Autophagy in periodontal health and disease

Abstract

Autophagy, also known as “self-eating”, is a catabolic trafficking mechanism for energy metabolism, amino acid and fatty acid recycling, and nucleotide synthesis. It occurs constitutively at a low housekeeping level and may be upregulated when cells are exposed to unfavorable conditions such as microbial infection, nutrition shortage, low oxygen level, growth factor withdrawal, and so on. As stated in various reviews, autophagy is accepted to be related to various physiological and pathological cellular processes including neurodegeneration, metabolic disorders, cancer, and cardiovascular disorders. Recent studies have suggested the involvement of autophagy in periodontitis pathogenesis regarding apoptosis induction which the former biological processes attempt to save human gingival fibroblasts (HGFs), periodontal ligament (PDL) cells, and mononuclear cells from apoptotic death. Data also showed that autophagy in periodontal resident cells could be induced by hypoxia, lipopolysaccharides (LPS).

In this project, we hypothesized that autophagy is related to the progression of periodontitis and may be induced by tissue hypoxia. The project aimed to: i) evaluate the status of autophagy in healthy and diseased periodontal tissue; ii) examine whether hypoxia is critical in autophagy induction in HGFs and human gingival keratinocytes (HGKs); iii) investigate the relationship between autophagy in HGFs and type I collagen synthesis.

Human gingival biopsies were collected from periodontitis and periodontitis-free individuals, to detect the expressions of four autophagy markers including Beclin1 (BECN1), autophagy-related protein 7 (Apg7), microtubule-associated protein 1 light chain 3 (LC3), and sequestosome 1 (SQSTM1/p62). Data showed higher expressions of the four markers in the connective tissue in periodontitis tissue while in the epithelial layer the expression was barely detectable. Protein and RNA levels of the markers were also detected using freshly extracted proteins and RNA from periodontitis and healthy tissues. Formation of autophagosome in periodontal tissues was observed under transmission electron microscopy (TEM).

Primary HGFs and HGKs were cultured and exposed to hypoxia with or without LPS for different time periods. Autophagy in HGFs detected by western blotting (WB), real-time polymerase chain reaction (Real-Time PCR), and TEM were noticed to be hypoxia-related while in HGKs autophagy remained unchanged in a normoxic or hypoxic environment with or without LPS.

Collagen synthesis was then preliminarily studied using HGFs cultured under hypoxic (1% O2) or normoxic (18% O2) conditions. Timepoints were set to be 6, 12, and 24 hours. Addition to WB and real-time PCR detection, immunofluorescence and Masson's trichrome staining were performed to localize COL-1 protein and observe the changes of the total collagen content in HGFs, respectively.

Autophagy appeared to be relevant in human periodontitis, evidence being increased expressions of markers (BECN1, Apg7, LC3, and SQSTM1/p62) in diseased periodontal connective tissue. However, in epithelia of the healthy or diseased periodontium, expressions of the autophagy markers are hardly observable. Autophagy was at a more significant level in HGFs and found to be hypoxia-dependent regardless the presence of LPS. Regarding in vitro autophagic reaction, hypoxia and LPS seem not effective in HGKs. In HGFs, hypoxia, not LPS, appeared to be related to autophagic changes. In collagen synthesis study, metabolism of type I collagen was found to be obviously inhibited by autophagy inhibitor 3-Methyladenine (3-MA) under hypoxic environment, suggesting perhaps a positive role of hypoxia-related autophagy in HGF type I collagen formation.

Herein we conclude that autophagy is apparently a connective tissue cellular reaction that takes place in chronic periodontitis while hypoxic state may be of the mechanisms inducing such biological function. Upregulated autophagy is at least partly associated with type I collagen synthesis enhancement in HGFs, under hypoxic condition.