Homer2 and Homer3 Modulate RANKL-Induced Nfatc1 Signaling in Osteoclastogenesis and Bone Metabolism
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Roles of Homer2/3 in 242:3 241–249 Endocrinology osteoclastogenesis RESEARCH Homer2 and Homer3 modulate RANKL-induced NFATc1 signaling in osteoclastogenesis and bone metabolism Aran Son1,*, Namju Kang1,2,*, Sue Young Oh1, Ki Woo Kim1, Shmuel Muallem3, Yu-Mi Yang1 and Dong Min Shin1,2 1Department of Oral Biology, Yonsei University College of Dentistry, Seoul, Korea 2BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Korea 3Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA Correspondence should be addressed to Y-M Yang or D M Shin: [email protected] or [email protected] *(A Son and N Kang contributed equally to this work) Abstract The receptor activator of nuclear factor-kappa B ligand (RANKL) induces Key Words osteoclastogenesis by induction of Ca2+ oscillation, calcineurin activation and f RANKL translocation into the nucleus of nuclear factor of activated T cells type c1 (NFATc1). f osteoclast differentiation Homer proteins are scaffold proteins. They regulate Ca2+ signaling by modulating f scaffold protein the activity of multiple Ca2+ signaling proteins. Homers 2 and 3, but not Homer1, also f osteoporosis independently affect the interaction between NFATc1 and calcineurin. However, to date, f NFATc1 whether and how the Homers are involved in osteoclastogenesis remains unknown. In the present study, we investigated Homer2 and Homer3 roles in Ca2+ signaling and NFATc1 function during osteoclast differentiation. Deletion of Homer2/Homer3 (Homer2/3) markedly decreased the bone density of the tibia, resulting in bone erosion. RANKL-induced osteoclast differentiation is greatly facilitated in Homer2/3 DKO bone marrow-derived monocytes/macrophages (BMMs) due to increased NFATc1 expression and nuclear translocation. However, these findings did not alter RANKL-induced Ca2+ oscillations. Of note, RANKL treatment inhibited Homer proteins interaction with NFATc1, but it was restored by cyclosporine A treatment to inhibit calcineurin. Finally, RANKL treatment of Homer2/3 DKO BMMs significantly increased the formation of multinucleated cells. These findings suggest a novel potent mode of bone homeostasis regulation through osteoclasts differentiation. Specifically, we found that Homer2 and Homer3 regulate NFATc1 function through its interaction with calcineurin to regulate Journal of Endocrinology RANKL-induced osteoclastogenesis and bone metabolism. (2019) 242, 241–249 Introduction Bone is a dynamic organ. It is constantly renewing through can be found. Examples are autoimmune arthritis, a bone remodeling process, mediated by osteoblastic osteoporosis, periodontitis, osteopetrosis and bone bone formation and osteoclastic bone resorption. In tumors (Takayanagi 2007, Zaidi 2007). Multinucleated numerous bone diseases, an imbalance in this process mature osteoclasts originate from bone marrow-derived https://joe.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/JOE-19-0123 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 07:42:04PM via free access -19-0123 Journal of A Son, N Kang et al. Roles of Homer2/3 in 242:3 242 Endocrinology osteoclastogenesis monocytes/macrophages (BMMs) through two essential (Brakeman et al. 1997, Xiao et al. 1998, Bottai et al. 2002). factors. One is receptor activator of nuclear factor- Homer1a predominantly consists of the N-terminal kappa B ligand (RANKL) secreted by osteoblasts. The Ena/VASP homology 1 (EVH) protein-binding domain. other is macrophage-colony-stimulating factor (M-CSF) The remaining Homer proteins (i.e., Homer1b/c, Homer2 (Takayanagi 2007). RANK receptor’s activation by RANKL and Homer3, referred to as ‘long Homers’) are composed leads to oscillations in intracellular Ca2+ concentration of an EVH domain and a C-terminus. The latter includes 2+ ([Ca ]i). The latter is linked to the activation of Gq. In a coiled-coil multimerization domain and leucine zipper turn, Gq generates inositol 1,4,5-triphosphate (IP3) to (Tu et al. 1998, Worley et al. 2007). Of note, the EVH 2+ activate the IP3 receptors (IP3Rs), Ca release from the ER domain binds to several G protein coupled-receptors and activation of Ca2+ influx from the extracellular space (GPCRs). Specifically, these are as follows: canonical by the Orai channels and transient receptor potential TRP (TRPC) channels, IP3Rs, ryanodine receptors (RyRs) (TRP) channels. Ultimately, as previously reported, this and the Shank family of scaffold proteins (Tu et al. generates an increase in intracellular reactive oxygen 1998, 1999, Xiao et al. 1998, Feng et al. 2002, Shin species (Yang et al. 2009, 2013, Kim et al. 2010, Son et al. 2003, Yuan et al. 2003). Homer proteins were et al. 2012, Park et al. 2013). Subsequently, the RANKL- previously identified as synaptic proteins regulating 2+ mediated [Ca ]i oscillations increase NFATc1’s nuclear synaptic activity in neurons. However, earlier studies translocation, multinucleated cells’ formation and bone have reported on the importance of the Homer family resorption (Takayanagi et al. 2002, Yang et al. 2009, Kim in other tissues (Brakeman et al. 1997, Tu et al. 1998, et al. 2010). 