Molecular mechanosensors in osteocytes

Abstract

Osteocytes, the most abundant and long-lived cells in bone, are the master regulators of bone remodeling. In addition to their functions in endocrine regulation and calcium and phosphate metabolism, osteocytes are the major responsive cells in force adaptation due to mechanical stimulation. Mechanically induced bone formation and adaptation, disuse-induced bone loss and skeletal fragility are mediated by osteocytes, which sense local mechanical cues and respond to these cues in both direct and indirect ways. The mechanotransduction process in osteocytes is a complex but exquisite regulatory process between cells and their environment, between neighboring cells, and between different functional mechanosensors in individual cells. Over the past two decades, great efforts have focused on finding various mechanosensors in osteocytes that transmit extracellular mechanical signals into osteocytes and regulate responsive gene expression. The osteocyte cytoskeleton, dendritic processes, Integrin-based focal adhesions, connexin-based intercellular junctions, primary cilium, ion channels, and extracellular matrix are the major mechanosensors in osteocytes reported so far with evidence from both in vitro and in vitro studies. This review aims to give a systematic introduction to osteocyte mechanobiology, provide details of osteocyte mechanosensors, and discuss the roles of osteocyte mechanosensitive signaling pathways in the regulation of bone homeostasis.

References

[1] Bonewald LF. The amazing osteocyte. J. Bone Min. Res., 2011, 26:229-238

[2] Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev., 2000, 21:115-137

[3] Schaffler MB, Kennedy OD. Osteocyte signaling in bone. Curr. Osteoporos. Rep., 2012, 10:118-125

[4] Schaffler MB, Cheung WY, Majeska R, Kennedy O. Osteocytes: master orchestrators of bone. Calcif. Tissue Int., 2014, 94:5-24

[5] Florencio-Silva R, Sasso GR, Sasso-Cerri E, Simoes MJ, Cerri PS. Biology of bone tissue: structure, function, and factors that influence bone cells. Biomed. Res. Int., 2015, 2015:421746

[6] Dallas SL, Prideaux M, Bonewald LF. The osteocyte: an endocrine cell… and more. Endocr. Rev., 2013, 34:658-690

[7] Han Y, You X, Xing W, Zhang Z, Zou W. Paracrine and endocrine actions of bone-the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts. Bone Res., 2018, 6:16

[8] Bonewald LF, Wacker MJ. FGF23 production by osteocytes. Pediatr. Nephrol., 2013, 28:563-568

[9] Rochefort GY, Pallu S, Benhamou CL. Osteocyte: the unrecognized side of bone tissue. Osteoporos. Int., 2010, 21:1457-1469

[10] Uda Y, Azab E, Sun N, Shi C, Pajevic PD. Osteocyte mechanobiology. Curr. Osteoporos. Rep., 2017, 15:318-325

[11] Yavropoulou MP, Yovos JG. The molecular basis of bone mechanotransduction. J. Musculoskelet. Neuronal Interact., 2016, 16:221-236

[12] Wang JH, Thampatty BP. An introductory review of cell mechanobiology. Biomech. Model Mechanobiol., 2006, 5:1-16

[13] Wolfenson H, Yang B, Sheetz MP. Steps in mechanotransduction pathways that control cell morphology. Annu Rev. Physiol., 2019, 81:585-605

[14] Wolff, J. Das Gesetz der Transformation der Knochen. (Berlin, A. Hirschwald, 1892).

[15] Frost HM. Bone “mass” and the “mechanostat”: a proposal. Anat. Rec., 1987, 219:1-9

[16] Jacobs CR, Temiyasathit S, Castillo AB. Osteocyte mechanobiology and pericellular mechanics. Annu Rev. Biomed. Eng., 2010, 12:369-400

[17] Tatsumi S et al. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab., 2007, 5:464-475

[18] Thompson WR, Rubin CT, Rubin J. Mechanical regulation of signaling pathways in bone. Gene, 2012, 503:179-193

[19] Wassermann F, Yaeger JA. Fine structure of the osteocyte capsule and of the wall of the lacunae in bone. Z. für Zellforsch. und Mikroskopische Anat., 1965, 67:636-652

[20] McNamara LM, Majeska RJ, Weinbaum S, Friedrich V, Schaffler MB. Attachment of osteocyte cell processes to the bone matrix. Anat. Rec., 2009, 292:355-363

[21] Sharma D et al. Alterations in the osteocyte lacunar-canalicular microenvironment due to estrogen deficiency. Bone, 2012, 51:488-497

[22] Wang Y, McNamara LM, Schaffler MB, Weinbaum S. A model for the role of integrins in flow induced mechanotransduction in osteocytes. Proc. Natl Acad. Sci. USA, 2007, 104:15941-15946

[23] Geoghegan IP, Hoey DA, McNamara LM. Integrins in osteocyte biology and mechanotransduction. Curr. Osteoporos. Rep., 2019, 17:195-206

