Modulation of TRPM6 and Na(+)/Mg(2+) exchange in mammary epithelial cells in response to variations of magnesium availability.

Mammary epithelial cells (HC11) chronically adapted to grow in a low-magnesium (0.05 mM vs. 0.5 mM) or in a high-magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non-physiological magnesium availability. Magnesium influx was higher in low-magnesium cells compared to control or high-magnesium cells, whereas magnesium efflux was higher in high-magnesium cells compared to control and low-magnesium cells. Magnesium efflux was partially inhibited by imipramine, inhibitor of the Na(+)/Mg(2+) exchange. Using a monoclonal antibody detecting a approximately 70 kDa protein associated with Na(+)/Mg(2+) exchange activity, we found that the expression levels of this protein were proportional to magnesium efflux capacity, that is, high-magnesium cells > control cells > low-magnesium cells. As for magnesium influx, this was abolished by Co(III)hexaammine, inhibitor of magnesium channels. Surprisingly, we found that cells grown in low magnesium upregulated mRNA for the magnesium channel TRPM6, but not for other channels like TRPM7 or MagT1. TRPM6 mRNA was also rapidly upregulated or downregulated in HC11 cells deprived of magnesium or in low-magnesium cells re-added with magnesium, respectively. TRPM6 protein levels, as assessed by Western blot and immunofluorescence, underwent similar changes under comparable conditions. We propose that mammary epithelial cells adapt to decreased magnesium availability by upregulating magnesium influx via TRPM6, and counteract increased magnesium availability by increasing magnesium efflux primarily via Na(+)/Mg(2+) exchange. These results show, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells, similar to what is known for intestine and kidney.

Magnesium deficiency affects mammary epithelial cell proliferation: involvement of oxidative stress.

Low Mg availability reversibly inhibited the growth of mammary epithelial HC11 cells by increasing the number of cells in the G0/G1 phase of the cell cycle. Because low Mg has been reported to promote oxidative reactions, we considered that low Mg-dependent growth arrest was mediated by oxidative stress. Surprisingly, both dichlorofluorescein-detectable reactive oxygen species and hydrogen peroxide-induced oxidative DNA damage were found to be lower in cells cultured in low Mg than in cells grown under control or high-Mg conditions. Gene expression profiling of low- and high-Mg cells showed the modulation of several genes, some regulating cell proliferation. In addition, low Mg cells also displayed overexpression of glutathione S-transferase (GST), leading to increased enzymatic activity. Of note, GST has been shown to modulate cell growth; therefore, we suggest that in low-Mg cells, GST upregulation might have a dual role in protecting against oxidative stress and in modulating cell proliferation.

Magnesium deficiency and endothelial dysfunction: is oxidative stress involved?

Low magnesium (Mg) has been associated with oxidative stress, an important player in aging, atherosclerosis and other vascular diseases. In vivo, low Mg and immune system activation seem to cooperate to promote endothelial dysfunction. We therefore evaluated whether exposure of human endothelial cells to low Mg in vitro determines oxidative stress features. We therefore measured intracellular reactive oxygen species (ROS) by dichlorofluorescein (DCF) fluorescence after Mg deprivation with or without treatment with H2O2 While we did not observe any alteration of DCF-detectable intracellular ROS under basal conditions, we show that, early after exposure to low Mg (2 h), endothelial cells are more sensitive to the oxidant action of H2O2 than the controls cultured in physiologic concentrations of Mg. This increase of ROS in Mg deprived cells is transient and followed by a stable reduction of DCF-fluorescence below the levels measured in the controls. We also evaluated oxidative DNA damage and observed higher 8-hydroxy-deoxyguanine levels early (2 h) after Mg deprivation in respect to the controls, both in basal conditions and after treatment with H2O2 Mg deficiency in vivo associates with the onset of an inflammatory response leading to increased circulating levels of cytokines, which trigger an oxidative response in endothelial cells. We here show that exposure to IL-1 and IL-6 significantly increased the levels of DCF-detectable ROS in cells cultured in physiologic concentrations of Mg, but not in Mg-deprived cells. We conclude that low Mg transiently leads to pro-oxidant effects. We suggest that different molecules, including pro-inflammatory cytokines, might be involved in promoting endothelial dysfunction.

Insights into the mechanisms involved in magnesium-dependent inhibition of primary tumor growth.

We have previously shown that a low Magnesium (Mg)-containing diet reversibly inhibits the growth of primary tumors that develop after the injection of Lewis lung carcinoma (LLC) cells in mice. Here we investigate some of the mechanisms responsible for the Mg-dependent regulation of tumor development by studying cell cycle regulation, tumor angiogenesis, and gene expression under Mg deficiency. The inhibition of LLC tumor growth in Mg-deficient mice is due to a direct effect of low Mg on LLC cell proliferation and to an impairment of the angiogenic switch. We also observed an increase of nitric oxide synthesis and oxidative DNA damage. Complementary DNA arrays reveal that Mg deficiency modulates tumor expression of genes involved in the control of cell cycle, stress response, proteolysis, and adhesion. Our results suggest that Mg has multiple and complex roles in tumor development.