Life sciences · 2010
Magnesium deprivation inhibits a MEK-ERK cascade and cell proliferation in renal epithelial Madin-Darby canine kidney cells.
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Ikari Akira, Atomi Kosuke, Kinjo Keishi, Sasaki Yohei, Sugatani Junko
Аннотация
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.
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.