Advertisement

Differentially expressed proteins identified by TMT proteomics analysis in bone marrow microenvironment of osteoporotic patients

  • Q. Zhou
  • F. Xie
  • B. Zhou
  • J. Wang
  • B. Wu
  • L. Li
  • Y. Kang
  • R. DaiEmail author
  • Y. Jiang
Original Article

Abstract

Summary

We applied tandem mass tag (TMT)-based proteomics to investigate protein changes in bone marrow microenvironment of osteoporotic patients undergoing spine fusion. Multiple bioinformatics tools were used to identify and analyze 219 differentially expressed proteins. These proteins may be associated with the pathogenesis of osteoporosis.

Introduction

Bone marrow microenvironment is indispensable for the maintenance of bone homeostasis. We speculated that alterations of some factors in the microenvironment of osteoporotic subjects might influence the homeostasis. This study aimed to investigate the changes in the expression of protein factors in the bone marrow environment of osteoporosis.

Methods

We performed a proteomics analysis in the vertebral body-derived bone marrow supernatant fluid from 8 Chinese patients undergoing posterior lumbar interbody fusion (4 osteoporotic vs. 4 non-osteoporotic) and used micro-CT to analyze the microstructural features of spinous processes from these patients. We further performed western blotting to validate the differential expressions of some proteins.

Results

There was deteriorated bone microstructure in osteoporotic patients. Based on proteomics analysis, 172 upregulated and 47 downregulated proteins were identified. These proteins had multiple biological functions associated with osteoblast differentiation, lipid metabolism, and cell migration, and formed a complex protein–protein interaction network. We identified five major regulatory mechanisms, splicing, translation, protein degradation, cytoskeletal organization, and lipid metabolism, involved in the pathogenesis of osteoporosis.

Conclusions

There are various protein factors, such as DDX5, PSMC2, CSNK1A1, PLIN1, ILK, and TPM4, differentially expressed in the bone marrow microenvironment of osteoporotic patients, providing new ideas for finding therapeutic targets for osteoporosis.

Keywords

Bone marrow microenvironment Bone marrow supernatant fluid Bone microstructure Osteoporosis Proteomics 

Notes

Acknowledgments

We thank Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (Shanghai, China) for providing us with SkyScan1176 to perform micro-CT and Jingjie PTM BioLab (Hangzhou, China) for the technical support.

Funding Information

This research is supported by the National Natural Science Foundation of China (81670804), the Science and Technology Program of Hunan Province (2016WK2020) and the Clinical Big Data Project of Central South University.

Compliance with ethical standards

This study obtained the ethics approval from the ethical committee of the Second Xiangya Hospital.

Conflicts of interest

None.

