Skip to main content

Prednisolone induces osteocytes apoptosis by promoting Notum expression and inhibiting PI3K/AKT/GSK3β/β-catenin pathway

Abstract

The apoptosis of mature osteocytes is the main factor causing damage to the microstructure of cortical bone in glucocorticoid-induced osteoporosis (GIOP). Our previous research found damaged areas and empty osteocytes lacunae in the tibial cortical bone of GIOP mice. However, the specific mechanism has not been clarified. Recently, a study showed that the quality of the cortical bone significantly increased by knocking out Notum, a gene encoding α/β hydrolase. However, it is not clear whether Notum affects cortical bone remodeling by participating in glucocorticoids (GCs)-induced apoptosis of osteocytes. The present study aimed to explore the correlation between Notum, osteocytes apoptosis, and cortical bone quality in GIOP. Prednisolone acetate was intragastrically administered to mice for two weeks. Histochemical staining was applied to evaluate changes in GIOP and Notum expression. Osteocytes were stimulated with prednisolone, and cell viability was assessed via CCK8. Hoechst 33342/PI staining, flow cytometry, RT-PCR, and western blot were used to detect osteocytes apoptosis, siRNA transfection efficiency, and expressions of pathway related factors. The results showed that the number of empty osteocytes lacunae increased in GIOP mice. TUNEL-stained apoptotic osteocytes and Notum immuno-positive osteocytes were also observed. Furthermore, prednisolone was found to promote Notum expression and osteocytes apoptosis in vitro. Knocking down Notum via siRNA partially restored osteocytes apoptosis and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3β (GSK3β)/β-catenin pathway. These findings showed GCs-induced osteocytes apoptosis by promoting Notum expression and inhibiting PI3K/AKT/GSK3β/β-catenin pathway. Thus, Notum might be a potential therapeutic target for the treatment of GIOP.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. Baron R, Kneissel M (2013) WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med 19:179–192. https://doi.org/10.1038/nm.3074

    CAS  Article  PubMed  Google Scholar 

  2. Bodine PV (2008) Wnt signaling control of bone cell apoptosis. Cell Res 18:248–253. https://doi.org/10.1038/cr.2008.13

    CAS  Article  PubMed  Google Scholar 

  3. Brommage R et al (2019) NOTUM inhibition increases endocortical bone formation and bone strength. Bone Res 7:2. https://doi.org/10.1038/s41413-018-0038-3

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Cao J, Venton L, Sakata T, Halloran BP (2003) Expression of RANKL and OPG correlates with age-related bone loss in male C57BL/6 mice. J Bone Miner Res 18:270–277. https://doi.org/10.1359/jbmr.2003.18.2.270

    CAS  Article  PubMed  Google Scholar 

  5. Carvas JS et al (2010) A single dose of zoledronic acid reverses the deleterious effects of glucocorticoids on titanium implant osseointegration. Osteoporos Int 21:1723–1729. https://doi.org/10.1007/s00198-009-1125-5

    CAS  Article  PubMed  Google Scholar 

  6. Compston JE (2007) Emerging consensus on prevention and treatment of glucocorticoid-induced osteoporosis. Curr Rheumatol Rep 9:78–84. https://doi.org/10.1007/s11926-007-0026-x

    CAS  Article  PubMed  Google Scholar 

  7. Compston J (2010) Management of glucocorticoid-induced osteoporosis. Nat Rev Rheumatol 6:82–88. https://doi.org/10.1038/nrrheum.2009.259

    CAS  Article  PubMed  Google Scholar 

  8. Dalle Carbonare L, Arlot ME, Chavassieux PM, Roux JP, Portero NR, Meunier PJ (2001) Comparison of trabecular bone microarchitecture and remodeling in glucocorticoid-induced and postmenopausal osteoporosis. J Bone Miner Res 16:97–103. https://doi.org/10.1359/jbmr.2001.16.1.97

