ABSTRACTd-a-Tocopherol, but not d-jJ-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-c-Tocopherol inhibits protein kinase C (PKC) activity, whereas d-13-tocopherol is ineffective. Furthermore d-18-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-ctocopherol. The negative regulation by d-cv-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-a-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-c-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation.Vascular smooth muscle cell (vascular SMC) proliferation represents a significant event in a number of diseases such as arteriosclerosis and hypertension (1-4). Smooth muscle proliferation is controlled by growth factors released from blood cells (1, 2, 5), by inhibitors or stimulants produced by the vessel wall cells (6, 7), by tocopherols, and by active oxygen species (8, 9). Evidence indicates that experimental atherosclerosis and foam cell formation can be effectively retarded by antioxidants (10-12). In addition, supplementation of human subjects with antioxidants has been shown to increase the resistance of their low density lipoproteins to oxidation and to protect against arteriosclerosis (13-16). As antioxidants, tocopherols may stimulate in some cases cell proliferation by removing inhibitory lipid peroxides (17-22). However, d-atocopherol has also a direct effect as cell-growth inhibitor, and this effect is not obviously mediated by its reduction-oxidation properties (23)(24)(25).PKC participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters, and growth factors (26). Activation of PKC by phorbol esters may be responsible for their growth-promoting activity. d-aTocopherol has been shown to inhibit PKC activity in a number of cell lines and, in particular, in SMC. The mechanism of this inhibition has not yet been clarified (23)(24)(25).In the present study PKC inhibition has been found to be the basis of the inhibition of cell proliferation by d-a-tocopherol.Moreover PKC inhibition has been found to be cell cycle dependent, a result inconsistent with a direct interaction between PKC and d-a-tocopherol. Finally, the inhibitory specificity of d-a-tocopherol versus d-3-tocopherol and their mutual competition suggest a nonantioxidant mechanism to be at the basis of its action.
MATERIALS AND METHODSGrowth media and serum were from GIBCO; A7r5 rat aortic SMC were from the American Type Culture Collection; phorbol 12-myristate 13-acetate (PMA) and streptolysin-O (25,000 units) were from Sigma; calphostin C, calyculin A, and okadaic acid were from LC Services (
