Sex-Linked Skeletal Phenotype of Lysyl Oxidase Like-1 Mutant Mice
- 503 Downloads
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.
KeywordsLysyl oxidases Genetic animal model Micro-comuted tomography Collagen Bone histomorphometry
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.
- 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
- 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
- 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
- 25.Gere JM, Goodno BJ (2009) Mechanics of materials. Cengage Learning Inc., IndependenceGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- 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
- 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