Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5？/？), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5G171V) and their WT littermates (WTLrp5, WTHBM). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5G171V mice compared to their WTHBM littermates, and lower in Lrp5？/？ cells. Cells from female LRP5G171V mice grew more rapidly than those from males, whereas cells from female Lrp5？/？ mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5？/？ mice than in those from WTHBM or LRP5G171V mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the ‘threshold’ at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro.
References
[1]
Ehrlich PJ, Lanyon LE (2002) Mechanical strain and bone cell function: a review. Osteoporos Int 13: 688–700.
[2]
Bennett CN, Longo KA, Wright WS, Suva LJ, Lane TF, et al. (2005) Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci U S A 102: 3324–3329.
[3]
Day TF, Guo X, Garrett-Beal L, Yang Y (2005) Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell 8: 739–750.
[4]
Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, et al. (2002) Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 157: 303–314.
[5]
Krishnan V, Bryant HU, Macdougald OA (2006) Regulation of bone mass by Wnt signaling. J Clin Invest 116: 1202–1209.
[6]
Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, et al. (2001) LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107: 513–523.
[7]
Babij P, Zhao W, Small C, Kharode Y, Yaworsky PJ, et al. (2003) High bone mass in mice expressing a mutant LRP5 gene. J Bone Miner Res 18: 960–974.
[8]
Bodine PV, Zhao W, Kharode YP, Bex FJ, Lambert AJ, et al. (2004) The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice. Mol Endocrinol 18: 1222–1237.
[9]
Clement-Lacroix P, Ai M, Morvan F, Roman-Roman S, Vayssiere B, et al. (2005) Lrp5-independent activation of Wnt signaling by lithium chloride increases bone formation and bone mass in mice. Proc Natl Acad Sci U S A 102: 17406–17411.
[10]
Frontali M, Stomeo C, Dallapiccola B (1985) Osteoporosis-pseudoglioma syndrome: report of three affected sibs and an overview. American Journal of Medical Genetics 22: 35–47.
[11]
Gong Y, Vikkula M, Boon L, Liu J, Beighton P, et al. (1996) Osteoporosis-pseudoglioma syndrome, a disorder affecting skeletal strength and vision, is assigned to chromosome region 11q12–13. Am J Hum Genet 59: 146–151.
[12]
Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, et al. (2002) High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 346: 1513–1521.
[13]
Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, et al. (2002) A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 70: 11–19.
[14]
Johnson ML, Gong G, Kimberling W, Recker SM, Kimmel DB, et al. (1997) Linkage of a gene causing high bone mass to human chromosome 11 (11q12-13). Am J Hum Genet 60: 1326–1332.
[15]
Sawakami K, Robling AG, Ai M, Pitner ND, Liu D, et al. (2006) The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J Biol Chem 281: 23698–23711.
[16]
Saxon LK, Jackson BF, Sugiyama T, Lanyon LE, Price JS (2011) 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 49: 184–193.
[17]
Zaman G, Suswillo RF, Cheng MZ, Tavares IA, Lanyon LE (1997) Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture. J Bone Miner Res 12: 769–777.
[18]
Yadav VK, Ryu JH, Suda N, Tanaka KF, Gingrich JA, et al. (2008) Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 135: 825–837.
[19]
Rawlinson SC, el-Haj AJ, Minter SL, Tavares IA, Bennett A, et al. (1991) Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: a role for prostacyclin in adaptive bone remodeling? J Bone Miner Res 6: 1345–1351.
[20]
Rawlinson SC, McKay IJ, Ghuman M, Wellmann C, Ryan P, et al. (2009) Adult rat bones maintain distinct regionalized expression of markers associated with their development. PLoS One 4: e8358.
[21]
Koshihara Y, Hirano M, Kawamura M, Oda H, Higaki S (1991) Mineralization ability of cultured human osteoblast-like periosteal cells does not decline with aging. J Gerontol 46: B201–206.
[22]
Fedarko NS, Vetter UK, Weinstein S, Robey PG (1992) Age-related changes in hyaluronan, proteoglycan, collagen, and osteonectin synthesis by human bone cells. J Cell Physiol 151: 215–227.
[23]
Fedarko NS, D'Avis P, Frazier CR, Burrill MJ, Fergusson V, et al. (1995) Cell proliferation of human fibroblasts and osteoblasts in osteogenesis imperfecta: influence of age. J Bone Miner Res 10: 1705–1712.
[24]
Battmann A, Jundt G, Schulz A (1997) Endosteal human bone cells (EBC) show age-related activity in vitro. Exp Clin Endocrinol Diabetes 105: 98–102.
[25]
Kassem M, Ankersen L, Eriksen EF, Clark BF, Rattan SI (1997) Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts. Osteoporos Int 7: 514–524.
[26]
Yudoh K, Matsuno H, Nakazawa F, Katayama R, Kimura T (2001) Reconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblasts. J Bone Miner Res 16: 1453–1464.
[27]
Bergman RJ, Gazit D, Kahn AJ, Gruber H, McDougall S, et al. (1996) Age-related changes in osteogenic stem cells in mice. J Bone Miner Res 11: 568–577.
[28]
Stenderup K, Justesen J, Clausen C, Kassem M (2003) Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone 33: 919–926.
[29]
Jiang D, Fei RG, Pendergrass WR, Wolf NS (1992) An age-related reduction in the replicative capacity of two murine hematopoietic stroma cell types. Exp Hematol 20: 1216–1222.
