Activin Regulation of Osteoclast Formation, Motility, Function, and Survival

The process of bone resorption is regulated at multiple levels. Current bone-active therapies target bone resorption, whereas targets such as the formation of osteoclasts (OC) and their motility, fusion and lifespan have been largely unexplored. We and others have shown that RANKL and Activin A (ActA) are potent stimulators of murine bone marrow osteoclastogenesis. However, ActA treatment does not induce osteoclastogenesis in purified populations of murine bone marrow macrophages (BMM), but inhibits RANKLNinduced OC development. We hypothesized that these factors differentially regulate the migration of both OC precursors and mature OC. We determined the effects of ActA and RANKL on BMM motility and bone resorption using time-lapse video microscopy. BMMs were plated onto culture dishes with 25 ng/ml mCSF alone for 24hr, prior to the addition of 100 ng/mL RANKL or 50 ng/mL ActA, alone or together, for up to 4d. Other BMMs were cultured on bone slices for 5d in the presence of mCSF+RANKL to stimulate OC differentiation prior to treatment with RANKL or ActA, alone or together for an additional 1N2d. The 4hr movement of ten OC precursors were tracked and analyzed with NIH ImageJ. Parameters calculated for each cell included cumulative track length, migration rate, maximum instantaneous velocity, range of velocity, and percent total displacement. BMMs migrated at a constant velocity and distance from d1N4. RANKL significantly increased parameters on d2 and d3, an effect that was lost by d4. In contrast, ActA treatment decreased all parameters on d1N4. Surprisingly, when added with RANKL, ActA treatment completely blocked all RANKL stimulated OC precursor motility on plastic, as well as mature RANKLNstimulated OC motility on bone slices. ActA treatment of mature OC (d5) also suppressed RANKLN stimulated bone resorption, measured by pit formation. The suppression was associated with a decrease in OC lifespan, since ActA increased activated Caspase 3, and caused a decrease in actin ring formation in mature OC on bone slices. Collectively these data indicate a dual modulatory function of ActA on OCs. ActA indirectly stimulates murine whole bone marrow osteoclastogenesis, but it also directly inhibits RANKLNinduced murine OC differentiation, motility, and resorption and enhances OC apoptosis.

Year	Entry
1983	Jee WS, Wronski TJ, Morey ER, Kimmel DB. Effects of spaceflight on trabecular bone in rats. Am J Physiol Regul Integr Comp Physiol. 1983;244(3):R310-R314.
2005	Bellido T, Ali AA, Gubrij I, Plotkin LI, Fu Q, O’Brien CA, Manolagas SC, Jilka RL. Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hormonal control of osteoblastogenesis. Endocrinology. November 2005;146(11):4577-4583.
2004	van Bezooijen RL, Roelen BAJ, Visser A, van der Wee-Pals L, de Wilt E, Karperien M, Hamersma H, Papapoulos SE, ten Dijke P, Löwik CWGM. Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J Exp Med. March 15, 2004;199(6):805-814.
1998	Lacey DL, Timms E, Tan H-L, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian Y-X, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. April 17, 1998;93(2):165-176.
2001	Toyosawa S, Shintani S, Fujiwara T, Ooshima T, Sato A, Ijuhin N, Komori T. Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts. J Bone Miner Res. November 2001;16(11):2017-2026.
2002	Riggs BL, Khosla S, Melton LJ III. Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev. June 2002;23(3):279-302.
1995	Klein-Nulend J, Semeins CM, Ajubi NE, Nijweide P, Burger EH. Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts - correlation with prostaglandin upregulation. Biochem Biophys Res Commun. December 14, 1995;217(2):640-648.
1997	Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsent G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. May 30, 1997;89(5):747-754.
1999	Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev. June 1999;20(3):345-357.
2006	Rubin J, Rubin C, Jacobs CR. Molecular pathways mediating mechanical signaling in bone. Gene. February 15, 2006;367:1-16.
2003	Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nat Rev Genet. August 2003;4(8):638-649.
1999	Kong Y-Y, Yoshida H, Sarosi I, Tan H-L, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. January 28, 1999;397(6717):315-323.
1988	Pead MJ, Skerry TM, Lanyon LE. Direct transformation from quiescence to bone formation in the adult periosteum following a single brief period of bone loading. J Bone Miner Res. December 1988;3(6):647-656.
2002	Zhang M, Xuan S, Bouxsein ML, von Stechow D, Akeno N, Faugere MC, Malluche H, Zhao G, Rosen CJ, Efstratiadis A, Clemens TL. Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem. November 15, 2002;277(46):44005-44012.
1981	Rodan GA, Martin TJ. Role of osteoblasts in hormonal control of bone resorption: a hypothesis. Calcif Tiss Int. December 1981;33(1):349-351.
2002	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. May 2002;17(5):907-914.
1986	Globus RK, Bikle DD, Morey-Holton E. The temporal response of bone to unloading. Endocrinology. February 1986;118(2):733-742.
2005	Hill TP, Später D, Taketo MM, Birchmeier W, Hartmann C. Canonical Wnt/β-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell. May 2005;8(5):727-738.
2003	Noble BS, Peet N, Stevens HY, Brabbs A, Mosley JR, Reilly GC, Reeve J, Skerry TM, Lanyon LE. Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. Am J Physiol Cell Physiol. April 2003;284(4):C934-C943.
1998	Turner CH, Pavalko FM. Mechanotransduction and functional response of the skeleton to physical stress: the mechanisms and mechanics of bone adaptation. J Orthop Sci. November 1998;3(6):346-355.
2008	Kogianni G, Mann V, Noble BS. Apoptotic bodies convey activity capable of initiating osteoclastogenesis and localized bone destruction. J Bone Miner Res. June 2008;23(6):915-927.
2003	Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, Shpektor D, Jonas M, Kovacevich BR, Staehling-Hampton K, Appleby M, Brunkow ME, Latham JA. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J. January 2003;22(23):6267-6276.
2008	You L, Temiyasathit S, Lee P, Kim CH, Tummala P, Yao W, Kingery W, Malone AM, Kwon RY, Jacobs CR. Osteocytes as mechanosensors in the inhibition of bone resorption due to mechanical loading. Bone. January 2008;42(1):172-179.
2000	Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev. April 2000;21(2):115-137.

Year

Entry

1983

Jee WS, Wronski TJ, Morey ER, Kimmel DB. Effects of spaceflight on trabecular bone in rats. Am J Physiol Regul Integr Comp Physiol. 1983;244(3):R310-R314.

2005

Bellido T, Ali AA, Gubrij I, Plotkin LI, Fu Q, O’Brien CA, Manolagas SC, Jilka RL. Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hormonal control of osteoblastogenesis. Endocrinology. November 2005;146(11):4577-4583.

2004

van Bezooijen RL, Roelen BAJ, Visser A, van der Wee-Pals L, de Wilt E, Karperien M, Hamersma H, Papapoulos SE, ten Dijke P, Löwik CWGM. Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J Exp Med. March 15, 2004;199(6):805-814.

1998

Lacey DL, Timms E, Tan H-L, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian Y-X, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. April 17, 1998;93(2):165-176.

2001

Toyosawa S, Shintani S, Fujiwara T, Ooshima T, Sato A, Ijuhin N, Komori T. Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts. J Bone Miner Res. November 2001;16(11):2017-2026.