Modulation of vascular smooth muscle cell growth by magnesium—role of mitogen—activated protein kinases

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Abstract
We tested the hypothesis that Mg2+ influences growth of vascular smooth muscle cells (VSMCs) by modulating cell cycle activation through mitogen-activated protein (MAP) kinase-dependent pathways. Rat VSMCs were grown in culture medium containing normal Mg2+ (1.02 mmol/L, control) and increasing concentrations of Mg2+ (2–4 mmol/L) for 1–8 days. Effects of varying extracellular Mg2+ concentration ([Mg2+]e) on intracellular free Mg2+ concentration ([Mg2+]i) were assessed using mag-fura. Growth actions of Mg2+ were evaluated by measuring cell cycle activation, DNA synthesis, and protein synthesis. Expression of cell cycle promoters, cyclin D1, cyclin E, Cdk2, and Cdk4 was assessed by immunoblotting. Phosphorylation of cell cycle inhibitors p21cip1 and p27kip1 and MAP kinases, ERK1/2, p38MAP kinase, and JNK was evaluated using phospho-specific antibodies. [Mg2+]i increased in a dose-dependent manner in response to increasing [Mg2+]e. These effects were evident within 2 days and maximal responses were obtained after 6 days. High [Mg2+]e induced cell cycle activation with a lower proportion of cells in G1 phase (75 ± 1.0%) and a higher fraction of cells in S phase (12 ± 0.7%) versus control (G1, 88.5 ± 1.4%; S, 6.8 ± 1.2%; P < 0.05). This was associated with increased protein content of cyclin D1 and Cdk4 and decreased activation of p21cip1 and p27kip1. In cells exposed to 2 mmol/L Mg2+, DNA and protein synthesis was increased ∼threefold. Phosphorylation of MEK1/2 and ERK1/2 was enhanced two to threefold in cells grown in 2 mmol/L Mg2+. These effects were rapid, occurring within 2 days. Phosphorylation of MEK3/6, p38 MAP kinase, and JNK was unaltered by increasing [Mg2]e. PD98059 (10−5 mol/L), specific MEK1/2 inhibitor, but not SB202190 (10−5 mol/L) (specific p38 MAP kinase inhibitor), attenuated Mg2+-induced growth actions. These data demonstrate the novel findings that cell cycle activation and growth regulation by Mg2+ occurs via ERK1/2-dependent, p38 MAP kinase-independent pathways. J. Cell. Physiol. 197: 326–335, 2003

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