Calcified Tissue International

, Volume 76, Issue 1, pp 39–49 | Cite as

Skeletal Phenotype of Growing Transgenic Mice that Express a Function-Perturbing Form of β1 Integrin in Osteoblasts

  • R. K. Globus
  • D. Amblard
  • Y. Nishimura
  • U. T. Iwaniec
  • J.-B. Kim
  • E. A. C. Almeida
  • C. D. Damsky
  • T. J. Wronski
  • M. C. H. van der Meulen


Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of β1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for β1 integrin. To determine the influence of β1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35–90 days old TG mice were less curved than those of WT mice (72–92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they demonstrated differences in osteoblast rather than osteoclast parameters. Together, these results suggest that integrin signals generated during growth enhance the acquisition of a skeletal mass, structure, and strength to withstand the mechanical loads generated by weight-bearing.



We thank Dr. Randall Kramer (UCSF) for the kind gift of β1 integrin antibodies. We are also grateful to Emily Morey-Holton for helpful discussions and critical reading of the manuscript. We thank Veronica Rocha and Julie Litzenberger for technical assistance at NASA Ames Research Center, Nathan Netravali for torsion tests and Heather Ansorge for bending tests at Cornell University, Neda Mitova-Caneva for technical assistance at the University of Florida, and Dr. Patricia Calarco and Mercedes Joaquin for assistance with the transgenic colony at UCSF. This research was supported by NASA grant #99-HEDS-062 and NIH grant #P60 DE13058.