[30]
Zha L, Hou N, Wang J, Yang G, Gao Y, et al. (2008) Collagen1[alpha]1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice. Journal of Genetics and Genomics 35: 525–530.
[31]
Kalajzic I, Kalajzic Z, Kaliterna M, Gronowicz G, Clark SH, et al. (2002) Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage. J Bone Miner Res 17: 15–25.
[32]
Armstrong VJ, Muzylak M, Sunters A, Zaman G, Saxon LK, et al. (2007) Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha. J Biol Chem 282: 20715–20727.
[33]
Sunters A, Armstrong VJ, Zaman G, Kypta RM, Kawano Y, et al. (2010) Mechano-transduction in osteoblastic cells involves strain-regulated estrogen receptor alpha-mediated control of insulin-like growth factor (IGF) I receptor sensitivity to Ambient IGF, leading to phosphatidylinositol 3-kinase/AKT-dependent Wnt/LRP5 receptor-independent activation of beta-catenin signaling. J Biol Chem 285: 8743–8758.
[34]
Shen L, Zhou S, Glowacki J (2009) Effects of age and gender on WNT gene expression in human bone marrow stromal cells. J Cell Biochem 106: 337–343.
[35]
Dwyer MA, Joseph JD, Wade HE, Eaton ML, Kunder RS, et al. (2010) WNT11 expression is induced by estrogen-related receptor alpha and beta-catenin and acts in an autocrine manner to increase cancer cell migration. Cancer Res 70: 9298–9308.
[36]
Almeida M, Han L, Bellido T, Manolagas SC, Kousteni S (2005) Wnt proteins prevent apoptosis of both uncommitted osteoblast progenitors and differentiated osteoblasts by beta-catenin-dependent and -independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase/AKT. J Biol Chem 280: 41342–41351.
[37]
Tobimatsu T, Kaji H, Sowa H, Naito J, Canaff L, et al. (2006) Parathyroid hormone increases beta-catenin levels through Smad3 in mouse osteoblastic cells. Endocrinology 147: 2583–2590.
[38]
Boutahar N, Guignandon A, Vico L, Lafage-Proust MH (2004) Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation. J Biol Chem 279: 30588–30599.
[39]
Cheng M, Zaman G, Rawlinson SC, Mohan S, Baylink DJ, et al. (1999) Mechanical strain stimulates ROS cell proliferation through IGF-II and estrogen through IGF-I. J Bone Miner Res 14: 1742–1750.
[40]
Damien E, Price JS, Lanyon LE (2000) Mechanical strain stimulates osteoblast proliferation through the estrogen receptor in males as well as females. J Bone Miner Res 15: 2169–2177.
[41]
Fermor B, Gundle R, Evans M, Emerton M, Pocock A, et al. (1998) Primary human osteoblast proliferation and prostaglandin E2 release in response to mechanical strain in vitro. Bone 22: 637–643.
[42]
Kaspar D, Seidl W, Neidlinger-Wilke C, Ignatius A, Claes L (2000) Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity. J Biomech 33: 45–51.
[43]
Neidlinger-Wilke C, Wilke HJ, Claes L (1994) Cyclic stretching of human osteoblasts affects proliferation and metabolism: a new experimental method and its application. J Orthop Res 12: 70–78.
[44]
Weyts FA, Bosmans B, Niesing R, van Leeuwen JP, Weinans H (2003) Mechanical control of human osteoblast apoptosis and proliferation in relation to differentiation. Calcif Tissue Int 72: 505–512.
[45]
Zhuang H, Wang W, Tahernia AD, Levitz CL, Luchetti WT, et al. (1996) Mechanical strain-induced proliferation of osteoblastic cells parallels increased TGF-beta 1 mRNA. Biochem Biophys Res Commun 229: 449–453.
[46]
Brighton CT, Fisher , Levine SE, Corsetti JR, Reilly T, et al. (1996) The biochemical pathway mediating the proliferative response of bone cells to a mechanical stimulus. J Bone Joint Surg Am 78: 1337–1347.
[47]
Lee KC, Jessop H, Suswillo R, Zaman G, Lanyon LE (2004) The adaptive response of bone to mechanical loading in female transgenic mice is deficient in the absence of oestrogen receptor-alpha and -beta. J Endocrinol 182: 193–201.
[48]
Case N, Ma M, Sen B, Xie Z, Gross TS, et al. (2008) Beta-catenin levels influence rapid mechanical responses in osteoblasts. J Biol Chem 283: 29196–29205.
[49]
Kamel MA, Picconi JL, Lara-Castillo N, Johnson ML (2010) 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 47: 872–881.
[50]
Cullen D, Akhter M, Johnson M, Morgan S, Recker R (2004) Ulna loading response altered by the HBM mutation. (Suppl.1).J Bone Miner Res 19. S396 p.
[51]
Akhter MP, Wells DJ, Short SJ, Cullen DM, Johnson ML, et al. (2004) Bone biomechanical properties in LRP5 mutant mice. Bone 35: 162–169.
[52]
Giladi M, Milgrom C, Simkin A, Danon Y (1991) Stress fractures. Identifiable risk factors. Am J Sports Med 19: 647–652.
[53]
Klein-Nulend J, van der Plas A, Semeins CM, Ajubi NE, Frangos JA, et al. (1995) Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9: 441–445.
