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Autophagy in periodontal ligament fibroblasts under biomechanical loading

Abstract

Autophagy (cellular self-consumption) is an adaptive stress response and an important aspect of adaption to mechanical loading. If mechanical forces are associated with autophagy regulation in periodontal ligament (PDL) fibroblasts is still unknown. The aim of this study was to analyze the influence of force magnitude on autophagy regulation and subsequently on cell death in human PDL fibroblasts. Autophagy-associated genes were analyzed with a specific PrimePCR assay after 24 h of stimulation with high (STSH) and low magnitudes (STSL) of static tensile strain applied to PDL fibroblasts. Based on the results, targets were selected for further real-time PCR analysis. The autophagic flux was assessed by immunoblotting for autophagy marker microtubule-associated protein 1, light chain 3, and by autophagosome staining. Cell death was determined by TUNEL assay and Cell Death Detection ELISAPLUS. Autophagy was induced pharmacologically by rapamycin and inhibited by chloroquine. For statistical analysis, the Kruskal Wallis test followed by the post-hoc Dunnett’s test was used. Static tensile strain had regulatory effects on mRNA expression of multiple autophagy-associated targets. Stimulation with STSH induced mRNA expression changes in more autophagy-associated targets than STSL. The autophagic flux was induced by STSH while STSL had no significant effect on autophagosome formation. Furthermore, autophagy inhibition led to increased cell death. Low magnitudes of tensile strain seem to have cell-protective properties. Taken together, our findings provide novel insights about autophagy regulation by biomechanical loading in human PDL fibroblasts. Our results suggest a gradual response of autophagy to static tensile strain in human PDL fibroblasts.

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References

  1. Basdra EK, Komposch G (1997) Osteoblast-like properties of human periodontal ligament cells: an in vitro analysis. Eur J Orthod 19:615–621

    CAS  Article  Google Scholar 

  2. Chen H, Chen L, Cheng B, Jiang C (2015) Cyclic mechanical stretching induces autophagic cell death in tenofibroblasts through activation of prostaglandin E2 production. Cell Physiol Biochem 36:24–33

    Article  Google Scholar 

  3. Deschner B, Rath B, Jäger A, Deschner J, Denecke B, Memmert S, Götz W (2012) Gene analysis of signal transduction factors and transcription factors in periodontal ligament cells following application of dynamic strain. J Orofac Orthop 73:486–495, 497

    CAS  Article  Google Scholar 

  4. Glick D, Barth S, Macleod KF (2010) Autophagy: cellular and molecular mechanisms. J Pathol 221:3–12

    CAS  Article  Google Scholar 

  5. Guo S, Liang Y, Murphy SF, Huang A, Shen H, Kelly DF, Sobrado P, Sheng Z (2015) A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications. Autophagy 11:560–572

    Article  Google Scholar 

  6. Huang WP, Klionsky DJ (2002) Autophagy in yeast: a review of the molecular machinery. Cell Struct Funct 27:409–420

    CAS  Article  Google Scholar 

  7. King JS (2012) Mechanical stress meets autophagy: potential implications for physiology and pathology. Trends Mol Med 18:583–588

    CAS  Article  Google Scholar 

  8. King JS, Veltman DM, Insall RH (2011) The induction of autophagy by mechanical stress. Autophagy 7:1490–1499

    CAS  Article  Google Scholar 

  9. Kirschneck C, Batschkus S, Proff P, Köstler J, Spanier G, Schröder A (2017) Valid gene expression normalization by RT-qPCR in studies on hPDL fibroblasts with focus on orthodontic tooth movement and periodontitis. Sci Rep 7:14751

    Article  Google Scholar 

  10. Klionsky DJ, Abdelmohsen K, Abe A et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1–222

    Article  Google Scholar 

  11. Krishnan V, Davidovitch Z, Bahar H (2015) Biological mechanisms of tooth movement, 2nd Edition. Wiley-Blackwell, Chichester

    Book  Google Scholar 

  12. Kroemer G, Mariño G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40:280–293

    CAS  Article  Google Scholar 

  13. Li M, Khambu B, Zhang H, Kang JH, Chen X, Chen D, Vollmer L, Liu PQ, Vogt A, Yin XM (2013) Suppression of lysosome function induces autophagy via a feedback down-regulation of MTOR complex 1 (MTORC1) activity. J Biol Chem 288:35769–35780

    CAS  Article  Google Scholar 

  14. Li Y, Jacox LA, Little SH, Ko CC (2018) Orthodontic tooth movement: the biology and clinical implications. Kaohsiung J Med Sci 34:207–214

    Article  Google Scholar 

  15. Lippai M, Szatmári Z (2017) Autophagy-from molecular mechanisms to clinical relevance. Cell Biol Toxicol 33:145–168

    CAS  Article  Google Scholar 

  16. Ma Y, Galluzzi L, Zitvogel L, Kroemer G (2013) Autophagy and cellular immune responses. Immunity 39:211–227

    CAS  Article  Google Scholar 

  17. Marchesan JT, Scanlon CS, Soehren S, Matsuo M, Kapila YL (2011) Implications of cultured periodontal ligament cells for the clinical and experimental setting: a review. Arch Oral Biol 56:933–943