[24] Kalajzic I et al. In vitro and in vivo approaches to study osteocyte biology. Bone, 2013, 54:296-306

[25] Robling AG et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J. Biol. Chem., 2008, 283:5866-5875

[26] Rubin C, Xu G, Judex S. The anabolic activity of bone tissue, suppressed by disuse, is normalized by brief exposure to extremely low-magnitude mechanical stimuli. FASEB J., 2001, 15:2225-2229

[27] De Souza RL et al. Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: a new model to study cortical and cancellous compartments in a single loaded element. Bone, 2005, 37:810-818

[28] Spatz JM et al. Sclerostin antibody inhibits skeletal deterioration due to reduced mechanical loading. J. Bone Min. Res., 2013, 28:865-874

[29] Robling AG, Hinant FM, Burr DB, Turner CH. Improved bone structure and strength after long-term mechanical loading is greatest if loading is separated into short bouts. J. Bone Min. Res., 2002, 17:1545-1554

[30] Tu X et al. Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading. Bone, 2012, 50:209-217

[31] Ko FC et al. In vivo cyclic compression causes cartilage degeneration and subchondral bone changes in mouse tibiae. Arthritis Rheum., 2013, 65:1569-1578

[32] Lynch ME et al. Tibial compression is anabolic in the adult mouse skeleton despite reduced responsiveness with aging. Bone, 2011, 49:439-446

[33] Lee KC, Maxwell A, Lanyon LE. Validation of a technique for studying functional adaptation of the mouse ulna in response to mechanical loading. Bone, 2002, 31:407-412

[34] Fritton JC, Myers ER, Wright TM, van der Meulen MC. Loading induces site-specific increases in mineral content assessed by microcomputed tomography of the mouse tibia. Bone, 2005, 36:1030-1038

[35] Lynch ME et al. Cancellous bone adaptation to tibial compression is not sex dependent in growing mice. J. Appl Physiol., 2010, 109:685-691

[36] Jackson JR et al. Satellite cell depletion does not inhibit adult skeletal muscle regrowth following unloading-induced atrophy. Am. J. Physiol. Cell Physiol., 2012, 303:C854-C861

[37] Tidball JG, Wehling-Henricks M. Macrophages promote muscle membrane repair and muscle fibre growth and regeneration during modified muscle loading in mice in vivo. J. Physiol., 2007, 578:327-336

[38] Brocca L et al. FoxO-dependent atrogenes vary among catabolic conditions and play a key role in muscle atrophy induced by hindlimb suspension. J. Physiol., 2017, 595:1143-1158

[39] Ajubi NE et al. Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes–a cytoskeleton-dependent process. Biochem Biophys. Res. Commun., 1996, 225:62-68

[40] Klein-Nulend J, Burger EH, Semeins CM, Raisz LG, Pilbeam CC. Pulsating fluid flow stimulates prostaglandin release and inducible prostaglandin G/H synthase mRNA expression in primary mouse bone cells. J. Bone Min. Res., 1997, 12:45-51

[41] Sterck JG, Klein-Nulend J, Lips P, Burger EH. Response of normal and osteoporotic human bone cells to mechanical stress in vitro. Am. J. Physiol., 1998, 274:E1113-E1120

[42] Li J, Rose E, Frances D, Sun Y, You L. Effect of oscillating fluid flow stimulation on osteocyte mRNA expression. J. Biomech., 2012, 45:247-251

[43] Spatz JM et al. The Wnt inhibitor sclerostin is up-regulated by mechanical unloading in osteocytes in vitro. J. Biol. Chem., 2015, 290:16744-16758

[44] Slyfield CR, Tkachenko EV, Wilson DL, Hernandez CJ. Three-dimensional dynamic bone histomorphometry. J. Bone Min. Res., 2012, 27:486-495

[45] Morrell AE et al. Mechanically induced Ca(2+) oscillations in osteocytes release extracellular vesicles and enhance bone formation. Bone Res., 2018, 6:6

[46] Kuttenberger J, Polska E, Schaefer BM. A novel three-dimensional bone chip organ culture. Clin. Oral. Investig., 2013, 17:1547-1555

[47] Sun Q et al. Ex vivo 3D osteocyte network construction with primary murine bone cells. Bone Res., 2015, 3:15026

[48] Michael Sheetz, H. Y. The Cell as a Machine 1 edn, 0–434 (Cambridge university press, 2019).

[49] Pegoraro, A. F., Janmey, P. & Weitz, D. A. Mechanical Properties of the Cytoskeleton and Cells. Cold Spring Harb. Perspect. Biol 9, a022038 (2017).

