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Calcified Tissue International

, Volume 98, Issue 2, pp 172–185 | Cite as

Sex-Linked Skeletal Phenotype of Lysyl Oxidase Like-1 Mutant Mice

  • Loai Alsofi
  • Eileen Daley
  • Ian Hornstra
  • Elise F. Morgan
  • Zachary D. Mason
  • Jesus F. Acevedo
  • R. Ann Word
  • Louis C. Gerstenfeld
  • Philip C. TrackmanEmail author
Original Research

Abstract

Lysyl oxidases are required for collagen and elastin cross-linking and extracellular matrix maturation including in bone. The lysyl oxidase family consists of lysyl oxidase (LOX) and 4 isoforms (LOXL1-4). Here we investigate whether deletion of LOXL1, which has been linked primarily to elastin maturation, leads to skeletal abnormalities. Left femurs (n = 8), L5 vertebrae (n = 8), and tibiae (n = 8) were analyzed by micro-computed tomography in 13-week-old wild-type (WT) and LOXL1/− male and female mice. Right femurs (n = 8) were subjected to immunohistochemistry for LOXL1, and histochemical/histology analyses of osteoclasts and growth plates. Sera from all mice were analyzed for bone turnover markers. Results indicate strong expression of LOXL1 in wild-type growth plates in femurs. Significant deterioration of trabecular bone structure in long bones and vertebrae from female was observed but not from male, mutant mice compared with WT. Decreases in BV/TV, Conn.D, trabecular thickness, and number in the femoral distal metaphysis were observed in female, but not in male, mutant mice. Trabecular spacing was increased significantly in femurs of female mutant mice. Findings were similar in trabeculae of L5 vertebrae from female mutant mice. The number of TRAP positive osteoclasts at the trabecular bone surface was increased in female mutant mice compared with WT females, consistent with increased serum RANKL and decreased OPG levels. Analysis of bone turnover markers confirmed increased bone resorption as indicated by significantly elevated CTX-1 in the serum of female LOXL1/− mice compared to their wild-type counterparts, as well as decreased bone formation as measured by decreased serum levels of PINP. Picrosirius red staining revealed a loss of heterogeneity in collagen organization in female LOXL1/− mice only, with little to no yellow and orange birefringence. Organization was also impaired in chondrocyte columns in both female and male LOXL1/− mice, but to a greater extent in females. Data indicate that LOXL1/− mutant mice develop appendicular and axial skeletal phenotypes characterized by decreased bone volume fraction and compromised trabecular microstructure, predominantly in females.

Keywords

Lysyl oxidases Genetic animal model Micro-comuted tomography Collagen Bone histomorphometry 

Notes

Acknowledgments

This study was supported by DE14066 (PCT), and AG028048 (RAW).

Compliance with Ethical Standards

Conflicts of Interest

Loai Alsofi, Eileen Daley, Ian Hornstra, Elise F. Morgan, Zachary D. Mason, Jesus F. Acevedo, R. Ann Word, Louis C. Gerstenfeld, and Philip C. Trackman declare no conflicts of interest.

Human and Animal Rights and Informed Consent

The Institutional Animal Care and Use Committees of Boston University Medical Center and of the University of Texas Southwestern Medical Center approved the protocols regarding the use of mice in the reported experiments.