References

  1. 1.
    Rachner TD, Khosla S, Hofbauer LC (2011) Osteoporosis: now and the future. Lancet 377(9773):1276–1287.  https://doi.org/10.1016/S0140-6736(10)62349-5 CrossRefGoogle Scholar
  2. 2.
    Collin-Osdoby P (1994) Role of vascular endothelial cells in bone biology. J Cell Biochem 55(3):304–309.  https://doi.org/10.1002/jcb.240550306 CrossRefGoogle Scholar
  3. 3.
    Li J, Liu X, Zuo B, Zhang L (2016) The role of bone marrow microenvironment in governing the balance between osteoblastogenesis and adipogenesis. Aging Dis 7(4):514–525.  https://doi.org/10.14336/AD.2015.1206 CrossRefGoogle Scholar
  4. 4.
    Feng X, McDonald JM (2011) Disorders of bone remodeling. Annu Rev Pathol 6:121–145.  https://doi.org/10.1146/annurev-pathol-011110-130203 CrossRefGoogle Scholar
  5. 5.
    Moore KA, Lemischka IR (2006) Stem cells and their niches. Science 311(5769):1880–1885.  https://doi.org/10.1126/science.1110542 CrossRefGoogle Scholar
  6. 6.
    Bai XC, Lu D, Bai J, Zheng H, Ke ZY, Li XM, Luo SQ (2004) Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB. Biochem Biophys Res Commun 314(1):197–207CrossRefGoogle Scholar
  7. 7.
    Gillet C, Dalla Valle A, Gaspard N, Spruyt D, Vertongen P, Lechanteur J, Rigutto S, Dragan ER, Heuschling A, Gangji V, Rasschaert J (2017) Osteonecrosis of the femoral head: lipotoxicity exacerbation in MSC and modifications of the bone marrow fluid. Endocrinology 158(3):490–502.  https://doi.org/10.1210/en.2016-1687 Google Scholar
  8. 8.
    Elbaz A, Wu X, Rivas D, Gimble JM, Duque G (2010) Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro. J Cell Mol Med 14(4):982–991.  https://doi.org/10.1111/j.1582-4934.2009.00751.x CrossRefGoogle Scholar
  9. 9.
    Xie F, Zhou B, Wang J, Liu T, Wu X, Fang R, Kang Y, Dai R (2018) Microstructural properties of trabecular bone autografts: comparison of men and women with and without osteoporosis. Arch Osteoporos 13(1):18.  https://doi.org/10.1007/s11657-018-0422-z CrossRefGoogle Scholar
  10. 10.
    Zeng Y, Zhang L, Zhu W, He H, Sheng H, Tian Q, Deng FY, Zhang LS, Hu HG, Deng HW (2017) Network based subcellular proteomics in monocyte membrane revealed novel candidate genes involved in osteoporosis. Osteoporos Int 28(10):3033–3042.  https://doi.org/10.1007/s00198-017-4146-5 CrossRefGoogle Scholar
  11. 11.
    Nielson CM, Wiedrick J, Shen J, Jacobs J, Baker ES, Baraff A, Piehowski P, Lee CG, Baratt A, Petyuk V, McWeeney S, Lim JY, Bauer DC, Lane NE, Cawthon PM, Smith RD, Lapidus J, Orwoll ES, Osteoporotic Fractures in Men Study Research G (2017) Identification of hip BMD loss and fracture risk markers through population-based serum proteomics. J Bone Miner Res 32(7):1559–1567.  https://doi.org/10.1002/jbmr.3125 CrossRefGoogle Scholar
  12. 12.
    Zhu W, Shen H, Zhang JG, Zhang L, Zeng Y, Huang HL, Zhao YC, He H, Zhou Y, Wu KH, Tian Q, Zhao LJ, Deng FY, Deng HW (2017) Cytosolic proteome profiling of monocytes for male osteoporosis. Osteoporos Int 28(3):1035–1046.  https://doi.org/10.1007/s00198-016-3825-y CrossRefGoogle Scholar
  13. 13.
    Zhang L, Liu YZ, Zeng Y, Zhu W, Zhao YC, Zhang JG, Zhu JQ, He H, Shen H, Tian Q, Deng FY, Papasian CJ, Deng HW (2016) Network-based proteomic analysis for postmenopausal osteoporosis in Caucasian females. Proteomics 16(1):12–28.  https://doi.org/10.1002/pmic.201500005 CrossRefGoogle Scholar
  14. 14.
    Xie Y, Gao Y, Zhang L, Chen Y, Ge W, Tang P (2018) Involvement of serum-derived exosomes of elderly patients with bone loss in failure of bone remodeling via alteration of exosomal bone-related proteins. Aging Cell 17(3):e12758.  https://doi.org/10.1111/acel.12758 CrossRefGoogle Scholar
