Modulation of phosphorylation of tocopherol and phosphatidylinositol by hTAP1/SEC14L2-mediated lipid exchange.

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-32P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, ßT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains.

Modulation of gene expression by α-tocopherol and α-tocopheryl phosphate in THP-1 monocytes.

The natural vitamin E analog α-tocopheryl phosphate (αTP) modulates atherosclerotic and inflammatory events more efficiently than the unphosphorylated α-tocopherol (αT). To investigate the molecular mechanisms involved, we have measured plasma levels of αTP and compared the cellular effects of αT and αTP in THP-1 monocytes. THP-1 cell proliferation is slightly increased by αT, whereas it is inhibited by αTP. CD36 surface expression is inhibited by αTP within hours without requiring transport of αTP into cells, suggesting that αTP may bind to CD36 and/or trigger its internalization. As assessed by gene expression microarrays, more genes are regulated by αTP than by αT. Among a set of confirmed genes, the expression of vascular endothelial growth factor is induced by αTP as a result of activating protein kinase B (PKB/Akt) and is associated with increased levels of reactive oxygen species (ROS). Increased Akt(Ser473) phosphorylation and induction of ROS by αTP occur in a wortmannin-sensitive manner, indicating the involvement of phosphatidylinositol kinases. The induction of Akt(Ser473) phosphorylation and ROS production by αTP can be attenuated by αT. It is concluded that αTP and αT influence cell proliferation, ROS production, and Akt(Ser473) phosphorylation in an antagonistic manner, most probably by modulating phosphatidylinositol kinases.

Effect of tocopheryl phosphate on key biomarkers of inflammation: Implication in the reduction of atherosclerosis progression in a hypercholesterolaemic rabbit model.

1. Many studies have evaluated the effectiveness of alpha-tocopherol (vitamin E) in the development and progression of cardiovascular diseases, with conflicting results reported on the protective effect of this anti-oxidant. 2. The present study examined the effectiveness of a novel tocopheryl phosphate mixture (TPm) compared with that of alpha-tocopherol (TA) on key pro-inflammatory markers involved in atherogenesis, including interleukin (IL)-1beta, IL-6, IL-8, plasminogen activator inhibitor-1, tumour necrosis factor-alpha and C-reactive protein (CRP), as well as vascular function and lesion development in rabbits fed a 2% cholesterol diet. 3. Treatment with TPm, incorporated into the rabbit food at four doses ranging from 60 to 360 mg/kg chow, resulted in a significant reduction in plasma levels of all pro-inflammatory cytokines and biomarkers that appeared to be somewhat dose dependent. Conversely, treatment with TA, at a dose equivalent to the highest dose of TPm used, only decreased plasma levels of CRP, IL-6 and IL-8. Both TPm and TA treatment significantly improved vascular function to a similar extent, although TPm was more effective in reducing lesion development. 4. The reduction in these key pro-inflammatory markers appears to follow the improvement in the atherogenic state of the animals, indicating that the anti-inflammatory properties of TPm may be potentially beneficial in inflammatory disease states.

Vitamin E and cancer.

Protection by vitamin E against free radical-induced DNA mutations appears not to be an effective occurrence. On the other hand, in vitro evidence that different tocopherols slow down cell proliferation is an accepted observation. However, such an event may not be sufficient to result in beneficial clinical outcomes. Tocopheryl phosphate, a more active, natural derivative of tocopherol, endowed with prevention and therapeutic potential, represents a possible key to the understanding of the present conflict between laboratory and clinical results.

Modulation of cell proliferation and gene expression by alpha-tocopheryl phosphates: relevance to atherosclerosis and inflammation.

The effect of a mixture of alpha-tocopheryl phosphate and di-alpha-tocopheryl phosphate (TPm) was studied in vitro on two cell lines, RASMC (from rat aortic smooth muscle) and human THP-1 monocytic leukaemia cells. Inhibition of cell proliferation by TPm was shown in both lines and occurred with TPm at concentrations lower than those at which alpha-tocopherol was equally inhibitory. TPm led in non-stimulated THP-1 cells to inhibition of CD36 mRNA and protein expression, to inhibition of oxidized low density lipoprotein surface binding and oxLDL uptake. In non-stimulated THP-1 cells, alpha-tocopherol had only very weak effects on these events. Contrary to alpha-tocopherol, TPm was cytotoxic to THP-1 cells at high concentrations. Thus, TPm is able to inhibit the major aggravating elements involved in the progression of atherosclerosis. The higher potency of TPm may be due to a better uptake of the molecule and to its intracellular hydrolysis, providing more alpha-tocopherol to sensitive sites. Alternatively, a direct effect of the phosphate ester on specific cell targets may be considered.

Modulation of cell proliferation and gene expression by alpha-tocopheryl phosphates: relevance to atherosclerosis and inflammation.

The effect of a mixture of alpha-tocopheryl phosphate plus di-alpha-tocopheryl phosphate (TPm) was studied in vitro on two cell lines, RASMC (from rat aortic smooth muscle) and human THP-1 monocytic leukemia cells. Inhibition of cell proliferation by TPm was shown in both lines and occurred with TPm at concentrations lower than those at which alpha-tocopherol was equally inhibitory. TPm led in nonstimulated THP-1 cells to inhibition of CD36 mRNA and protein expression, to inhibition of oxidized low-density lipoprotein surface binding and oxLDL uptake. In nonstimulated THP-1 cells, alpha-tocopherol had only very weak effects on these events.