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LRP receptors in chondrocytes are modulated by simulated microgravity and cyclic hydrostatic pressure


Autoři: Rachel C. Nordberg aff001;  Liliana F. Mellor aff002;  Andrew R. Krause aff003;  Henry J. Donahue aff004;  Elizabeth G. Loboa aff001
Působiště autorů: College of Engineering, University of Missouri, Columbia, Missouri, United States of America aff001;  Spanish National Cancer Research Centre, Madrid, Spain aff002;  Sport Health and Physical Education, Vancouver Island University, Nanaimo, British Columbia, Canada aff003;  Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Penn State College of Medicine, Hershey, Pennsylvania, United States of America aff004;  Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America aff005
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0223245

Souhrn

Mechanical loading is essential for the maintenance of musculoskeletal homeostasis. Cartilage has been demonstrated to be highly mechanoresponsive, but the mechanisms by which chondrocytes respond to mechanical stimuli are not clearly understood. The goal of the study was to determine how LRP4, LRP5, and LRP6 within canonical Wnt-signaling are regulated in simulated microgravity and cyclic hydrostatic pressure, and to investigate the potential role of LRP 4/5/6 in cartilage degeneration. Rat chondrosacroma cell (RCS) pellets were stimulated using either cyclic hydrostatic pressure (1Hz, 7.5 MPa, 4hr/day) or simulated microgravity in a rotating wall vessel (RWV) bioreactor (11RPM, 24hr/day). LRP4/5/6 mRNA expression was assessed by RT-qPCR and LRP5 protein expression was determined by fluorescent immunostaining. To further evaluate our in vitro findings in vivo, mice were subjected to hindlimb suspension for 14 days and the femoral heads stained for LRP5 expression. We found that, in vitro, LRP4/5/6 mRNA expression is modulated in a time-dependent manner by mechanical stimulation. Additionally, LRP5 protein expression is upregulated in response to both simulated microgravity and cyclic hydrostatic pressure. LRP5 is also upregulated in vivo in the articular cartilage of hindlimb suspended mice. This is the first study to examine how LRP4/5/6, critical receptors within musculoskeletal biology, respond to mechanical stimulation. Further elucidation of this mechanism could provide significant clinical benefit for the identification of pharmaceutical targets for the maintenance of cartilage health.

Klíčová slova:

Cartilage – Hydrostatic pressure – Chondrocytes – Mouse models – Osteoarthritis – Wnt signaling cascade – Artificial gravity – Articular cartilage


Zdroje

1. Robling AG, Turner CH. Mechanical signaling for bone modeling and remodeling. Crit Rev Eukaryot Gene Expr. 2009;19: 319–338. 19817708

2. Robinson JA, Chatterjee-Kishore M, Yaworsky PJ, Cullen DM, Zhao W, Li C, et al. Wnt/beta-catenin signaling is a normal physiological response to mechanical loading in bone. J Biol Chem. 2006;281: 31720–31728. doi: 10.1074/jbc.M602308200 16908522

3. Kang KS, Robling AG. New Insights into Wnt-Lrp5/6-beta-Catenin Signaling in Mechanotransduction. Front Endocrinol (Lausanne). 2015;5: 246.

4. LeBlanc A, Schneider V, Shackelford L, West S, Oganov V, Bakulin A, et al. Bone mineral and lean tissue loss after long duration space flight. J Musculoskelet Neuronal Interact. 2000;1: 157–160. 15758512

5. LeBlanc A, Lin C, Shackelford L, Sinitsyn V, Evans H, Belichenko O, et al. Muscle volume, MRI relaxation times (T2), and body composition after spaceflight. J Appl Physiol (1985). 2000;89: 2158–2164.

6. Liphardt AM, Mundermann A, Koo S, Backer N, Andriacchi TP, Zange J, et al. Vibration training intervention to maintain cartilage thickness and serum concentrations of cartilage oligometric matrix protein (COMP) during immobilization. Osteoarthritis Cartilage. 2009;17: 1598–1603. doi: 10.1016/j.joca.2009.07.007 19747585

7. Roos EM, Herzog W, Block JA, Bennell KL. Muscle weakness, afferent sensory dysfunction and exercise in knee osteoarthritis. Nat Rev Rheumatol. 2011;7: 57–63. doi: 10.1038/nrrheum.2010.195 21119605

8. Rehan Youssef A, Longino D, Seerattan R, Leonard T, Herzog W. Muscle weakness causes joint degeneration in rabbits. Osteoarthritis Cartilage. 2009;17: 1228–1235. doi: 10.1016/j.joca.2009.03.017 19427403

