calpain and midostaurin

Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca²(+) influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca²(+) import through hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis-inducing factor (AIF)-mediated apoptosis. Downregulation of HCN2 prevented the drug-induced Ca²(+) increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca²(+) entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412-induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C-terminal domain of HCN2 revealed that dephosphorylation of Thr⁵⁴⁹ was critical for the prolonged Ca²(+) entry required for AIF-mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calcium; Calpain; Caspases; Cell Line; Cell Line, Tumor; Cells, Cultured; Enzyme Inhibitors; Gene Expression; HEK293 Cells; Humans; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Ion Channels; Neurons; Phosphorylation; Potassium Channels; Protein Kinase C; Rats; Rats, Sprague-Dawley; Staurosporine

To investigate the interrelationship between erythrocyte Na+/H+ exchange rate and free cytosolic Ca2+ concentration, the effect of the (non)selective protein kinase C inhibitors staurosporine, Ro 31-8220 and CGP 41251 (1 micromol/L) and of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA, 1 micromol/L) was studied in vitro on these variables. PKC depleted erythrocytes were obtained after 24 h PMA down-regulation of the cells and intracellular Ca2+ clamping was obtained using quin-2 AM and fluo-3 AM. PMA increased (P < .05) the erythrocyte Na+/H+ exchange activity and this rise was accompanied by an increase in the free cytosolic Ca2+ concentration. When staurosporine and Ro 31-8220 were added to erythrocytes in suspension, a decrease in free cytosolic Ca2+ concentration was also found, whereas no significant change was observed after CGP 41251 administration. The Na+/H+ exchange rate was decreased in the 24 h PMA down-regulated erythrocytes as well as in Ca2+-clamped cells. Addition of Ca2+ in a concentration range of 0 to 1 mmol/L in the presence of calcimycin resulted (P < .001) in a stimulation of Na+/H+ exchange by 74%. Calcium increased the Vmax for cellular pHi or external Na+ activation of Na+/H+ exchange, whereas it did not affect the Km for H+(i) or external Na+ activation. However, in PKC down-regulated cells, calcium did not activate the Na+/H+ exchange in erythrocytes and the calpain inhibitor E-64d did not prevent this inactivation. Our data show a concomitant increase in free cytosolic Ca2+ concentration and Na+/H+-exchange rate upon protein kinase C activation and a corresponding decrease in both variables upon PKC inhibition, indicating a Ca2+ requirement for protein kinase C activation of Na+/H+ exchange.

Topics: Calcium; Calpain; Carcinogens; Cytosol; Enzyme Inhibitors; Erythrocytes; Humans; Protein Kinase C; Sodium; Sodium-Hydrogen Exchangers; Staurosporine; Tetradecanoylphorbol Acetate