Skip to main content
SpringerLink
Log in

Modulation of unloading-induced bone loss in mice with altered ERK signaling

  • Published:
Mammalian Genome Aims and scope Submit manuscript

Abstract

Genetic variations mediate skeletal responsiveness to mechanical unloading, with individual space travelers exhibiting large variations in the extent of bone loss. We previously identified genomic regions harboring several hundred genes that can modulate the magnitude of skeletal adaptation to mechanical unloading. Here, bioinformatic filters aided in shortlisting 30 genes with bone-related and mechanoregulatory roles. The genes CD44, FGF2, NOD2, and Fas, all associated with ERK signaling, were then functionally tested in hindlimb-unloaded (HLU) knockout (KO) mice. Compared to their respective normally ambulating wildtype (WT) controls, all KO strains, except Fas mice, had lower trabecular bone volume, bone volume fraction, and/or trabecular number. For cortical bone and compared to ambulatory WT mice, CD44−/− had impaired properties while FGF2−/− showed enhanced indices. NOD2−/− and Fas−/− did not have a cortical phenotype. In all KO and WT groups, HLU resulted in impaired trabecular and cortical indices, primarily due to trabecular tissue loss and mitigation of cortical bone growth. The difference in trabecular separation between HLU and ambulatory controls was significantly greater in CD44−/− and NOD2−/− mice than in WT mice. In cortical bone, differences in cortical thickness, total pore volume, and cortical porosity between HLU and controls were aggravated in CD44−/− mice. In contrast, deletion of NOD2 and Fas genes mitigated the differences in Po.V between HLU and control mice. Together, we narrowed a previous list of QTL-derived candidate genes from over 300 to 30, and showed that CD44, NOD2, and Fas have distinct functions in regulating changes in trabecular and cortical bone indices during unloading.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Allen MR, Bloomfield SA (2003) Hindlimb unloading has a greater effect on cortical compared with cancellous bone in mature female rats. J Appl Physiol 94:642–650

    Article  PubMed  Google Scholar 

  • Amblard D, Lafage-Proust MH, Laib A, Thomas T, Rüegsegger P, Alexandre C, Vico L (2003) Tail suspension induces bone loss in skeletally mature mice in the C57BL/6 J strain but not in the C3H/HeJ strain. J Bone Miner Res 18:561–569

    Article  PubMed  Google Scholar 

  • Baba HA, Stypmann J, Grabellus F, Kirchhof P, Sokoll A, Schäfers M, Takeda A, Wilhelm MJ, Scheld HH, Takeda N (2003) Dynamic regulation of MEK/Erks and Akt/GSK-3β in human end-stage heart failure after left ventricular mechanical support: myocardial mechanotransduction-sensitivity as a possible molecular mechanism. Cardiovasc Res 59:390–399

    Article  CAS  PubMed  Google Scholar 

  • Baron R, Kneissel M (2013) WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med 19:179–192

    Article  CAS  PubMed  Google Scholar 

  • Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Müller R (2010) Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res 25:1468–1486

    Article  PubMed  Google Scholar 

  • Castillo AB, Blundo JT, Chen JC, Lee KL, Yereddi NR, Jang E, Kumar S, Tang WJ, Zarrin S, Kim J-B (2012) Focal adhesion kinase plays a role in osteoblast mechanotransduction in vitro but does not affect load-induced bone formation in vivo. PLoS ONE 7:e43291

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chikazu D, Hakeda Y, Ogata N, Nemoto K, Itabashi A, Takato T, Kumegawa M, Nakamura K, Kawaguchi H (2000) Fibroblast growth factor (FGF)-2 directly stimulates mature osteoclast function through activation of FGF receptor 1 and p42/p44 MAP kinase. J Biol Chem 275:31444–31450

    Article  CAS  PubMed  Google Scholar 

  • Coffin J, Florkiewicz R, Neumann J, Mort-Hopkins T, Gn Dorn, Lightfoot P, German R, Howles P, Kier A, O’toole B (1995) Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice. Mol Biol Cell 6:1861–1873

