Pathological Cyclic Strain-Induced Apoptosis in Human Periodontal Ligament Cells through the RhoGDIα/Caspase-3/PARP Pathway

Li Wang,Meng Song,Jinsong Pan,Wantao Chen,Tingle Wang,Philip C Trackman

doi:10.1371/journal.pone.0075973

Abstract

AimHuman periodontal ligament (PDL) cells incur changes in morphology and express proteins in response to cyclic strain. However, it is not clear whether cyclic strain, especially excessive cyclic strain, induces PDL cell apoptosis and if so, what mechanism(s) are responsible. The aim of the present study was to elucidate the molecular mechanisms by which pathological levels of cyclic strain induce human PDL cell apoptosis.Materials and MethodsHuman PDL cells were obtained from healthy premolar tissue. After three to five passages in culture, the cells were subjected to 20% cyclic strain at a frequency of 0.1 Hz for 6 or 24 h using an FX-5000T system. Morphological changes of the cells were assessed by inverted phase-contrast microscopy, and apoptosis was detected by fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide staining followed by flow cytometry. Protein expression was evaluated by Western blot analysis.ResultsThe number of apoptotic human PDL cells increased in a time-dependent manner in response to pathological cyclic strain. The stretched cells were oriented parallel to each another with their long axes perpendicular to the strain force vector. Cleaved caspase-3 and poly-ADP-ribose polymerase (PARP) protein levels increased in response to pathological cyclic strain over time, while Rho GDP dissociation inhibitor alpha (RhoGDIα) decreased. Furthermore, knock-down of RhoGDIα by targeted siRNA transfection increased stretch-induced apoptosis and upregulated cleaved caspase-3 and PARP protein levels. Inhibition of caspase-3 prevented stretch-induced apoptosis, but did not change RhoGDIα protein levels.ConclusionThe overall results suggest that pathological-level cyclic strain not only influenced morphology but also induced apoptosis in human PDL cells through the RhoGDIα/caspase-3/PARP pathway. Our findings provide novel insight into the mechanism of apoptosis induced by pathological cyclic strain in human PDL cells.

Highlights

During occlusal load or orthodontic tooth movement, the cells in the periodontal ligament (PDL) are directly subjected to mechanical stress
The number of apoptotic human PDL cells increased in a time-dependent manner in response to pathological cyclic strain
Cleaved caspase-3 and poly-ADP-ribose polymerase (PARP) protein levels increased in response to pathological cyclic strain over time, while Rho GDP dissociation inhibitor alpha (RhoGDIa) decreased

Summary

Introduction

During occlusal load or orthodontic tooth movement, the cells in the periodontal ligament (PDL) are directly subjected to mechanical stress. The PDL is a complex soft tissue that connects teeth to the surrounding bone, and a common assumption is that it acts as the major element in tooth mobility and stress distribution to supporting tissues [5,6]. Apoptosis induced by cyclic strain is an important determinant of connective tissue destruction in periodontal disease [7]. The application of light orthodontic force causes direct resorption of alveolar bone and tooth mobility, while the application of excessive orthodontic force results in excessive cyclic strain, which induces local ischaemia, tissue hyalinisation, and cell death in the PDL [8]. Programmed cell death (apoptosis) [10,11] plays a key role in the regulation of tissue turnover in long-lived mammals that must integrate multiple physiological as well as pathological death signals

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