9. Clements KM, Bee ZC, Crossingham GV, Adams MA, Sharif M. How severe must repetitive loading be to kill chondrocytes in articular cartilage? Osteoarthritis Cartilage. 2001;9: 499–507. doi: 10.1053/joca.2000.0417 11467899

10. Horisberger M, Fortuna R, Valderrabano V, Herzog W. Long-term repetitive mechanical loading of the knee joint by in vivo muscle stimulation accelerates cartilage degeneration and increases chondrocyte death in a rabbit model. Clin Biomech (Bristol, Avon). 2013;28: 536–543. doi: 10.1016/j.clinbiomech.2013.04.009 23701865

11. Sharma L, Chang A. Overweight: advancing our understanding of its impact on the knee and the hip. Ann Rheum Dis. 2007;66: 141–142. doi: 10.1136/ard.2006.059931 17242017

12. Bader DL, Salter DM, Chowdhury TT. Biomechanical influence of cartilage homeostasis in health and disease. Arthritis. 2011;2011: 979032. doi: 10.1155/2011/979032 22046527

13. Roddy E, Zhang W, Doherty M. Aerobic walking or strengthening exercise for osteoarthritis of the knee? A systematic review. Ann Rheum Dis. 2005;64: 544–548. doi: 10.1136/ard.2004.028746 15769914

14. Manninen P, Riihimaki H, Heliovaara M, Suomalainen O. Physical exercise and risk of severe knee osteoarthritis requiring arthroplasty. Rheumatology (Oxford). 2001;40: 432–437.

15. Elder BD, Athanasiou KA. Hydrostatic pressure in articular cartilage tissue engineering: from chondrocytes to tissue regeneration. Tissue Eng Part B Rev. 2009;15: 43–53. doi: 10.1089/ten.teb.2008.0435 19196119

16. Fioravanti A, Moretti E, Scapigliati G, Cervone R, Galeazzi M, Collodel G. Morphological, immunocytochemical and biochemical studies in human osteoarthritic chondrocytes exposed to IL-1b and cyclical hydrostatic pressure. Clin Exp Rheumatol. 2007;25: 690–695. 18078615

17. Fioravanti A, Collodel G, Petraglia A, Nerucci F, Moretti E, Galeazzi M. Effect of hydrostatic pressure of various magnitudes on osteoarthritic chondrocytes exposed to IL-1beta. Indian J Med Res. 2010;132: 209–217. 20716822

18. Karamesinis K, Spyropoulou A, Dalagiorgou G, Katsianou MA, Nokhbehsaim M, Memmert S, et al. Continuous hydrostatic pressure induces differentiation phenomena in chondrocytes mediated by changes in polycystins, SOX9, and RUNX2. J Orofac Orthop. 2017;78: 21–31. doi: 10.1007/s00056-016-0061-1 27909759

19. Yu B, Yu D, Cao L, Zhao X, Long T, Liu G, et al. Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway. Biochem Biophys Res Commun. 2011;414: 412–418. doi: 10.1016/j.bbrc.2011.09.103 21971552

20. Mayer-Wagner S, Hammerschmid F, Redeker JI, Schmitt B, Holzapfel BM, Jansson V, et al. Simulated microgravity affects chondrogenesis and hypertrophy of human mesenchymal stem cells. Int Orthop. 2014;38: 2615–2621. doi: 10.1007/s00264-014-2454-3 25030964

21. Emin N, Koc A, Durkut S, Elcin AE, Elcin YM. Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture. Artif Cells Blood Substit Immobil Biotechnol. 2008;36: 123–137. doi: 10.1080/10731190801932116 18437589

22. Mellor LF, Steward AJ, Nordberg RC, Taylor MA, Loboa EG. Comparison of Simulated Microgravity and Hydrostatic Pressure for Chondrogenesis of hASC. Aerosp Med Hum Perform. 2017;88: 377–384. doi: 10.3357/AMHP.4743.2017 28518000

23. Angele P, Yoo JU, Smith C, Mansour J, Jepsen KJ, Nerlich M, et al. Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. J Orthop Res. 2003;21: 451–457. doi: 10.1016/S0736-0266(02)00230-9 12706017

24. Miyanishi K, Trindade MC, Lindsey DP, Beaupre GS, Carter DR, Goodman SB, et al. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro. Tissue Eng. 2006;12: 1419–1428. doi: 10.1089/ten.2006.12.1419 16846340

25. Miyanishi K, Trindade MC, Lindsey DP, Beaupre GS, Carter DR, Goodman SB, et al. Dose- and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro. Tissue Eng. 2006;12: 2253–2262. doi: 10.1089/ten.2006.12.2253 16968165

