Genetic analysis of osteoblast activity identifies Zbtb40 as a regulator of osteoblast activity and bone mass

Autoři: Madison L. Doolittle aff001;  Gina M. Calabrese aff002;  Larry D. Mesner aff002;  Dana A. Godfrey aff004;  Robert D. Maynard aff001;  Cheryl L. Ackert-Bicknell aff001;  Charles R. Farber aff002
Působiště autorů: Center for Musculoskeletal Research, University of Rochester, Rochester, New York, United States of America aff001;  Center for Public Health Genomics, University of Virginia, Charlottesville, Virginia, United States of America aff002;  Department of Public Health Sciences, University of Virginia, Charlottesville, Virginia, United States of America aff003;  Department of Orthopedics, University of Colorado, Aurora, Colorado, United States of America aff004;  Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, United States of America aff005
Vyšlo v časopise: Genetic analysis of osteoblast activity identifies Zbtb40 as a regulator of osteoblast activity and bone mass. PLoS Genet 16(6): e32767. doi:10.1371/journal.pgen.1008805
Kategorie: Research Article


Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

Klíčová slova:

Alizarin staining – Bone fracture – Genetic loci – Genome-wide association studies – Mouse models – Osteoblast differentiation – Osteoblasts – Osteoporosis


1. Black DM, Rosen CJ. Postmenopausal Osteoporosis. N Engl J Med. 2016;374(21):2096–7.

2. Sozen T, Ozisik L, Basaran NC. An overview and management of osteoporosis. Eur J Rheumatol. 2017;4(1):46–56. doi: 10.5152/eurjrheum.2016.048 28293453

3. Johnell O, Kanis JA. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int. 2006;17(12):1726–33. doi: 10.1007/s00198-006-0172-4 16983459

4. Cummings SR, Melton LJ. Epidemiology and outcomes of osteoporotic fractures. Lancet. 2002;359(9319):1761–7. doi: 10.1016/S0140-6736(02)08657-9 12049882

5. Kanis JA, Johnell O, Oden A, Sembo I, Redlund-Johnell I, Dawson A, et al. Long-term risk of osteoporotic fracture in Malmo. Osteoporos Int. 2000;11(8):669–74. doi: 10.1007/s001980070064 11095169

6. Melton LJ 3rd, Atkinson EJ, O'Connor MK, O'Fallon WM, Riggs BL. Bone density and fracture risk in men. J Bone Miner Res. 1998;13(12):1915–23. doi: 10.1359/jbmr.1998.13.12.1915 9844110

7. Melton LJ 3rd, Chrischilles EA, Cooper C, Lane AW, Riggs BL. Perspective. How many women have osteoporosis? J Bone Miner Res. 1992;7(9):1005–10. doi: 10.1002/jbmr.5650070902 1414493

8. Ackert-Bicknell CL, Karasik D, Li Q, Smith RV, Hsu YH, Churchill GA, et al. Mouse BMD quantitative trait loci show improved concordance with human genome-wide association loci when recalculated on a new, common mouse genetic map. J Bone Miner Res. 2010;25(8):1808–20. doi: 10.1002/jbmr.72 20200990

9. Farber CR, Kelly SA, Baruch E, Yu D, Hua K, Nehrenberg DL, et al. Identification of quantitative trait loci influencing skeletal architecture in mice: emergence of Cdh11 as a primary candidate gene regulating femoral morphology. J Bone Miner Res. 2011;26(9):2174–83. doi: 10.1002/jbmr.436 21638317

10. Ralston SH, Uitterlinden AG. Genetics of osteoporosis. Endocr Rev. 2010;31(5):629–62. doi: 10.1210/er.2009-0044 20431112

11. Boudin E, Fijalkowski I, Hendrickx G, Van Hul W. Genetic control of bone mass. Mol Cell Endocrinol. 2016;432:3–13. doi: 10.1016/j.mce.2015.12.021 26747728

12. Richards JB, Zheng HF, Spector TD. Genetics of osteoporosis from genome-wide association studies: advances and challenges. Nat Rev Genet. 2012;13(8):576–88. doi: 10.1038/nrg3228 22805710