References

  1. 1.
    Csiszar K (2001) Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol 70:1–32CrossRefPubMedGoogle Scholar
  2. 2.
    Khosravi R, Sodek KL, Xu WP, Bais MV, Saxena D, Faibish M, Trackman PC (2014) A novel function for lysyl oxidase in pluripotent mesenchymal cell proliferation and relevance to inflammation-associated osteopenia. PLoS One 9:e100669PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Perryman L, Erler JT (2014) Lysyl oxidase in cancer research. Future Oncol 10:1709–1717CrossRefPubMedGoogle Scholar
  4. 4.
    Kenyon K, Contente S, Trackman PC, Tang J, Kagan HM, Friedman RM (1991) Lysyl oxidase and rrg messenger RNA. Science 253:802CrossRefPubMedGoogle Scholar
  5. 5.
    Liu G, Daneshgari F, Li M, Lin D, Lee U, Li T, Damaser MS (2007) Bladder and urethral function in pelvic organ prolapsed lysyl oxidase like-1 knockout mice. Br J Urol Int 100:414–418CrossRefGoogle Scholar
  6. 6.
    Panchenko MV, Stetler-Stevenson WG, Trubetskoy OV, Gacheru SN, Kagan HM (1996) Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase. J Biol Chem 271:7113–7119CrossRefPubMedGoogle Scholar
  7. 7.
    Trackman PC, Bedell-Hogan D, Tang J, Kagan HM (1992) Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor. J Biol Chem 267:8666–8671PubMedGoogle Scholar
  8. 8.
    Uzel MI, Scott IC, Babakhanlou-Chase H, Palamakumbura AH, Pappano WN, Hong HH, Greenspan DS, Trackman PC (2001) Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures. J Biol Chem 276:22537–22543CrossRefPubMedGoogle Scholar
  9. 9.
    Liu X, Zhao Y, Gao J, Pawlyk B, Starcher B, Spencer JA, Yanagisawa H, Zuo J, Li T (2004) Elastic fiber homeostasis requires lysyl oxidase-like 1 protein. Nat Genet 36:178–182CrossRefPubMedGoogle Scholar
  10. 10.
    Wieslander CK, Rahn DD, McIntire DD, Acevedo JF, Drewes PG, Yanagisawa H, Word RA (2009) Quantification of pelvic organ prolapse in mice: vaginal protease activity precedes increased MOPQ scores in fibulin 5 knockout mice. Biol Reprod 80:407–414PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Kagan HM, Trackman PC (1991) Properties and function of lysyl oxidase. Am J Respir Cell Mol Biol 5:206–210CrossRefPubMedGoogle Scholar
  12. 12.
    Smith-Mungo LI, Kagan HM (1998) Lysyl oxidase: properties, regulation and multiple functions in biology. Matrix Biol J Int Soc Matrix Biol 16:387–398CrossRefGoogle Scholar
  13. 13.
    Siegel RC (1979) Lysyl oxidase. Int Rev Connect Tissue Res 8:73–118CrossRefPubMedGoogle Scholar
  14. 14.
    Kagan HM (1986) Characterization and regulation of lysyl oxidase. In: Mecham RP (ed) Regulation of matrix accumulation. Academic Press, San Diego, pp 321–398Google Scholar
  15. 15.
    Hornstra IK, Birge S, Starcher B, Bailey AJ, Mecham RP, Shapiro SD (2003) Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem 278:14387–14393CrossRefPubMedGoogle Scholar
  16. 16.
    Maki JM, Rasanen J, Tikkanen H, Sormunen R, Makikallio K, Kivirikko KI, Soininen R (2002) Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. Circulation 106:2503–2509CrossRefPubMedGoogle Scholar
  17. 17.
    Pischon N, Maki JM, Weisshaupt P, Heng N, Palamakumbura AH, N’Guessan P, Ding A, Radlanski R, Renz H, Bronckers TA, Myllyharju J, Kielbassa AM, Kleber BM, Bernimoulin JP, Trackman PC (2009) Lysyl oxidase (lox) gene deficiency affects osteoblastic phenotype. Calcif Tissue Int 85:119–126PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Simon LS (2007) Osteoporosis. Rheum Dis Clin N Am 33:149–176CrossRefGoogle Scholar
