ABSTRACT
The effect of alpha-tocopherol (vitamin E) on gene expression in rat vascular smooth muscle cells was studied by the differential display technique. One gene out of about 1000 genes analyzed, identified as alpha-tropomyosin, showed an increased transcription level caused by alpha-tocopherol treatment. Northern and Western blot analysis revealed a time-dependent transient up-regulation of the amount of mRNA (peak between 2 and 3 h) and protein (peak at 5 h) in alpha-tocopherol-treated cells. No effect was observed in cells treated with beta-tocopherol.
Subject(s)
Muscle, Smooth, Vascular/drug effects , Tropomyosin/biosynthesis , Tropomyosin/genetics , Vitamin E/pharmacology , Animals , Blood , Culture Media , Gene Expression Regulation , RNA, Messenger/analysis , RatsABSTRACT
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.
Subject(s)
Cell Division/drug effects , Muscle, Smooth/cytology , Vitamin E/pharmacology , Cell Line , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Humans , Immunosorbent Techniques , Isoenzymes/antagonists & inhibitors , Male , Muscle, Smooth, Vascular/cytology , Phosphorylation , Prostatic Neoplasms , Protein Kinase C/antagonists & inhibitors , Tropomyosin/genetics , Tumor Cells, CulturedABSTRACT
Cytochrome c oxidase (COX) has a complex modular structure in eukaryotes. Depending on growth conditions, interchangeable isoforms of selected subunits are synthesized and combined to the evolutionarily conserved catalytic core of the enzyme. In Dictyostelium this structural make-up is regulated by oxygen and involves two forms of the smallest subunit, termed VIIe and VIIs. Here we show that, in spite of a considerable sequence divergency, they are encoded by adjacent genes, linked 'tail to head' by only 800 bp. Deletion analyses reveal the presence of a short intergenic segment acting as an oxygen transcriptional switch. This structural organization and the different stability of the two subunit isoforms offer a molecular explanation for the extraordinary sensitivity to oxygen of the switching mechanism.