    CAS  Article  PubMed  Google Scholar 

  9. de Oliveira MA, Asahi DA, Silveira CAE, Lima L, Glick M, Gallottini M (2015) The effects of zoledronic acid and dexamethasone on osseointegration of endosseous implants: histological and histomorphometrical evaluation in rats. Clin Oral Implant Res 26:e17–e21. https://doi.org/10.1111/clr.12335

    Article  Google Scholar 

  10. Dong J, Xu X, Zhang Q, Yuan Z, Tan B (2020) The PI3K/AKT pathway promotes fracture healing through its crosstalk with Wnt/β-catenin. Exp Cell Res 394:112137. https://doi.org/10.1016/j.yexcr.2020.112137

    CAS  Article  PubMed  Google Scholar 

  11. Fowler TW et al (2017) Glucocorticoid suppression of osteocyte perilacunar remodeling is associated with subchondral bone degeneration in osteonecrosis. Sci Rep 7:44618. https://doi.org/10.1038/srep44618

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Gifre L, Ruiz-Gaspà S, Monegal A, Nomdedeu B, Filella X, Guañabens N, Peris P (2013) Effect of glucocorticoid treatment on Wnt signalling antagonists (sclerostin and Dkk-1) and their relationship with bone turnover. Bone 57:272–276. https://doi.org/10.1016/j.bone.2013.08.016

    CAS  Article  PubMed  Google Scholar 

  13. Häcker U, Nybakken K, Perrimon N (2005) Heparan sulphate proteoglycans: the sweet side of development. Nat Rev Mol Cell Biol 6:530–541. https://doi.org/10.1038/nrm1681

    CAS  Article  PubMed  Google Scholar 

  14. Janda CY, Waghray D, Levin AM, Thomas C, Garcia KC (2012) Structural basis of Wnt recognition by Frizzled. Science 337:59–64. https://doi.org/10.1126/science.1222879

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Kakugawa S et al (2015) Notum deacylates Wnt proteins to suppress signalling activity. Nature 519:187–192. https://doi.org/10.1038/nature14259

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Kenanidis E, Potoupnis ME, Kakoulidis P, Leonidou A, Sakellariou GT, Sayegh FE, Tsiridis E (2015) Management of glucocorticoid-induced osteoporosis: clinical data in relation to disease demographics, bone mineral density and fracture risk. Expert Opin Drug Saf 14:1035–1053. https://doi.org/10.1517/14740338.2015.1040387

    CAS  Article  PubMed  Google Scholar 

  17. Kitase Y, Barragan L, Qing H, Kondoh S, Jiang JX, Johnson ML, Bonewald LF (2010) Mechanical induction of PGE2 in osteocytes blocks glucocorticoid-induced apoptosis through both the β-catenin and PKA pathways. J Bone Miner Res 25:2657–2668. https://doi.org/10.1002/jbmr.168

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Kogianni G, Mann V, Ebetino F, Nuttall M, Nijweide P, Simpson H, Noble B (2004) Fas/CD95 is associated with glucocorticoid-induced osteocyte apoptosis. Life Sci 75:2879–2895. https://doi.org/10.1016/j.lfs.2004.04.048

    CAS  Article  PubMed  Google Scholar 

  19. Lerner UH, Ohlsson C (2015) The WNT system: background and its role in bone. J Int Med 277:630–649. https://doi.org/10.1111/joim.12368

    CAS  Article  Google Scholar 

  20. Li M et al (2013) Histological examination on osteoblastic activities in the alveolar bone of transgenic mice with induced ablation of osteocytes. Histol Histopathol 28:327–335. https://doi.org/10.14670/hh-28.327

    Article  PubMed  Google Scholar 

  21. Luo X, Li L, Xu W, Cheng Y, Xie Z (2020) HLY78 attenuates neuronal apoptosis via the LRP6/GSK3β/β-catenin signaling pathway after subarachnoid hemorrhage in rats. Neurosci Bull 36:1171–1181. https://doi.org/10.1007/s12264-020-00532-4