[50] Klein-Nulend J, Bacabac RG, Bakker AD. Mechanical loading and how it affects bone cells: the role of the osteocyte cytoskeleton in maintaining our skeleton. Eur. Cell Mater., 2012, 24:278-291

[51] Kardas D, Nackenhorst U, Balzani D. Computational model for the cell-mechanical response of the osteocyte cytoskeleton based on self-stabilizing tensegrity structures. Biomech. Modeling Mechanobiol., 2013, 12:167-183

[52] Tanaka-Kamioka K, Kamioka H, Ris H, Lim SS. Osteocyte shape is dependent on actin filaments and osteocyte processes are unique actin-rich projections. J. Bone Min. Res., 1998, 13:1555-1568

[53] Lyons, J. S. et al. Microtubules tune mechanotransduction through NOX2 and TRPV4 to decrease sclerostin abundance in osteocytes. Sci. Signal 10, 5748 (2017).

[54] Moorer MC, Buo AM, Garcia-Pelagio KP, Stains JP, Bloch RJ. Deficiency of the intermediate filament synemin reduces bone mass in vivo. Am. J. Physiol. Cell Physiol., 2016, 311:C839-C845

[55] Zhang K et al. E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation. Mol. Cell Biol., 2006, 26:4539-4552

[56] Prideaux M, Loveridge N, Pitsillides AA, Farquharson C. Extracellular matrix mineralization promotes E11/gp38 glycoprotein expression and drives osteocytic differentiation. PLoS ONE, 2012, 7

[57] Staines KA et al. Conditional deletion of E11/podoplanin in bone protects against load-induced osteoarthritis. BMC Musculoskelet. Disord., 2019, 20

[58] Burra S et al. Dendritic processes of osteocytes are mechanotransducers that induce the opening of hemichannels. Proc. Natl Acad. Sci. USA, 2010, 107:13648-13653

[59] Wu D, Schaffler MB, Weinbaum S, Spray DC. Matrix-dependent adhesion mediates network responses to physiological stimulation of the osteocyte cell process. Proc. Natl Acad. Sci. USA, 2013, 110:12096-12101

[60] Thi MM, Suadicani SO, Schaffler MB, Weinbaum S, Spray DC. Mechanosensory responses of osteocytes to physiological forces occur along processes and not cell body and require αVβ3 integrin. Proc. Natl Acad. Sci. USA, 2013, 110:21012-21017

[61] Terenzio M, Schiavo G, Fainzilber M. Compartmentalized signaling in neurons: from cell biology to neuroscience. Neuron, 2017, 96:667-679

[62] Davenport JR, Yoder BK. An incredible decade for the primary cilium: a look at a once-forgotten organelle. Am. J. Physiol. Ren. Physiol., 2005, 289:F1159-F1169

[63] Temiyasathit S, Jacobs CR. Osteocyte primary cilium and its role in bone mechanotransduction. Ann. N. Y. Acad. Sci., 2010, 1192:422-428

[64] Huber C, Cormier-Daire V. Ciliary disorder of the skeleton. Am. J. Med. Genet C. Semin. Med. Genet., 2012, 160C:165-174

[65] Hoey DA, Chen JC, Jacobs CR. The primary cilium as a novel extracellular sensor in bone. Front. Endocrinol., 2012, 3:75

[66] Hoey DA, Tormey S, Ramcharan S, O’Brien FJ, Jacobs CR. Primary cilia-mediated mechanotransduction in human mesenchymal stem cells. Stem Cells, 2012, 30:2561-2570

[67] Ascenzi MG et al. Effect of localization, length and orientation of chondrocytic primary cilium on murine growth plate organization. J. Theor. Biol., 2011, 285:147-155

[68] Xiao Z et al. Cilia-like structures and polycystin-1 in osteoblasts/osteocytes and associated abnormalities in skeletogenesis and Runx2 expression. J. Biol. Chem., 2006, 281:30884-30895

[69] Malone AM et al. Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism. Proc. Natl Acad. Sci. USA, 2007, 104:13325-13330

[70] Uzbekov RE et al. Centrosome fine ultrastructure of the osteocyte mechanosensitive primary cilium. Microsc. Microanal., 2012, 18:1430-1441

[71] Coughlin TR, Voisin M, Schaffler MB, Niebur GL, McNamara LM. Primary cilia exist in a small fraction of cells in trabecular bone and marrow. Calcif. Tissue Int., 2015, 96:65-72

[72] Kwon RY, Temiyasathit S, Tummala P, Quah CC, Jacobs CR. Primary cilium-dependent mechanosensing is mediated by adenylyl cyclase 6 and cyclic AMP in bone cells. FASEB J., 2010, 24:2859-2868

[73] Lehti MS et al. Cilia-related protein SPEF2 regulates osteoblast differentiation. Sci. Rep., 2018, 8

[74] Temiyasathit S et al. Mechanosensing by the primary cilium: deletion of Kif3A reduces bone formation due to loading. PLoS ONE, 2012, 7

[75] Qiu N et al. Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia. J. Cell Sci., 2012, 125:1945-1957

[76] Lee KL et al. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo. FASEB J., 2014, 28:1157-1165

[77] Oliazadeh N, Gorman KF, Eveleigh R, Bourque G, Moreau A. Identification of elongated primary cilia with impaired mechanotransduction in idiopathic scoliosis patients. Sci. Rep., 2017, 7