1999, Xiao et al. 1998). In previous studies, it has been Members of the NFAT family of transcription factors shown that Homer2 modulates the intensity of the play a central role in immune responses and in the GPCRs stimulus. Of note, this is done by regulating the development of cardiac and skeletal muscles, bone and the GAP activity of RGS proteins and of PLCβ in pancreas nervous system. In these tissues, the NFAT family members acinar cells (Shin et al. 2003). In the skeletal muscle, are expressed in most cells. The NFAT family consists of Homer2 enhances the NFATc1-dependent signaling five members (NFATc1-5). NFATc1 through NFATc4 are pathway by increasing the release of RyR-dependent regulated by Ca2+ signaling. Specifically, Ca2+ activates Ca2+ during myogenic differentiation (Stiber et al. calcineurin by calmodulin. This dephosphorylates NFAT 2005). It has been demonstrated that Homer3’s deletion to expose its nuclear import signal (Olson & Williams induces severe autoimmune phenotypes in the lung and 2000, Crabtree & Olson 2002, Hogan et al. 2003, liver. Additionally, Homer3’s phosphorylation by the Takayanagi 2007). Ca2+/calmodulin-dependent protein kinase (CaMK) II Ca2+ is a universal intracellular messenger that is regulates Purkinje cells synaptic activity (Huang et al. involved in various cellular functions and processes, 2008, Mizutani et al. 2008). Of note, Homer2 and Homer3 such as cell proliferation, differentiation and apoptosis have been shown to regulate T cell activation through (Bootman et al. 2001, Berridge et al. 2003). Disruption the interaction with NFATc2. Specifically, this is done of Ca2+ signaling is one mechanism by which RANKL by competing with calcineurin to bind with NFATc2 function, NFATc1 activation and the associated bone (Huang et al. 2008). Additionally, Homer2 identified metabolism can be altered. An additional less explored a co-localization with NFATc1 at the neuromuscular mechanism NFAT function’s regulation is through their junction. The latter is part of the calcineurin-NFATc1 interaction with adapter proteins. As for example SH3 signaling pathway (Salanova et al. 2011). However, to domain-binding protein 2 (3BP2), NFAT-interacting date, there is a poor understanding of the characteristics protein (NIP45) and most prominently the Homer of the RANKL-induced NFATc1 signaling pathway during proteins (Takayanagi et al. 2002, Foucault et al. 2005, osteoclastogenesis and whether and how it is regulated Huang et al. 2008, GuezGuez et al. 2010, Shanmugarajan by Homer proteins. et al. 2012). The Homer family of scaffolding proteins In the present study, we investigated the role of consists of Homer1, Homer2 and Homer3 and several Homer proteins in the RANKL-induced NFATc1 signaling splice variants. The short Homer1a’s gene coding was pathway, osteoclast differentiation and bone metabolism identified as an immediate early gene transcriptionally with the use of Homer2 and Homer3 (Homer2/3) upregulated by synaptic activity in neuronal cells double-knockout (DKO) mice. https://joe.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/JOE-19-0123 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 07:42:04PM via free access Research Journal of A Son, N Kang et al. Roles of Homer2/3 in 242:3 243 Endocrinology osteoclastogenesis Materials and methods male mice were injected twice with calcein (15 mg/kg intraperitoneally) 2 days apart. The animals were killed Cell culture and reagents on day 4, and undecalcified bones were embedded in methylmethacrylate. Longitudinal sections (10 m The Homer2/3 DKO mice were generously provided μ thickness) of the tibias were prepared, and new bone by Dr Paul F Worley (Johns Hopkins University School formation was assessed by confocal microscopy (model of Medicine, Baltimore, MD) (Huang et al. 2008) and LSM 510; Carl Zeiss) by capturing images of calcein green. all animal care and experimental procedures complied with institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) 2+ [Ca ]i measurement in Yonsei University (IACUC approval no. 2014-0067). The cells were seeded