BMP-induced Atoh8 attenuates osteoclastogenesis by suppressing Runx2 transcriptional activity and reducing the Rankl/Opg expression ratio in osteoblasts

Abstract

Adult bone structural integrity is maintained by remodeling via the coupling of osteoclastic bone resorption and osteoblastic bone formation. Osteocytes or osteoblasts express receptor activator of nuclear factor κ-B ligand (Rankl) or osteoprotegerin (Opg) to promote or inhibit osteoclastogenesis, respectively. Bone morphogenetic protein (BMP) is a potent bone inducer, but its major role in adult bone is to induce osteocytes to upregulate sclerostin (Sost) and increase the Rankl/Opg expression ratio, resulting in promotion of osteoclastogenesis. However, the precise effect of BMP-target gene(s) in osteoblasts on the Rankl/Opg expression ratio remains unclear. In the present study, we identified atonal homolog 8 (Atoh8), which is directly upregulated by the BMP-Smad1 axis in osteoblasts. In vivo, Atoh8 was detected in osteoblasts but not osteocytes in adult mice. Although global Atoh8-knockout mice showed only a mild phenotype in the neonate skeleton, the bone volume was decreased and osteoclasts were increased in the adult phase. Atoh8-null marrow stroma cells were more potent than wild-type cells in inducing osteoclastogenesis in marrow cells. Atoh8 loss in osteoblasts increased Runx2 expression and the Rankl/Opg expression ratio, while Runx2 knockdown normalized the Rankl/Opg expression ratio. Moreover, Atoh8 formed a protein complex with Runx2 to inhibit Runx2 transcriptional activity and decrease the Rankl/Opg expression ratio. These results suggest that bone remodeling is regulated elaborately by BMP signaling; while BMP primarily promotes bone resorption, it simultaneously induces Atoh8 to inhibit Runx2 and reduce the Rankl/Opg expression ratio in osteoblasts, suppressing osteoclastogenesis and preventing excessive BMP-mediated bone resorption.

References

[1] Suchacki KJ et al. Skeletal energy homeostasis: a paradigm of endocrine discovery. J. Endocrinol., 2017, 234:R67-R79

[2] Crockett JC, Rogers MJ, Coxon FP, Hocking LJ, Helfrich MH. Bone remodelling at a glance. J. Cell Sci., 2011, 124:991-998

[3] Sims NA, Gooi JH. Bone remodeling: multiple cellular interactions required for coupling of bone formation and resorption. Semin Cell Dev. Biol., 2008, 19:444-451

[4] Matsuo K, Irie N. Osteoclast-osteoblast communication. Arch. Biochem Biophys., 2008, 473:201-209

[5] Komori T et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell, 1997, 89:755-764

[6] Fakhry M, Hamade E, Badran B, Buchet R, Magne D. Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts. World J. Stem Cells, 2013, 5:136-148

[7] Boyce BF, Hughes DE, Wright KR, Xing L, Dai A. Recent advances in bone biology provide insight into the pathogenesis of bone diseases. Lab Investig., 1999, 79:83-94

[8] Crockett JC, Mellis DJ, Scott DI, Helfrich MH. New knowledge on critical osteoclast formation and activation pathways from study of rare genetic diseases of osteoclasts: focus on the RANK/RANKL axis. Osteoporos. Int., 2011, 22:1-20

[9] Boyce BF, Xing L. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch. Biochem. Biophys., 2008, 473:139-146

[10] Urist MR. Bone: formation by autoinduction. Science, 1965, 150:893-899

[11] Miyazono K, Maeda S, Imamura T. BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine Growth Factor Rev., 2005, 16:251-263

[12] Lee KS, Hong SH, Bae SC. Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-β and bone morphogenetic protein. Oncogene, 2002, 21:7156-7163

[13] Franceschi RT, Xiao G. Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. J. Cell Biochem., 2003, 88:446-454

[14] Afzal F et al. Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription. J. Cell Physiol., 2005, 204:63-72

[15] Biswas S et al. BMPRIA is required for osteogenic differentiation and RANKL expression in adult bone marrow mesenchymal stromal cells. Sci. Rep., 2018, 8

[16] Tachi K et al. Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts. Cell Tissue Res., 2010, 342:213-220

[17] Mishina Y et al. Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling. J. Biol. Chem., 2004, 279:27560-27566

[18] Kamiya N et al. Disruption of BMP signaling in osteoblasts through type IA receptor (BMPRIA) increases bone mass. J. Bone Min. Res., 2008, 23:2007-2017

[19] Kamiya N et al. BMP signaling negatively regulates bone mass through sclerostin by inhibiting the canonical Wnt pathway. Development, 2008, 135:3801-3811

[20] Holmen SL et al. Essential role of β-catenin in postnatal bone acquisition. J. Biol. Chem., 2005, 280:21162-21168

[21] Winkler DG et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J., 2003, 22:6267-6276

[22] Nakashima T et al. Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat. Med., 2011, 17:1231-1234

[23] Owen R, Reilly GC. In vitro models of bone remodelling and associated disorders. Front Bioeng. Biotechnol., 2018, 6:134

[24] Otsuka E, Yamaguchi A, Hirose S, Hagiwara H. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid. Am. J. Physiol., 1999, 277:C132-C138

[25] Shioi A, Ross FP, Teitelbaum SL. Enrichment of generated murine osteoclasts. Calcif. Tissue Int., 1994, 55:387-394