  15. 15.
    Miranda M, Pino AM, Fuenzalida K, Rosen CJ, Seitz G, Rodriguez JP (2016) Characterization of fatty acid composition in bone marrow fluid from postmenopausal women: modification after hip fracture. J Cell Biochem 117(10):2370–2376.  https://doi.org/10.1002/jcb.25534 CrossRefGoogle Scholar
  16. 16.
    Pino AM, Rios S, Astudillo P, Fernandez M, Figueroa P, Seitz G, Rodriguez JP (2010) Concentration of adipogenic and proinflammatory cytokines in the bone marrow supernatant fluid of osteoporotic women. J Bone Miner Res 25(3):492–498.  https://doi.org/10.1359/jbmr.090802 CrossRefGoogle Scholar
  17. 17.
    Wiig H, Berggreen E, Borge BA, Iversen PO (2004) Demonstration of altered signaling responses in bone marrow extracellular fluid during increased hematopoiesis in rats using a centrifugation method. Am J Physiol Heart Circ Physiol 286(5):H2028–H2034.  https://doi.org/10.1152/ajpheart.00934.2003 CrossRefGoogle Scholar
  18. 18.
    Moulder R, Bhosale SD, Goodlett DR, Lahesmaa R (2017) Analysis of the plasma proteome using iTRAQ and TMT-based isobaric labeling. Mass Spectrom Rev 37:583–606.  https://doi.org/10.1002/mas.21550 CrossRefGoogle Scholar
  19. 19.
    Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75(8):1895–1904CrossRefGoogle Scholar
  20. 20.
    Chabot B, Shkreta L (2016) Defective control of pre-messenger RNA splicing in human disease. J Cell Biol 212(1):13–27.  https://doi.org/10.1083/jcb.201510032 CrossRefGoogle Scholar
  21. 21.
    Dejaeger M, Bohm AM, Dirckx N, Devriese J, Nefyodova E, Cardoen R, St-Arnaud R, Tournoy J, Luyten FP, Maes C (2017) Integrin-linked kinase regulates bone formation by controlling cytoskeletal organization and modulating BMP and Wnt signaling in Osteoprogenitors. J Bone Miner Res 32(10):2087–2102.  https://doi.org/10.1002/jbmr.3190 CrossRefGoogle Scholar
  22. 22.
    Higuchi C, Nakamura N, Yoshikawa H, Itoh K (2009) Transient dynamic actin cytoskeletal change stimulates the osteoblastic differentiation. J Bone Miner Metab 27(2):158–167.  https://doi.org/10.1007/s00774-009-0037-y CrossRefGoogle Scholar
  23. 23.
    Novack DV, Faccio R (2011) Osteoclast motility: putting the brakes on bone resorption. Ageing Res Rev 10(1):54–61.  https://doi.org/10.1016/j.arr.2009.09.005 CrossRefGoogle Scholar
  24. 24.
    Wang F, Canadeo LA, Huibregtse JM (2015) Ubiquitination of newly synthesized proteins at the ribosome. Biochimie 114:127–133.  https://doi.org/10.1016/j.biochi.2015.02.006 CrossRefGoogle Scholar
  25. 25.
    Schaefer A, Nethe M, Hordijk PL (2012) Ubiquitin links to cytoskeletal dynamics, cell adhesion and migration. Biochem J 442(1):13–25.  https://doi.org/10.1042/BJ20111815 CrossRefGoogle Scholar
  26. 26.
    Gravina GL, Tortoreto M, Mancini A, Addis A, Di Cesare E, Lenzi A, Landesman Y, McCauley D, Kauffman M, Shacham S, Zaffaroni N, Festuccia C (2014) XPO1/CRM1-selective inhibitors of nuclear export (SINE) reduce tumor spreading and improve overall survival in preclinical models of prostate cancer (PCa). J Hematol Oncol 7:46.  https://doi.org/10.1186/1756-8722-7-46 CrossRefGoogle Scholar
  27. 27.
    Ramanathan N, Lim N, Stewart CL (2015) DDX5/p68 RNA helicase expression is essential for initiating adipogenesis. Lipids Health Dis 14:160.  https://doi.org/10.1186/s12944-015-0163-6 CrossRefGoogle Scholar
  28. 28.
    Jensen ED, Niu L, Caretti G, Nicol SM, Teplyuk N, Stein GS, Sartorelli V, van Wijnen AJ, Fuller-Pace FV, Westendorf JJ (2008) p68 (Ddx5) interacts with Runx2 and regulates osteoblast differentiation. J Cell Biochem 103(5):1438–1451.  https://doi.org/10.1002/jcb.21526 CrossRefGoogle Scholar