  19. 19.
    Shih MS, Cook MA, Spence CA, Palnitkar S, McElroy H, Parfitt AM (1993) Relationship between bone formation rate and osteoblast surface on different subdivisions of the endosteal envelope in aging & osteoporosis. Bone 14:519–521CrossRefPubMedGoogle Scholar
  20. 20.
    Sanada H, Shikata J, Hamamoto H, Ueba Y, Yamamuro T, Takeda T (1978) Changes in collagen cross-linking and lysyl oxidase by estrogen. Biochim Biophys Acta 541:408–413CrossRefPubMedGoogle Scholar
  21. 21.
    Saito M, Marumo K (2010) Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus. Osteoporos Int 21:195–214CrossRefPubMedGoogle Scholar
  22. 22.
    Atsawasuwan P, Mochida Y, Parisuthiman D, Yamauchi M (2005) Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells. Biochem Biophys Res Commun 327:1042–1046CrossRefPubMedGoogle Scholar
  23. 23.
    Assaggaf MA, Kantarci A, Sume SS, Trackman PC (2015) Prevention of phenytoin-induced gingival overgrowth by lovastatin in mice. Am J Pathol 185:1588–1599CrossRefPubMedGoogle Scholar
  24. 24.
    Morgan EF, Mason ZD, Chien KB, Pfeiffer AJ, Barnes GL, Einhorn TA, Gerstenfeld LC (2009) Micro-computed tomography assessment of fracture healing: relationships among callus structure, composition, and mechanical function. Bone 44:335–344PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Gere JM, Goodno BJ (2009) Mechanics of materials. Cengage Learning Inc., IndependenceGoogle Scholar
  26. 26.
    Buie HR, Campbell GM, Klinck RJ, MacNeil JA, Boyd SK (2007) Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis. Bone 41:505–515CrossRefPubMedGoogle Scholar
  27. 27.
    Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R (2010) Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res 25:1468–1486CrossRefPubMedGoogle Scholar
  28. 28.
    Junqueira LC, Bignolas G, Brentani RR (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11:447–455CrossRefPubMedGoogle Scholar
  29. 29.
    Vasikaran S, Eastell R, Bruyere O, Foldes AJ, Garnero P, Griesmacher A, McClung M, Morris HA, Silverman S, Trenti T, Wahl DA, Cooper C, Kanis JA (2011) Markers of bone turnover for the prediction of fracture risk and monitoring of osteoporosis treatment: a need for international reference standards. Osteoporos Int 22:391–420CrossRefPubMedGoogle Scholar
  30. 30.
    Iftikhar M, Hurtado P, Bais MV, Wigner N, Stephens DN, Gerstenfeld LC, Trackman PC (2011) Lysyl oxidase-like-2 (LOXL2) is a major isoform in chondrocytes and is critically required for differentiation. J Biol Chem 286:909–918PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Martin A, Salvador F, Moreno-Bueno G, Floristan A, Ruiz-Herguido C, Cuevas EP, Morales S, Santos V, Csiszar K, Dubus P, Haigh JJ, Bigas A, Portillo F, Cano A (2015) Lysyl oxidase-like 2 represses Notch1 expression in the skin to promote squamous cell carcinoma progression. EMBO J 34:1090–1109CrossRefPubMedGoogle Scholar
  32. 32.
    Tommasini SM, Morgan TG, van der Meulen M, Jepsen KJ (2005) Genetic variation in structure-function relationships for the inbred mouse lumbar vertebral body. J Bone Miner Res 20:817–827CrossRefPubMedGoogle Scholar
  33. 33.
