 at its conserved serine residues necessary for nuclear mixed with FuGENE 6 and transfected into the cells following the man- export and promotes nuclear exit (15). It also has been demon- ufacturer’s protocol. After 48 h of transfection, the cells were washed b twice with PBS and then lysed in passive lysis buffer (Promega). Lucif- strated that the phosphorylation of the Ser-Pro repeats by GSK3 erase activity was measured using a dual-luciferase reporter assay system inhibits the ability of NFATc1 to bind DNA and thereby inhibits (Promega) according to the manufacturer’s instructions. NFATc1-dependent gene expression (16). These results prompted us to test the possibility that Akt could induce osteoclast differen- Results tiation through regulating the GSK3b/NFATc1 signaling cascade. PI3K plays a role in RANKL-induced osteoclastogenesis PI3K/Akt activation inhibits GSK3b by phosphorylation, and We ﬁrst examined the effect of the PI3K signaling pathway on this inhibition of GSK3b leads to the nuclear localization of RANKL-induced osteoclast differentiation. BMMs were cultured NFATc1, resulting in enhanced osteoclastogenesis. The enhanced 2 2 with M-CSF and RANKL in the absence or presence of LY294002, osteoclastogenesis appears to be consistent in SHIP / mice,

a specific inhibitor of PI3K. Consistent with previous results (12), Downloaded from where PI3K/Akt signaling is upregulated and the expression level treatment with LY294002 inhibited RANKL-induced osteoclast of phospho-GSK3b (inactive form of GSK3b) is elevated. Our formation in a dose-dependent manner (Fig. 1A,1B). studies suggest that the PI3K/Akt/GSK3b/NFATc1 signaling axis It has been shown that RANKL-induced activation of early plays an important role in RANKL-induced osteoclastogenesis. signaling pathways is important for osteoclast formation (3, 6). Because treatment of LY294002 attenuated RANKL-induced Materials and Methods osteoclastogenesis, we investigated whether LY294002 could af- Mice fect other RANKL-induced signaling pathways. BMMs were http://www.jimmunol.org/ SHIP-deficient mice were purchased from The Jackson Laboratory. starved and stimulated with RANKL for the indicated times. Breeding and genotyping were performed as described previously (9, 18). Consistent with previous results (23, 24), RANKL activated Akt, k Reagents and plasmids JNK, p38, and NF- B in 10 min. The treatment with LY294002 LY294002 was purchased from Calbiochem. The OSCAR reporter construct and expression construct encoding NFATc1 were described previously (19). A human constitutively active form of Akt (pECE-myr-Akt-D4- 129) was provided by J.K. Jung (Seoul National University). Expression constructs encoding wild type (WT) of GSK3b (GSK3b-WT) and a ki- by guest on September 25, 2021 nase-inactive mutant of GSK3b (GSK3b-KD) were provided by K.Y. Lee (Chonnam National University) (20). A constitutively active form of GSK- 3b, GSK3b-S9A (serine 9 of human GSK3b was replaced with alanine), was generated by the QuickChange method of site-directed mutagenesis (Stratagene) (21). Osteoclast formation Murine osteoclasts were prepared from bone marrow cells as described previously (22). Bone marrow cells were cultured in a-MEM containing 10% FBS with M-CSF (5 ng/ml) for 16 h. Nonadherent cells were harvested and cultured for 3 d in the presence of M-CSF (30 ng/ml). Floating cells were removed and adherent cells were used as osteoclast precursors (bone marrow-derived macrophages [BMMs]). To generate osteoclasts, BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 3 d. Cultured cells were fixed and stained for TRAP. TRAP-positive multinuclear cells (TRAP+ mononuclear cells [MNCs]) containing more than three nuclei were counted as osteoclasts. Retroviral infection To generate retroviral stocks, retroviral vectors were transfected into the packaging cell line Plat E using FuGENE 6 (Roche Applied Sciences). Viral supernatant was collected from culture media 24–48 h after transfection. BMMs were incubated with viral supernatant for 8 h in the presence of FIGURE 1. PI3K plays a role in RANKL-induced osteoclastogenesis. A polybrene (10 mg/ml). After removing the viral