    CAS  Article  PubMed  Google Scholar 

  22. Maruotti N, Corrado A, Cantatore FP (2010) Glucocorticoid induced risk of fractures. Panminerva Med 52:339–343

    CAS  PubMed  Google Scholar 

  23. Migliaccio S, Brama M, Fornari R, Greco EA, Spera G, Malavolta N (2007) Glucocorticoid-induced osteoporosis: an osteoblastic disease. Aging Clin Exp Res 19:5–10

    PubMed  Google Scholar 

  24. Necela BM, Cidlowski JA (2004) Mechanisms of glucocorticoid receptor action in noninflammatory and inflammatory cells. Proc Am Thorac Soc 1:239–246. https://doi.org/10.1513/pats.200402-005MS

    CAS  Article  PubMed  Google Scholar 

  25. Nong J, Kang K, Shi Q, Zhu X, Tao Q, Chen YG (2021) Phase separation of Axin organizes the β-catenin destruction complex. J Cell Biol. https://doi.org/10.1083/jcb.202012112

    Article  PubMed  Google Scholar 

  26. O’Brien CA et al (2004) Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 145:1835–1841. https://doi.org/10.1210/en.2003-0990

    CAS  Article  PubMed  Google Scholar 

  27. Parfitt AM et al (1987) Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2:595–610. https://doi.org/10.1002/jbmr.5650020617

    CAS  Article  PubMed  Google Scholar 

  28. Pederson L, Ruan M, Westendorf JJ, Khosla S, Oursler MJ (2008) Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate. Proc Natl Acad Sci USA 105:20764–20769. https://doi.org/10.1073/pnas.0805133106

    Article  PubMed  PubMed Central  Google Scholar 

  29. Sato AY et al (2016) Protection from glucocorticoid-induced osteoporosis by anti-catabolic signaling in the absence of sost/sclerostin. J Bone Miner Res 31:1791–1802. https://doi.org/10.1002/jbmr.2869

    CAS  Article  PubMed  Google Scholar 

  30. Sun B et al (2016) Immunolocalization of MMP 2, 9 and 13 in prednisolone induced osteoporosis in mice. Histol Histopathol 31:647–656. https://doi.org/10.14670/hh-11-702

    CAS  Article  PubMed  Google Scholar 

  31. Tu X et al (2015) Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone. Proc Natl Acad Sci USA 112:E478-486. https://doi.org/10.1073/pnas.1409857112

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Wang FS et al (2005) Secreted frizzled-related protein 1 modulates glucocorticoid attenuation of osteogenic activities and bone mass. Endocrinology 146:2415–2423. https://doi.org/10.1210/en.2004-1050

    CAS  Article  Google Scholar 

  33. Wang T, Yu X, He C (2019) Pro-inflammatory cytokines: cellular and molecular drug targets for glucocorticoid-induced-osteoporosis via osteocyte. Curr Drug Targets 20:1–15. https://doi.org/10.2174/1389450119666180405094046

    CAS  Article  PubMed  Google Scholar 

  34. Weivoda MM et al (2016) Wnt signaling inhibits osteoclast differentiation by activating canonical and noncanonical cAMP/PKA pathways. J Bone Miner Res 31:65–75. https://doi.org/10.1002/jbmr.2599

    CAS  Article  PubMed  Google Scholar 

  35. Wen Q et al (2020) Runx2 regulates mouse tooth root development via activation of WNT inhibitor NOTUM. J Bone Miner Res 35:2252–2264

    CAS  Article  Google Scholar 

  36. Xia X et al (2010) Glucocorticoid-induced autophagy in osteocytes. J Bone Miner Res 25:2479–2488. https://doi.org/10.1002/jbmr.160

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Yoon SH, Grynpas M, Mitchell J (2019) Intermittent PTH treatment improves bone and muscle in glucocorticoid treated Mdx mice: a model of Duchenne Muscular Dystrophy. Bone 121:232–242. https://doi.org/10.1016/j.bone.2019.01.028

