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Autophagy facilitates type I collagen synthesis in periodontal ligament cells.

ABSTRACT: Autophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

SUBMITTER: Nakamura T

PROVIDER: S-EPMC7809284 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Autophagy facilitates type I collagen synthesis in periodontal ligament cells.

Nakamura Tomomi T Yamashita Motozo M Ikegami Kuniko K Suzuki Mio M Yanagita Manabu M Kitagaki Jirouta J Kitamura Masahiro M Murakami Shinya S

Scientific reports 20210114 1

Autophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue co ...[more]

PMID: 33446772

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Project description:Periodontal ligament contains periodontal ligament stem cells that maintain tissue homeostasis. Targeting hPDLSCs (human periodontal ligament cells) is a promising strategy for repair and regeneration of bone tissue destroyed by periodontal diseases. However, the mechanisms by which PDLSCs differentiate into osteoblasts to form a mineralized matrix is unclear. In this study, we demonstrate for the first time the molecular events that contribute to osteogenic differentiation of PDLSCs. Dentin matrix protein 1 (DMP1) and its receptor, Glucose regulated protein-78 (GRP78), are localized in the progenitor cells of the PDL. Our overall goal is to demonstrate the formation of DMP1-GRP78 complex at the plasma membrane and subsequent protein trafficking and nuclear localization to promote osteogenic differentiation. To study the internalization and routing of the complex, we mimic an in vivo differentiation scenario by stimulating cells with DMP1 and culturing them in the presence of osteogenic differentiation conditions. We first demonstrate the translocation of the ER chaperone protein GRP78 to the plasma membrane during the differentiation process. Total internal reflection microscopy imaging demonstrates the formation and internalization of the receptor- ligand (GRP78-DMP1) complex. Confocal microscopy results show the internalization of the GRP78-DMP1 complex specifically through the caveolin pathway and trafficked through the cell with various endocytic markers such as Rab5 and 7 GTPases to early and late endosomes respectively. DMP1 is ultimately transported to the nucleus where it functions to promote osteogenic differentiation as demonstrated by quantitative Real-Time PCR. This observation is the first report that suggests DMP1 and GRP78 can interact at the plasma membrane, then packaged in vesicles and ultimately DMP1 is routed to the nucleus where it aids in osteogenic differentiation of PDLSCs. Characterizing the osteogenic potential of PDLSCs would favor the development of therapeutic strategies for reconstruction of mineralized tissues destroyed by periodontal diseases.

Project description:The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self-renewal, lineage commitment and apoptosis. Trans-4-[(1R)-aminoethyl]-N-(4-pyridinyl)cylohexanecarboxamidedihydrochloride (Y-27632), Rho-associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti-apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound-healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the effects of Y-27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y-27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y-27632 induced PDLSCs proliferation through extracellular-signal-regulated kinase (ERK) signalling cascade. Therefore, control of Rho-kinase activity may enhance the efficiency of stem cell-based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.

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Autophagy facilitates type I collagen synthesis in periodontal ligament cells.

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Autophagy facilitates type I collagen synthesis in periodontal ligament cells.

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