supernatant, BMMs were and B, BMMs were derived from bone marrow cells by culturing for 3 d in cultured further with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 3 d. the presence of M-CSF. BMMs were cultured for an additional 3 d in the Fractionation and Western blot analysis presence of M-CSF and RANKL with increasing concentrations (0.5–2.5 mM) of LY294002, an inhibitor of PI3K, as indicated. A, Cultured cells Cultured cells were harvested after washing with ice-cold PBS and then were fixed and stained for TRAP. Original magnification 3100. B, TRAP+ lysed in extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MNCs were counted as osteoclasts. *p , 0.01, **p , 0.001 versus pos- EDTA, 0.5% Nonidet P-40, 0.01% protease inhibitor mixture). Cells were itive control. C, BMMs were starved and pretreated with 2.5 mM fractionated using Nuclear and Cytoplasmic Extraction Reagents (Pierce) according to the manufacturer’s protocol. Cytoplasmic and nuclear extracts LY294002 or DMSO (vehicle) for 2 h before exposure to RANKL for the were subjected to SDS-PAGE and Western blotting. Primary Abs used indicated times. D and E, BMMs were cultured with M-CSF and RANKL included NFATc1 (Santa Cruz), OSCAR, c-Fos (Calbiochem), IkB, in the presence of 2.5 mM LY294002 or DMSO for the indicated times. phospho-p38, p38, phospho-JNK, JNK, phospho-Akt, Akt, phospho- C–E, Whole cell extracts were subjected to Western blot analysis with GSK3b, GSK3b (Cell Signaling Technology), actin (Sigma-Aldrich), and specific Abs as indicated. The Journal of Immunology 165 strongly blocked RANKL-induced Akt activation, whereas other signaling pathways such as JNK, p38, and NF-kB were not affected by LY294002 (Fig. 1C). Thus, our data confirmed that LY294002 could specifically inhibit PI3K/Akt pathway among RANKL-induced early signaling pathways and that PI3K is important for RANKL-induced osteoclastogenesis. To identify how PI3K regulates osteoclast differentiation, we examined the gene expression patterns during osteoclastogenesis. The expression of c-Fos, an AP-1 family member, was induced after 12 h exposure to RANKL and strongly increased till 24 h (Fig. 1D). This RANKL-induced expression of c-Fos was not affected by treatment with LY294002. However, NFATc1 induction mediated by RANKL was strongly attenuated by blockage of PI3K (Fig. 1E). We also observed the downregulation of OSCAR, an osteoclast-specific gene, by treatment with LY294002. As a result, PI3K can regulate NFATc1 gene expression, not c-Fos, during RANKL-induced osteoclastogenesis.

PI3K/Akt signaling cascade regulates NFATc1 expression Downloaded from during RANKL-induced osteoclastogenesis To investigate the role of Akt in osteoclast differentiation, we overexpressed constitutively active Akt (Ca-Akt) in BMMs using retrovirus. Ectopic expression of Ca-Akt alone did not induce osteoclast differentiation in the presence of M-CSF (Fig. 2A, upper

panel). However, RANKL-induced osteoclast formation was sig- http://www.jimmunol.org/ nificantly enhanced by overexpression of Akt (Fig. 2A,2B). We have demonstrated that LY294002 treatment blocks Akt activation and attenuates RANKL-induced osteoclastogenesis. FIGURE 2. The PI3K/Akt signaling cascade regulates NFATc1 expression Therefore, we examined whether overexpression of Akt could during RANKL-induced osteoclastogenesis. A and B, BMMs were transduced rescue osteoclast formation in LY294002-treated cells. LY294002 with pMX-IRES-EGFP (control) or retrovirus containing sequence for a con- + treatment reduced the formation of TRAP MNCs from BMMs stitutively active form of Akt (Ca-Akt). Cells were cultured for 3 d with M- in empty vector-infected cells (Fig. 2C,2D). Retroviral over- CSF and in the absence or presence of RANKL. A, Cultured cells were fixed expression of Akt, however, overcame the inhibitory effect of and stained for TRAP. Original magnification 3100. B,TRAP+ MNCs were LY294002 on osteoclastogenesis. These data suggest that a PI3K/ counted as osteoclasts. **p , 0.001 versus positive control. C and D,BMMs by guest on September 25, 2021 Akt axis is important for RANKL-induced osteoclast formation. were transduced with