    Lee UJ, Gustilo-Ashby AM, Daneshgari F, Kuang M, Vurbic D, Lin DL, Flask CA, Li T, Damaser MS (2008) Lower urogenital tract anatomical and functional phenotype in lysyl oxidase like-1 knockout mice resembles female pelvic floor dysfunction in humans. Am J Physiol Renal Physiol 295:F545–F555CrossRefPubMedGoogle Scholar
  34. 34.
    Kapczuk K, Sowinska-Przepiera E, Friebe Z (2003) Osteoprotegerin/RANKL/RANK system and postmenopausal osteoporosis. The possible therapeutic aspects. Ginekol Pol 74:323–331PubMedGoogle Scholar
  35. 35.
    Bouxsein ML, Myers KS, Shultz KL, Donahue LR, Rosen CJ, Beamer WG (2005) Ovariectomy-induced bone loss varies among inbred strains of mice. J Bone Mine Res 20:1085–1092CrossRefGoogle Scholar
  36. 36.
    Bord S, Ireland DC, Beavan SR, Compston JE (2003) The effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts. Bone 32:136–141CrossRefPubMedGoogle Scholar
  37. 37.
    Boyce BF, Xing L (2007) Biology of RANK, RANKL, and osteoprotegerin. Arthritis Res Ther 9(Suppl 1):S1PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Bronson RE, Calaman SD, Traish AM, Kagan HM (1987) Stimulation of lysyl oxidase (EC 1.4.3.13) activity by testosterone and characterization of androgen receptors in cultured calf aorta smooth-muscle cells. Biochem J 244:317–323PubMedCentralCrossRefPubMedGoogle Scholar
  39. 39.
    Ballanti P, Minisola S, Pacitti MT, Scarnecchia L, Rosso R, Mazzuoli GF, Bonucci E (1997) Tartrate-resistant acid phosphate activity as osteoclastic marker: sensitivity of cytochemical assessment and serum assay in comparison with standardized osteoclast histomorphometry. Osteoporos Int 7:39–43CrossRefPubMedGoogle Scholar
  40. 40.
    Biver E (2012) Use of bone turnover markers in clinical practice. Curr Opin Endocrinol Diabetes Obes 19:468–473CrossRefPubMedGoogle Scholar
  41. 41.
    Garnero P, Ferreras M, Karsdal MA, Nicamhlaoibh R, Risteli J, Borel O, Qvist P, Delmas PD, Foged NT, Delaissé JM (2003) The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation. J Bone Miner Res 18:859–867CrossRefPubMedGoogle Scholar
  42. 42.
    Koivula M-K, Risteli L, Risteli J (2012) Measurement of aminoterminal propeptide of type I procollagen (PINP) in serum. Clin Biochem 45:920–927CrossRefPubMedGoogle Scholar
  43. 43.
    Christiansen DL, Huang EK, Silver FH (2000) Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties. Matrix Biol 19:409–420CrossRefPubMedGoogle Scholar
  44. 44.
    Muiznieks LD, Keeley FW (2013) Molecular assembly and mechanical properties of the extracellular matrix: a fibrous protein perspective. Biochim Biophys Acta 1832:866–875CrossRefPubMedGoogle Scholar
  45. 45.
    Ottani V, Raspanti M, Ruggeri A (2001) Collagen structure and functional implications. Micron 32:251–260CrossRefPubMedGoogle Scholar
  46. 46.
    Gualeni B, Rajpar MH, Kellogg A, Bell PA, Arvan P, Boot-Handford RP, Briggs MD (2013) A novel transgenic mouse model of growth plate dysplasia reveals that decreased chondrocyte proliferation due to chronic ER stress is a key factor in reduced bone growth. Dis Models Mech 6:1414–1425CrossRefGoogle Scholar
  47. 47.
    Wang G, Woods A, Agoston H, Ulici V, Glogauer M, Beier F (2007) Genetic ablation of Rac1 in cartilage results in chondrodysplasia. Dev Biol 306:612–623CrossRefPubMedGoogle Scholar
  48. 48.
