Hydrogen peroxide induces expression and activation of AMP-activated protein kinase in a dental pulp cell line

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.

RPC-C2A cells derived from rat dental pulp were maintained in MEM supplemented with 10% FBS at 37 degrees 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 alpha1 and regulatory beta1 and gamma1 subunit isoforms. The effect of silencing AMPKalpha1 on cell viability was determined using siRNA.

Exposure to H(2)O(2) decreased cell viability in a time- and dose-dependent manner. The catalytic AMPKalpha1 subunit and its activated form, phospho-AMPKalpha, increased with exposure to H(2)O(2) in a time- and dose-dependent manner, whereas the regulatory beta1 and gamma1 subunits showed no change. Downregulation of AMPKalpha1 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).

Reactive oxygen induced energy fuel gauge enzyme AMPKalpha 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.