pMX-IRES-EGFP (control) or Ca-Akt retrovirus and Next, we investigated which genes can be regulated by over- cultured for 3 d in the presence of M-CSF and RANKL with increasing m expression of Akt during osteoclastogenesis. RANKL-mediated c- concentrations (0–2.5 M) of LY294002 as indicated. C, Cultured cells were fixed and stained for TRAP. Original magnification 3100. D,TRAP+ MNCs Fos induction was comparable in empty vector- and Akt-infected were counted as osteoclasts. #p , 0.05, **p , 0.001 versus positive control. E samples (Fig. 2E). However, overexpression of Akt strongly in- and F, BMMs were transduced with pMX-IRES-EGFP (control) or Ca-Akt creased the expression of NFATc1 and OSCAR compared with retrovirus and cultured for the indicated times. Whole cell extracts were control (Fig. 2F). Collectively, the PI3K/Akt signaling cascade subjected to Western blot analysis with specific Abs as indicated. plays a role in RANKL-induced osteoclastogenesis via regulating NFATc1 expression without affecting expression of c-Fos. than control vector-infected osteoclasts (Fig. 3C, lower panel). Overexpression of Akt enhances phosphorylation of GSK3b When we examined the localization of NFATc1 in osteoclasts, and nuclear localization of NFATc1 overexpressed Akt strongly induced enrichment of NFATc1 in the nucleus region of osteoclasts, rather than in cytoplasmic region b It has been shown that GSK3 is one of the known downstream (Fig. 3D). Our data suggest that Akt can enhance the level of b targets of Akt and that GSK3 enhances nuclear export of NFAT GSK3b phosphorylation and nuclear localization of NFATc1 b proteins (14, 15). Therefore, we hypothesized that GSK3 might during RANKL-induced osteoclastogenesis. have a role in Akt-mediated NFATc1 induction during osteoclastogenesis. When Akt was transfected into 293T cells, overex- b pressed Akt increased the level of phosphorylated GSK3b, Overexpression of GSK3 attenuates osteoclast formation whereas total level of GSK3b was not changed (Fig. 3A). To in- through downregulation of NFATc1 vestigate whether Akt regulates GSK3b in osteoclast cells, we first To investigate the effect of GSK3b on RANKL-induced osteo- examined the effect of Akt on the phosphorylation level of clastogenesis, we used the retroviral vector expressing the non- GSK3b in BMMs. RANKL stimulation induced phosphoryla- phosphorylatable constitutively active mutant of GSK3b (GSK3b- tion of GSK3b in 10 min, and the level of RANKL-induced S9A) where the serine residue at position 9 was mutated to GSK3b phosphorylation was further enhanced by overexpres- alanine. Compared with control vector-infected samples, over- sion of Akt (Fig. 3B). Next, we compared the status of GSK3b expression of GSK3b-S9A significantly attenuated RANKL- phosphorylation in empty vector-infected and Akt-overexpressed induced osteoclast differentiation (Fig. 4A,4B). osteoclasts. Compared with control, the ratio of phosphorylated When we examined the NFATc1 expression by Western blot GSK3b/GSK3b was strongly increased by overexpression of Akt analysis, overexpression of GSK3b-S9A downregulated RANKL- in osteoclasts (Fig. 3C, middle panel). We could observe the induced NFATc1 expression in osteoclasts (Fig. 4C, lower panel). stronger induction of NFATc1 in Akt-overexpressed osteoclasts By fractionation analysis, we confirmed that overexpression of 166 ROLE OF THE Akt/GSK3b/NFATc1 AXIS DURING OSTEOCLASTOGENESIS Downloaded from

FIGURE 3. Overexpression of Akt enhances phosphorylation of GSK3b and nuclear localization of NFATc1. A, 293T cells were transfected with empty vector (Mock) or Akt. B, BMMs were transduced with pMX-IRES- http://www.jimmunol.org/ EGFP (control) or Ca-Akt retrovirus. Cells were starved and stimulated with RANKL for the indicated times. C, BMMs were transduced with FIGURE 4. Overexpression of GSK3b attenuates osteoclast formation pMX-IRES-EGFP (control) or Ca-Akt retrovirus and cultured for 2 d with through downregulation of NFATc1. BMMs were transduced with pMX- M-CSF and RANKL. A–C, Whole cell extracts were subjected to Western IRES-EGFP (control) or retrovirus containing sequence for a constitutively blot analysis