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 7 beta -hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7 beta -hydroxycholesterol induced Ca2+ oscillations with a frequency of approximate to 0.3-0.4 min(-1). A few hours later, thapsigargin-sensitive Ca2+ pools were depleted, indicating that 7 beta -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 7 beta -hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol were more potent in inducing apoptosis than 7 beta -hydroxycholesterol and cholesterol-5 alpha ,6 alpha -epoxide, as determined by TUNEL staining. Addition of TNF alpha. (10 ng/ml) and IFN gamma (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 7 beta -hydroxycholesterol but not cholesterol inhibited protein synthesis at 4-8 h as determined by [S-35]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 7 beta -hydroxycholesterol, (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.