Sheppard K E
Molecular Physiology Laboratory, Baker Medical Research Institute, Prahran, Vic., Australia.
J Steroid Biochem Mol Biol. 1994 Mar;48(4):337-45. doi: 10.1016/0960-0760(94)90073-6.
The effect of increasing intracellular free calcium and activating protein kinase C on glucocorticoid receptor (GR) expression was investigated in AtT-20 cells, a mouse corticotrope tumor cell line. Treatment of AtT-20 cells with the calcium ionophore A23187 induced a rapid time- and dose-dependent decrease in [3H]dexamethasone ([3H]DEX) binding when measured in intact cells. Binding fell to 16% of control following 3 h of treatment with 10 microM A23187. In contrast, A23187 did not acutely effect steady state levels of GR mRNA, although levels fell to 76 +/- 1% of control after 8-15 h of treatment. Scatchard analysis of A23187 treated cultures demonstrated a decrease in GR binding capacity but no change in affinity for [3H]DEX. Acute inhibition of protein synthesis with cycloheximide had no effect on [3H]DEX binding, suggesting that the calcium-dependent decrease was not simply due to inhibition of GR protein synthesis. In contrast to the A23187 induced decrease in [3H]DEX binding in intact cells, when binding was measured in cytosol extract from A23187 treated cultures there was no decrease. These data suggest that the A23187 induced decrease in GR binding in whole cells is not due to a decrease in GR protein but reversible conversion of the receptor to a non-binding form. Inducing calcium influx only through L-type voltage-dependent calcium channels with BAY K8644 also decreased [3H]DEX binding at AtT-20 cells, though the effect was less than that induced by A23187. Although activation of protein kinase C with phorbol ester transiently increases intracellular free calcium in AtT-20 cells, when cells were treated for 0.5 to 22 h with phorbol 12-myristate 13-acetate, there was no acute fall in [3H]DEX binding, and only a small decrease following 16 h of treatment. These data demonstrate that sustained increases in intracellular calcium in corticotropes can induce a rapid and marked decrease in GR binding. The mechanism is post-translational and involves the reversible conversion of the receptor to a non-binding form. In addition, the cellular milieu is clearly important in conferring non-binding status on GR since once the cell is disrupted GR binding is restored.