with specific Abs as indicated. D, BMMs were transduced active form of GSK3b (GSK3b-S9A). Cells were cultured for 3 d in the with pMX-IRES-EGFP (control) or Ca-Akt retrovirus and cultured for 2 d presence of M-CSF with increasing concentrations of RANKL as indi- with M-CSF and RANKL. Whole cell extracts, cytoplasmic fractions, and cated. A, Cultured cells were fixed and stained for TRAP. Original mag- nuclear fractions were harvested from cultured cells and subjected to nification 3100. B, TRAP+ MNCs were counted as osteoclasts. *p , 0.01 Western blot analysis with specific Abs as indicated. Abs for actin and versus positive control. C, BMMs were transduced with pMX-IRES-EGFP lamin B1 were used for the normalization of cytoplasmic and nuclear (control) or GSK3b-S9A retrovirus and cultured for 2 d with M-CSF and by guest on September 25, 2021 extracts, respectively. RANKL. Whole cell extracts, cytoplasmic fractions, and nuclear fractions were harvested from cultured cells and subjected to Western blot analysis b with specific Abs as indicated. Abs for actin and lamin B1 were used for GSK3 -S9A strongly induced nuclear export of NFATc1 in the normalization of cytoplasmic and nuclear extracts, respectively. D, osteoclasts (Fig. 4C, upper panel). 293T cells were transfected with an OSCAR reporter construct, NFATc1, Next, we investigated the effect of GSK3b on transcriptional WT GSK3b (GSK-WT), and a kinase-inactive form of GSK3b (GSK-KD) activity of NFATc1 using a reporter assay. Consistent with pre- as indicated. Luciferase activity was measured using a dual-luciferase vious results (19, 25), cotransfection of the OSCAR reporter reporter assay system. Data represent means 6 SDs of triplicate samples, construct and NFATc1 resulted in approximately a 4-fold increase and the results shown are representative of at least three independent sets in promoter activity (Fig. 4D). This NFATc1-mediated trans- of similar experiments. activation of OSCAR was strongly attenuated by wild type of GSK3b (GSK3b-WT), but not by a kinase-inactive mutant upper panel). We could also observe that RANKL induced more GSK3b (GSK3b-KD). Collectively, these data suggest that phosphorylation of GSK3b in SHIP KO mice than that in WT GSK3b attenuates RANKL-induced osteoclast differentiation by littermates (Fig. 5D, middle panel). When we examined the ex- downregulation of NFATc1. pression patterns of important genes such as c-Fos and NFATc1 during osteoclastogenesis, RANKL-mediated c-Fos induction was Deficiency of SHIP enhances osteoclastogenesis through comparable in WT and SHIP KO mice (Fig. 5E). However, in- activating the Akt/GSK3b/NFATc1 signaling cascade duction of NFATc1 mediated by RANKL was stronger in SHIP It has been shown that SHIP negatively regulates growth factor KO mice than in WT littermates (Fig. 5F). Our data suggest that receptor-mediated Akt activation in hematopoietic cells (9). deficiency of SHIP enhances osteoclastogenesis through activating Therefore, we investigated whether SHIP could regulate the Akt/ the Akt/GSK3b/NFATc1 signaling cascade. GSK3b/NFATc1 signaling cascade during RANKL-induced os- b teoclast differentiation. We first examined the physiologic role of Overexpression of GSK3 attenuates osteoclast formation via SHIP in osteoclast formation using SHIP knockout (KO) mice. downregulation of NFATc1 in SHIP KO mice Consistent with previous results (10), the formation of TRAP+ Given our observation that inactivation of GSK3b was strongly MNCs was significantly increased in SHIP KO mice compared induced by RANKL in BMMs from SHIP KO mice, we tested with WT littermates (Fig. 5A,5B). The RANKL-induced early whether a constitutively active form of GSK3b (GSK3b-S9A) signaling pathways such as activation of JNK, p38, and NF-kB could attenuate osteoclast formation through downregulation of were comparable in WT and SHIP KO mice (Fig. 5C). However, NFATc1 in SHIP KO mice. BMMs from SHIP KO mice were Akt activation mediated by RANKL in BMMs from SHIP KO transduced with control or GSK3b-S9A retrovirus and cultured mice was much stronger than that in BMMs from WT (Fig. 5D, with M-CSF and RANKL for 3 d. Compared with controls, The Journal of Immunology 167 Downloaded from http://www.jimmunol.org/

