Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mechanical loading activates the YAP/TAZ pathway and chemokine expression in the MLO-Y4 osteocyte-like cell line

Abstract

Osteocytes are mechanosensitive cells that control bone remodeling in response to mechanical loading. To date, specific signaling pathways modulated by mechanical loading in osteocytes are not well understood. Yes associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), the main effectors of the Hippo pathway, are reported to play a role in mechanotransduction and during osteoblastogenesis. Here, we hypothesized that YAP/TAZ signaling mediates osteocyte mechanosensing to target genes of the bone remodeling process. We aimed to investigate the contribution of YAP/TAZ in modulating the gene expression in an osteocyte-like cell line MLO-Y4. We developed a 3D osteocyte compression culture model from an MLO-Y4 osteocyte cell line embedded in concentrated collagen hydrogel. 3D-mechanical loading led to the increased expression of mechanosensitive genes and a subset of chemokines, including M-csf, Cxcl1, Cxcl2, Cxcl3, Cxcl9, and Cxcl10. The transcription regulators YAP and TAZ translocated to the nucleus and upregulated their target genes and proteins. RNAseq analysis revealed that YAP/TAZ knockdown mediated the regulation of several genes including osteocyte dendrite formation. Use of YAP/TAZ knockdown partially blunted the increase in M-csf and Cxcl3 levels in response to MLO-Y4 compression. These findings demonstrate that YAP/TAZ signaling is required for osteocyte-like cell mechano-transduction, regulates the gene expression profiles and controls chemokine expression.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Analysis of YAP/TAZ activation and nuclear translocation.
Fig. 2: YAP/TAZ immunostaining in trabecular and cortical bone.
Fig. 3: Principal component analysis and MA (ratio intensity) plots.
Fig. 4: Effect of YAP/TAZ knockdown on chemokine expression after mechanical loading evaluated by RT-qPCR.

Data availability

Datasets will be available from the authors upon reasonable request.

References

  1. 1.

    Robling, A. G., Castillo, A. B. & Turner, C. H. Biomechanical and molecular regulation of bone remodeling. Annu. Rev. Biomed. Eng. 8, 455–498 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Schaffler, M. B., Cheung, W.-Y., Majeska, R. & Kennedy, O. Osteocytes: master orchestrators of bone. Calcif. Tissue Int. 94, 5–24 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Xiao, Z. & Quarles, L. D. Physiological mechanisms and therapeutic potential of bone mechanosensing. Rev. Endocr. Metab. Disord. 16, 115–129 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Yavropoulou, M. P. & Yovos, J. G. The molecular basis of bone mechanotransduction. J. Musculoskelet Neuronal. Interact 16, 221–236 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Taylor, A. F. et al. Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions. Am. J. Physiol. Cell Physiol. 292, C545–C552 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Cherian, P. P. et al. Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin. Mol. Biol. Cell 16, 3100–3106 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Genetos, D. C., Kephart, C. J., Zhang, Y., Yellowley, C. E. & Donahue, H. J. Oscillating fluid flow activation of GAP junction hemichannels induces ATP release from MLO-Y4 osteocytes. J. Cell Physiol. 212, 207–214 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Bao, L., Locovei, S. & Dahl, G. Pannexin membrane channels are mechanosensitive conduits for ATP. FEBS Lett. 572, 65–68 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. 9.

    Fox, S. W., Chambers, T. J. & Chow, J. W. Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulation. Am. J. Physiol. 270, E955–E960 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Zaman, G. et al. Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in Osteocytes. J. Bone Miner. Res. 14, 1123–1131 (1999).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Tan, S. D. et al. Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption. Bone 41, 745–751 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Liao, C. et al. Shear stress inhibits IL-17A-mediated induction of osteoclastogenesis via osteocyte pathways. Bone 101, 10–20 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    You, L. et al. Osteocytes as mechanosensors in the inhibition of bone resorption due to mechanical loading. Bone 42, 172–179 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Kulkarni, R. N., Bakker, A. D., Everts, V. & Klein-Nulend, J. Inhibition of osteoclastogenesis by mechanically loaded osteocytes: Involvement of MEPE. Calcif. Tissue Int. 87, 461–468 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Xiong, J., Piemontese, M., Onal, M. & Campbell, J. Osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling bone. PLoS ONE 10, e0138189 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  16. 16.

