EGF stimulates Mg²⁺ influx in mammary epithelial cells

Highlights: • EGF stimulation potentiates Mg²⁺ influx into epithelial cells. • EGF-induced Mg²⁺ influx does not depend on the concomitantly induced Ca²⁺ signal. • EGF-induced Ca²⁺ signal is dependent on the presence of extracellular Mg²⁺. • New players in EGF-mediated signaling might be exploited as therapeutic targets. - Abstract: Magnesium is well established as a fundamental factor that regulates cell proliferation. However, the molecular mechanisms linking mitogenic signals, extracellular magnesium availability and intracellular effectors are still largely unknown. In the present study we sought to determine whether EGF regulates magnesium homeostasis in normal HC11 mammary epithelial cells. To this end, we measured Mg²⁺ and Ca²⁺ fluxes by confocal imaging in live cells loaded with specific fluorescent ion indicators (Mag-Fluo-4 and Fluo-4, respectively). EGF stimulation induces a rapid and sustained increase in intracellular Mg²⁺, concomitantly with a rise in intracellular calcium. The increase in intracellular Mg²⁺ derives from an influx from the extracellular compartment, and does not depend on Ca²⁺. On the contrary, the increase in intracellular Ca²⁺ derives from intracellular stores, and is impaired in the absence of extracellular magnesium. Inhibition of the EGF receptor tyrosine kinase by Tyrphostin AG1478 markedly inhibits EGF-induced Mg²⁺ and Ca²⁺ signals. These findings demonstrate that not only does Mg²⁺ influx represent an important step in the physiological response of epithelial cells to EGF, but unexpectedly the EGF-induced Mg²⁺ influx is essential for the Ca²⁺ signal to occur