FIGURE 6. Overexpression of GSK3b attenuates osteoclast formation via downregulation of NFATc1 in SHIP KO mice. A and B, BMMs from SHIP KO mice were transduced with pMX-IRES-EGFP (control) or FIGURE 5. Deficiency of SHIP enhances osteoclastogenesis through GSK3b-S9A retrovirus and cultured for 3 d with M-CSF and RANKL. A, activating the Akt/GSK3b/NFATc1 signaling cascade. A and B, BMMs Cultured cells were fixed and stained for TRAP. Original magnification from WT or SHIP knockout mice were cultured for 3 d in the presence of 3100. B, TRAP+ MNCs were counted as osteoclasts. #p , 0.05, *p , 0.01 M-CSF with increasing concentrations of RANKL as indicated. A, Cul- versus positive control. C–E, BMMs from SHIP KO mice were transduced tured cells were fixed and stained for TRAP. Original magnification 3100. with pMX-IRES-EGFP (control) or GSK3b-S9A retrovirus and cultured B, TRAP+ MNCs were counted as osteoclasts. *p , 0.01, **p , 0.001 with M-CSF and RANKL for the indicated times. Whole cell extracts, by guest on September 25, 2021 versus positive control. C and D, BMMs from WT or SHIP KO mice were cytoplasmic fractions, and nuclear fractions were harvested from cultured starved in 0.5% FBS for 4 h and stimulated with RANKL for the indicated cells and subjected to Western blot analysis with specific Abs as indicated. times. Whole cell extracts were subjected to Western blot analysis with Abs for actin and lamin B1 were used for the normalization of cytoplasmic specific Abs as indicated. E and F, BMMs from WT or SHIP knockout and nuclear extracts, respectively. mice were cultured with M-CSF and RANKL for the indicated times. Whole cell extracts were subjected to Western blot analysis with specific Abs as indicated. mura et al. (28) showed that wortmannin, an irreversible PI3K inhibitor, inhibits ruffled border and actin ring formation and thus overexpression of GSK3b-S9A in BMMs strongly attenuated reduces osteoclastic bone resorption. Although previous studies RANKL-induced osteoclast formation (Fig. 6A,6B). Next, we have confirmed the crucial role of PI3K/Akt signaling pathway in compared the expression levels of c-Fos and NFATc1 in both osteoclastogenesis, the precise mechanism of how Akt regulates samples during osteoclastogenesis. As shown in Fig. 6C, the ex- the differentiation of osteoclasts has remained elusive. However, pression levels of c-Fos are comparable in control vector- or several studies have revealed that GSK3b is a major effector of GSK3b-S9A–infected samples from SHIP KO mice. Over- Akt and NFATc1 is a direct substrate of GSK3b (14, 15). Given expression of GSK3b-S9A, however, strongly downregulated the direct link among Akt, GSK3b, and NFATc1, we reasoned that expression of NFATc1 and OSCAR (Fig. 6D). When we examined the Akt/GSK3b/NFATc1 pathway may also pertain to osteoclasts the localization of NFATc1 in osteoclasts, overexpression of and that Akt would induce osteoclasts differentiation through this GSK3b-S9A strongly induced the export of NFATc1 from the pathway, which proved to be the case. nucleus (Fig. 6E). Collectively, our data suggest that over- PI3K primarily generates PIP3 after activation of receptors for b expression of an active form of GSK3 attenuates osteoclast cytokines and growth factors. PIP3 is a major modulator of cell formation via downregulation of NFATc1 in SHIP KO mice. functions and as such is an important point where many physiologic effects diverge. Thus, the signaling capacity of this molecule Discussion needs to be tightly controlled. This is done through the action of In osteoclasts, PI3K/Akt signaling cascade is activated as a critical several PIP3 phosphatases such as PTEN (phosphatase and tensin downstream effector from at least three cell surface receptors, homolog deleted on chromosome 10, a 39-phosphatase) and c-fms, aVb3 integrin, and RANK (13, 26, 27). The effector actions two 59-phosphatases, SHIP and SHIP2. SHIP is predominantly of PI3K/Akt signaling pathway are diverse; however, the pathway expressed in hematopoietic cells, and it blunts PI3K-initiated is mostly underscored in