[78] Geiger B, Spatz JP, Bershadsky AD. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol., 2009, 10:21-33

[79] Barczyk M, Carracedo S, Gullberg D. Integrins. Cell Tissue Res., 2010, 339:269-280

[80] Hughes DE, Salter DM, Dedhar S, Simpson R. Integrin expression in human bone. J. Bone Min. Res., 1993, 8:527-533

[81] Duong LT, Lakkakorpi P, Nakamura I, Rodan GA. Integrins and signaling in osteoclast function. Matrix Biol., 2000, 19:97-105

[82] Marie PJ, Hay E, Saidak Z. Integrin and cadherin signaling in bone: role and potential therapeutic targets. Trends Endocrinol. Metab., 2014, 25:567-575

[83] Cabahug-Zuckerman P et al. Potential role for a specialized β(3) integrin-based structure on osteocyte processes in bone mechanosensation. J. Orthop. Res., 2018, 36:642-652

[84] Litzenberger JB, Kim J-B, Tummala P, Jacobs CR. Beta1 integrins mediate mechanosensitive signaling pathways in osteocytes. Calcif. Tissue Int., 2010, 86:325-332

[85] Haugh MG, Vaughan TJ, McNamara LM. The role of integrin alpha(V)beta(3) in osteocyte mechanotransduction. J. Mech. Behav. Biomed. Mater., 2015, 42:67-75

[86] Stephens LE et al. Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality. Genes Dev., 1995, 9:1883-1895

[87] Shekaran A et al. The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype. Bone, 2014, 68:131-141

[88] Litzenberger JB, Tang WJ, Castillo AB, Jacobs CR. Deletion of β1 integrins from cortical osteocytes reduces load-induced bone formation. Cell. Mol. Bioeng., 2009, 2:416-424

[89] Zimmerman D, Jin F, Leboy P, Hardy S, Damsky C. Impaired bone formation in transgenic mice resulting from altered integrin function in osteoblasts. Dev. Biol., 2000, 220:2-15

[90] Smyth SS, Reis ED, Vaananen H, Zhang W, Coller BS. Variable protection of beta 3-integrin–deficient mice from thrombosis initiated by different mechanisms. Blood, 2001, 98:1055-1062

[91] Batra N, Kar R, Jiang JX. Gap junctions and hemichannels in signal transmission, function and development of bone. Biochim. Biophys. Acta, 2012, 1818:1909-1918

[92] Buo AM, Stains JP. Gap junctional regulation of signal transduction in bone cells. FEBS Lett., 2014, 588:1315-1321

[93] Moorer MC, Stains JP. Connexin43 and the intercellular signaling network regulating skeletal remodeling. Curr. Osteoporos. Rep., 2017, 15:24-31

[94] Lecanda F et al. Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J. Cell Biol., 2000, 151:931-944

[95] Davis HM et al. Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging. Aging Cell, 2017, 16:551-563

[96] Chung DJ et al. Low peak bone mass and attenuated anabolic response to parathyroid hormone in mice with an osteoblast-specific deletion of connexin43. J. Cell Sci., 2006, 119:4187-4198

[97] Bivi N et al. Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation. J. Bone Min. Res., 2012, 27:374-389

[98] Grimston SK, Brodt MD, Silva MJ, Civitelli R. Attenuated response to in vivo mechanical loading in mice with conditional osteoblast ablation of the connexin43 gene (Gja1). J. Bone Min. Res., 2008, 23:879-886

[99] Lloyd SA, Lewis GS, Zhang Y, Paul EM, Donahue HJ. Connexin 43 deficiency attenuates loss of trabecular bone and prevents suppression of cortical bone formation during unloading. J. Bone Min. Res., 2012, 27:2359-2372

[100] Pacheco-Costa R et al. Osteocytic connexin 43 is not required for the increase in bone mass induced by intermittent PTH administration in male mice. J. Musculoskelet. Neuronal Interact., 2016, 16:45-57

[101] Ma L et al. Connexin 43 hemichannels protect bone loss during estrogen deficiency. Bone Res., 2019, 7:11

[102] Cheng B et al. Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells. J. Bone Min. Res., 2001, 16:249-259

[103] Cheng B et al. PGE(2) is essential for gap junction-mediated intercellular communication between osteocyte-like MLO-Y4 cells in response to mechanical strain. Endocrinology, 2001, 142:3464-3473

[104] Alford AI, Jacobs CR, Donahue HJ. Oscillating fluid flow regulates gap junction communication in osteocytic MLO-Y4 cells by an ERK1/2 MAP kinase-dependent mechanism. Bone, 2003, 33:64-70

[105] Cherian PP et al. Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin. Mol. Biol. Cell, 2005, 16:3100-3106

[106] Zhang D et al. Extracellular matrix elasticity regulates osteocyte gap junction elongation: involvement of paxillin in intracellular signal transduction. Cell Physiol. Biochem., 2018, 51:1013-1026

