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Hydrogen peroxide induces expression and activation of AMP‐activated protein kinase in a dental pulp cell line
Author(s) -
Fukuyama Y.,
Ohta K.,
Okoshi R.,
Kizaki H.,
Nakagawa K.
Publication year - 2008
Publication title -
international endodontic journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.988
H-Index - 119
eISSN - 1365-2591
pISSN - 0143-2885
DOI - 10.1111/j.1365-2591.2007.01337.x
Subject(s) - viability assay , ampk , protein kinase a , chemistry , amp activated protein kinase , microbiology and biotechnology , downregulation and upregulation , phosphorylation , cell , biochemistry , biology , gene
Aim To investigate the effects of hydrogen peroxide on cell viability and expression and activation of AMP‐activated protein kinase (AMPK) in rat dental pulp cell line RPC‐C2A. Methodology RPC‐C2A cells derived from rat dental pulp were maintained in MEM supplemented with 10% FBS at 37 °C, in a humidified atmosphere at 5% CO 2 . Cells were cultured in the presence or absence of H 2 O 2 for up to 60 min at concentrations of from 0.1 to 3.0 mmol L −1 . Cell viability was analysed by WST‐1 reduction assay. Expression of AMPK subunit isoforms was analysed by Western blotting using antibodies to the catalytic α1 and regulatory β1 and γ1 subunit isoforms. The effect of silencing AMPKα1 on cell viability was determined using siRNA. Results Exposure to H 2 O 2 decreased cell viability in a time‐ and dose‐dependent manner. The catalytic AMPKα1 subunit and its activated form, phospho‐AMPKα, increased with exposure to H 2 O 2 in a time‐ and dose‐dependent manner, whereas the regulatory β1 and γ1 subunits showed no change. Downregulation of AMPKα1 resulted in a reduction in cell viability in H 2 O 2 ‐treated cells at a concentration of 0.1 mmol L −1 for 30 min incubation, indicating an increased sensitivity to H 2 O 2 . Conclusions Reactive oxygen induced energy fuel gauge enzyme AMPKα expression and its activation by phosphorylation in RPC‐C2A cells, suggesting that AMPK is essential for protection against H 2 O 2 ‐induced nonapoptotic cell death. Therefore, AMPK may be a therapeutic modulation target for treatment of the dentine–pulp complex injured by reactive oxygen.