26. Finger AR, Sargent CY, Dulaney KO, Bernacki SH, Loboa EG. Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure. Tissue Eng. 2007;13: 1151–1158. doi: 10.1089/ten.2006.0290 17518710

27. Ogawa R, Mizuno S, Murphy GF, Orgill DP. The effect of hydrostatic pressure on three-dimensional chondroinduction of human adipose-derived stem cells. Tissue Eng Part A. 2009;15: 2937–2945. doi: 10.1089/ten.TEA.2008.0672 19290804

28. Puetzer J, Williams J, Gillies A, Bernacki S, Loboa EG. The effects of cyclic hydrostatic pressure on chondrogenesis and viability of human adipose- and bone marrow-derived mesenchymal stem cells in three-dimensional agarose constructs. Tissue Eng Part A. 2013;19: 299–306. doi: 10.1089/ten.TEA.2012.0015 22871265

29. Ogawa R Md P, Orgill DP, Murphy GF, Mizuno S. Gene Expression Profile on Hydrostatic Pressure-Driven Three-Dimensional Cartilage Induction using Human Adipose-Derived Stem Cells and Collagen Gels. Tissue Eng Part A. 2014.

30. Jin L, Feng G, Reames DL, Shimer AL, Shen FH, Li X. The effects of simulated microgravity on intervertebral disc degeneration. Spine J. 2013;13: 235–242. doi: 10.1016/j.spinee.2012.01.022 23537452

31. Mellor LF, Baker TL, Brown RJ, Catlin LW, Oxford JT. Optimal 3D culture of primary articular chondrocytes for use in the rotating wall vessel bioreactor. Aviat Space Environ Med. 2014;85: 798–804. doi: 10.3357/ASEM.3905.2014 25199120

32. Milstead JR, Simske SJ, Bateman TA. Spaceflight and hindlimb suspension disuse models in mice. Biomed Sci Instrum. 2004;40: 105–110. 15133943

33. Takahashi I, Matsuzaki T, Kuroki H, Hoso M. Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis. Osteoarthritis Cartilage. 2019;27: 1084–1093. doi: 10.1016/j.joca.2019.03.001 30890456

34. Chun JS, Oh H, Yang S, Park M. Wnt signaling in cartilage development and degeneration. BMB Rep. 2008;41: 485–494. doi: 10.5483/bmbrep.2008.41.7.485 18682032

35. Usami Y, Gunawardena AT, Iwamoto M, Enomoto-Iwamoto M. Wnt signaling in cartilage development and diseases: lessons from animal studies. Lab Invest. 2016;96: 186–196. doi: 10.1038/labinvest.2015.142 26641070

36. Bonewald LF, Johnson ML. Osteocytes, mechanosensing and Wnt signaling. Bone. 2008;42: 606–615. doi: 10.1016/j.bone.2007.12.224 18280232

37. Papathanasiou I, Malizos KN, Tsezou A. Low-density lipoprotein receptor-related protein 5 (LRP5) expression in human osteoarthritic chondrocytes. J Orthop Res. 2010;28: 348–353. doi: 10.1002/jor.20993 19810105

38. Joiner DM, Less KD, Van Wieren EM, Hess D, Williams BO. Heterozygosity for an inactivating mutation in low-density lipoprotein-related receptor 6 (Lrp6) increases osteoarthritis severity in mice after ligament and meniscus injury. Osteoarthritis Cartilage. 2013;21: 1576–1585. doi: 10.1016/j.joca.2013.05.019 23756208

39. Asai N, Ohkawara B, Ito M, Masuda A, Ishiguro N, Ohno K. LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation. Biochem Biophys Res Commun. 2014.

40. Thomas RS, Clarke AR, Duance VC, Blain EJ. Effects of Wnt3A and mechanical load on cartilage chondrocyte homeostasis. Arthritis Res Ther. 2011;13: R203. doi: 10.1186/ar3536 22151902

41. Praxenthaler H, Kramer E, Weisser M, Hecht N, Fischer J, Grossner T, et al. Extracellular matrix content and WNT/beta-catenin levels of cartilage determine the chondrocyte response to compressive load. Biochim Biophys Acta Mol Basis Dis. 2018;1864: 851–859. doi: 10.1016/j.bbadis.2017.12.024 29277327

42. Dell'Accio F, De Bari C, El Tawil NM, Barone F, Mitsiadis TA, O'Dowd J, et al. Activation of WNT and BMP signaling in adult human articular cartilage following mechanical injury. Arthritis Res Ther. 2006;8: R139. doi: 10.1186/ar2029 16893455