13. Bonewald LF. The amazing osteocyte. J Bone Miner Res. 2011;26(2):229–38. doi: 10.1002/jbmr.320 21254230

14. Raggatt LJ, Partridge NC. Cellular and molecular mechanisms of bone remodeling. J Biol Chem. 2010;285(33):25103–8. doi: 10.1074/jbc.R109.041087 20501658

15. Sims NA, Gooi JH. Bone remodeling: Multiple cellular interactions required for coupling of bone formation and resorption. Semin Cell Dev Biol. 2008;19(5):444–51. doi: 10.1016/j.semcdb.2008.07.016 18718546

16. Eastell R, O'Neill TW, Hofbauer LC, Langdahl B, Reid IR, Gold DT, et al. Postmenopausal osteoporosis. Nat Rev Dis Primers. 2016;2:16069. doi: 10.1038/nrdp.2016.69 27681935

17. Khosla S. Pathogenesis of age-related bone loss in humans. J Gerontol A Biol Sci Med Sci. 2013;68(10):1226–35. doi: 10.1093/gerona/gls163 22923429

18. Patsch JM, Burghardt AJ, Yap SP, Baum T, Schwartz AV, Joseph GB, et al. Increased cortical porosity in type 2 diabetic postmenopausal women with fragility fractures. J Bone Miner Res. 2013;28(2):313–24. doi: 10.1002/jbmr.1763 22991256

19. Szafors P, Che H, Barnetche T, Morel J, Gaujoux-Viala C, Combe B, et al. Risk of fracture and low bone mineral density in adults with inflammatory bowel diseases. A systematic literature review with meta-analysis. Osteoporos Int. 2018;29(11):2389–97. doi: 10.1007/s00198-018-4586-6 29909470

20. Mehler PS, Cleary BS, Gaudiani JL. Osteoporosis in anorexia nervosa. Eat Disord. 2011;19(2):194–202. doi: 10.1080/10640266.2011.551636 21360368

21. Kim SM, Long J, Montez-Rath M, Leonard M, Chertow GM. Hip Fracture in Patients With Non-Dialysis-Requiring Chronic Kidney Disease. J Bone Miner Res. 2016;31(10):1803–9. doi: 10.1002/jbmr.2862 27145189

22. Yan C, Avadhani NG, Iqbal J. The effects of smoke carcinogens on bone. Curr Osteoporos Rep. 2011;9(4):202–9. doi: 10.1007/s11914-011-0068-x 21874290

23. Prada D, Zhong J, Colicino E, Zanobetti A, Schwartz J, Dagincourt N, et al. Association of air particulate pollution with bone loss over time and bone fracture risk: analysis of data from two independent studies. Lancet Planet Health. 2017;1(8):e337–e47. doi: 10.1016/S2542-5196(17)30136-5 29527596

24. Mikosch P. Alcohol and bone. Wien Med Wochenschr. 2014;164(1–2):15–24. doi: 10.1007/s10354-013-0258-5 24477631

25. Ackert-Bicknell CL, Demissie S, Marin de Evsikova C, Hsu YH, DeMambro VE, Karasik D, et al. PPARG by dietary fat interaction influences bone mass in mice and humans. J Bone Miner Res. 2008;23(9):1398–408. doi: 10.1359/jbmr.080419 18707223

26. Vakil N. Prescribing proton pump inhibitors: is it time to pause and rethink? Drugs. 2012;72(4):437–45. doi: 10.2165/11599320-000000000-00000 22356286

27. Cosman F, de Beur SJ, LeBoff MS, Lewiecki EM, Tanner B, Randall S, et al. Clinician's Guide to Prevention and Treatment of Osteoporosis. Osteoporos Int. 2014;25(10):2359–81. doi: 10.1007/s00198-014-2794-2 25182228

28. Buckley L, Guyatt G, Fink HA, Cannon M, Grossman J, Hansen KE, et al. 2017 American College of Rheumatology Guideline for the Prevention and Treatment of Glucocorticoid-Induced Osteoporosis. Arthritis Rheumatol. 2017;69(8):1521–37. doi: 10.1002/art.40137 28585373

29. Vestergaard P, Hermann P, Jensen JE, Eiken P, Mosekilde L. Effects of paracetamol, non-steroidal anti-inflammatory drugs, acetylsalicylic acid, and opioids on bone mineral density and risk of fracture: results of the Danish Osteoporosis Prevention Study (DOPS). Osteoporos Int. 2012;23(4):1255–65. doi: 10.1007/s00198-011-1692-0 21710339

