7β-Hydroxycholesterol induces Ca2+ oscillations, MAP kinase activation and apoptosis in human aortic smooth muscle cells

In the present study, we characterize the early cytotoxic effects of 7β-hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7β-hydroxycholesterol induced Ca2+ oscillations with a frequency of ≈0.3–0.4 min−1. A few hours later, thapsigargin-sensitive Ca2+ pools were depleted, indicating that 7β-hydroxycholesterol perturbs intracellular Ca2+ homeostasis. The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (but not JNK) were activated within 5 min after addition of 7β-hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol were more potent in inducing apoptosis than 7β-hydroxycholesterol and cholesterol-5α,6α-epoxide, as determined by TUNEL staining. Addition of TNFα (10 ng/ml) and IFNγ (20 ng/ml) enhanced the cytotoxicity of oxysterols and potentiated apoptosis. The cytokines alone were not toxic to smooth muscle cells at these concentrations. 25-Hydroxycholesterol and 7β-hydroxycholesterol but not cholesterol inhibited protein synthesis at 4–8 h as determined by [35S]methionine incorporation assay. Morphologically, oxysterol-induced cell death was characterized by disorganization of the ER and Golgi membranes. The Ca2+ and ERK signals preceded the ultrastructural changes induced by 7β-hydroxycholesterol.