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • de Vries TJ, Schoenmaker T, Beertsen W, van der Neut R, Everts V (2005) Effect of CD44 deficiency on in vitro and in vivo osteoclast formation. J Cell Biochem 94:954–966

    Article  CAS  PubMed  Google Scholar 

  • Drake TA, Hannani K, Kabo JM, Villa V, Krass K, Lusis AJ (2001) Genetic loci influencing natural variations in femoral bone morphometry in mice. J Orthop Res 19:511–517

    Article  CAS  PubMed  Google Scholar 

  • Dupont E, Stevens L, Cochon L, Falempin M, Bastide B, Canu M-H (2011) ERK is involved in the reorganization of somatosensory cortical maps in adult rats submitted to hindlimb unloading. PLoS ONE 6:e17564

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Faassen AE, Schrager JA, Klein DJ, Oegema TR, Couchman JR, McCarthy JB (1992) A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion. J Cell Biol 116:521–531

    Article  CAS  PubMed  Google Scholar 

  • Glatt V, Canalis E, Stadmeyer L, Bouxsein ML (2007) Age-related changes in trabecular architecture differ in female and male C57BL/6 J mice. J Bone Miner Res 22:1197–1207

    Article  PubMed  Google Scholar 

  • Goillot E, Raingeaud J, Ranger A, Tepper RI, Davis RJ, Harlow E, Sanchez I (1997) Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway. Proc Natl Acad Sci USA 94:3302–3307

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Goodison S, Urquidi V, Tarin D (1999) CD44 cell adhesion molecules. Mol Pathol 52:189

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gortazar AR, Martin-Millan M, Bravo B, Plotkin LI, Bellido T (2013) Crosstalk between caveolin-1/extracellular signal-regulated kinase (ERK) and β-catenin survival pathways in osteocyte mechanotransduction. J Biol Chem 288:8168–8175

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hake S, Rocheford T (2004) Exploiting quantitative trait loci in gene discovery. Genes Dev 18:597–601

    Article  CAS  PubMed  Google Scholar 

  • Hayer S, Steiner G, Görtz B, Reiter E, Tohidast-Akrad M, Amling M, Hoffmann O, Redlich K, Zwerina J, Skriner K (2005) CD44 is a determinant of inflammatory bone loss. J Exp Med 201:903–914

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Herishanu Y, Gibellini F, Njuguna N, Hazan-Halevy I, Keyvanfar K, Lee E, Wilson W, Wiestner A (2011) CD44 signaling via PI3 K/AKT and MAPK/ERK pathways protects CLL cells from spontaneous and drug induced apoptosis through MCL-1. Leuk Lymphoma 52:1758

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hurley M, Okada Y, Xiao L, Tanaka Y, Ito M, Okimoto N, Nakamura T, Rosen C, Doetschman T, Coffin J (2006) Impaired bone anabolic response to parathyroid hormone in Fgf2 −/− and Fgf2 ± mice. Biochem Biophys Res Commun 341:989–994

    Article  CAS  PubMed  Google Scholar 

  • Ichikawa S, Koller DL, Johnson ML, Lai D, Xuei X, Edenberg HJ, Klein RF, Orwoll ES, Hui SL, Foroud TM (2006) Human ALOX12, but not ALOX15, is associated with BMD in white men and women. J Bone Miner Res 21:556–564

    Article  CAS  PubMed  Google Scholar 

  • Ishijima M, Tsuji K, Rittling SR, Yamashita T, Kurosawa H, Denhardt DT, Nifuji A, Noda M (2002) Resistance to unloading-induced three-dimensional bone loss in osteopontin-deficient mice. J Bone Miner Res 17:661–667

    Article  PubMed  Google Scholar 

  • Judex S, Garman R, Squire M, Donahue LR, Rubin C (2004) Genetically based influences on the site-specific regulation of trabecular and cortical bone morphology. J Bone Miner Res 19:600–606

    Article  PubMed  Google Scholar 

  • Judex S, Zhang W, Donahue LR, Ozcivici E (2013) Genetic loci that control the loss and regain of trabecular bone during unloading and reambulation. J Bone Miner Res 28:1537–1549