[107] Batra N et al. Mechanical stress-activated integrin alpha5beta1 induces opening of connexin 43 hemichannels. Proc. Natl Acad. Sci. USA, 2012, 109:3359-3364

[108] Batra N, Jiang JX. “INTEGRINating” the connexin hemichannel function in bone osteocytes through the action of integrin α5. Commun. Integr. Biol., 2012, 5:516-518

[109] Giepmans BN. Gap junctions and connexin-interacting proteins. Cardiovasc. Res., 2004, 62:233-245

[110] Herve JC, Bourmeyster N, Sarrouilhe D, Duffy HS. Gap junctional complexes: from partners to functions. Prog. Biophys. Mol. Biol., 2007, 94:29-65

[111] Sorgen, P. L., Trease, A. J., Spagnol, G., Delmar, M. & Nielsen, M. S. Protein-protein interactions with connexin 43: regulation and function. Int. J. Mol. Sci. 19, 1428 (2018).

[112] Plotkin LI, Speacht TL, Donahue HJ. Cx43 and mechanotransduction in bone. Curr. Osteoporos. Rep., 2015, 13:67-72

[113] Robling AG, Turner CH. Mechanical signaling for bone modeling and remodeling. Crit. Rev. Eukaryot. Gene Expr., 2009, 19:319-338

[114] Davidson RM, Tatakis DW, Auerbach AL. Multiple forms of mechanosensitive ion channels in osteoblast-like cells. Pflug. Arch., 1990, 416:646-651

[115] Mikuni-Takagaki Y, Naruse K, Azuma Y, Miyauchi A. The role of calcium channels in osteocyte function. J. Musculoskelet. Neuronal Interact., 2002, 2:252-255

[116] Yu K et al. Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone. J. Orthop. Res., 2018, 36:653-662

[117] Rawlinson SC, Pitsillides AA, Lanyon LE. Involvement of different ion channels in osteoblasts’ and osteocytes’ early responses to mechanical strain. Bone, 1996, 19:609-614

[118] Zhao Q et al. Structure and mechanogating mechanism of the Piezo1 channel. Nature, 2018, 554:487-492

[119] Haselwandter, C. A. & MacKinnon, R. Piezo’s membrane footprint and its contribution to mechanosensitivity. Elife 7, e41968 (2018).

[120] Li, X. et al. Stimulation of Piezo1 by mechanical signals promotes bone anabolism. Elife 8, e49631 (2019).

[121] Sun, W. et al. The mechanosensitive Piezo1 channel is required for bone formation. Elife 8, e47454 (2019).

[122] Wang L et al. Mechanical sensing protein PIEZO1 regulates bone homeostasis via osteoblast-osteoclast crosstalk. Nat. Commun., 2020, 11

[123] Yang J et al. Blocking glucocorticoid signaling in osteoblasts and osteocytes prevents mechanical unloading-induced cortical bone loss. Bone, 2020, 130:115108

[124] Sasaki F et al. Mechanotransduction via the Piezo1-Akt pathway underlies Sost suppression in osteocytes. Biochem. Biophys. Res. Commun., 2020, 521:806-813

[125] el Haj AJ, Walker LM, Preston MR, Publicover SJ. Mechanotransduction pathways in bone: calcium fluxes and the role of voltage-operated calcium channels. Med. Biol. Eng. Comput., 1999, 37:403-409

[126] Thompson WR et al. Association of the α2δ1 subunit with Cav3.2 enhances membrane expression and regulates mechanically induced ATP release in MLO-Y4 osteocytes. J. Bone Miner. Res., 2011, 26:2125-2139

[127] Lu XL, Huo B, Chiang V, Guo XE. Osteocytic network is more responsive in calcium signaling than osteoblastic network under fluid flow. J. Bone Min. Res., 2012, 27:563-574

[128] Huo B et al. Fluid flow induced calcium response in bone cell network. Cell Mol. Bioeng., 2008, 1:58-66

[129] Li J, Duncan RL, Burr DB, Turner CH. L-type calcium channels mediate mechanically induced bone formation in vivo. J. Bone Min. Res., 2002, 17:1795-1800

[130] Lewis KJ et al. Osteocyte calcium signals encode strain magnitude and loading frequency in vivo. Proc. Natl Acad. Sci. USA, 2017, 114:11775-11780

[131] van Oers RFM, Wang H, Bacabac RG. Osteocyte shape and mechanical loading. Curr. Osteoporos. Rep., 2015, 13:61-66

[132] Wang X, Bank RA, TeKoppele JM, Agrawal CM. The role of collagen in determining bone mechanical properties. J. Orthop. Res., 2001, 19:1021-1026

[133] Kerschnitzki M et al. The organization of the osteocyte network mirrors the extracellular matrix orientation in bone. J. Struct. Biol., 2011, 173:303-311