regulating osteoclasts differentiation and signaling pathway. Notably, Takeshita et al. (10) showed that the function. Lee et al. (12) demonstrated that PI3K inhibition by its attenuated PIP3 degradation in SHIP deficiency leads to enhanced specific inhibitor LY294002 dramatically reduces RANKL/M- precursor proliferation, more robust differentiation of osteoclasts, CSF-dependent osteoclastogenesis. Consistent with this, Naka- and thus severe osteoporosis. The enhanced osteoclastogenesis 168 ROLE OF THE Akt/GSK3b/NFATc1 AXIS DURING OSTEOCLASTOGENESIS of SHIP insufficiency was repeated in our study. Moreover, the itor, was shown to antagonize the nuclear export of NFATc (34). experiments revealed that the accelerated osteoclasts differentia- Several other studies with murine primary T cells and bone tion in SHIP2/2 was mediated by the hyperactivation of Akt and marrow-derived mast cells also support the notion that GSK3b is consequential inactivation of GSK3b. These results were reca- a potential regulator of NFATc1 (35, 36). In fact, our results also pitulated at the level of NFATc1 expression where SHIP2/2 provide several lines of evidence to suggest GSK3b-dependent showed increased expression level. Consistent with these findings, regulation of NFATc1 in osteoclasts. The overexpression of the PTEN mutant with no phosphatase activity also has been reported constitutively active form of GSK3b (GSK3b-S9A) dramatically to increase RANKL-induced osteoclastogenesis (29). Ultimately, inhibited osteoclast formation through directly reducing the nu- these studies implicate that the net activity of lipid kinases and clear localization of NFATc1. GSK3b-S9A showed the same phosphatase is an important regulatory factor in determining the inhibitory effect on SHIP2/2 osteoclasts, where GSK3b-S9A degree of osteoclasts differentiation, at least, through regulating promoted nuclear exit of NFATc1 and thus reducing osteoclasts Akt/GSK3b/NFATc1 axis. formation. Recently, it has been shown that transgenic mice There are three Akt family members—Akt1, Akt2, and Akt3. expressing the GSK3b-S9A mutant show an osteopetrotic phe- Akt1 and Akt2, but not Akt3, are abundantly expressed in both notype because of impaired osteoclast differentiation. Further- bone cells: osteoblasts and osteoclasts (30). Although single KO more, osteoclast precursor cells from the transgenic mice showed mice of Akt isoform showed a mild phenotype, double KO mice defects in expression and nuclear localization of NFATc1 (37). of Akt1/Akt2 showed severely impaired bone development and These collective observations show that GSK3b has an important dwarfism (31). Kawamura et al. (30) reported that Akt1 is a crucial role in the regulation of NFATc1 during osteoclastogenesis. regulator of osteoblast and osteoclast by promoting their differ- The classic NFATc1 activation pathway requires an elevation of Downloaded from entiation and survival. They showed that Akt1 deficiency caused the intracellular calcium concentration. The calcium signals then impairment of bone resorption via cell autonomous dysfunction activate calcium/calmodulin-dependent protein phosphatase cal- in osteoclasts and the cell nonautonomous inhibition of osteo- cineurin, which in turn dephosphorylates cytosolic NFATc1 leading clastogenesis because of reduced RANKL expression in osteo- to nuclear translocation. However, a number of protein kinases blasts. Sugatani et al. (32) showed that knockdown of Akt1 and oppose calcineurin-mediated activation of NFATc1 by directly

Akt2 inhibited osteoclast differentiation because of downregula- phosphorylating and promoting nuclear exit of NFATc1. This http://www.jimmunol.org/ tion of RANKL-induced NF-kB p50 DNA binding activity. In this regulatory interplay between calcineurin and various NFATc1 study, however, PI3K inhibition by LY294002 abrogated RANKL- kinases determines the subcellular localization of NFATc1. In our induced Akt activation and phosphorylation of GSK3b (Supple- study, however, GSK3b appeared to be an important NFATc1 mental Fig. 1), but not other signaling pathways including NF-kB kinase in osteoclasts. GSK3b efficiently translocated