30. Karsenty G, Ferron M. The contribution of bone to whole-organism physiology. Nature. 2012;481(7381):314–20. doi: 10.1038/nature10763 22258610

31. Kemp JP, Morris JA, Medina-Gomez C, Forgetta V, Warrington NM, Youlten SE, et al. Identification of 153 new loci associated with heel bone mineral density and functional involvement of GPC6 in osteoporosis. Nat Genet. 2017;49(10):1468–75. doi: 10.1038/ng.3949 28869591

32. Morris JA, Kemp JP, Youlten SE, Laurent L, Logan JG, Chai RC, et al. An atlas of genetic influences on osteoporosis in humans and mice. Nat Genet. 2019;51(2):258–66. doi: 10.1038/s41588-018-0302-x 30598549

33. Farber CR, Bennett BJ, Orozco L, Zou W, Lira A, Kostem E, et al. Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis. PLoS Genet. 2011;7(4):e1002038. doi: 10.1371/journal.pgen.1002038 21490954

34. Russow G, Jahn D, Appelt J, Mardian S, Tsitsilonis S, Keller J. Anabolic Therapies in Osteoporosis and Bone Regeneration. Int J Mol Sci. 2018;20(1).

35. Mesner LD, Calabrese GM, Al-Barghouthi B, Gatti DM, Sundberg JP, Churchill GA, et al. Mouse genome-wide association and systems genetics identifies Lhfp as a regulator of bone mass. PLoS Genet. 2019;15(5):e1008123. doi: 10.1371/journal.pgen.1008123 31042701

36. Kang HM, Zaitlen NA, Wade CM, Kirby A, Heckerman D, Daly MJ, et al. Efficient control of population structure in model organism association mapping. Genetics. 2008;178(3):1709–23. doi: 10.1534/genetics.107.080101 18385116

37. Zheng HF, Forgetta V, Hsu YH, Estrada K, Rosello-Diez A, Leo PJ, et al. Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture. Nature. 2015;526(7571):112–7. doi: 10.1038/nature14878 26367794

38. Calabrese G, Bennett BJ, Orozco L, Kang HM, Eskin E, Dombret C, et al. Systems genetic analysis of osteoblast-lineage cells. PLoS Genet. 2012;8(12):e1003150. doi: 10.1371/journal.pgen.1003150 23300464

39. Ghazalpour A, Rau CD, Farber CR, Bennett BJ, Orozco LD, van Nas A, et al. Hybrid mouse diversity panel: a panel of inbred mouse strains suitable for analysis of complex genetic traits. Mamm Genome. 2012;23(9–10):680–92. doi: 10.1007/s00335-012-9411-5 22892838

40. Chengalvala MV, Bapat AR, Hurlburt WW, Kostek B, Gonder DS, Mastroeni RA, et al. Biochemical characterization of osteo-testicular protein tyrosine phosphatase and its functional significance in rat primary osteoblasts. Biochemistry. 2001;40(3):814–21. doi: 10.1021/bi0019996 11170399

41. Wheeler MA, Townsend MK, Yunker LA, Mauro LJ. Transcriptional activation of the tyrosine phosphatase gene, OST-PTP, during osteoblast differentiation. J Cell Biochem. 2002;87(4):363–76. doi: 10.1002/jcb.10297 12397596

42. Duncan EL, Danoy P, Kemp JP, Leo PJ, McCloskey E, Nicholson GC, et al. Genome-wide association study using extreme truncate selection identifies novel genes affecting bone mineral density and fracture risk. PLoS Genet. 2011;7(4):e1001372. doi: 10.1371/journal.pgen.1001372 21533022

43. Estrada K, Styrkarsdottir U, Evangelou E, Hsu YH, Duncan EL, Ntzani EE, et al. Genome-wide meta-analysis identifies 56 bone mineral density loci and reveals 14 loci associated with risk of fracture. Nat Genet. 2012;44(5):491–501. doi: 10.1038/ng.2249 22504420

44. Rivadeneira F, Styrkarsdottir U, Estrada K, Halldorsson BV, Hsu YH, Richards JB, et al. Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies. Nat Genet. 2009;41(11):1199–206. doi: 10.1038/ng.446 19801982