[134] Matsugaki A, Isobe Y, Saku T, Nakano T. Quantitative regulation of bone-mimetic, oriented collagen/apatite matrix structure depends on the degree of osteoblast alignment on oriented collagen substrates. J. Biomed. Mater. Res. Part A, 2015, 103:489-499

[135] Shah FA, Zanghellini E, Matic A, Thomsen P, Palmquist A. The orientation of nanoscale apatite platelets in relation to osteoblastic-osteocyte lacunae on trabecular bone surface. Calcif. Tissue Int., 2016, 98:193-205

[136] Addison WN et al. Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone. Bone, 2015, 71:244-256

[137] Burra S, Nicolella DP, Jiang JX. Dark horse in osteocyte biology. Communicative Integr. Biol., 2011, 4:48-50

[138] Wang L. Solute transport in the bone lacunar-canalicular system (LCS). Curr. Osteoporos. Rep., 2018, 16:32-41

[139] Thompson WR et al. Perlecan/Hspg2 deficiency alters the pericellular space of the lacunocanalicular system surrounding osteocytic processes in cortical bone. J. Bone Min. Res., 2011, 26:618-629

[140] Wijeratne SS et al. Single molecule force measurements of perlecan/HSPG2: a key component of the osteocyte pericellular matrix. Matrix Biol., 2016, 50:27-38

[141] Wang B et al. Perlecan-containing pericellular matrix regulates solute transport and mechanosensing within the osteocyte lacunar-canalicular system. J. Bone Min. Res., 2014, 29:878-891

[142] Pei S et al. Perlecan/Hspg2 deficiency impairs bone’s calcium signaling and associated transcriptome in response to mechanical loading. Bone, 2020, 131:115078

[143] Long F. Building strong bones: molecular regulation of the osteoblast lineage. Nat. Rev. Mol. Cell Biol., 2011, 13:27-38

[144] Briscoe J, Thérond PP. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat. Rev. Mol. Cell Biol., 2013, 14:416-429

[145] Andersson ER, Lendahl U. Therapeutic modulation of Notch signalling—are we there yet? Nat. Rev. Drug Discov., 2014, 13:357-378

[146] Su N, Jin M, Chen L. Role of FGF/FGFR signaling in skeletal development and homeostasis: learning from mouse models. Bone Res., 2014, 2:14003

[147] Rahman MS, Akhtar N, Jamil HM, Banik RS, Asaduzzaman SM. TGF-β/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation. Bone Res., 2015, 3:15005

[148] Luo Z et al. Notch signaling in osteogenesis, osteoclastogenesis, and angiogenesis. Am. J. Pathol., 2019, 189:1495-1500

[149] Laine CM et al. WNT1 mutations in early-onset osteoporosis and osteogenesis imperfecta. N. Engl. J. Med., 2013, 368:1809-1816

[150] Pyott SM et al. WNT1 mutations in families affected by moderately severe and progressive recessive osteogenesis imperfecta. Am. J. Hum. Genet., 2013, 92:590-597

[151] Maupin KA, Droscha CJ, Williams BO. A comprehensive overview of skeletal phenotypes associated with alterations in Wnt/β-catenin signaling in humans and mice. Bone Res., 2013, 1:27-71

[152] Burgers TA, Williams BO. Regulation of Wnt/β-catenin signaling within and from osteocytes. Bone, 2013, 54:244-249

[153] Bonewald LF, Johnson ML. Osteocytes, mechanosensing and Wnt signaling. Bone, 2008, 42:606-615

[154] Robinson JA et al. Wnt/β-catenin signaling is a normal physiological response to mechanical loading in bone. J. Biol. Chem., 2006, 281:31720-31728

[155] Krishnan V, Bryant HU, MacDougald OA. Regulation of bone mass by Wnt signaling. J. Clin. Investig., 2006, 116:1202-1209

[156] Baron R, Rawadi G. Wnt signaling and the regulation of bone mass. Curr. Osteoporos. Rep., 2007, 5:73-80

[157] Sawakami K et al. The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J. Biol. Chem., 2006, 281:23698-23711

[158] Akhter MP et al. Bone biomechanical properties in LRP5 mutant mice. Bone, 2004, 35:162-169

[159] Saxon LK, Jackson BF, Sugiyama T, Lanyon LE, Price JS. Analysis of multiple bone responses to graded strains above functional levels, and to disuse, in mice in vivo show that the human Lrp5 G171V High Bone Mass mutation increases the osteogenic response to loading but that lack of Lrp5 activity reduces it. Bone, 2011, 49:184-193

[160] Kang KS, Hong JM, Robling AG. Postnatal β-catenin deletion from Dmp1-expressing osteocytes/osteoblasts reduces structural adaptation to loading, but not periosteal load-induced bone formation. Bone, 2016, 88:138-145

[161] Kramer I et al. Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis. Mol. Cell Biol., 2010, 30:3071-3085

[162] Maurel DB et al. Beta-catenin haplo insufficient male mice do not lose bone in response to hindlimb unloading. PLoS ONE, 2016, 11