  29. 29.
    Kumar Y, Kapoor I, Khan K, Thacker G, Khan MP, Shukla N, Kanaujiya JK, Sanyal S, Chattopadhyay N, Trivedi AK (2015) E3 ubiquitin ligase Fbw7 negatively regulates osteoblast differentiation by targeting Runx2 for degradation. J Biol Chem 290(52):30975–30987.  https://doi.org/10.1074/jbc.M115.669531
  30. 30.
    Calviello G, Resci F, Serini S, Piccioni E, Toesca A, Boninsegna A, Monego G, Ranelletti FO, Palozza P (2007) Docosahexaenoic acid induces proteasome-dependent degradation of beta-catenin, down-regulation of survivin and apoptosis in human colorectal cancer cells not expressing COX-2. Carcinogenesis 28(6):1202–1209.  https://doi.org/10.1093/carcin/bgl254 CrossRefGoogle Scholar
  31. 31.
    Kimelman D, Xu W (2006) Beta-catenin destruction complex: insights and questions from a structural perspective. Oncogene 25(57):7482–7491.  https://doi.org/10.1038/sj.onc.1210055 CrossRefGoogle Scholar
  32. 32.
    Brakebusch C, Fassler R (2003) The integrin-actin connection an eternal love affair. EMBO J 22(10):2324–2333.  https://doi.org/10.1093/emboj/cdg245 CrossRefGoogle Scholar
  33. 33.
    Dossa T, Arabian A, Windle JJ, Dedhar S, Teitelbaum SL, Ross FP, Roodman GD, St-Arnaud R (2010) Osteoclast-specific inactivation of the integrin-linked kinase (ILK) inhibits bone resorption. J Cell Biochem 110(4):960–967.  https://doi.org/10.1002/jcb.22609 CrossRefGoogle Scholar
  34. 34.
    Gimona M, Kazzaz JA, Helfman DM (1996) Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects. Proc Natl Acad Sci U S A 93(18):9618–9623CrossRefGoogle Scholar
  35. 35.
    McMichael BK, Kotadiya P, Singh T, Holliday LS, Lee BS (2006) Tropomyosin isoforms localize to distinct microfilament populations in osteoclasts. Bone 39(4):694–705.  https://doi.org/10.1016/j.bone.2006.04.031 CrossRefGoogle Scholar
  36. 36.
    McMichael BK, Lee BS (2008) Tropomyosin 4 regulates adhesion structures and resorptive capacity in osteoclasts. Exp Cell Res 314(3):564–573.  https://doi.org/10.1016/j.yexcr.2007.10.018 CrossRefGoogle Scholar
  37. 37.
    Fazeli PK, Horowitz MC, MacDougald OA, Scheller EL, Rodeheffer MS, Rosen CJ, Klibanski A (2013) Marrow fat and bone—new perspectives. J Clin Endocrinol Metab 98(3):935–945.  https://doi.org/10.1210/jc.2012-3634 CrossRefGoogle Scholar
  38. 38.
    Brasaemle DL (2007) Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis. J Lipid Res 48(12):2547–2559.  https://doi.org/10.1194/jlr.R700014-JLR200
  39. 39.
    Grahn TH, Zhang Y, Lee MJ, Sommer AG, Mostoslavsky G, Fried SK, Greenberg AS, Puri V (2013) FSP27 and PLIN1 interaction promotes the formation of large lipid droplets in human adipocytes. Biochem Biophys Res Commun 432(2):296–301.  https://doi.org/10.1016/j.bbrc.2013.01.113 CrossRefGoogle Scholar
  40. 40.
    Lehman RA Jr, Kang DG, Wagner SC (2015) Management of osteoporosis in spine surgery. J Am Acad Orthop Surg 23(4):253–263.  https://doi.org/10.5435/JAAOS-D-14-00042 CrossRefGoogle Scholar

Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2019

Authors and Affiliations

  1. 1.Department of Metabolism and Endocrinology, Hunan Provincial Key Laboratory for Metabolic Bone Diseases, National Clinical Research Center for Metabolic Diseases, the Second Xiangya HospitalCentral South UniversityChangshaChina
  2. 2.Department of Spine Surgery, the Second Xiangya HospitalCentral South UniversityChangshaChina
  3. 3.Osteoporosis and Arthritis LabUniversity of MichiganAnn ArborUSA

Personalised recommendations