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Plcγ2 Regulates Osteoclastogenesis Via Its Interaction with ITAM Proteins and GAB2 Dailing Mao Washington University School of Medicine in St

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Plcγ2 Regulates Osteoclastogenesis Via Its Interaction with ITAM Proteins and GAB2 Dailing Mao Washington University School of Medicine in St Washington University School of Medicine Digital Commons@Becker Open Access Publications 2006 PLCγ2 regulates osteoclastogenesis via its interaction with ITAM proteins and GAB2 Dailing Mao Washington University School of Medicine in St. Louis Holly Epple Washington University School of Medicine in St. Louis Brian Uthgenannt Washington University School of Medicine in St. Louis Deborah V. Novack Washington University School of Medicine in St. Louis Roberta Faccio Washington University School of Medicine in St. Louis Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Mao, Dailing; Epple, Holly; Uthgenannt, Brian; Novack, Deborah V.; and Faccio, Roberta, ,"PLCγ2 regulates osteoclastogenesis via its interaction with ITAM proteins and GAB2." The ourJ nal of Clinical Investigation.,. 2869-2879. (2006). https://digitalcommons.wustl.edu/open_access_pubs/1551 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Downloaded on August 5, 2013. The Journal of Clinical Investigation. More information at www.jci.org/articles/view/28775 Research article PLCγ2 regulates osteoclastogenesis via its interaction with ITAM proteins and GAB2 Dailing Mao,1 Holly Epple,2 Brian Uthgenannt,1 Deborah V. Novack,3 and Roberta Faccio1 1Department of Orthopaedic Surgery and Cell Biology, 2Department of Medicine and Cell Biology, and 3Department of Medicine, Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri, USA. Excessive bone loss in arthritic diseases is mostly due to abnormal activation of the immune system leading to stimulation of osteoclasts. While phospholipase Cγ (PLCγ) isoforms are known modulators of T and B lympho- cyte–mediated immune responses, we found that blockade of PLCγ enzymatic activity also blocks early osteoclast development and function. Importantly, targeted deletion of Plcg2 in mice led to an osteopetrotic phenotype. PLCγ2, independent of PLCγ1, was required for receptor activator of NF-κB ligand–induced (RANKL-induced) osteoclastogenesis by differentially regulating nuclear factor of activated T cells c1 (NFATc1), activator pro- tein–1 (AP1), and NF-κB. Specifically, we show that NFATc1 upregulation is dependent on RANKL-mediated phosphorylation of PLCγ2 downstream of Dap12/Fc receptor γ (Dap12/FcRγ) receptors and is blocked by the PLCγ inhibitor U73122. In contrast, activation of JNK and NF-κB was not affected by U73122 or Dap12/FcRγ deletion. Interestingly, we found that in osteoclasts, PLCγ2 formed a complex with the regulatory adapter mol- ecule GAB2, was required for GAB2 phosphorylation, and modulated GAB2 recruitment to RANK. Thus, PLCγ2 mediates RANKL-induced osteoclastogenesis and is a potential candidate for antiresorptive therapy. Introduction nuclear factor of activated T cells c1 (NFATc1), a calcineurin- and Bone erosion is a major hallmark of rheumatoid arthritis and is calcium-regulated transcription factor, as a master transcription solely executed by the bone-resorbing cells, the osteoclasts (OCs) factor in this process (17, 18). The importance of NFATc1 in OC (1). These cells arise from macrophage precursors and differentiate development has been buttressed by the fact that NFATc1–/– embry- into the mature polykaryon after stimulation with receptor activa- onic stem cells cannot differentiate into OCs. Furthermore, over- tor of NF-κB ligand (RANKL) and M-CSF (2). OCs are recruited expression of the WT protein stimulates OC development from to sites of inflammation or differentiate at these sites owing to embryonic stem cells in a RANKL-independent manner (18). elevated levels of local RANKL secreted by cells in the inflamed Interestingly, the ability of NFATc1 to mediate osteoclastogenesis tissue. Levels of TNF-α, IL-1, and M-CSF are also elevated during is strictly connected to the c-Jun pathway (19). Transgenic mice inflammatory responses and they collaborate with RANKL to fur- expressing a dominant negative form of c-Jun have osteopetro- ther increase the rate of OC formation and bone resorption (3–6). sis due to defective osteoclastogenesis that cannot be rescued by Binding of RANKL to receptor activator of NF-κB (RANK) leads NFATc1 overexpression in OCs (19). to receptor trimerization, followed by the recruitment of TRAF6, Immune receptor tyrosine activation motifs (ITAMs) are impor- which acts as a key adapter to assemble signaling proteins that tant signaling components for a number of receptors in the immune direct OC-specific gene expression (2). Both