[163] Javaheri B et al. Deletion of a single β-catenin allele in osteocytes abolishes the bone anabolic response to loading. J. Bone Min. Res., 2014, 29:705-715

[164] Kamel MA, Picconi Jl Fau - Lara-Castillo N, Lara-Castillo N Fau - Johnson ML, Johnson ML. Activation of beta-catenin signaling in MLO-Y4 osteocytic cells versus 2T3 osteoblastic cells by fluid flow shear stress and PGE2: Implications for the study of mechanosensation in bone. Bone, 2010, 47:872-881

[165] Harburger DS, Calderwood DA. Integrin signalling at a glance. J. Cell Sci., 2009, 122:159-163

[166] Zaidel-Bar R, Itzkovitz S, Ma’ayan A, Iyengar R, Geiger B. Functional atlas of the integrin adhesome. Nat. Cell Biol., 2007, 9:858-867

[167] Wu C et al. Kindlin-2 controls TGF-β signalling and Sox9 expression to regulate chondrogenesis. Nat. Commun., 2015, 6

[168] Cao H et al. Focal adhesion protein Kindlin-2 regulates bone homeostasis in mice. Bone Res., 2020, 8:2

[169] Wang, Y. et al. Focal adhesion proteins Pinch1 and Pinch2 regulate bone homeostasis in mice. JCI insight 4, e131692 (2019).

[170] Turner CH, Forwood MR, Rho JY, Yoshikawa T. Mechanical loading thresholds for lamellar and woven bone formation. J. Bone Min. Res., 1994, 9:87-97

[171] Sun C et al. FAK promotes osteoblast progenitor cell proliferation and differentiation by enhancing Wnt signaling. J. Bone Min. Res., 2016, 31:2227-2238

[172] Plotkin LI et al. Mechanical stimulation prevents osteocyte apoptosis: requirement of integrins, Src kinases, and ERKs. Am. J. Physiol. Cell Physiol., 2005, 289:C633-C643

[173] Cardoso L et al. Osteocyte apoptosis controls activation of intracortical resorption in response to bone fatigue. J. Bone Min. Res., 2009, 24:597-605

[174] Cabahug-Zuckerman P et al. Osteocyte apoptosis caused by hindlimb unloading is required to trigger osteocyte RANKL production and subsequent resorption of cortical and trabecular bone in mice femurs. J. Bone Miner. Res., 2016, 31:1356-1365

[175] Verborgt O, Tatton NA, Majeska RJ, Schaffler MB. Spatial distribution of Bax and Bcl-2 in osteocytes after bone fatigue: complementary roles in bone remodeling regulation? J. Bone Miner. Res., 2002, 17:907-914

[176] Komori, T. Cell death in chondrocytes, osteoblasts, and osteocytes. Int. J. Mol. Sci. 17, 2045 (2016).

[177] Sherk VD, Rosen CJ. Senescent and apoptotic osteocytes and aging: exercise to the rescue? Bone, 2019, 121:255-258

[178] Farr JN et al. Identification of senescent cells in the bone microenvironment. J. Bone Min. Res., 2016, 31:1920-1929

[179] Farr JN, Khosla S. Cellular senescence in bone. Bone, 2019, 121:121-133

[180] Poole KE et al. Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation. FASEB J., 2005, 19:1842-1844

[181] Li X et al. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J. Biol. Chem., 2005, 280:19883-19887

[182] Sebastian A, Loots GG. Transcriptional control of Sost in bone. Bone, 2017, 96:76-84

[183] Semenov M, Tamai K, He X. SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. J. Biol. Chem., 2005, 280:26770-26775

[184] Lin C et al. Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/beta-catenin signaling. J. Bone Min. Res., 2009, 24:1651-1661

[185] Stegen S et al. Osteocytic oxygen sensing controls bone mass through epigenetic regulation of sclerostin. Nat. Commun., 2018, 9

[186] Low BC et al. YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth. FEBS Lett., 2014, 588:2663-2670

[187] Dupont S et al. Role of YAP/TAZ in mechanotransduction. Nature, 2011, 474:179-183

[188] Panciera T, Azzolin L, Cordenonsi M, Piccolo S. Mechanobiology of YAP and TAZ in physiology and disease. Nat. Rev. Mol. Cell Biol., 2017, 18:758-770

[189] Asaoka Y, Furutani-Seiki M. YAP mediated mechano-homeostasis—conditioning 3D animal body shape. Curr. Opin. Cell Biol., 2017, 49:64-70

[190] Xiong J, Almeida M, O’Brien CA. The YAP/TAZ transcriptional co-activators have opposing effects at different stages of osteoblast differentiation. Bone, 2018, 112:1-9

[191] Tang Y et al. MT1-MMP-dependent control of skeletal stem cell commitment via a β1-integrin/YAP/TAZ signaling axis. Dev. Cell, 2013, 25:402-416

[192] Kegelman CD et al. Skeletal cell YAP and TAZ combinatorially promote bone development. FASEB J., 2018, 32:2706-2721

[193] Kegelman, C. D. et al. YAP and TAZ mediate osteocyte perilacunar/canalicular remodeling. J. Bone Miner. Res. 35, 196–210 (2020).

