Magnesium deprivation inhibits a MEK–ERK cascade and cell proliferation in renal epithelial Madin-Darby canine kidney cells

Abstract

Aims Loss of magnesium (Mg 2+) inhibits cell proliferation and augments nephrotoxicant-induced renal injury, but the role of Mg 2+ has not been clarified in detail. We examined the effect of extracellular Mg 2+ deprivation on a MEK–ERK cascade and cell proliferation using a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells. Main methods MDCK cells were cultured in Mg 2+-containing or Mg 2+-free media. A HA-tagged constitutively active (CA)-MEK1 and a dominant negative (DN)-MEK1 were transfected into MDCK cells. The level of protein was examined by Western blotting. The intracellular free Mg 2+ concentration ([Mg 2+] i) was measured using a fluorescent dye, mag-fura 2. Cell proliferation was determined by WST-1 assay. Dead cells were identified by staining with annexin V-FITC and propidium iodide. Key findings In the presence of fetal calf serum (FCS), Mg 2+ deprivation decreased phosphorylated-ERK1/2 (p-ERK1/2) levels and [Mg 2+] i. Re-addition of Mg 2+ increased p-ERK1/2 levels, which were inhibited by U0126, a specific inhibitor of a MEK–ERK cascade. Glutathione-S-transferase pull-down and coimmunoprecipitation assays showed that CA-MEK1 and DN-MEK1 binds with ERK1/2 in the presence of Mg 2+. In contrast, neither CA-MEK1 nor DN-MEK1 bound to ERK1/2 in the absence of Mg 2+. These results indicate that the MEK–ERK cascade is regulated by [Mg 2+] i. Cell proliferation was increased by the treatment with FCS or the expression of CA-MEK1 in the presence of Mg 2+, but was inhibited by Mg 2+ deprivation. Mg 2+ deprivation did not increase the number of dead cells. Significance Mg 2+ is involved in the regulation of the MEK–ERK cascade and cell proliferation in MDCK cells.