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Continuous hydrostatic pressure induces differentiation phenomena in chondrocytes mediated by changes in polycystins, SOX9, and RUNX2

  • Konstantinos Karamesinis
  • Anastasia Spyropoulou
  • Georgia Dalagiorgou
  • Maria A. Katsianou
  • Marjan Nokhbehsaim
  • Svenja Memmert
  • James Deschner
  • Heleni Vastardis
  • Christina Piperi
Original Article

Abstract

Purpose

The present study aimed to investigate the long-term effects of hydrostatic pressure on chondrocyte differentiation, as indicated by protein levels of transcription factors SOX9 and RUNX2, on transcriptional activity of SOX9, as determined by pSOX9 levels, and on the expression of polycystin-encoding genes Pkd1 and Pkd2.

Materials and methods

ATDC5 cells were cultured in insulin-supplemented differentiation medium (ITS) and/or exposed to 14.7 kPa of hydrostatic pressure for 12, 24, 48, and 96 h. Cell extracts were assessed for SOX9, pSOX9, and RUNX2 using western immunoblotting. The Pkd1 and Pkd2 mRNA levels were detected by real-time PCR.

Results

Hydrostatic pressure resulted in an early drop in SOX9 and pSOX9 protein levels at 12 h followed by an increase from 24 h onwards. A reverse pattern was followed by RUNX2, which reached peak levels at 24 h of hydrostatic pressure-treated chondrocytes in ITS culture. Pkd1 and Pkd2 mRNA levels increased at 24 h of combined hydrostatic pressure and ITS treatment, with the latter remaining elevated up to 96 h.

Conclusions

Our data indicate that long periods of continuous hydrostatic pressure stimulate chondrocyte differentiation through a series of molecular events involving SOX9, RUNX2, and polycystins-1, 2, providing a theoretical background for functional orthopedic mechanotherapies.

Keywords

Chondrocytes Differentiation Hydrostatic pressure Polycystins RUNX2 SOX9 

Kontinuierlicher hydrostatischer Druck induziert Differenzierungsphänomene in Chondrozyten durch Änderungen in Polyzystinen, SOX9 und RUNX2

Zusammenfassung

Zielsetzung

Die vorgestellte Studie hatte zum Ziel, die langfristigen Auswirkungen von hydrostatischem Druck auf die Chondrozytendifferenzierung zu untersuchen. Parameter waren die Proteinkonzentrationen der Transkriptionsfaktoren SOX9 und RUNX2, die Transkriptionsaktivität von SOX9 und die Expression der Polyzytin-kodierenden Gene Pkd1 und Pkd2.

Material und Methoden

ATDC5-Zellen wurden in einem Insulin-supplementieren Differenzierungsmedium (ITS) kultiviert und/oder hydrostatischem Druck (14,7 kPA) über 12, 24, 48 und 96 h ausgesetzt. SOX9, pSOX9 und RUNX2 wurde in Zellextrakten mit Hilfe von Western-Immunoblot-Tests bestimmt, die Konzentrationen von Pkd1- und Pkd2-mRNA wurden ermittelt mittels real-time PCR.

Ergebnisse

Unter hydrostatischem Druck kam es zu einem frühen (12 h)Abfall der SOX9- und pSOX9-Proteinkonzentrationen, gefolgt von einer Erhöhung nach 24 h und danach. Ein umgekehrtes Muster war zu beobachten für RUNX2: Höchstmengen wurden nach 24 h hydrostatischem Druck in der ITS-Kultur beobachtet. Die Konzentrationen von Pkd1- und Pkd2-mRNA stiegen 24 h nach der Kombination von hydrostatischem Druck und ITS-Behandlung an, die Konzentration der Pkd2-mRNA blieb bis zu 96 h erhöht.

Schlussfolgerungen

Unsere Ergebnisse zeigen, dass langfristiger hydrostatischer Druck die Chondrozytendifferenzierung stimuliert. Dies geschieht über eine Reihe von Prozessen auf Molekularebene, an denen SOX9, RUNX2 sowie Polyzystin-1 und -2 beteiligt sind. Unsere Daten bieten somit einen theoretischen Hintergrund für funktionelle orthopädische Mechanotherapien.

Schlüsselwörter

Chondrozyten Differenzierung Hydrostatischer Druck Polyzystine RUNX2; SOX9 

Notes

Acknowledgements

This research project was funded by IKY-SIEMENS: Post-Doctoral Scholarship of Excellence 2013-2015 (to G. Dalagiorgou) and program IKYDA 2015 for the promotion of the exchange and scientific cooperation between Greece and Germany.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

K. Karamesinis, A. Spyropoulou, G. Dalagiorgou, M.A. Katsianou, M. Nokhbehsaim, S. Memmert, J. Deschner, H. Vastardis, and C. Piperi declare that they have no competing interests.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Konstantinos Karamesinis
    • 1
    • 2
  • Anastasia Spyropoulou
    • 1
  • Georgia Dalagiorgou
    • 1
  • Maria A. Katsianou
    • 1
  • Marjan Nokhbehsaim
    • 3
  • Svenja Memmert
    • 4
  • James Deschner
    • 3
  • Heleni Vastardis
    • 2
  • Christina Piperi
    • 1
  1. 1.Department of Biological ChemistryMedical School, National and Kapodistrian University of AthensAthensGreece
  2. 2.Department of OrthodonticsDental School, National and Kapodistrian University of AthensAthensGreece
  3. 3.Section of Experimental Dento-Maxillo-Facial MedicineUniversity of BonnBonnGermany
  4. 4.Department of Orthodontics Dento-Maxillo-Facial MedicineUniversity of BonnBonnGermany

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