    Robling, A. G. et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J. Biol. Chem. 283, 5866–5875 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Tu, X. et al. Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading. Bone 50, 209–217 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Spatz, J. M. et al. The Wnt-inhibitor sclerostin is up-regulated by mechanical unloading in Osteocytes in-vitro. J. Biol. Chem. 290, 16744–16758 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Lara-castillo, N. et al. In vivo mechanical loading rapidly activates β -catenin signaling in osteocytes through a prostaglandin mediated mechanism. Bone 76, 58–66 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Dong, J. et al. Elucidation of a universal size-control mechanism in drosophila and mammals. Cell 130, 1120–1133 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Pan, D. Hippo signaling in organ size control. Genes Dev. 21, 886–897 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Halder, G., Dupont, S. & Piccolo, S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat. Rev. Mol. Cell Biol. 13, 591–600 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Hansen, C. G., Moroishi, T. & Guan, K.-L. YAP and TAZ: a nexus for Hippo signaling and beyond. Trends Cell Biol. 25, 499–513 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Panciera, T., Azzolin, L., Cordenonsi, M. & Piccolo, S. Mechanobiology of YAP and TAZ in physiology and disease. Nat. Publ. Gr. 18, 758–770 (2017).

    CAS  Google Scholar 

  25. 25.

    Kim, M., Jang, J.-W. & Bae, S.-C. DNA binding partners of YAP/TAZ. BMB Rep. 51, 126–133 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Piccolo, S., Dupont, S. & Cordenonsi, M. The biology of YAP/TAZ: hippo signaling and beyond. Physiol. Rev. 94, 1287–1312 (2014).

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Morgan, J. T., Murphy, C. J. & Russell, P. What do mechanotransduction, Hippo, Wnt, and TGF β have in common? YAP and TAZ as key orchestrating molecules in ocular health and disease. Exp. Eye Res. 115, 1–12 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Azzolin, L. et al. YAP/TAZ incorporation in the B-catenin destruction complex orchestrates the Wnt response. Cell 158, 157–170 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Varelas, X. et al. The hippo pathway regulates Wnt/B-catenin signaling. Dev. Cell 18, 579–591 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Hong, J. et al. TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Sci. Rep. 309, 1074–1078 (2005).

    CAS  Google Scholar 

  31. 31.

    Tang, Y. & Weiss, S. J. Snail/Slug-YAP/TAZ complexes cooperatively regulate mesenchymal stem cell function and bone formation. Cell Cycle 16, 399–405 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Kim, K. M. et al. Shear stress induced by an interstitial level of slow flow increases the osteogenic differentiation of mesenchymal stem cells through TAZ activation. PLoS ONE 9, e9427 (2014).

    Google Scholar 

  33. 33.

    McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K. & Chen, C. S. Cell shape, cytoskeletal tension, and RhoA regulate stemm cell lineage commitment. Dev. Cell 6, 483–495 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Chen, Z., Luo, Q., Lin, C. & Song, G. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells through down regulating the transcriptional co-activator TAZ. Biochem. Biophys. Res. Commun. 468, 21–26 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Li, X. et al. Stimulation of Piezo1 by mechanical signals promotes bone anabolism. Elife 8, e49631 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Xiong, J., Almeida, M. & O’Brien, C. A. The YAP/TAZ transcriptional co-activators have opposing effects at different stages of osteoblast differentiation. Bone 112, 1–9 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Kato, Y., Windle, J., Koop, B., Mundy, G. & Bonewald, L. Establishment of an Osteocyte-like Cell Line, MLO-Y4. Am. Soc. Bone Miner. Res 12, 2014–2023 (1997).

    CAS  Article  Google Scholar 

  38. 38.

    Banes, A. J., Gilbert, J., Taylor, D. & Monbureau, O. A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro. J. Cell Sci. 75, 35–42 (1985).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Gilbert, J. A., Weinhold, P. S., Banes, A. J., Link, G. W. & Jones, G. L. Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro. J. Biomech. 27, 1169–1177 (1994).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Hens, J. R. et al. TOPGAL mice show that the canonical Wnt signaling pathway is active during bone development and growth and is activated by mechanical loading in vitro. J. Bone Min. Res. 20, 1103–1113 (2005).

    CAS  Article  Google Scholar 

  41. 41.

    Fermor, B. et al. The effects of static and intermittent compression on nitric oxide production in articular cartilage explants. J. Orthop. Res. 19, 729–737 (2001).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Hara, M. et al. Construction of collagen gel scaffolds for mechanical stress analysis. Biosci. Biotechnol. Biochem. 78, 458–461 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    Sardroodian, M., Madeleine, P., Voigt, M. & Hansen, E. A. Freely chosen stride frequencies during walking and running are not correlated with freely chosen pedalling frequency and are insensitive to strength training. Gait Posture 42, 60–64 (2015).

    PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    Ewels, P., Magnusson, M., Lundin, S. & Max, K. Data and text mining MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32, 3047–3048 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic rna-seq quantification. Nat. Biotechnol. 34, 525–527 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Compeau, P. E. C., Pevzner, P. A. & Tesler, G. Why are de Bruijn graphs useful for genome assembly? Nat. Biotechnol. 29, 987–991 (2017).

    Article  CAS  Google Scholar 

  47. 47.

    Soneson, C., Love, M. I. & Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res 4, 1521 (2015).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  48. 48.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  49. 49.