[194] Langdahl BL et al. Romosozumab (sclerostin monoclonal antibody) versus teriparatide in postmenopausal women with osteoporosis transitioning from oral bisphosphonate therapy: a randomised, open-label, phase 3 trial. Lancet, 2017, 390:1585-1594

[195] Prasadam I et al. Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes: role of integrinbeta1 and focal adhesion kinase signaling cues. Arthritis Res. Ther., 2013, 15:R150

[196] Claes L, Recknagel S, Ignatius A. Fracture healing under healthy and inflammatory conditions. Nat. Rev. Rheumatol., 2012, 8:133-143

[197] Metzger CE, Narayanan SA. The role of osteocytes in inflammatory bone loss. Front. Endocrinol., 2019, 10:285

[198] Wang, W., Sarazin, B. A., Kornilowicz, G. & Lynch, M. E. Mechanically-loaded breast cancer cells modify osteocyte mechanosensitivity by secreting factors that increase osteocyte dendrite formation and downstream resorption. Front. Endocrinol. 9, 352 (2018).

[199] Ross, M. H. et al. Bone-induced expression of integrin β3 enables targeted nanotherapy of breast cancer metastases. Cancer Res. 77, 6299–6312 (2017).

[200] Holguin N, Brodt MD, Silva MJ. Activation of Wnt signaling by mechanical loading is impaired in the bone of old mice. J. Bone Miner. Res., 2016, 31:2215-2226

[201] Hemmatian H, Bakker AD, Klein-Nulend J, van Lenthe GH. Aging, osteocytes, and mechanotransduction. Curr. Osteoporos. Rep., 2017, 15:401-411

[202] Findlay DM, Kuliwaba JS. Bone–cartilage crosstalk: a conversation for understanding osteoarthritis. Bone Res., 2016, 4:16028

[203] Jaiprakash A et al. Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones: a possible pathological role in subchondral bone sclerosis. Int. J. Biol. Sci., 2012, 8:406-417

[204] Wen CY et al. Collagen fibril stiffening in osteoarthritic cartilage of human beings revealed by atomic force microscopy. Osteoarthr. Cartil., 2012, 20:916-922

[205] Gao, H. et al. Lipoatrophy and metabolic disturbance in mice with adipose-specific deletion of kindlin-2. JCI Insight 4, e128405 (2019).

[206] Upadhyay J, Farr OM, Mantzoros CS. The role of leptin in regulating bone metabolism. Metabolism, 2015, 64:105-113

[207] Lories RJ et al. Articular cartilage and biomechanical properties of the long bones in Frzb-knockout mice. Arthritis Rheum., 2007, 56:4095-4103

[208] Mosley JR, Lanyon LE. Strain rate as a controlling influence on adaptive modeling in response to dynamic loading of the ulna in growing male rats. Bone, 1998, 23:313-318

[209] Mosley JR, March BM, Lynch J, Lanyon LE. Strain magnitude related changes in whole bone architecture in growing rats. Bone, 1997, 20:191-198

[210] Bonnet N et al. The matricellular protein periostin is required for sost inhibition and the anabolic response to mechanical loading and physical activity. J. Biol. Chem., 2009, 284:35939-35950

[211] Akhter MP, Cullen DM, Pedersen EA, Kimmel DB, Recker RR. Bone response to in vivo mechanical loading in two breeds of mice. Calcif. Tissue Int., 1998, 63:442-449

[212] Moustafa A et al. Mechanical loading-related changes in osteocyte sclerostin expression in mice are more closely associated with the subsequent osteogenic response than the peak strains engendered. Osteoporos. Int., 2012, 23:1225-1234

[213] Sugiyama T et al. Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1-34) on trabecular and cortical bone in mice. Bone, 2008, 43:238-248

[214] Rucci N et al. Lipocalin 2: a new mechanoresponding gene regulating bone homeostasis. J. Bone Min. Res., 2015, 30:357-368

[215] Baehr LM et al. Muscle-specific and age-related changes in protein synthesis and protein degradation in response to hindlimb unloading in rats. J. Appl Physiol., 2017, 122:1336-1350

[216] Ajubi NE, Klein-Nulend J, Alblas MJ, Burger EH, Nijweide PJ. Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes. Am. J. Physiol., 1999, 276:E171-E178

[217] Joldersma M, Burger EH, Semeins CM, Klein-Nulend J. Mechanical stress induces COX-2 mRNA expression in bone cells from elderly women. J. Biomech., 2000, 33:53-61

[218] Kulkarni RN, Bakker AD, Everts V, Klein-Nulend J. Inhibition of osteoclastogenesis by mechanically loaded osteocytes: involvement of MEPE. Calcif. Tissue Int., 2010, 87:461-468