    Article  CAS  PubMed  Google Scholar 

  • Kania JR, Kehat-Stadler T, Kupfer SR (1997) CD44 antibodies inhibit osteoclast formation. J Bone Miner Res 12:1155–1164

    Article  CAS  PubMed  Google Scholar 

  • Katavić V, Lukić IK, Kovačić N, Grčević D, Lorenzo JA, Marušić A (2003) Increased bone mass is a part of the generalized lymphoproliferative disorder phenotype in the mouse. J Immunol 170:1540–1547

    Article  PubMed  Google Scholar 

  • Kleeberger SR, Schwartz DA (2005) From quantitative trait locus to gene: a work in progress. Am J Respir Crit Care Med 171:804–805

    Article  PubMed  Google Scholar 

  • Klein RF, Turner RJ, Skinner LD, Vartanian KA, Serang M, Carlos AS, Shea M, Belknap JK, Orwoll ES (2002) Mapping quantitative trait loci that influence femoral cross-sectional area in mice. J Bone Miner Res 17:1752–1760

    Article  CAS  PubMed  Google Scholar 

  • Klein RF, Allard J, Avnur Z, Nikolcheva T, Rotstein D, Carlos AS, Shea M, Waters RV, Belknap JK, Peltz G, Orwoll ES (2004) Regulation of bone mass in mice by the lipoxygenase gene Alox15. Science 303:229–232

    Article  CAS  PubMed  Google Scholar 

  • Kovacic N, Grcevic D, Katavic V, Lukic IK, Grubisic V, Mihovilovic K, Cvija H, Croucher PI, Marusic A (2010) Fas receptor is required for estrogen deficiency-induced bone loss in mice. Lab Invest 90:402–413

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kovačić N, Lukić IK, Grčević D, Katavić V, Croucher P, Marušić A A (2007) The Fas/Fas ligand system inhibits differentiation of murine osteoblasts but has a limited role in osteoblast and osteoclast apoptosis. J Immunol 178:3379–3389

    Article  PubMed Central  PubMed  Google Scholar 

  • Lang T, LeBlanc A, Evans H, Lu Y, Genant H, Yu A (2004) Cortical and trabecular bone mineral loss from the spine and hip in long-duration spaceflight. J Bone Miner Res 19:1006–1012

    Article  PubMed  Google Scholar 

  • LeBlanc A, Schneider V, Shackelford L, West S, Oganov V, Bakulin A, Voronin L (2000) Bone mineral and lean tissue loss after long duration space flight. J Musculoskelet Neuronal Interact 1:157–160

    CAS  PubMed  Google Scholar 

  • LeBlanc A, Spector E, Evans H, Sibonga J (2007) Skeletal responses to space flight and the bed rest analog: a review. J Musculoskelet Neuronal Interact 7:33

    CAS  PubMed  Google Scholar 

  • Li X, Masinde G, Gu W, Wergedal J, Mohan S, Baylink DJ (2002) Genetic dissection of femur breaking strength in a large population (MRL/MpJ × SJL/J) of F2 mice: single QTL effects, epistasis, and pleiotropy. Genomics 79:734–740

    Article  CAS  PubMed  Google Scholar 

  • Li Q, Chen M, Liu H, Yang L, Yang T, He G (2013) The dual role of ERK signaling in the apoptosis of neurons. Front BioSci (Landmark Edit) 19:1411–1417

    Article  Google Scholar 

  • Liang H, Pun S, Wronski T (1999) Bone anabolic effects of basic fibroblast growth factor in ovariectomized rats 1. Endocrinology 140:5780–5788

    CAS  PubMed  Google Scholar 

  • Lindberg MK, Movérare S, Skrtic S, Gao H, Dahlman-Wright K, Gustafsson J-AK, Ohlsson C (2003) Estrogen receptor (ER)-β reduces ERα-regulated gene transcription, supporting a “Ying Yang” relationship between ERα and ERβ in mice. Mol Endocrinol 17:203–208

    Article  CAS  PubMed  Google Scholar 

  • Lu Z, Xu S (2006) ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life 58:621–631

