Autophagy Regulates Extracellular Matrix Production by Human Periodontal Ligament Cells

Objectives: To maintain the homeostasis in periodontal tissue, various extracellular matrix (ECM) proteins are produced by periodontal ligament (PDL) cells. Balancing the synthesis of ECM proteins and proteolysis of misfolded ECM proteins is required for the proper ECM protein turnover. Autophagy is a lysosomal protein degradation system in which the cell self-digests its protein components and organelles. Defects in autophagy have been reported in the pathogenesis of age-related chronic degenerative disease. However, little is known about the role of autophagy in the pathogenesis of periodontitis. In this study, we examined the physiological function of autophagy in PDL cells with focusing on the ECM biosynthesis.
Methods: The formation of autophagosomes in human PDL (HPDL) cells was observed by the transmission electron microscopy (TEM). HPDL cells were cultured in the ossification inducing medium including L-ascorbic acid and β-glycelophosphate in the presence or absence of autophagy inhibitor, E-64d, Pepstatin A (PSA). Cellular distribution of type I collagen was estimated by immunocytochemical staining. Expression of type I / III collagen, fibronectin and hyaluronan was analyzed by ELISA, western blotting method and RT-qPCR.
Results: Induction of autophagy in HPDL cells was established in in vitro system. TEM analysis revealed that the nutrient starvation induced autophagic vacuoles formation. E-64d and PSA treatment induced the accumulation of collagen aggregates inside the HPDL cells. In contrast, the distribution of collagen in the extracellular body was decreased. E-64d and PSA treatment inhibited the production of mature form of type I collagen and mRNA expression of type I / III collagen and fibronectin. Conversely, hyaluronan production was not influenced.
Conclusions: Autophagy regulates ECM production of HPDL cells. Accumulation of collagen aggregates induced by the defects in autophagy may turn into the failure of ECM synthesis. This suggests that autophagy system is indispensable for maintaining the homeostasis in periodontal tissue.