    Lever, J., Krzywinski, M. & Altman, N. Principal component analysis. Nat. Methods 14, 641–642 (2017).

    CAS  Article  Google Scholar 

  50. 50.

    Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, R14 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. 51.

    Oshlack, A. & Wakefield, M. J. Transcript length bias in RNA-seq data confounds systems biology. Biol. Direct. 4, 14 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. 52.

    Stoltz, J. et al. Influence of mechanical forces on bone: introduction to mechanobiology and mechanical adaptation concept. J. Cell. Immunother. 4, 10–12 (2018).

    Article  Google Scholar 

  53. 53.

    Kaneko, K., Ito, M., Naoe, Y., Lacy-Hulbert, A. & Ikeda, K. Integrin αv in the mechanical response of osteoblast lineage cells. Biochem. Biophys. Res. Commun. 447, 352–357 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    Kegelman, C. D. et al. YAP and TAZ mediate osteocyte perilacunar/canalicular remodeling. J. Bone Min. Res. 35, 196–210 (2020).

    CAS  Article  Google Scholar 

  55. 55.

    Kegelman, C. D. et al. Skeletal cell YAP and TAZ combinatorially promote bone development. FASEB J. 32, 2706–2721 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Govey, P. M. et al. Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways. J. Biomech. 47, 1838–1845 (2014).

    PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Govey, P. M., Imamura, Y. & Donahue, H. J. Mapping the osteocytic cell response to fluid flow using RNA-Seq. J. Biomech. 48, 4327–4332 (2015).

    PubMed  Article  PubMed Central  Google Scholar 

  58. 58.

    Chen, W. et al. Gene expression patterns of osteocyte-like MLO-Y4 cells in response to cyclic compressive force stimulation. Cell Biol. Int. 34, 425–432 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  59. 59.

    Ha, J. et al. CXC chemokine ligand 2 induced by receptor activator of NF-kB ligand enhances osteoclastogenesis. J. Immunol. 184, 4717–4724 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  60. 60.

    Ha, J., Lee, Y. & Kim, H. H. CXCL2 mediates lipopolysaccharide-induced osteoclastogenesis in RANKL-primed precursors. Cytokine 55, 48–55 (2011).

    CAS  PubMed  Article  Google Scholar 

  61. 61.

    Hardaway, A. L., Herroon, M. K., Rajagurubandara, E. & Podgorski, I. Marrow adipocyte-derived CXCL1 and CXCL2 contribute to osteolysis in metastatic prostate cancer. Clin. Exp. Metastasis 32, 353–368 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. 62.

    Kwak, H. B. et al. Monokine induced by interferon-gamma is induced by receptor activator of nuclear factor kappa B ligand and is involved in osteoclast adhesion and migration. Blood 105, 2963–2969 (2005).

    CAS  PubMed  Article  Google Scholar 

  63. 63.

    Liu, P. et al. Loss of menin in osteoblast lineage affects osteocyte-osteoclast crosstalk causing osteoporosis. Cell Death Differ. 24, 672–682 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  64. 64.

    Amarasekara, D. S. et al. Regulation of osteoclast differentiation by cytokine networks. Immune Netw. 18, 1–18 (2018).

    Article  Google Scholar 

  65. 65.

    Wood, C. L., Pajevic, P. D. & Gooi, J. H. Osteocyte secreted factors inhibit skeletal muscle differentiation. Bone Rep. 6, 74–80 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We thank Dr Lynda F Bonewald for providing the MLO-Y4 osteocyte cell line. We thank Dr Kristina Kampmann from GATC Biotech for RNAseq experiments and analysis. We also thank Laura Smales for excellent editing of the paper.

Funding

The study was supported by SYBIL European consortium.

Author information

Affiliations

Authors

Contributions

Conception and design (Z.M., E.F., S.J.M., R.F., H.E., C.S.M.); analysis and interpretation of the data (Z.M., S.D., H.E., C.S.M.); drafting of the article (Z.M., S.D., E.F., S.J.M., H.E., C.S.M.); critical revision of the article for important intellectual content (Z.M., S.D., S.J.M., H.E., C.S.M.); final approval of the article (all authors); provision of study materials or patients (E.F., R.F., H.C., C.S.M.); statistical expertize (Z.M., D.S.); obtaining funding (C.S.M.); administrative, technical, or logistical support (E.F., H.C., B.M., R.F.); collection and assembly of data (Z.M., C.S.M.).

Corresponding author

Correspondence to Martine Cohen-Solal.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zarka, M., Etienne, F., Bourmaud, M. et al. Mechanical loading activates the YAP/TAZ pathway and chemokine expression in the MLO-Y4 osteocyte-like cell line. Lab Invest (2021). https://doi.org/10.1038/s41374-021-00668-5

Download citation

Search

Quick links