    Article  CAS  PubMed  Google Scholar 

  • Lloyd SA, Lang CH, Zhang Y, Paul EM, Laufenberg LJ, Lewis GS, Donahue HJ (2014) Interdependence of muscle atrophy and bone loss induced by mechanical unloading. J Bone Miner Res 29:1118–1130

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lublinsky S, Ozcivici E, Judex S (2007) An automated algorithm to detect the trabecular-cortical bone interface in micro-computed tomographic images. Calcif Tissue Int 81:285–293

    Article  CAS  PubMed  Google Scholar 

  • Meyers VE, Zayzafoon M, Gonda SR, Gathings WE, McDonald JM (2004) Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells. J Cell Biochem 93:697–707

    Article  CAS  PubMed  Google Scholar 

  • Montero A, Okada Y, Tomita M, Ito M, Tsurukami H, Nakamura T, Doetschman T, Coffin JD, Hurley MM (2000) Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. J Clin Investig 105:1085–1093

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Morey-Holton ER, Globus RK (2002) Hindlimb unloading rodent model: technical aspects. J Appl Physiol 92:1367–1377

    Article  PubMed  Google Scholar 

  • Motulsky HJ, Brown RE (2006) Detecting outliers when fitting data with nonlinear regression—a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinform 7:123

    Article  CAS  Google Scholar 

  • Nagata S, Golstein P (1995) The Fas death factor. Science 267:1449–1456

    Article  CAS  PubMed  Google Scholar 

  • Nedvetzki S, Gonen E, Assayag N, Reich R, Williams RO, Thurmond RL, Huang J-F, Neudecker BA, Wang F-S, Turley EA (2004) RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: a different interpretation of redundancy. Proc Natl Acad Sci USA 101:18081–18086

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nigam R, Laulederkind SJ, Hayman GT, Smith JR, Wang S-J, Lowry TF, Petri V, De Pons J, Tutaj M, Liu W (2013) Rat Genome Database: a unique resource for rat, human, and mouse quantitative trait locus data. Physiol Genomics 45:809–816

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Oganov V, Cann C, Rakhmanov A, Ternovoĭ S (1989) Study of the musculoskeletal system of the spine in humans after long-term space flights by the method of computerized tomography. Kosmicheskaia Biol i Aviakosmicheskaia Med 24:20–21

    Google Scholar 

  • Ozcivici E, Zhang W, Donahue LR, Judex S (2014) Quantitative trait loci that modulate trabecular bone’s risk of failure during unloading and reloading. Bone 64:25–32

    Article  PubMed  Google Scholar 

  • Rogner UC, Avner P (2003) Congenic mice: cutting tools for complex immune disorders. Nat Rev Immunol 3:243–252

    Article  CAS  PubMed  Google Scholar 

  • Rubin J, Murphy TC, Fan X, Goldschmidt M, Taylor WR (2002) Activation of extracellular signal-regulated kinase is involved in mechanical strain inhibition of RANKL expression in bone stromal cells. J Bone Miner Res 17:1452–1460

    Article  CAS  PubMed  Google Scholar 

  • Rubin J, Rubin C, Jacobs CR (2006) Molecular pathways mediating mechanical signaling in bone. Gene 367:1–16

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sartor MA, Mahavisno V, Keshamouni VG, Cavalcoli J, Wright Z, Karnovsky A, Kuick R, Jagadish H, Mirel B, Weymouth T (2010) ConceptGen: a gene set enrichment and gene set relation mapping tool. Bioinformatics 26:456–463

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Saxon L, Galea G, Meakin L, Price J, Lanyon L (2012) Estrogen receptors α and β have different gender-dependent effects on the adaptive responses to load bearing in cancellous and cortical bone. Endocrinology 153:2254–2266

    Article  CAS  PubMed  Google Scholar 

  • Sobue T, Naganawa T, Xiao L, Okada Y, Tanaka Y, Ito M, Okimoto N, Nakamura T, Coffin J, Hurley M (2005) Over-expression of fibroblast growth factor-2 causes defective bone mineralization and osteopenia in transgenic mice. J Cell Biochem 95:83–94

