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Regulation of the autophagy-marker Sequestosome 1 in periodontal cells and tissues by biomechanical loading

Regulation des Autophagie-Markers Sequestosom 1 durch biomechanische Kräfte in parodontalen Zellen und Geweben

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Abstract

Purpose

Orthodontic treatment is based on the principle of force application to teeth and subsequently to the surrounding tissues and periodontal cells. Sequestosome 1 (SQSTM1) is a well-known marker for autophagy, which is an important cellular mechanism of adaptation to stress. The aim of this study was to analyze whether biomechanical loading conditions regulate SQSTM1 in periodontal cells and tissues, thereby providing further information on the role of autophagy in orthodontic tooth movement.

Methods

Periodontal ligament (PDL) fibroblasts were exposed to cyclic tensile strain of low magnitude (3%, CTSL), and the regulation of autophagy-associated targets was determined with an array-based approach. SQSTM1 was selected for further biomechanical loading experiments with dynamic and static tensile strain and assessed via real-time polymerase chain reaction (RT-PCR) and immunoblotting. Signaling pathways involved in SQSTM1 activation were analyzed by using specific inhibitors, including an autophagy inhibitor. Finally, SQSTM1 expression was analyzed in gingival biopsies and histological sections of rats in presence and absence of orthodontic forces.

Results

Multiple autophagy-associated targets were regulated by CTSL in PDL fibroblasts. All biomechanical loading conditions tested increased the SQSTM1 expression significantly. Stimulatory effects of CTSL on SQSTM1 expression were diminished by inhibition of the c‑Jun N‑terminal kinase (JNK) pathway and of autophagy. Increased SQSTM1 levels after CTSL were confirmed by immunoblotting. Orthodontic force application also led to significantly elevated SQTSM1 levels in the gingiva and PDL of treated animals as compared to control.

Conclusions

Our in vitro and in vivo findings provide evidence of a role of SQSTM1 and thereby autophagy in orthodontic tooth movement.

Zusammenfassung

Ziel

Der kieferorthopädischen Zahnbewegung liegt das Prinzip einer Kraftapplikation auf Zähne und somit auf umgebende parodontale Zellen und Gewebe zugrunde. Sequestosom 1 (SQSTM1) gilt als Marker des wichtigen zellulären Adaptationsmechanismus Autophagie. Ziel dieser Studie war es, die Regulation von SQSTM1 bei der Adaptation an biomechanische Belastung in parodontalen Zellen und Geweben zu untersuchen und damit die Rolle der Autophagie bei der kieferorthopädischen Zahnbewegung zu eruieren.

Methoden

Parodontale Ligament(PDL)-Zellen wurden einer dynamischen Belastung mit geringen Kräften (3 %, „cyclic tensile strain of low magnitude“, CTSL) ausgesetzt und die Regulation von autophagieassoziierten Zielgenen wurde mittels Arrays bestimmt. SQSTM1 wurde als Zielgen für weitere Untersuchungen ausgewählt. Die Regulation wurde nach Applikation dynamischer und statischer Belastungen durch „real-time polymerase chain reaction“ (RT-PCR) und Immunoblotting untersucht. Die an der Regulation beteiligten Signalwege wurden mittels spezifischer Inhibition, einschließlich eines Autophagie-Hemmers, analysiert. Weiterhin wurde die SQSTM1-Expression in gingivalen Biopsien und in histologischen Schnitten von Ratten nach kieferorthopädisch Zahnbewegung ermittelt und mit entsprechenden Kontrollen verglichen.

Ergebnisse

Eine Vielzahl an Genen wurde durch die Applikation von CTSL in PDL-Zellen reguliert. Alle getesteten biomechanischen Belastungszustände steigerten die SQSTM1-Genexpression signifikant. Die CTSL-induzierte Steigerung der SQSTM1-Genexpression konnte sowohl durch eine Hemmung des „c‑Jun N‑terminal kinase“(JNK)-Signalwegs als auch der Autophagie inhibiert werden. Auf Proteinebene konnte ebenfalls eine Zunahme von SQSTM1 durch CTSL nachgewiesen werden. Durch Applikation kieferorthopädischer Kräfte führten sowohl zu einer erhöhten SQSTM1-Genexpression in der Gingiva als auch im PDL der behandelten Tiere.

Schlussfolgerungen

Die Ergebnisse unserer Studie legen nahe, dass SQSTM1 und damit die Autophagie eine zentrale Rolle bei der kieferorthopädischen Zahnbewegung spielen.

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Acknowledgements

The authors would like to thank Ms. Ramona Menden, Ms. Silke van Dyck, Ms. Inka Bay and Ms. Jana Marciniak for their valuable support. This study was supported by the Medical Faculty of the University of Bonn, the German Research Foundation (DFG; ME 4798/1‑1, ME 4798/1-2) and the German Orthodontic Society (DGKFO).

Funding

This study was supported by the Medical Faculty of the University of Bonn, the German Orthodontic Society (DGKFO) and the German Research Foundation (DFG, ME 4798/1–1, ME 4798/1–2).

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Authors and Affiliations

  1. Department of Orthodontics, Center of Dento-Maxillo-Facial Medicine, University of Bonn, Welschnonnenstr. 17, 53111, Bonn, Germany

    S. Memmert, B. Rath-Deschner, W. Götz & A. Jäger

  2. Section of Experimental Dento-Maxillo-Facial Medicine, Center of Dento-Maxillo-Facial Medicine, University of Bonn, Bonn, Germany

    S. Memmert & M. Nokhbehsaim

  3. Department of Periodontology and Operative Dentistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany

    A. V. B. Nogueira, A. Damanaki & J. Deschner

  4. Department of Orthodontics and Orofacial Orthopedics, University of Erlangen, Erlangen, Germany

    L. Gölz

  5. Institute of Human Genetics, University Hospital of Bonn, Bonn, Germany

    L. Gölz

  6. Department of Diagnosis and Surgery, School of Dentistry at Araraquara, University Estadual Paulista—UNESP, Araraquara, Brazil

    J. A. Cirelli

  7. Institute of Reconstructive Neurobiology, Life and Brain Center, University of Bonn, Bonn, Germany

    A. Till

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  1. S. Memmert
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Correspondence to S. Memmert.

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Conflict of interest

S. Memmert, A.V.B. Nogueira, A. Damanaki, M. Nokhbehsaim, B. Rath-Deschner, W. Götz, L. Gölz, J.A. Cirelli, A. Till, A. Jäger and J. Deschner declare that they have no competing interests.

Ethical standards

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of the University of Bonn (#117/15) and with the 1964 Helsinki declaration and its later amendments. All procedures of animal experiments were performed in accordance with the ethical standards of the Ethical Committee on Animal Experimentation (protocol number: 23/2012) from the School of Dentistry at Araraquara, University Estadual Paulista – UNESP and in accordance with the recommendations of the ARRIVE guidelines. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Informed consent was obtained from all individual participants included in the study.

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Memmert, S., Nogueira, A.V.B., Damanaki, A. et al. Regulation of the autophagy-marker Sequestosome 1 in periodontal cells and tissues by biomechanical loading. J Orofac Orthop 81, 10–21 (2020). https://doi.org/10.1007/s00056-019-00197-3

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