    CAS  Article  PubMed  Google Scholar 

  38. Yun SI, Yoon HY, Jeong SY, Chung YS (2009) Glucocorticoid induces apoptosis of osteoblast cells through the activation of glycogen synthase kinase 3beta. J Bone Miner Metab 27:140–148. https://doi.org/10.1007/s00774-008-0019-5

    CAS  Article  PubMed  Google Scholar 

  39. Zarrinkalam MR, Mulaibrahimovic A, Atkins GJ, Moore RJ (2012) Changes in osteocyte density correspond with changes in osteoblast and osteoclast activity in an osteoporotic sheep model. Osteoporos Int 23:1329–1336. https://doi.org/10.1007/s00198-011-1672-4

    CAS  Article  PubMed  Google Scholar 

  40. Zhang X et al (2015) Notum is required for neural and head induction via Wnt deacylation, oxidation, and inactivation. Dev Cell 32:719–730. https://doi.org/10.1016/j.devcel.2015.02.014

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Zhang C, Wei W, Chi M, Wan Y, Li X, Qi M, Zhou Y (2019a) FOXO1 mediates advanced glycation end products induced mouse osteocyte-like MLO-Y4 cell apoptosis and dysfunctions. J Diabetes Res. https://doi.org/10.1155/2019/6757428

    Article  PubMed  PubMed Central  Google Scholar 

  42. Zhang Y et al (2019b) Electromagnetic field treatment increases purinergic receptor P2X7 expression and activates its downstream Akt/GSK3β/β-catenin axis in mesenchymal stem cells under osteogenic induction. Stem Cell Res Ther 10:407. https://doi.org/10.1186/s13287-019-1497-1

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Zhao C et al (2020) GOLPH3 promotes angiogenesis of lung adenocarcinoma by regulating the Wnt/β-catenin signaling pathway. Onco Targets Ther 13:6265–6277. https://doi.org/10.2147/ott.s249994

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Zhu M, Li D, Wu Y, Huang X, Wu M (2014) TREM-2 promotes macrophage-mediated eradication of Pseudomonas aeruginosa via a PI3K/Akt pathway. Scand J Immunol 79:187–196. https://doi.org/10.1111/sji.12148

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 81972072) to Li M, the National Natural Science Foundation of China (Nos. 81970964, 81771108) to Guo J, the National Natural Science Foundation of China (No. 81800982) to Liu H, and the Construction Engineering Special Fund of “Taishan Young Scholars” of Shandong Province (No. tsqn 202103177) to Liu H.

Author information

Affiliations

Authors

Contributions

Conceptualization: CL, PY, BL; Methodology: CL, PY, HL, TH and JB; Formal analysis and investigation: CL, PY and ML; Writing—original draft preparation: CL; Writing—review and editing: CL; Data curation: CL and PY; Funding acquisition: ML, JG and HL; Resources: ML, JG and HS; Supervision: ML and JG; Project administration: ML; Software: CL; Validation: CL and ML; Visualization: CL. All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

Corresponding authors

Correspondence to Haipeng Si or Minqi Li.

Ethics declarations

Conflict of interest

The authors have no conflicts of interest to declare.

Ethical approval

All experimental procedures complied with the ARRIVE guidelines and were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. And all animal experiments and the use of the cell line were approved by the ethics committee of School and Hospital of Stomatology, Shandong University (No. 20210116).

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, C., Yang, P., Liu, B. et al. Prednisolone induces osteocytes apoptosis by promoting Notum expression and inhibiting PI3K/AKT/GSK3β/β-catenin pathway. J Mol Histol (2021). https://doi.org/10.1007/s10735-021-10006-0

Download citation

Keywords

  • GIOP
  • Cortical bone
  • Notum
  • Osteocytes apoptosis
  • PI3K/AKT/GSK3β/β-catenin