    Article  CAS  PubMed  Google Scholar 

  • Squire M, Donahue L-R, Rubin C, Judex S (2004) Genetic variations that regulate bone morphology in the male mouse skeleton do not define its susceptibility to mechanical unloading. Bone 35:1353–1360

    Article  PubMed  Google Scholar 

  • Strober W, Murray PJ, Kitani A, Watanabe T (2006) Signalling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol 6:9–20

    Article  CAS  PubMed  Google Scholar 

  • Stupakov G, Kazeĭkin V, Kozlovskiĭ A, Korolev V (1983) Evaluation of the changes in the bone structures of the human axial skeleton in prolonged space flight. Kosmicheskaia Biol i Aviakosmicheskaia Med 18:33–37

    Google Scholar 

  • Suzuki K, Zhu B, Rittling S, Denhardt D, Goldberg H, McCulloch C, Sodek J (2002) Colocalization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts. J Bone Miner Res 17:1486–1497

    Article  CAS  PubMed  Google Scholar 

  • Thompson WR, Rubin CT, Rubin J (2012) Mechanical regulation of signaling pathways in bone. Gene 503:179–193

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Underhill C (1992) CD44: the hyaluronan receptor. J Cell Sci 103:293–298

    CAS  PubMed  Google Scholar 

  • Volkman SK, Galecki AT, Burke DT, Paczas MR, Moalli MR, Miller RA, Goldstein SA (2003) Quantitative trait loci for femoral size and shape in a genetically heterogeneous mouse population. J Bone Miner Res 18:1497–1505

    Article  CAS  PubMed  Google Scholar 

  • Wada T, Nakashima T, Hiroshi N, Penninger JM (2006) RANKL–RANK signaling in osteoclastogenesis and bone disease. Trends Mol Med 12:17–25

    Article  CAS  PubMed  Google Scholar 

  • Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, Franz M, Grouios C, Kazi F, Lopes CT (2010) The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 38:W214–W220

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xiao L, Liu P, Li X, Doetschman T, Coffin JD, Drissi H, Hurley MM (2009) Exported 18-kDa isoform of fibroblast growth factor-2 is a critical determinant of bone mass in mice. J Biol Chem 284:3170–3182

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xing L, Xiu Y, Boyce BF (2012) Osteoclast fusion and regulation by RANKL-dependent and independent factors. World J Orthop 3:212

    Article  PubMed Central  PubMed  Google Scholar 

  • Yuan H, Zelkha S, Burkatovskaya M, Gupte R, Leeman SE, Amar S (2013) Pivotal role of NOD2 in inflammatory processes affecting atherosclerosis and periodontal bone loss. Proc Natl Acad Sci USA 110:E5059–E5068

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zielenski J, Corey M, Rozmahel R, Markiewicz D, Aznarez I, Casals T, Larriba S, Mercier B, Cutting GR, Krebsova A (1999) Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13. Nat Genet 22:128–129

    Article  CAS  PubMed  Google Scholar 

  • Zubilewicz A, Hecquet C, Jeanny J-C, Soubrane G, Courtois Y, Mascarelli F (2001) Two distinct signalling pathways are involved in FGF2-stimulated proliferation of choriocapillary endothelial cells: a comparative study with VEGF. Oncogene 20:1403–1413

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors are grateful for financial support by NASA (NNX12AL25G). The authors thank Alyssa Tuthill, Tee Pamon, and Sherin Kuriakose for technical assistance.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY, 11794-5281, USA

    Jeyantt S. Sankaran, Bing Li & Stefan Judex

  2. Department of Orthopedics, Tianjin Hospital, Tianjin, 300211, China

    Bing Li

  3. The Jackson Laboratory, Bar Harbor, ME, 04609, USA

    Leah Rae Donahue

Authors
  1. Jeyantt S. Sankaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bing Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leah Rae Donahue
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stefan Judex
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan Judex.

Ethics declarations

Conflict of interest

All authors have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sankaran, J.S., Li, B., Donahue, L.R. et al. Modulation of unloading-induced bone loss in mice with altered ERK signaling. Mamm Genome 27, 47–61 (2016). https://doi.org/10.1007/s00335-015-9611-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00335-015-9611-x

Keywords

Search

Navigation