NF-κB and activa- system, and they have been recently identified as costimulatory tor protein–1 (AP1) are rapidly induced in response to RANKL, signals in RANKL-induced osteoclastogenesis (20). The 2 ITAM- following the activation of IκB kinase 1/2 (IKK1/2) and JNK, containing adapters, Dap12 and the γ chain of Fc receptor (FcRγ), respectively (2, 7–9). Interfering with IKK1/2 or JNK1 signaling have a profound impact on OCs by modulating RANKL-mediated pathways blocks in vitro and in vivo osteoclastogenesis (10–12). calcium influx and NFATc1 expression. In humans, mutation in Targeted mutagenesis of the p50/p52 components of NF-κB (13, the Dap12 gene is responsible for Nasu-Hakola disease, character- 14) as well as the c-Fos component of AP1 (15) results in osteo- ized by multiple bone cysts associated with a unique form of neuro- petrosis due to a block in osteoclastogenesis. Further, a recent degeneration (21). Deletion of Dap12 and FcRγ (Dap12–/–FcRγ–/–), in finding indicates that activation of JNK and NF-κB pathways mice, leads to severe osteopetrosis due to defective OC development requires the phosphorylation and the recruitment of the Grb2- (20–22). In vitro triggering of either receptor by crosslinking with binding adapter protein, GAB2, to RANK (16). Consistent with an antibody accelerates RANKL-induced OC differentiation (23). these observations, GAB2–/– mice exhibit an osteopetrotic pheno- However, in the absence of RANKL, these receptor-mediated signals type due to aberrant OC differentiation (16). alone are not sufficient to induce OC differentiation. In addition to the known roles of AP1 and NF-κB pathways in The phospholipase C (PLC) family regulates PKC activation, osteoclastogenesis, recent studies have highlighted the role of the intracellular calcium levels, and NFAT expression in hematopoi- etic systems in response to the stimulation of immune receptors Nonstandard abbreviations used: AP1, activator protein–1; BMM, BM macrophage; (24–27). PLC cleaves the membrane phospholipid phosphatidyl FcRγ, Fc receptor γ; IP3, inositol-1,4,5-triphosphate; ITAM, immune receptor tyrosine inositol-4,5-biphosphate (PIP2) into the second messenger mol- activation motif; NFATc1, nuclear factor of activated T cells c1; OB, osteoblast; OC, osteoclast; PLC, phospholipase C; RANK, receptor activator of NF-κB; RANKL, RANK ecules inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). ligand; SFK, Src family kinase; TRAP, tartrate-resistant acid phosphatase. IP3 directly increases intracellular calcium levels by inducing the Conflict of interest: The authors have declared that no conflict of interest exists. release of endoplasmic reticulum calcium stores, while DAG acti- Citation for this article: J. Clin. Invest. 116:2869–2879 (2006). doi:10.1172/JCI28775. vates PKC at the plasma membrane. The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 11 November 2006 2869 Downloaded on August 5, 2013. The Journal of Clinical Investigation. More information at www.jci.org/articles/view/28775 research article Figure 1 PLCγ inhibition blocks osteoclastogenesis and actin ring formation. (A) WT OCs were generated with RANKL (100 ng/ml) and M-CSF (10 ng/ml) in the presence of the PLC inhibitor U73122 (5 μM) for 4 days on plastic. In some wells, the media with the inhibitor was replaced with fresh osteo- clastogenic media, and cells were allowed to differentiate for 4 more days (Withdrawal U73122). (B) WT OCs were grown on dentin with or without U73122 for 10 days. Cells were then removed and pits stained with hematoxylin red (Magnification, ×200). CTR, control. (C) WT OCs were gen- erated on dentin in the absence of the inhibitor, then treated with vehicle or U73122 for 1 hour. Cells were fixed and actin stained using FITC-phalloidin (Magnification, ×200). The PLCγ family consists of 2 members, PLCγ1 and PLCγ2 block OC formation and bone resorption. Addition of U73122 to (24–26). Both isoforms require phosphorylation on specific tyro- the culture media blocked OC development, as demonstrated by sine residues for their catalytic activity. In addition, they contain the absence of tartrate-resistant acid phosphatase–positive (TRAP- 2 SH2 and 1 SH3 domains through which they can interact with positive) cells, compared with the numerous multinucleated OCs other signaling proteins, suggesting adapter function. PLCγ1 is obtained in wells cultured only with RANKL and M-CSF (Figure widely distributed, while PLCγ2 is primarily limited to cells of 1A). Replacing the media with fresh osteoclastogenic media lack- hematopoietic lineage (24). Mice deficient in PLCγ1 develop nor- ing the inhibitor restored the ability of the cells to differentiate, mally to embryonic day 8.5 but die soon after due to undefined indicating that the compound
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