    Article  Google Scholar 

  18. Mariño G, Niso-Santano M, Baehrecke EH, Kroemer G (2014) Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol 15:81–94

    Article  Google Scholar 

  19. Mariotti A, Cochran DL (1990) Characterization of fibroblasts derived from human periodontal ligament and gingiva. J Periodontol 61:103–111

    CAS  Article  Google Scholar 

  20. Memmert S, Damanaki A, Nogueira AVB, Eick S, Nokhbehsaim M, Papadopoulou AK, Till A, Rath B, Jepsen S, Götz W, Piperi C, Basdra EK, Cirelli JA, Jäger A, Deschner J (2017) Role of cathepsin S in periodontal inflammation and infection. Mediat Inflamm 2017:4786170

    CAS  Article  Google Scholar 

  21. Memmert S, Nogueira AVB, Damanaki A, Nokhbehsaim M, Eick S, Divnic-Resnik T, Spahr A, Rath-Deschner B, Till A, Götz W, Cirelli JA, Jäger A, Deschner J (2018) Damage-regulated autophagy modulator 1 in oral inflammation and infection. Clin Oral Investig 22:2933–2941

    Article  Google Scholar 

  22. Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741

    CAS  Article  Google Scholar 

  23. Nakamura Y, Hirashita A, Kuwabara Y (1984) The localization of acid phosphatase activity in osteoblasts incident to experimental tooth movement. Acta Histochem Cytochem 17:581–582

    Google Scholar 

  24. Natali AN, Pavan PG, Scarpa C (2004) Numerical analysis of tooth mobility: formulation of a non-linear constitutive law for the periodontal ligament. Dent Mater 20:623–629

    Article  Google Scholar 

  25. Noda T, Ohsumi Y (1998) Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J Biol Chem 273:3963–3966

    CAS  Article  Google Scholar 

  26. Pietrocola F, Izzo V, Niso-Santano M, Vacchelli E, Galluzzi L, Maiuri MC, Kroemer G (2013) Regulation of autophagy by stress-responsive transcription factors. Semin Cancer Biol 23:310–322

    CAS  Article  Google Scholar 

  27. Salabei JK, Hill BG (2015) Autophagic regulation of smooth muscle cell biology. Redox Biol 4:97–103

    CAS  Article  Google Scholar 

  28. Towers CG, Thorburn A (2016) Therapeutic targeting of autophagy. EBioMedicine 14:15–23

    Article  Google Scholar 

  29. Wesselborg S, Stork B (2015) Autophagy signal transduction by ATG proteins: from hierarchies to networks. Cell Mol Life Sci 72:4721–4757

    CAS  Article  Google Scholar 

  30. Wu Y, Zhao D, Zhuang J, Zhang F, Xu C (2016) Caspase-8 and Caspase-9 functioned differently at different stages of the cyclic stretch-induced apoptosis in human periodontal ligament cells. PLoS One 11:e0168268

    Article  Google Scholar 

  31. Xu C, Hao Y, Wei B, Ma J, Li J, Huang Q, Zhang F (2011) Apoptotic gene expression by human periodontal ligament cells following cyclic stretch. J Periodontal Res 46:742–748

    CAS  Article  Google Scholar 

  32. Xu HG, Yu YF, Zheng Q, Zhang W, Wang CD, Zhao XY, Tong WX, Wang H, Liu P, Zhang XL (2014) Autophagy protects end plate chondrocytes from intermittent cyclic mechanical tension induced calcification. Bone 66:232–239

    Article  Google Scholar 

  33. Zhao D, Wu Y, Xu C, Zhang F (2017) Cyclic-stretch induces apoptosis in human periodontal ligament cells by activation of caspase-5. Arch Oral Biol 73:129–135

    CAS  Article  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Michaela Rinneburger, Dr. Benedikt Kleineidam, Ms. Ramona Menden, Ms. Silke van Dyck, and Ms. Inka Bay for their valuable support.

Funding statement

This study was supported by the Medical Faculty of the University of Bonn, the German Research Foundation (DFG, ME 4798/1-1) and the German Orthodontic Society (DGKFO).

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Correspondence to Svenja Memmert.

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Research involving human participants and/or animals

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

PDL cells were taken after the approval of the Ethics Committee of the University of Bonn was given (#117/15). Patients or their legal guardians provided written informed consent before PDL cells were harvested.

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Informed consent was obtained from all individual participants included in the study.

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Memmert, S., Damanaki, A., Weykopf, B. et al. Autophagy in periodontal ligament fibroblasts under biomechanical loading. Cell Tissue Res 378, 499–511 (2019). https://doi.org/10.1007/s00441-019-03063-1

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Keywords

  • Periodontal ligament
  • Autophagy
  • Biomechanical loading
  • Orthodontic tooth movement
  • Cell death