43. Yingst S, Bloxham K, Warner LR, Brown RJ, Cole J, Kenoyer L, et al. Characterization of collagenous matrix assembly in a chondrocyte model system. J Biomed Mater Res A. 2009;90: 247–255. doi: 10.1002/jbm.a.32078 18496861

44. Stevens JW. Swarm chondrosarcoma: a continued resource for chondroblastic-like extracellular matrix and chondrosarcoma biology research. Connect Tissue Res. 2013;54: 252–259. doi: 10.3109/03008207.2013.806913 23758266

45. Brown RJ, Mallory C, McDougal OM, Oxford JT. Proteomic analysis of Col11a1-associated protein complexes. Proteomics. 2011;11: 4660–4676. doi: 10.1002/pmic.201100058 22038862

46. Chen Y, Sumiyoshi H, Oxford JT, Yoshioka H, Ramirez F, Morris NP. Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the alpha1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices. Matrix Biol. 2001;20: 589–599. 11731275

47. Freeman PM, Natarajan RN, Kimura JH, Andriacchi TP. Chondrocyte cells respond mechanically to compressive loads. J Orthop Res. 1994;12: 311–320. doi: 10.1002/jor.1100120303 8207584

48. Hanson AD, Marvel SW, Bernacki SH, Banes AJ, van Aalst J, Loboa EG. Osteogenic effects of rest inserted and continuous cyclic tensile strain on hASC lines with disparate osteodifferentiation capabilities. Ann Biomed Eng. 2009;37: 955–965. doi: 10.1007/s10439-009-9648-7 19229619

49. Collette NM, Genetos DC, Murugesh D, Harland RM, Loots GG. Genetic evidence that SOST inhibits WNT signaling in the limb. Dev Biol. 2010;342: 169–179. doi: 10.1016/j.ydbio.2010.03.021 20359476

50. Parkinson IH, Parsons MA, Moore RJ. The Histochemical Localization of Osteoclasts in EDTA Decalcified Bone. Journal of Histotechnology. 1990;13.

51. 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: 402–408. doi: 10.1006/meth.2001.1262 11846609

52. Shin Y, Huh YH, Kim K, Kim S, Park KH, Koh JT, et al. Low-density lipoprotein receptor-related protein 5 governs Wnt-mediated osteoarthritic cartilage destruction. Arthritis Res Ther. 2014;16: R37. doi: 10.1186/ar4466 24479426

53. Sakai S, Mishima H, Ishii T, Akaogi H, Yoshioka T, Ohyabu Y, et al. Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage. J Orthop Res. 2009;27: 517–521. doi: 10.1002/jor.20566 18932231

54. Vunjak-Novakovic G, Obradovic B, Martin I, Freed LE. Bioreactor studies of native and tissue engineered cartilage. Biorheology. 2002;39: 259–268. 12082288

55. Baker TL, Goodwin TJ. Three-dimensional culture of bovine chondrocytes in rotating-wall vessels. In Vitro Cell Dev Biol Anim. 1997;33: 358–365. doi: 10.1007/s11626-997-0006-5 9196894

56. Sawakami K, Robling AG, Ai M, Pitner ND, Liu D, Warden SJ, et al. The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J Biol Chem. 2006;281: 23698–23711. doi: 10.1074/jbc.M601000200 16790443

57. Akhter MP, Alvarez GK, Cullen DM, Recker RR. Disuse-related decline in trabecular bone structure. Biomech Model Mechanobiol. 2011;10: 423–429. doi: 10.1007/s10237-010-0244-4 20683635

58. Lau KH, Kapur S, Kesavan C, Baylink DJ. Up-regulation of the Wnt, estrogen receptor, insulin-like growth factor-I, and bone morphogenetic protein pathways in C57BL/6J osteoblasts as opposed to C3H/HeJ osteoblasts in part contributes to the differential anabolic response to fluid shear. J Biol Chem. 2006;281: 9576–9588. doi: 10.1074/jbc.M509205200 16461770

59. Lodewyckx L, Luyten FP, Lories RJ. Genetic deletion of low-density lipoprotein receptor-related protein 5 increases cartilage degradation in instability-induced osteoarthritis. Rheumatology (Oxford). 2012;51: 1973–1978. doi: 10.1093/rheumatology/kes178 22850184

60. Chan BY, Fuller ES, Russell AK, Smith SM, Smith MM, Jackson MT, et al. Increased chondrocyte sclerostin may protect against cartilage degradation in osteoarthritis. Osteoarthritis Cartilage. 2011;19: 874–885. doi: 10.1016/j.joca.2011.04.014 21619935

