Combined effects of starvation and butyrate on autophagy-dependent gingival epithelial cell death.
OBJECTIVE
Bacteria in the dental biofilm surrounding marginal gingival grooves cause periodontal diseases. Numerous bacteria within the biofilm consume nutrients from the gingival crevicular fluid. Furthermore, some gram-negative bacteria in mature dental biofilms produce butyrate. Thus, gingival epithelial cells in close proximity to mature dental biofilms are at risk of both starvation and exposure to butyrate. In the present study, we determined the combined effects of starvation and butyrate exposure on gingival epithelial cell death and the underlying mechanisms.


METHODS
The Ca9-22 cell line was used as an in vitro counterpart of gingival epithelial cells. Cell death was measured as the amount of total DNA in the dead cells using SYTOX Green dye, which penetrates through membranes of dead cells and emits fluorescence when it intercalates into double-stranded DNA. AMP-activated protein kinase (AMPK) activity, the amount of autophagy, and acetylation of histone H3 were determined using western blot. Gene expression levels of microtubule-associated protein 1 light chain 3b (lc3b) were determined using quantitative reverse transcription-polymerase chain reaction.


RESULTS
Butyrate-induced cell death occurred in a dose-dependent manner whether cells were starved or fed. However, the induction of cell death was two to four times higher when cells were placed under starvation conditions compared to when they were fed. Moreover, both starvation and butyrate exposure induced AMPK activity and autophagy. While AMPK inactivation resulted in decreased autophagy and butyrate-induced cell death under conditions of starvation, AMPK activation resulted in butyrate-induced cell death when cells were fed. Combined with the results of our previous report, which demonstrated butyrate-induced autophagy-dependent cell death, the results of this study suggest that the combination of starvation and butyrate exposure activates AMPK inducing autophagy and subsequent cell death. Notably, this combination markedly induced LC3B production and the induction was attenuated by AMPK inhibition. LC3B knockdown, in turn, significantly decreased butyrate-induced cell death. Therefore, AMPK-dependent LC3B induction apparently plays an important role in butyrate-induced cell death. There was a lack of correspondence between the levels of AMPK activation and LC3B induction; this may reflect the histone deacetylase-inhibitory capacity of butyrate on histone proteins.


CONCLUSIONS
Taken together, starvation and butyrate exposure promote autophagy via AMPK signaling, while the histone deacetylase-inhibitory effects of butyrate alter chromatin to transcriptionally active state, resulting in strong LC3B induction and subsequent cell death. These findings may help improve the understanding of the cellular processes underlying periodontal disease initiation.
Nihon University School of Dentistry, Tokyo, Japan.
M
M
Evans
Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan.
T
T
Murofushi
Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan.|Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan.
H
H
Tsuda
Division of Microscopic Anatomy, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Y
Y
Mikami
Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan.|Department of Endodontics, School of Dentistry, Shandong University, Jinan, Shandong, China.
N
N
Zhao
Department of Oral Microbiology, Nihon University School of Dentistry, Tokyo, Japan.|Divisions of Immunology and Pathobiology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan.
K
K
Ochiai
Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
T
T
Kurita-Ochiai
Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
M
M
Yamamoto
Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan.|Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan.
K
K
Otsuka
Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan.|Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan.
N
N
Suzuki
D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology*; D005881:Gingiva / Q000187:drug effects / Q000503:physiopathology*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013217:Starvation / Q000503:physiopathology
D002087:Butyrates; D053148:Caspase 3; D053179:Caspase 7
AMP-activated protein kinase; autophagy; butyrate; gingival epithelium