Mechanisms of diethylstilbestrol-induced calcium movement in MG63 human osteosarcoma cells

The effect of the estrogen diethylstilbestrol (DES) on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in MG63 human osteoblasts was explored by using fura-2 as a Ca²⁺ indicator. DES at concentrations between 5-20 μM induced an immediate increase in [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ of 10 μM. Removing extracellular Ca²⁺ reduced the Ca²⁺ signal by 70%. Pretreatment with 50 μM La³⁺ or 10 μM of nifedipine, verapamil and diltiazem did not change 20 μM DES-induced [Ca²⁺]_i increases. Addition of 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with 20 μM DES in Ca²⁺-free medium. Pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) to deplete the endoplasmic reticulum Ca²⁺ store partly inhibited 20 μM DES-induced Ca²⁺ release, but addition of carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and thapsigargin together abolished DES-induced Ca²⁺ release. Conversely, pretreatment with 20 μM DES abrogated CCCP- and thapsigargin-induced Ca²⁺ release. Inhibition of phospholipase C activity with 2 μM U73122 did not alter 20 μM DES-induced Ca2+ release. Another estrogen 17β-estradiol also increased [Ca²⁺]i in a concentration-dependent manner with an EC50 of 7 μM. Together, the data indicate that in human osteoblasts, DES increased [Ca²⁺]_i via causing Ca²⁺ release from both mitochondria and the endoplasmic reticulum in a phospholipase C-independent manner, and by causing Ca²⁺ influx.