  1. 1.
    Duncan, RL, Hruska, KA 1994Chronic, intermittent loading alters mechanosensitive channel characteristics in osteoblast-like cellsAm J Physiol267F909916PubMedGoogle Scholar
  2. 2.
    Cheng, MZ, Rawlinson, SC, Pitsillides, AA, Zaman, G, Mohan, S, Baylink, DJ, Lanyon, LE 2002Human osteoblasts’ proliferative responses to strain and 17beta-estradiol are mediated by the estrogen receptor and the receptor for insulin-like growth factor IJ Bone Miner Res17593602PubMedGoogle Scholar
  3. 3.
    Mikuni-Takagaki, Y, Susuki, Y, Kawase, T, Saito, S 1996Distinct responses of different populations of bone cells to mechanical stressEndocrinology199620282035CrossRefGoogle Scholar
  4. 4.
    Lean, JM, Jagger, CJ, Chambers, TJ, Chow, JW 1995Increased insulin-like growth factor I mRNA expression in rat osteocytes in response to mechanical stimulationAm J Physiol268E318327PubMedGoogle Scholar
  5. 5.
    Fox, S, Chambers, T, Chow, J 1996Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulationAm J Physiol270E955960PubMedGoogle Scholar
  6. 6.
    Chow, J, Chambers, T 1994Indomethacin has distinct early and late actions on bone formation induced by mechanical loadingAm J Physiol267E287292PubMedGoogle Scholar
  7. 7.
    Pavalko, FM, Norvell, SM, Burr, DB, Turner, CH, Duncan, RL, Bidwell, JP 2003A model for mechanotransduction in bone cells: the load-bearing mechanosomesJ Cell Biochem88104112CrossRefPubMedGoogle Scholar
  8. 8.
    Martin, KH, Slack, JK, Boerner, SA, Martin, CC, Parsons, JT 2002Integrin connections map: to infinity and beyondScience29616521653CrossRefPubMedGoogle Scholar
  9. 9.
    Damsky, CH, Ilic, D 2002Integrin signaling: it’s where the action isCurr Opin Cell Biol14594602CrossRefPubMedGoogle Scholar
  10. 10.
    Bennett, JH, Moffatt, S, Horton, M 2001Cell adhesion molecules in human osteoblasts: structure and functionHistol Histopathol16603611PubMedGoogle Scholar
  11. 11.
    Gohel, A, Hand, A, Gronowicz, G 1995Immunogold localization of beta 1-integrin in bone: effect of glucocorticoids and insulin-like growth factor I on integrins and osteocyte formationJ Histochem Cytochem4310851096PubMedGoogle Scholar
  12. 12.
    Moursi, A, Globus, R, Damsky, C 1997Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitroJ Cell Sci11021872196PubMedGoogle Scholar
  13. 13.
    Carvalho, RS, Scott, JE, Yen, EH 1995The effects of mechanical stimulation on the distribution of beta 1 integrin and expression of beta 1-integrin mRNA in TE-85 human osteosarcoma cellsArch Oral Biol40257264CrossRefPubMedGoogle Scholar
  14. 14.
    Ingber, D 1991Integrins as mechanochemical transducersCurr Biol3841848CrossRefGoogle Scholar
  15. 15.
    Hynes, RO 2002Integrins: bidirectional, allosteric signaling machinesCell110673687CrossRefPubMedGoogle Scholar
  16. 16.
    Takeuchi, Y, Nakayama, K, Matsumoto, T 1996Differentiation and cell surface expression of transforming growth factor-beta receptors are regulated by interaction with matrix collagen in murine osteoblastic cellsJ Biol Chem27139383944Google Scholar
  17. 17.
    Jikko, A, Harris, SE, Chen, D, Mendrick, DL, Damsky, CH 1999Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2J Bone Miner Res1410751083PubMedGoogle Scholar
  18. 18.
    Salter, D, Robb, J, Wright, M 1997Electrophysical reponses of human bone cells to mechanical stimulation: evidence for specific integrin function in mechanotransductionJ Bone Miner Res1211331141PubMedGoogle Scholar
  19. 19.
    Pommerenke, H, Schmidt, C, Durr, F, Nebe, B, Luthen, F, Muller, P, Rychly, J 2002The mode of mechanical integrin stressing controls intracellular signaling in osteoblastsJ Bone Miner Res17603611PubMedGoogle Scholar
  20. 20.
    Stephens, L, Sutherland, A, Klimanskaya, I, Andrieux, A, Meneses, J, Pedersen, R, Damsky, C 1995Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethalityGenes Dev91883l895PubMedGoogle Scholar
  21. 21.
    Fassler, R, Meyer, M 1995Consequences of lack of beta 1 integrin gene expression in miceGenes Dev918961908PubMedGoogle Scholar
  22. 22.
    Aszodi, A, Hunziker, EB, Brakebusch, C, Fassler, R 2003Beta 1 integrins regulate chondrocyte rotation, G1 progression, and cytokinesisGenes Dev1724652479CrossRefPubMedGoogle Scholar
  23. 23.
    Zimmerman, D, Jin, F, Leboy, P, Hardy, S, Damsky, C 2000Impaired bone formation in transgenic mice resulting from altered integrin function in osteoblastsDev Biol220215CrossRefPubMedGoogle Scholar
  24. 24.
    Baker, AR, Hollingshead, PG, Pitts-Meek, S, Hansen, S, Taylor, R, Stewart, TA 1992Osteoblast-specific expression of growth hormone stimulates bone growth in transgenic miceMol Cell Biol1255415547PubMedGoogle Scholar
  25. 25.
    Akiyama, SK, Yamada, SS, Yamada, KM, LaFlamme, SE 1994Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimerasJ Biol Chem2691596115964Google Scholar
  26. 26.
    Lukashev, ME, Sheppard, D, Pytela, R 1994Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed beta 1 integrin cytoplasmic domainJ Biol Chem2691831118314Google Scholar
  27. 27.
    Stephens, LE, Sonne, JE, Fitzgerald, ML, Damsky, CH 1993Targeted deletion of β1 integrins in F9 embryonal carcinoma cells affects morphological differentiation but not tissue-specific gene expressionJ Cell Biol12316071620CrossRefPubMedGoogle Scholar
  28. 28.
    Hardy, S, Kitamura, M, Harris-Stansil, T, Dai, Y, Phipps, M 1997Construction of adenovirus vectors through Cre-Lox recombinationJ Virol7118421849PubMedGoogle Scholar
  29. 29.
    Chen, MS, Almeida, EA, Huovila, AP, Takahashi, Y, Shaw, LM, Mercurio, AM, White, JM 1999Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAMJ Cell Biol144549561CrossRefPubMedGoogle Scholar
  30. 30.
    Biewener, AA 1983Allometry of quadrupedal locomotion: the scaling of duty factor, bone curvature and limb orientation to body sizeJ Exp Biol105147171PubMedGoogle Scholar
  31. 31.
    Mikic, B, Meulen, MC, Kingsley, DM, Carter, DR 1995Long bone geometry and strength in adult BMP-5 deficient miceBone16445454PubMedGoogle Scholar
  32. 32.
    Meulen, MCH, Netravali, NA 2003Considerations for mechanical testing of mouse long bonesTrans Orthop Res Soc28432Google Scholar
  33. 33.
    Baron, R, Vignery, A, Neff, L, Silverglate, A, Maria, AS 1983