    Lucero HA, Ravid K, Grimsby JL, Rich CB, DiCamillo SJ, Maki JM, Myllyharju J, Kagan HM (2008) Lysyl oxidase oxidizes cell membrane proteins and enhances the chemotactic response of vascular smooth muscle cells. J Biol Chem 283:24103–24117PubMedCentralCrossRefPubMedGoogle Scholar
  49. 49.
    Atsawasuwan P, Mochida Y, Katafuchi M, Kaku M, Fong KS, Csiszar K, Yamauchi M (2008) Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity. J Biol Chem 283:34229–34240PubMedCentralCrossRefPubMedGoogle Scholar
  50. 50.
    Li W, Nugent MA, Zhao Y, Chau AN, Li SJ, Chou IN, Liu G, Kagan HM (2003) Lysyl oxidase oxidizes basic fibroblast growth factor and inactivates its mitogenic potential. J Cell Biochem 88:152–164CrossRefPubMedGoogle Scholar
  51. 51.
    Le Provost GS, Debret R, Cenizo V, Aimond G, Pez F, Kaniewski B, Andre V, Sommer P (2010) Lysyl oxidase silencing impairs keratinocyte differentiation in a reconstructed-epidermis model. Exp Dermatol 19:1080–1087CrossRefPubMedGoogle Scholar
  52. 52.
    Palamakumbura AH, Vora SR, Nugent MA, Kirsch KH, Sonenshein GE, Trackman PC (2009) Lysyl oxidase propeptide inhibits prostate cancer cell growth by mechanisms that target FGF-2-cell binding and signaling. Oncogene 28:3390–3400PubMedCentralCrossRefPubMedGoogle Scholar
  53. 53.
    Kirschmann DA, Seftor EA, Fong SF, Nieva DR, Sullivan CM, Edwards EM, Sommer P, Csiszar K, Hendrix MJ (2002) A molecular role for lysyl oxidase in breast cancer invasion. Cancer Res 62:4478–4483PubMedGoogle Scholar
  54. 54.
    Li J, Gu X, Ma Y, Calicchio ML, Kong D, Teng YD, Yu L, Crain AM, Vartanian TK, Pasqualini R, Arap W, Libermann TA, Snyder EY, Sidman RL (2010) Nna1 mediates Purkinje cell dendritic development via lysyl oxidase propeptide and NF-kappaB signaling. Neuron 68:45–60PubMedCentralCrossRefPubMedGoogle Scholar
  55. 55.
    Vora SR, Palamakumbura AH, Mitsi M, Guo Y, Pischon N, Nugent MA, Trackman PC (2010) Lysyl oxidase propeptide inhibits FGF-2-induced signaling and proliferation of osteoblasts. J Biol Chem 285:7384–7393PubMedCentralCrossRefPubMedGoogle Scholar
  56. 56.
    Uveges TE, Collin-Osdoby P, Cabral WA, Ledgard F, Goldberg L, Bergwitz C, Forlino A, Osdoby P, Gronowicz GA, Marini JC (2008) Cellular mechanism of decreased bone in Brtl mouse model of OI: imbalance of decreased osteoblast function and increased osteoclasts and their precursors. J Bone Miner Res 23:1983–1994PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Loai Alsofi
    • 1
    • 2
  • Eileen Daley
    • 1
  • Ian Hornstra
    • 3
  • Elise F. Morgan
    • 4
  • Zachary D. Mason
    • 4
  • Jesus F. Acevedo
    • 5
  • R. Ann Word
    • 5
  • Louis C. Gerstenfeld
    • 6
  • Philip C. Trackman
    • 1
    Email author
  1. 1.Department of Molecular and Cell Biology, Henry M. Goldman School of Dental MedicineBoston UniversityBostonUSA
  2. 2.Department of Endodontics, Faculty of DentistryKing Abdulaziz UniversityJeddahSaudi Arabia
  3. 3.Division of DermatologyWashington University School of MedicineSaint LouisUSA
  4. 4.Department of Mechanical EngineeringBoston UniversityBostonUSA
  5. 5.Department of Obstetrics and GynecologyUniversity of Texas Southwestern Medical CenterDallasUSA
  6. 6.Department of Orthopaedic SurgeryBoston University School of MedicineBostonUSA

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