NFATc1 activation. We also observed the RANKL-induced hyperactivation from the nucleus of both WT and SHIP2/2 osteoclasts, thus re- of Akt and phosphorylation of GSK3b in SHIP KO osteoclasts, sulting in reduced osteoclasts formation. However, given the fact although activation of other signaling pathways such as NF-kB, that there are large numbers of phosphorylation sites on the p38, and JNK is comparable to WT osteoclasts. These data suggest NFATc1 protein (38), it is possible that other NFATc1 kinases that PI3K/Akt regulates osteoclast differentiation primarily through besides GSK3b participate in the regulation of NFATc1 locali- by guest on September 25, 2021 GSK3b rather than the NF-kB signaling cascade. zation in osteoclasts. The more detailed analysis of the regulatory GSK3b is a critical downstream effector of the PI3K/Akt sig- network of NFATc1 may help us to better understand the kinase- naling pathway. GSK3b is ubiquitously expressed and constitu- phosphatase–mediated regulation of NFATc1 localization. tively active in resting cells. Thus, it is regulated through In this study, we demonstrated that Akt induces osteoclasto- inhibition of its activity by Akt-mediated phosphorylation at ser- genesis through GSK3b/NFATc1 signaling cascade. Furthermore, ine 9 (14). The wide distribution of GSK3b in all cell types our data revealed insights into the regulation of NFATc1 in confers a Akt/GSK3b signaling pathway capable of controlling osteoclasts, in which GSK3b regulated NFATc1 in the phos- diverse cellular functions such as cell cycle regulation, glycogen phorylation-dependent manner. However, future studies with and protein synthesis regulation, and transcription factor modu- GSK3b transgenic mice or GSK3b conditional KO mice will lation (33). Whether the pathway is also involved in the regulation clarify and extend our understanding of the role of GSK3b and of osteoclasts differentiation and functions has not been eluci- its regulation of NFATc1 nuclear presence in osteoclasts. dated; however, our observation that the overexpression of constitutively active form of Akt increased the expression of phospho- GSK3b suggests that GSK3b is a bona fide target of Akt in Acknowledgments osteoclasts. We thank T. Kitamura for Plat E cells. Although GSK3b is an important downstream target of Akt, we did not observe complete abrogation of RANKL-induced osteo- Disclosures clastogenesis by overexpression of a constitutively active form of The authors have no financial conflicts of interest. GSK3b in BMMs from SHIP KO mice. This finding could be the result of imperfect retroviral infection efficiency, because we have References demonstrated 50–70% infection in BMMs. Another possibility is 1. Boyle, W. J., W. S. Simonet, and D. L. Lacey. 2003. Osteoclast differentiation that other kinases have a role in the Akt-NFATc1 signaling axis in and activation. Nature 423: 337–342. osteoclasts. 2. Suda, T., N. Takahashi, N. Udagawa, E. Jimi, M. T. Gillespie, and T. J. Martin. b 1999. Modulation of osteoclast differentiation and function by the new members of GSK3 has been shown to regulate NFATc1 signaling pathway the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20: 345–357. as demonstrated in several independent studies. Beals et al. (15) 3. Walsh, M. C., N. Kim, Y. Kadono, J. Rho, S. Y. Lee, J. Lorenzo, and Y. Choi. identified GSK3b as the principal cellular kinase responsible 2006. Osteoimmunology: interplay between the immune system and bone metabolism. Annu. Rev. Immunol. 24: 33–63. for the phosphorylation of NFATc at conserved serine residues 4. Kodama, H., M. Nose, S. Niida, and A. Yamasaki. 1991. Essential role of in COS cells. Upon phosphorylation by GSK3b, NFATc was macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells. J. Exp. Med. 173: 1291–1294. exported from the nucleus and its transcriptional activity was 5. Kong, Y. Y., H. Yoshida, I. Sarosi, H. L. Tan, E. Timms, C. Capparelli, ceased. However, LiCl treatment, which acts as a GSK3b inhib- S. Morony, A. J. Oliveira-dos-Santos, G. Van, A. Itie, et al. 1999. OPGL is a key The Journal of Immunology 169

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