61. Holmen SL, Giambernardi TA, Zylstra CR, Buckner-Berghuis BD, Resau JH, Hess JF, et al. Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6. J Bone Miner Res. 2004;19: 2033–2040. doi: 10.1359/JBMR.040907 15537447

62. Choi HY, Dieckmann M, Herz J, Niemeier A. Lrp4, a novel receptor for Dickkopf 1 and sclerostin, is expressed by osteoblasts and regulates bone growth and turnover in vivo. PLoS One. 2009;4: e7930. doi: 10.1371/journal.pone.0007930 19936252

63. Ahn Y, Sims C, Murray MJ, Kuhlmann PK, Fuentes-Antras J, Weatherbee SD, et al. Multiple modes of Lrp4 function in modulation of Wnt/beta-catenin signaling during tooth development. Development. 2017;144: 2824–2836. doi: 10.1242/dev.150680 28694256

64. Cheleschi S, De Palma A, Pecorelli A, Pascarelli NA, Valacchi G, Belmonte G, et al. Hydrostatic Pressure Regulates MicroRNA Expression Levels in Osteoarthritic Chondrocyte Cultures via the Wnt/beta-Catenin Pathway. Int J Mol Sci. 2017;18: doi: 10.3390/ijms18010133 28085114

65. Niu Q, Li F, Zhang L, Xu X, Liu Y, Gao J, et al. Role of the Wnt/beta-catenin signaling pathway in the response of chondrocytes to mechanical loading. Int J Mol Med. 2016;37: 755–762. doi: 10.3892/ijmm.2016.2463 26821383

66. Semenov M, Tamai K, He X. SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. J Biol Chem. 2005;280: 26770–26775. doi: 10.1074/jbc.M504308200 15908424

67. Frings-Meuthen P, Boehme G, Liphardt AM, Baecker N, Heer M, Rittweger J. Sclerostin and DKK1 levels during 14 and 21 days of bed rest in healthy young men. J Musculoskelet Neuronal Interact. 2013;13: 45–52. 23445914

68. Spatz JM, Fields EE, Yu EW, Divieti Pajevic P, Bouxsein ML, Sibonga JD, et al. Serum sclerostin increases in healthy adult men during bed rest. J Clin Endocrinol Metab. 2012;97: E1736–40. doi: 10.1210/jc.2012-1579 22767636

69. Robling AG, Niziolek PJ, Baldridge LA, Condon KW, Allen MR, Alam I, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J Biol Chem. 2008;283: 5866–5875. doi: 10.1074/jbc.M705092200 18089564

70. Tu X, Rhee Y, Condon KW, Bivi N, Allen MR, Dwyer D, et al. Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading. Bone. 2012;50: 209–217. doi: 10.1016/j.bone.2011.10.025 22075208

71. Spatz JM, Wein MN, Gooi JH, Qu Y, Garr JL, Liu S, et al. The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro. J Biol Chem. 2015;290: 16744–16758. doi: 10.1074/jbc.M114.628313 25953900

72. Lin C, Jiang X, Dai Z, Guo X, Weng T, Wang J, et al. Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/beta-catenin signaling. J Bone Miner Res. 2009;24: 1651–1661. doi: 10.1359/jbmr.090411 19419300

73. Spatz JM, Ellman R, Cloutier AM, Louis L, van Vliet M, Suva LJ, et al. Sclerostin antibody inhibits skeletal deterioration due to reduced mechanical loading. J Bone Miner Res. 2013;28: 865–874. doi: 10.1002/jbmr.1807 23109229

74. Chu CR, Szczodry M, Bruno S. Animal models for cartilage regeneration and repair. Tissue Eng Part B Rev. 2010;16: 105–115. doi: 10.1089/ten.TEB.2009.0452 19831641

75. Moran CJ, Ramesh A, Brama PA, O'Byrne JM, O'Brien FJ, Levingstone TJ. The benefits and limitations of animal models for translational research in cartilage repair. J Exp Orthop. 2016;3: 1-015-0037-x. Epub 2016 Jan 6. doi: 10.1186/s40634-015-0037-x 26915001

76. MacDonald BT, Semenov MV, Huang H, He X. Dissecting molecular differences between Wnt coreceptors LRP5 and LRP6. PLoS One. 2011;6: e23537. doi: 10.1371/journal.pone.0023537 21887268

77. Krishnan V, Bryant HU, Macdougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest. 2006;116: 1202–1209. doi: 10.1172/JCI28551 16670761


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