[26] Yamaguchi A et al. Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochem. Biophys. Res. Commun., 1996, 220:366-371

[27] Imamura K et al. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation. J. Biol. Chem., 2014, 289:9865-9879

[28] Pederson L, Ruan M, Westendorf JJ, Khosla S, Oursler MJ. Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate. Proc. Natl. Acad. Sci. USA, 2008, 105:20764-20769

[29] Mizrahi O et al. BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells. Gene Ther., 2013, 20:370-377

[30] Korchynskyi O, Ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J. Biol. Chem., 2002, 277:4883-4891

[31] Morikawa M et al. ChIP-seq reveals cell type-specific binding patterns of BMP-specific Smads and a novel binding motif. Nucleic Acids Res., 2011, 39:8712-8727

[32] Morikawa M et al. The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses and protects against the development of pulmonary arterial hypertension. Sci. Signal, 2019, 12

[33] Boing M, Brand-Saberi B, Napirei M. Murine transcription factor Math6 is a regulator of placenta development. Sci. Rep., 2018, 8

[34] Schroeder N, Wuelling M, Hoffmann D, Brand-Saberi B, Vortkamp A. Atoh8 acts as a regulator of chondrocyte proliferation and differentiation in endochondral bones. PLoS ONE, 2019, 14

[35] Kawao N, Kaji H. Interactions between muscle tissues and bone metabolism. J. Cell Biochem, 2015, 116:687-695

[36] Geoffroy V, Kneissel M, Fournier B, Boyde A, Matthias P. High bone resorption in adult aging transgenic mice overexpressing Cbfa1/Runx2 in cells of the osteoblastic lineage. Mol. Cell Biol., 2002, 22:6222-6233

[37] Enomoto H et al. Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-κ B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2−/− mice by RANKL transgene. J. Biol. Chem., 2003, 278:23971-23977

[38] Ducy P et al. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev., 1999, 13:1025-1036

[39] Harada H et al. Cbfa1 isoforms exert functional differences in osteoblast differentiation. J. Biol. Chem., 1999, 274:6972-6978

[40] Inoue C et al. Math6, a bHLH gene expressed in the developing nervous system, regulates neuronal versus glial differentiation. Genes Cells, 2001, 6:977-986

[41] Chen J et al. Diversification and molecular evolution of ATOH8, a gene encoding a bHLH transcription factor. PLoS ONE, 2011, 6

[42] Wang B, Balakrishnan-Renuka A, Napirei M, Theiss C, Brand-Saberi B. Spatiotemporal expression of Math6 during mouse embryonic development. Histochem. Cell Biol., 2015, 143:575-582

[43] Yao J et al. Atoh8, a bHLH transcription factor, is required for the development of retina and skeletal muscle in zebrafish. PLoS ONE, 2010, 5

[44] Lynn FC, Sanchez L, Gomis R, German MS, Gasa R. Identification of the bHLH factor Math6 as a novel component of the embryonic pancreas transcriptional network. PLoS ONE, 2008, 3

[45] Rawnsley DR et al. The transcription factor Atonal homolog 8 regulates Gata4 and Friend of Gata-2 during vertebrate development. J. Biol. Chem., 2013, 288:24429-24440

[46] Ejarque M et al. Generation of a conditional allele of the transcription factor Atonal homolog 8 (Atoh8). PLoS ONE, 2016, 11

[47] Balakrishnan-Renuka A et al. ATOH8, a regulator of skeletal myogenesis in the hypaxial myotome of the trunk. Histochem. Cell Biol., 2014, 141:289-300

[48] Guttsches AK et al. ATOH8: a novel marker in human muscle fiber regeneration. Histochem. Cell Biol., 2015, 143:443-452

[49] Liu W et al. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J. Cell Biol., 2001, 155:157-166

[50] Takizawa T, Ochiai W, Nakashima K, Taga T. Enhanced gene activation by Notch and BMP signaling cross-talk. Nucleic Acids Res., 2003, 31:5723-5731

[51] Itoh F et al. Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells. EMBO J., 2004, 23:541-551

[52] Seo S, Lim JW, Yellajoshyula D, Chang LW, Kroll KL. Neurogenin and NeuroD direct transcriptional targets and their regulatory enhancers. EMBO J., 2007, 26:5093-5108

[53] Bialek P et al. A twist code determines the onset of osteoblast differentiation. Dev. Cell, 2004, 6:423-435

[54] McLarren KW et al. The mammalian basic helix loop helix protein HES-1 binds to and modulates the transactivating function of the runt-related factor Cbfa1. J. Biol. Chem., 2000, 275:530-538

[55] Ejarque M, Altirriba J, Gomis R, Gasa R. Characterization of the transcriptional activity of the basic helix-loop-helix (bHLH) transcription factor Atoh8. Biochim. Biophys. Acta, 2013, 1829:1175-1183

[56] Shirakawa K et al. CCAAT/enhancer-binding protein homologous protein (CHOP) regulates osteoblast differentiation. Mol. Cell Biol., 2006, 26:6105-6116

[57] Morikawa M et al. BMP sustains embryonic stem cell self-renewal through distinct functions of different Kruppel-like factors. Stem Cell Rep., 2016, 6:64-73

Funding

MEXT | Japan Society for the Promotion of Science (JSPS)(15K10486)