Daniel Boscoboinik

α-Tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity

Abstract α-Tocopherol (vitamin E) protects against free radical damage, which has been implicated in aging, cancer initiation, and atherosclerosis. We have found that physiological concentrations of α-tocopherol specifically inhibited aorta smooth muscle cell (VSMC, line A7r5) proliferation and protein kinase C (PKC) activity. Other water and lipid soluble antioxidants were inactive, α-Tocopherol inhibition of PKC and of VSMC proliferation may represent a physiological mechanism, relevant to the onset of diseased states such as atherosclerosis.

Inhibition of smooth muscle cell proliferation and protein kinase C activity by tocopherols and tocotrienols

alpha-Tocopherol, the most active form of vitamin E, causes a dose-dependent inhibition of serum-induced proliferation of smooth muscle cells (A7r5) in culture. Some tocopherol-related compounds exhibiting various degrees of antioxidant potency have also been tested on cellular proliferation. No direct correlation between the antioxidant activity of these compounds and their effect on smooth muscle cell growth could be observed. While most of the derivatives employed were not effective in inhibiting protein kinase C, in the case of alpha-tocopherol the antiproliferative effect was found to be parallel to the inhibition of protein kinase C activity, as measured in streptolysin-O permeabilized cells.

Vitamin E: a sensor and an information transducer of the cell oxidation state

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.

Suramin, an anti-cancer drug, inhibits protein kinase C and induces differentiation in neuroblastoma cell clone NB2A.

Protein kinase C purified from rat brain was found to be inhibited by suramin, a substance used originally in the therapy of antitrypanosomic infections and more recently proposed as antineoplastic agent. The inhibition of suramin was competitive with one of the substrates of the enzyme, ATP with a K i of 10 μM. At concentrations adequate to inhibit the isolated enzyme, suramin was shown to slow the rate of proliferation of neuroblastoma NB2A cells in vitro and to induce their differentiation as evidenced by typical morphological changes.

d-α-Tocopherol inhibits low density lipoprotein induced proliferation and protein kinase C activity in vascular smooth muscle cells

Native and malondialdehyde modified low density lipoproteins have been shown to stimulate smooth muscle cell proliferation (A7r5) in vitro. The stimulation is associated with an increase of protein kinase C activity. d‐α‐Tocopherol, at physiological concentrations, has been found to inhibit both protein kinase C activity and cell proliferation.

Hydrogen peroxide-and fetal bovine serum-induced DNA synthesis in vascular smooth muscle cells: positive and negative regulation by protein kinase C isoforms

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.

d-α-tocopherol control of cell proliferation

Uncontrolled cell growth is at the basis of neoplastic proliferation and arteriosclerotic lesions. In vitro proliferation of vascular smooth muscle cells, Balb c/3T3 fibroblasts, retinal neuroepithelial cells and neuroblastoma cells is inhibited by d-α-tocopherol. On the contrary Chinese hamster ovary cells, osteosarcoma cells and macrophages are not sensitive. PDGF-BB activated proliferation is highly d-α-tocopherol sensitive while lysophosphatidic acid induced growth is poorly inhibited. d-β-Tocopherol, an analogue of d-α-tocopherol, with similar antioxidant properties, does not inhibit proliferation. Protein kinase C activity is inhibited by d-α-tocopherol but not by d-β-tocopherol, suggesting a central role of this enzyme in the control of cell proliferation by d-α-tocopherol. Activation of the transcription activation complex AP-1 (but not NFϰB) is prevented by d-α-tocopherol and not by d-β-tocopherol.

Vitamin E mediated response of smooth muscle cell to oxidant stress

Oxidant stress is associated with diminution of antioxidant molecules, such as alpha-tocopherol. Alpha-tocopherol specifically decreases, in a concentration dependent way, the proliferation of vascular smooth muscle cells. At the same concentrations (10-50 microM) it induces inhibition of protein kinase C (PKC) activity. The latter event is not due to a decrease in PKC level or to alpha-tocopherol binding to PKC, but it results from increase of protein phosphatase 2A1 activity. In vitro data, as well as at a cellular level, demonstrates that protein phosphatase 2A1 is activated, in its trimeric structure--but not as a dimer by alpha-tocopherol. This activation is followed by PKC-alpha dephosphorylation. The activation of protein phosphatase 2A1 and deactivation of PKC-alpha affect the AP1 transcription factor, resulting in a change in the composition and the binding of this factor to DNA. By transfecting smooth muscle cell with a construct containing three TRE (TPA responsive elements), the promoter thymidine kinase and the reporter gene chloramphenicol-acetyl-transferase a modulation of gene expression by alpha-tocopherol is observed. Beta-tocopherol does not cause any of the responses observed with alpha-tocopherol and R,R,R-alpha-tocopherol is twice as potent as all-rac-alpha-tocopherol. When added together, beta-tocopherol prevents the effects of alpha-tocopherol indicating that the mechanism involved is not related to the radical-scavenging properties of these two molecules, which are essentially equal. By differential display analysis it has been found that several genes of smooth muscle cells are differentially transcribed in the presence of alpha-tocopherol but not beta-tocopherol. In particular, the gene of alpha-tropomyosin shows a transient enhancement of transcription as a function of the cell cycle time. Alpha-tropomyosin translation is also increased by alpha-tocopherol and not by beta-tocopherol. Because no changes of mRNA stability can be observed in the presence of alpha-tocopherol, the data supports the conclusion of a transcriptional control exerted by alpha-tocopherol on alpha-tropomyosin. Generally, the data strongly suggests the existence of a ligand/receptor type of mechanism at the basis of alpha-tocopherol action. It is concluded that an oxidative stress-induced diminution of alpha-tocopherol in smooth muscle cell activates a reaction cascade leading to changes in gene expression and increase in cell proliferation by a non-antioxidant mechanism.

Inhibition of protein kinase C activity and vascular smooth muscle cell growth by d-α-tocopherol

Abstract The inhibition by d-α-tocopherol of protein kinase C activity has been studied in synchronised A7r5 rat smooth muscle cells during the cell cycle. Cell protein kinase C activity has been found to oscillate, with a minimum in the G 0 phase, a maximum in the late G 1 phase and a new minimum in the S phase. An inhibition of protein kinase C activity by d-α-tocopherol appears to be at the basis of cell growth inhibition. Nevertheless, the amount of the different protein kinase C isoenzymes present in smooth muscle cells, measured by their specific antibodies, does not change during the cell cycle in both untreated and d-α-tocopherol-treated cells. The possible mechanisms of protein kinase C modulation during the cell cycle and of its inhibition by d-α-tocopherol are discussed.

The phosphorylation state of MAP-kinases modulates the cytotoxic response of smooth muscle cells to hydrogen peroxide

Micromolar concentrations of hydrogen peroxide induced the phosphorylation of mitogen‐activated protein (MAP) kinases and a lethal response in growth‐arrested smooth muscle cells (A7r5). The H2O2‐induced phosphorylation of MAP‐kinases was markedly lower in the presence of protein tyrosine kinase (PTK) inhibitors or in protein kinase C (PKC) down‐regulated cells. Similarly, the toxicity of H2O2 was diminished by concomitant addition of either PKC or PTK inhibitors and was also lower in PKC down‐regulated cells. These results are consistent with the possibility that phosphorylation of MAP‐kinases is a critical event in the toxic response of cultured smooth muscle cells to H2O2.