論文の公開元へCigarette smoke extract impairs gingival epithelial barrier function
概要
Title
Cigarette smoke extract impairs gingival
epithelial barrier function
Author(s)
Yamaga, Shunsuke; Tanigaki, Keita; Nakamura,
Eriko et al.
Citation
Scientific Reports. 2023, 13(1), p. 9228
Version Type VoR
URL
rights
https://hdl.handle.net/11094/92682
This article is licensed under a Creative
Commons Attribution 4.0 International License.
Note
Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University
www.nature.com/scientificreports
OPEN
Cigarette smoke extract impairs
gingival epithelial barrier function
Shunsuke Yamaga 1,5, Keita Tanigaki 1,5, Eriko Nakamura 1, Naoko Sasaki 3, Yuta Kato 1,
Masae Kuboniwa 1, Michiya Matsusaki 3,4, Atsuo Amano 1 & Hiroki Takeuchi 2,5*
We previously showed that junctional adhesion molecule 1 (JAM1) and coxsackievirus and adenovirus
receptor (CXADR), tight junction-associated proteins, have important roles to maintain epithelial
barrier function in gingival tissues. Smoking is considered to be a significant risk factor for periodontal
disease. The present study was conducted to examine the effects of cigarette smoke extract (CSE)
on JAM1 and CXADR in human gingival epithelial cells. CSE was found to cause translocation of
JAM1 from the cellular surface to EGFR-positive endosomes, whereas CXADR did not. Using a threedimensional multilayered gingival epithelial tissue model, CSE administration was found to increase
permeability to lipopolysaccharide and peptidoglycan, whereas overexpression of JAM1 in the tissue
model prevented penetration by those substrates. Furthermore, vitamin C increased JAM1 expression,
and inhibited penetration of LPS and PGN induced by CSE. These findings strongly suggest that CSE
disrupts gingival barrier function via dislocation of JAM1, thus allowing bacterial virulence factors
to penetrate into subepithelial tissues. Furthermore, they indicate that vitamin C increases JAM1
expression and prevents disruption of gingival barrier function by CSE.
Periodontal disease is a chronic infectious disease caused by complex actions of periodontal bacteria in oral
biofilm, with cigarette smoking recognized as significant among the many known risk factors1–3. Essentially,
both natural tobacco leaves and smoke formed from burning tobacco contain several toxic chemicals. Studies
of cigarette smoke extract (CSE) have shown that it causes increased permeability in respiratory epithelium and
human bronchial epithelial cells, leading to impairment of epithelial barrier f unction4,5. As for the oral region,
cigarette smoking reportedly inhibits gingival epithelial cell g rowth6. Furthermore, as compared to non-smokers,
cigarette smokers are known to show worse response to periodontal treatment7,8. On the other hand, the molecular mechanisms associated with the negative influence of smoking on periodontal tissues are not well understood.
Human mucosal surfaces are exposed to abundant microbiota and their virulence factors. Lipopolysaccharides (LPS), endotoxins of gram-negative bacteria, and peptidoglycan (PGN) are prototypical representatives of
pathogen-associated molecular patterns recognized by innate immunity factors9. Using a gingival epithelial tissue
model, we previously showed that the periodontal pathogen Porphyromonas gingivalis specifically degraded two
tight junction-associated proteins, junctional adhesion molecule 1 (JAM1) and coxsackievirus and adenovirus
receptor (CXADR), which allowed the pathogen to successfully break down the gingival epithelial barrier, thus
increasing epithelial permeability to LPS and PGN, which likely leads to onset of periodontal d
isease10–12. JAM1
and CXADR are thought to play important roles in maintenance of the epithelial barrier to prevent periodontal
diseases, thus the effects of cigarette smoking on these two molecules are of interest.
The Nutrition Examination Survey (NHANES III) cross-sectional study conducted in the United States13
found that low intake of vitamin C, an essential dietary requirement for humans, is a risk factor for periodontal
disease. Furthermore, gingival bleeding in vitamin C-deficient patients has been shown to be improved by vitamin C supplementation14,15. The amount of vitamin C in the blood of smokers is known to be lower as compared
to nonsmokers16, thus elucidation of the molecular basis for the effects of vitamin C on onset and progression
of periodontal disease in smokers is important.
