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 (

The mechanism of protein kinase C (PKC) regulation by alpha-tocopherol has been investigated in smooth-muscle cells. Treatment of rat aortic A7r5 smooth-muscle cells with alpha-tocopherol resulted in a time- and dose-dependent inhibition of PKC. The inhibition was not related to a direct interaction of alpha-tocopherol with the enzyme nor with a diminution of its expression. Western analysis demonstrated the presence of PKCalpha, beta, delta, epsilon, zeta and micro isoforms in these cells. Autophosphorylation and kinase activities of the different isoforms have shown that only PKCalpha was inhibited by alpha-tocopherol. The inhibitory effects were not mimicked by beta-tocopherol, an analogue of alpha-tocopherol with similar antioxidant properties. The inhibition of PKCalpha by alpha-tocopherol has been found to be associated with its dephosphorylation. Moreover the finding of an activation of protein phosphatase type 2A in vitro by alpha-tocopherol suggests that this enzyme might be responsible for the observed dephosphorylation and subsequent deactivation of PKCalpha. It is therefore proposed that PKCalpha inhibition by alpha-tocopherol is linked to the activation of a protein phosphatase, which in turn dephosphorylates PKCalpha and inhibits its activity.

Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C 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. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn-l,2-diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor-promoting activity. The cellular localization of the different calcium-activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.The eukaryotic cell is a highly regulated entity, responding to its immediate intracellular environment as well as to external stimuli. Most regulations are mediated, either directly or indirectly, by conformational changes in proteins, and the equilibrium between active and inactive conformational states can be altered by both allosteric and covalent mechanisms. A most common covalent means of regulating protein activity is protein phosphorylation, which is particularly prominent for the role that it serves in signal transduction and is important in many other cellular responses (Edelman et al., 1987). Signals impinging on cells have their effects amplified and distributed by a network of protein phosphorylation and dephosphorylation reactions.

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Rat and human vascular smooth muscle cell proliferation is specifically sensitive to alpha-tocopherol, but not beta-tocopherol. The former, but not the latter, is capable of limiting proliferation and inhibiting protein kinase C activity in a dose-dependent manner. The phenomenon occurs at concentrations in the range 10-50 microM. beta-tocopherol addition together with alpha-tocopherol, prevents both cell growth and protein kinase C inhibition. alpha-tocopherol increases de novo synthesis of protein kinase C molecules. The enzyme specific activity, however, is diminished, due to a decreased phosphorylation of protein kinase C, occurring in the presence of alpha-tocopherol. Experiments with protein kinase C isoform-specific inhibitors and precipitating antibodies show that the only isoform affected by alpha-tocopherol is protein kinase C-alpha. The effect of alpha-tocopherol is prevented by okadaic acid indicating a phosphatase of the PP2A type as responsible for protein kinase C-alpha dephosphorylation produced in the presence of alpha-tocopherol. At a gene level alpha-tocopherol but not beta-tocopherol induces a transient activation of alpha-tropomyosin gene transcription and protein expression. It is proposed that, by inhibiting protein kinase C activity via an activation of a phosphatase PP2A, alpha-tocopherol controls smooth muscle cell proliferation through changes in gene expression.

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We studied the effects of RRR-alpha-tocopherol and RRR-beta-tocopherol in smooth muscle cells from rat (line A7r5) and human aortas. RRR-alpha-Tocopherol, but not RRR-beta-tocopherol, inhibited smooth muscle cell proliferation in a dose-dependent manner at concentrations in the range from 10 to 50 mumol/L. RRR-beta-Tocopherol added simultaneously with RRR-alpha-tocopherol prevented growth inhibition. The earliest event brought about by RRR-alpha-tocopherol in the signal transduction cascade controlling receptor-mediated cell growth was the activation of the transcription factor AP-1. RRR-beta-tocopherol alone was without effect but in combination with RRR-alpha-tocopherol prevented the AP-1 activating effect of the latter. Protein kinase C was inhibited by RRR-alpha-tocopherol and not by RRR-beta-tocopherol, which also in this case prevented the effect of RRR-alpha-tocopherol. Calyculin A, a protein phosphatase inhibitor, prevented the effect of RRR-alpha-tocopherol on protein kinase C. The data can be rationalized by a model in which a tocopherol-binding protein discriminates between RRR-alpha-tocopherol and RRR-beta-tocopherol and initiates a cascade of events at the level of cell signal transduction that leads to the inhibition of cell proliferation.