    Processing of undecalcified bone specimens for bone histomorphometry

    Recker, R eds. Bone histomorphometry: techniques and interpretationCRC PressBoca Raton1335
    Google Scholar
  34. 34.
    Parfitt, A, Drezner, M, Glorieaux, F, Kanis, J, Malluche, H, Meunier, P, Ott, S, Recker, R 1987Bone histomorphometry: standardization of nomenclature, symbols, and unitsJ Bone Miner Res2595610PubMedGoogle Scholar
  35. 35.
    Mikic, B, Meulen, MC, Kingsley, DM, Carter, DR 1996Mechanical and geometric changes in the growing femora of BMP-5 deficient miceBone18601607CrossRefPubMedGoogle Scholar
  36. 36.
    Keller, TS, Spengler, DM 1989Regulation of bone stress and strain in the immature and mature rat femurJ Biomech2211151127Google Scholar
  37. 37.
    Silva, MJ, Brodt, MD, Ettner, SL 2002Long bones from the senescence accelerated mouse SAMP6 have increased size but reduced whole-bone strength and resistance to fractureJ Bone Miner Res1715971603PubMedGoogle Scholar
  38. 38.
    Frenkel, B, Capparelli, C, Auken, M, Baran, D, Bryan, J, Stein, JL, Stein, GS, Lian, JB 1997Activity of the osteocalcin promoter in skeletal sites of transgenic mice and during osteoblast differentiation in bone marrow-derived stromal cell cultures: effects of age and sexEndocrinology13821092116CrossRefPubMedGoogle Scholar
  39. 39.
    Kasai, R, Bianco, P, Robey, PG, Kahn, AJ 1994Production and characterization of an antibody against the human bone GLA protein (BGP/osteocalcin) propeptide and its use in immunocytochemistry of bone cellsBone Miner25167182PubMedGoogle Scholar
  40. 40.
    Candeliere, GA, Liu, F, Aubin, JE 2001Individual osteoblasts in the developing calvaria express different gene repertoiresBone28351361CrossRefPubMedGoogle Scholar
  41. 41.
    Miao, D, Bai, X, Panda, D, McKee, M, Karaplis, A, Goltzman, D 2001Osteomalacia in hyp mice is associated with abnormal phex expression and with altered bone matrix protein expression and depositionEndocrinology142926939CrossRefPubMedGoogle Scholar
  42. 42.
    Biewener, AA, Bertram, JE 1994Structural response of growing bone to exercise and disuseJ Appl Physiol76946955PubMedGoogle Scholar
  43. 43.
    Lanyon, LE 1980The influence of function on the development of bone curvatureJ Zool (Lond)192457466Google Scholar
  44. 44.
    LaFlamme, SE, Thomas, LA, Yamada, SS, Yamada, KM 1994Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assemblyJ Cell Biol12612871298CrossRefPubMedGoogle Scholar
  45. 45.
    Colvin, JS, Bohne, BA, Harding, GW, McEwen, DG, Ornitz, DM 1996Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3Nat Genet12390397CrossRefPubMedGoogle Scholar
  46. 46.
    Biewener, AA 1991Musculoskeletal design in relation to body sizeJ Biomech 24 Suppl11929Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • R. K. Globus
    • 1
    • 2
  • D. Amblard
    • 1
    • 2
  • Y. Nishimura
    • 1
    • 2
  • U. T. Iwaniec
    • 3
  • J.-B. Kim
    • 2
  • E. A. C. Almeida
    • 1
    • 2
  • C. D. Damsky
    • 2
  • T. J. Wronski
    • 3
  • M. C. H. van der Meulen
    • 4
  1. 1.Life Sciences DivisionNASA Ames Research CenterMoffett FieldUSA
  2. 2.Department of StomatologyUniversity of CaliforniaSan FranciscoUSA
  3. 3.Department of Physiological SciencesUniversity of FloridaGainesvilleUSA
  4. 4.Sibley School of Mechanical and Aerospace EngineeringCornell UniversityIthacaUSA

Personalised recommendations