45. Styrkarsdottir U, Halldorsson BV, Gretarsdottir S, Gudbjartsson DF, Walters GB, Ingvarsson T, et al. Multiple genetic loci for bone mineral density and fractures. N Engl J Med. 2008;358(22):2355–65. doi: 10.1056/NEJMoa0801197 18445777

46. Zhang L, Choi HJ, Estrada K, Leo PJ, Li J, Pei YF, et al. Multistage genome-wide association meta-analyses identified two new loci for bone mineral density. Hum Mol Genet. 2014;23(7):1923–33. doi: 10.1093/hmg/ddt575 24249740

47. Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res. 1992;7(6):683–92. doi: 10.1002/jbmr.5650070613 1414487

48. Ichida F, Nishimura R, Hata K, Matsubara T, Ikeda F, Hisada K, et al. Reciprocal roles of MSX2 in regulation of osteoblast and adipocyte differentiation. J Biol Chem. 2004;279(32):34015–22. doi: 10.1074/jbc.M403621200 15175325

49. Stark K, Vainio S, Vassileva G, McMahon AP. Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature. 1994;372(6507):679–83. doi: 10.1038/372679a0 7990960

50. Kuhn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science. 1995;269(5229):1427–9. doi: 10.1126/science.7660125 7660125

51. Logan M, Martin JF, Nagy A, Lobe C, Olson EN, Tabin CJ. Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis. 2002;33(2):77–80. doi: 10.1002/gene.10092 12112875

52. Beamer WG, Shultz KL, Coombs HF 3rd, DeMambro VE, Reinholdt LG, Ackert-Bicknell CL, et al. BMD regulation on mouse distal chromosome 1, candidate genes, and response to ovariectomy or dietary fat. J Bone Miner Res. 2011;26(1):88–99. doi: 10.1002/jbmr.200 20687154

53. Shultz KL, Donahue LR, Bouxsein ML, Baylink DJ, Rosen CJ, Beamer WG. Congenic strains of mice for verification and genetic decomposition of quantitative trait loci for femoral bone mineral density. J Bone Miner Res. 2003;18(2):175–85. doi: 10.1359/jbmr.2003.18.2.175 12568393

54. Goncalves L, Filipe M, Marques S, Salgueiro AM, Becker JD, Belo JA. Identification and functional analysis of novel genes expressed in the Anterior Visceral Endoderm. Int J Dev Biol. 2011;55(3):281–95. doi: 10.1387/ijdb.103273lg 21553379

55. Esen E, Chen J, Karner CM, Okunade AL, Patterson BW, Long F. WNT-LRP5 signaling induces Warburg effect through mTORC2 activation during osteoblast differentiation. Cell Metab. 2013;17(5):745–55. doi: 10.1016/j.cmet.2013.03.017 23623748

56. Moverare-Skrtic S, Henning P, Liu X, Nagano K, Saito H, Borjesson AE, et al. Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures. Nat Med. 2014;20(11):1279–88. doi: 10.1038/nm.3654 25306233

57. Okamoto M, Udagawa N, Uehara S, Maeda K, Yamashita T, Nakamichi Y, et al. Noncanonical Wnt5a enhances Wnt/beta-catenin signaling during osteoblastogenesis. Sci Rep. 2014;4:4493. doi: 10.1038/srep04493 24670389

58. Karner CM, Long F. Wnt signaling and cellular metabolism in osteoblasts. Cell Mol Life Sci. 2017;74(9):1649–57. doi: 10.1007/s00018-016-2425-5 27888287

59. Westendorf JJ, Kahler RA, Schroeder TM. Wnt signaling in osteoblasts and bone diseases. Gene. 2004;341:19–39. doi: 10.1016/j.gene.2004.06.044 15474285

60. Yavropoulou MP, Yovos JG. The role of the Wnt signaling pathway in osteoblast commitment and differentiation. Hormones (Athens). 2007;6(4):279–94.

