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.

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Cells depend on specific stimuli, such as trophic factors, for survival and in the absence of such stimuli, undergo apoptosis. How do cells initiate apoptosis in response to the withdrawal of trophic factors or other dependent stimuli? Recent studies of apoptosis induction by neurotrophin withdrawal argue for a novel form of pro-apoptotic signal transduction ±`negative signal transduction' ± in which the absence of ligand-receptor interaction induces cell death. We have found that the prototype for this form of signaling ± the common neurotrophin receptor, p75 NTR ± creates a state of cellular dependence (or addiction) on neurotrophins, and that this effect requires an`addiction/dependence domain' (ADD) in the intracytoplasmic region of p75 NTR . We have recently found other receptors that include dependence domains, arguing that dependence receptors, and their associated dependence domains, may be involved in a rather general mechanism to create cellular states of dependence on trophic factors, cytokines, adhesion, electrical activity and other dependent stimuli.

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.

The effects of hydrogen peroxide, D-a-tocopherol and of D-P-tocopherol on proliferation, protein kinase C and activator protein-1 (AP-1) activation have been studied in vascular smooth muscle cells. Cell proliferation, when activated by foetal calf serum, was inhibited by D-a-tocopherol. Protein kinase C activity was stimulated by hydrogen peroxide in a manner similar to phorbol myristate acetate; in the latter case, but not in the former, D-a-tocopherol inhibited the reaction. Hydrogen peroxide prevented phorbol-myristate-acetate-stimulated AP-1 binding to DNA but stimulated it if protein kinase C was down-regulated or inhibited. D-a-Tocopherol promoted AP-1 activation in quiescent cells but prevented its activation by phorbol myristate acetate.None of the described effects of D-a-tocopherol were shared by D-P-tocopherol, suggesting a non-antioxidant mechanism as the basis of its action. The data show that hydrogen peroxide and Da-tocopherol affect more than one element in the cell signal-transduction cascade.

Hydrogen peroxide and fetal bovine serum stimulate DNA synthesis in growth-arrested smooth muscle cells with remarkably similar kinetics and cell density dependence. However, while stimulation with fetal bovine serum results in cell proliferation, that by H2O2 is followed by cell death. Depletion of conventional and novel protein kinase C isoforms, resulting from a long treatment with phorbol-12-myristate-13-acetate, further increases H2O2-induced DNA synthesis. On the other hand, the specific protein kinase C inhibitor calphostin C abolished the increased DNA synthesis promoted by fetal bovine serum or H2O2. H2O2 increases protein kinase C activity in smooth muscle cells. This effect is markedly reduced, but not abolished, by down-regulation of the alpha, delta and epsilon protein kinase C isoforms. Thus, the zeta isoform of protein kinase C, which is not down-regulated, may be responsible for the residual H2O2 stimulation of protein kinase C. In conclusion, the results obtained show that H2O2 stimulates protein kinase C activity and DNA synthesis in growth-arrested smooth muscle cells: these events are not followed by cell proliferation but rather by cell death. This H2O2 stimulated DNA synthesis appears to be negatively controlled by alpha, delta and epsilon isoforms and positively controlled by the zeta isoform of protein kinase C.

During inflammatory reactions in the central nervous system (CNS), resident macrophages, the microglia, are exposed to Th1 cell‐derived cytokines and pro‐apoptotic Fas ligand (FasL). Despite the presence of TNF‐α and IFN‐γ, both being capable of sensitzing microglia to FasL, apoptosis of microglia is not a hallmark of inflammatory diseases of the CNS. In the present study, TGF‐β is found to counteract the effect of TNF‐α and IFN‐γ to sensitize microglia to FasL‐mediated apoptosis. Resistance to Fas‐mediated apoptosis by TGF‐β does not correlate with a down‐regulation of Fas expression. As a key inhibitor of Fas‐mediated apoptosis, we found expression of the cellular FLICE‐inhibitory protein (c‐FLIP) to be induced by TGF‐β in resting as well as in activated microglia. Induction of FLIP was found to depend on a mitogen‐activated protein kinase kinase (MKK)‐dependent pathway as shown by the use of the specific MKK‐inhibitor PD98059. The presence of FLIP strongly interfered with FasL‐induced activation of caspase‐8 and caspase‐3 preventing subsequent cell death. The presented data provide the first evidence for a TGF‐β‐mediated FLIP in macrophage‐like cells and suggest a mode of action for the anti‐apoptotic role of TGF‐β in the CNS.