Based on results of the present study, it is suggested that CSE disrupts the barrier function of gingival epithelium via JAM1 translocation, thus allowing for penetration of bacterial virulence factors into subepithelial
tissues. Additionally, they provide the molecular basis for cigarette smoking as a risk factor for periodontal
1
Department of Preventive Dentistry, Graduate School of Dentistry, Osaka University, Suita‑Osaka 565‑0871,
Japan. 2Department of Preventive Dentistry, Osaka University Dental Hospital, 1–8 Yamadaoka,
Suita‑Osaka 565‑0871, Japan. 3Joint Research Laboratory (TOPPAN) for Advanced Cell Regulatory Chemistry,
Graduate School of Engineering, Osaka University, Suita‑Osaka 565‑0871, Japan. 4Department of Applied
Chemistry, Graduate School of Engineering, Osaka University, Suita‑Osaka 565‑0871, Japan. 5These authors
contributed equally: Shunsuke Yamaga, Keita Tanigaki and Hiroki Takeuchi. *email: takeuchi.hiroki.dent@
osaka-u.ac.jp
Scientific Reports |
(2023) 13:9228
| https://doi.org/10.1038/s41598-023-36366-z
1
Vol.:(0123456789)
www.nature.com/scientificreports/
disease development. In addition, vitamin C is indicated as a potential nutrient to increase the barrier function
of gingival epithelium exposed to cigarette smoke.
Results
CSE causes loss of JAM1, but not CXADR, on surface of gingival epithelial cells. The effects of
CSE on JAM1 and CXADR distribution were initially examined. At 1 h after administration of CSE derived from
Kent, Marlboro, or Seven Stars brand cigarettes, JAM1 was found to have disappeared from the cellular surface
(Fig. 1a), while negligible effects were observed in regard to CXADR localization (Supplementry Figure 1). In
addition, the findings confirmed that JAM1 and CXADR levels were not altered by CSE (Fig. 1b). The same
trends were confirmed in examinations of primary gingival epithelial cells (Supplementary Figure 2). Hence,
CSE affects JAM1 localization, but not the level of that protein.
The distribution of JAM1 in human gingival tissues of smoker and non-smoker subjects was also examined.
In gingival epithelium specimens obtained from the non-smokers, JAM1 showed a grid-liked pattern, while scattered localization was noted in specimens from the smokers (Fig. 2). Even the specimen obtained from Smoker
#1, who was without bleeding on probing and showed periodontal pockets ≥ 4 mm, JAM1 had lost its original
localization. These results prompted us to examine whether cigarette smoking diminishes intercellular JAM1
localization in human gingival epithelia.
JAM1 translocation from cell surface to EGFR‑positive endosomes induced by CSE. We previ-
ously showed that JAM1 is transported to the plasma membrane via an endomembrane system10, which consists
of different membranes suspended in cytoplasm within a eukaryotic cell. It was thus speculated that CSE induces
JAM1 translocation to intracellular organelles. Following CSE administration, IHGE cells were stained with
anti-EGFR as a marker for the endocytosis p
athway17. At 1 h after CSE administration, JAM1 was clearly found
located in EGFR-positive endosomes (Fig. 3). In general, endocytosis does not occur below 10 °C18. To confirm
whether CSE induces translocation of JAM1 from the plasma membrane via the endocytic pathway, IHGE cells
were treated with CSE at 4 °C, though JAM1 localization remained on the plasma membrane for up to 1 h
(Fig. 4). These findings were also confirmed in primary gingival epithelial cells (Supplementary Figure 3). Additionally, fractionation of IHGE cells showed that JAM1 and CXADR could be detected in membrane compartments including plasma membrane and endosomes, but not in cytosol compartments, in either the CSE-added
or non-added cells (Supplementary Figure 4a). Isolation of plasma membrane fraction of IHGE cells showed
that CSE treatment decreased JAM1 levels, but not of CXADR, in the plasma membrane fraction (Supplementary Figure 4b). Together, these results suggest that JAM1 selectively internalized in the plasma membrane is
induced by CSE to translocate to gingival epithelial cells.
CSE induces penetration of LPS and PGN into gingival epithelium. To assess the effects of CSE
on JAM1 localization in deeper epithelium, a 3D-tissue model of gingival epithelium was generated using a
cell-accumulation technique19 (Fig. 5a). JAM1 proteins were found localized in phalloidin-stained plasma membranes in the model without CSE (Fig. 5b). At 1 h after CSE administration, JAM1 was found to have translocated from cell surfaces to intracellular space in the tissues up to 3–4 layers below the surface, which was
effectively compensated by JAM1 overexpression. These results suggest that CSE causes loss of surface JAM1 in
human gingival epithelial tissues.
It was previously reported that cigarette smoke facilitates allergen penetration through human bronchial
epithelial cell m
onolayers4. Bronchial epithelium has a pseudostratified ciliated columnar structure, whereas
gingival epithelium has a stratified squamous form, thus we attempted to clarify the biological implications of
loss of JAM1 on the gingival epithelium surface induced by CSE. 3D-tissue models of IHGE wild type (WT) cells
or those overexpressing JAM1 were generated, then permeability assays using fluorescein isothiocyanate (FITC)labeled P. gingivalis LPS and PGN were performed (Fig. 6a). At 3 h after administration, permeability to both
LPS (Fig. 6b,d,f) and PGN (Fig. 6c,e,g) in the examined tissues was significantly increased by CSEs. ...
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