Konstantinos Karamesinis1,2, Anastasia Spyropoulou1, Georgia Dalagiorgou1, Maria A Katsianou1, Marjan Nokhbehsaim3, Svenja Memmert4, James Deschner3, Heleni Vastardis2, Christina Piperi5. 1. Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, 75, M. Asias Street, 11527, Athens, Greece. 2. Department of Orthodontics, Dental School, National and Kapodistrian University of Athens, 11527, Athens, Greece. 3. Section of Experimental Dento-Maxillo-Facial Medicine, University of Bonn, Welschnonnenstrasse 17, 53111, Bonn, Germany. 4. Department of Orthodontics Dento-Maxillo-Facial Medicine, University of Bonn, Welschnonnenstrasse 17, 53111, Bonn, Germany. 5. Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, 75, M. Asias Street, 11527, Athens, Greece. cpiperi@med.uoa.gr.
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.
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.
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