61. Yu B, Chang J, Liu Y, Li J, Kevork K, Al-Hezaimi K, et al. Wnt4 signaling prevents skeletal aging and inflammation by inhibiting nuclear factor-kappaB. Nat Med. 2014;20(9):1009–17. doi: 10.1038/nm.3586 25108526

62. Beaulieu AM, Sant'Angelo DB. The BTB-ZF family of transcription factors: key regulators of lineage commitment and effector function development in the immune system. J Immunol. 2011;187(6):2841–7. doi: 10.4049/jimmunol.1004006 21900183

63. Siggs OM, Beutler B. The BTB-ZF transcription factors. Cell Cycle. 2012;11(18):3358–69. doi: 10.4161/cc.21277 22894929

64. Mei B, Wang Y, Ye W, Huang H, Zhou Q, Chen Y, et al. LncRNA ZBTB40-IT1 modulated by osteoporosis GWAS risk SNPs suppresses osteogenesis. Hum Genet. 2019;138(2):151–66. doi: 10.1007/s00439-019-01969-y 30661131

65. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 1997;89(5):755–64. doi: 10.1016/s0092-8674(00)80258-5 9182763

66. Matsubara T, Kida K, Yamaguchi A, Hata K, Ichida F, Meguro H, et al. BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation. J Biol Chem. 2008;283(43):29119–25. doi: 10.1074/jbc.M801774200 18703512

67. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. 2002;108(1):17–29. doi: 10.1016/s0092-8674(01)00622-5 11792318

68. Kobayashi A, Stewart CA, Wang Y, Fujioka K, Thomas NC, Jamin SP, et al. beta-Catenin is essential for Mullerian duct regression during male sexual differentiation. Development. 2011;138(10):1967–75. doi: 10.1242/dev.056143 21490063

69. Dacic S, Kalajzic I, Visnjic D, Lichtler AC, Rowe DW. Col1a1-driven transgenic markers of osteoblast lineage progression. J Bone Miner Res. 2001;16(7):1228–36. doi: 10.1359/jbmr.2001.16.7.1228 11450698

70. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5. doi: 10.1038/nmeth.2089 22930834

71. Lariviere WR, Mogil JS. The genetics of pain and analgesia in laboratory animals. Methods Mol Biol. 2010;617:261–78. doi: 10.1007/978-1-60327-323-7_20 20336428

72. Yang H, Ding Y, Hutchins LN, Szatkiewicz J, Bell TA, Paigen BJ, et al. A customized and versatile high-density genotyping array for the mouse. Nat Methods. 2009;6(9):663–6. doi: 10.1038/nmeth.1359 19668205

73. Turner SD. qqman: an R package for visualizing GWAS results using Q-Q and manhattan plots. bioRxiv Cold Spring Harbor Laboratory. 2014;005165.

74. Calabrese GM, Mesner LD, Stains JP, Tommasini SM, Horowitz MC, Rosen CJ, et al. Integrating GWAS and Co-expression Network Data Identifies Bone Mineral Density Genes SPTBN1 and MARK3 and an Osteoblast Functional Module. Cell Syst. 2017;4(1):46–59 e4. doi: 10.1016/j.cels.2016.10.014 27866947

75. Mesner LD, Hamlin JL. Specific signals at the 3' end of the DHFR gene define one boundary of the downstream origin of replication. Genes Dev. 2005;19(9):1053–66. doi: 10.1101/gad.1307105 15879555

76. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. doi: 10.1006/meth.2001.1262 11846609

77. Canalis E, Yu J, Schilling L, Yee SP, Zanotti S. The lateral meningocele syndrome mutation causes marked osteopenia in mice. J Biol Chem. 2018;293(36):14165–77. doi: 10.1074/jbc.RA118.004242 30042232

Článek vyšel v časopise

PLOS Genetics

2020 Číslo 6
Nejčtenější tento týden
Nejčtenější v tomto čísle

Zvyšte si kvalifikaci online z pohodlí domova

Důležitost adherence při depresivním onemocnění
nový kurz
Autoři: MUDr. Eliška Bartečková, Ph.D.

Koncepce osteologické péče pro gynekology a praktické lékaře
Autoři: MUDr. František Šenk

Sekvenční léčba schizofrenie
Autoři: MUDr. Jana Hořínková, Ph.D.

Hypertenze a hypercholesterolémie – synergický efekt léčby
Autoři: prof. MUDr. Hana Rosolová, DrSc.

Multidisciplinární zkušenosti u pacientů s diabetem
Autoři: Prof. MUDr. Martin Haluzík, DrSc., prof. MUDr. Vojtěch Melenovský, CSc., prof. MUDr. Vladimír Tesař, DrSc.

Všechny kurzy
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se