ABSTRACT
Vascular endothelial growth factor (VEGF) is a potent vascular endothelial cell-specific mitogen that modulates endothelial cell function. In the present study, we show that VEGF induces manganese-superoxide dismutase (MnSOD) mRNA and protein in human coronary artery endothelial cells (HCAEC) and pulmonary artery endothelial cells. VEGF-mediated induction of MnSOD mRNA was inhibited by pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI), and 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not with the nitric oxide synthase inhibitor L-NAME (N-monomethyl-L-arginine) or the xanthine oxidase inhibitor allopurinol. VEGF stimulation of MnSOD was also inhibited by adenoviral-mediated overexpression of catalase Cu, Zn-SOD and a dominant-negative form of the small GTPase component of NADPH oxidase Rac1 (Rac1N17). Treatment of HCAEC with VEGF resulted in a transient increase in ROS production at 20 min, as measured by 2,7-dichlorodihydrofluorescein oxidation. This effect was abrogated by expression of Rac1N17. Taken together, these findings suggest that VEGF induces MnSOD by an NADPH oxidase-dependent mechanism and that VEGF signaling in the endothelium is coupled to the redox state of the cell.
Subject(s)
Endothelial Growth Factors/pharmacology , Endothelium, Vascular/drug effects , Lymphokines/pharmacology , NADPH Oxidases/metabolism , Superoxide Dismutase/drug effects , rac1 GTP-Binding Protein/physiology , Adenoviridae/genetics , Blotting, Northern , Blotting, Western , Catalase/genetics , Catalase/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Free Radical Scavengers/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Genetic Vectors/genetics , Humans , NADPH Oxidases/antagonists & inhibitors , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , rac1 GTP-Binding Protein/geneticsABSTRACT
NADPH oxidase has been shown to play an important role in cardiovascular biology. The goal of the present study was to determine whether NADPH oxidase activity is important for endothelial cell growth and migration. In proliferation assays, growth factor- or serum-induced DNA synthesis in three different types of human endothelial cells was abrogated by inhibitors of NADPH oxidase, but not by inhibitors of xanthine oxidase or nitric oxide synthase. Moreover, vascular endothelial growth factor-induced migration of human endothelial cells was suppressed in the presence of NADPH oxidase inhibitors. These results support a potential role for NADPH oxidase in mediating angiogenesis.
Subject(s)
Endothelium, Vascular/metabolism , NADPH Oxidases/metabolism , Acetophenones/pharmacology , Allopurinol/pharmacology , Cell Division/drug effects , Cell Division/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Dose-Response Relationship, Drug , Endothelial Growth Factors/pharmacology , Endothelium, Vascular/cytology , Enzyme Inhibitors/pharmacology , Fluorescent Dyes , Humans , Lymphokines/pharmacology , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Sulfones/pharmacology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Xanthine Oxidase/antagonists & inhibitorsABSTRACT
Ribonucleotide reductase synthesizes dNDPs, a specific and limiting step in DNA synthesis, and can participate in neoplastic transformation when overexpressed. The small subunit (ribonucleotide reductase 2 (RNR2)) was cloned as a major product in a subtraction library from eukaryotic initiation factor 4E (eIF4E)-transformed cells (Chinese hamster ovary-4E (CHO-4E)). CHO-4E cells have 20-40-fold elevated RNR2 protein, reflecting an increased distribution of RNR2 mRNA to the heavy polysomes. CHO-4E cells display an altered cell cycle with shortened S phase, similar to cells selected for RNR2 overexpression with hydroxyurea. The function of ribonucleotide reductase as a checkpoint component of S progression was studied in yeast in which elevated eIF4E rescued S-arrested rnr2-68(ts) cells, by increasing recruitment of its mRNA to polysomes. Crosses between rnr2-68(ts) and mutant eIF4E (cdc33-1(ts)) engendered conditional synthetic lethality, with extreme sensitivity to hydroxyurea and the microtubule depolymerizing agent, benomyl. The double mutant (cdc33-1 rnr2-68) also identified a unique terminal phenotype, arrested with small bud and a randomly distributed single nucleus, which is distinct from those of both parental single mutants. This phenotype defines eIF4E and RNR2 as determinants in an important cell cycle checkpoint, in early/mid-S phase. These results also provide a link between protein and DNA synthesis and provide an explanation for cell cycle alterations induced by elevated eIF4E.
Subject(s)
DNA Replication , Gene Expression Regulation , Peptide Initiation Factors/metabolism , Protein Biosynthesis , Ribonucleotide Reductases/genetics , Transcription, Genetic , Animals , Benomyl/pharmacology , CHO Cells , Cell Cycle/physiology , Cloning, Molecular , Cricetinae , Eukaryotic Initiation Factor-4E , Fibroblast Growth Factors/genetics , Gene Expression Regulation/drug effects , Hydroxyurea/pharmacology , Microtubules/drug effects , Microtubules/ultrastructure , Ornithine Decarboxylase/genetics , Peptide Initiation Factors/genetics , Polyribosomes/metabolism , RNA, Messenger/genetics , Recombinant Proteins/metabolism , Ribonucleotide Reductases/biosynthesis , S Phase , Saccharomyces cerevisiae/genetics , TransfectionABSTRACT
Deoxyhypusine synthase catalyzes the NAD+-dependent formation of deoxyhypusine in the eIF-5A precursor protein by transferring the 4-aminobutyl moiety of spermidine. This enzyme has recently been shown to be essential for cell viability and growth of yeast [Sasaki, K., Abid, M.R., and Miyazaki, M. (1996) FEBS Lett. 384, 151 154]. We have purified and characterized the enzyme from the yeast Saccharomyces carlsbergensis. The yeast and recombinant enzymes had a specific activity of 1.21 to 1.26 pmol per min per pmol of protein, and recognized both the eIF-5A precursor proteins almost equally as judged from their similar K(m) and V(max) values. Size exclusion chromatography and SDS-PAGE indicated that the active form of the enzyme is a homotetramer consisting of 43-kDa subunits. The enzyme showed a strict specificity for its substrates, NAD+, spermidine and eIF-5A precursor protein. Among all the substrates tested, only NAD+ showed a protective effect against heat inactivation of the enzyme suggesting that NAD+ initiates some conformational change in the enzyme. NADH exhibited a strong non-competitive inhibition (product inhibition). Unexpectedly, FAD, FMN, and riboflavin showed a moderate competitive inhibition. The competitive inhibition by diamines was maximal with compounds resembling spermidine in carbon chain length. 1,3-Diaminopropane inhibited the enzyme strongly in a competitive manner (product inhibition). On the other hand, putrescine did not inhibit the enzyme or act as a substrate. A polyclonal antibody raised against the yeast recombinant enzyme specifically inhibited deoxyhypusine synthase activity. The cross-reactivity (by Western blotting) of this antibody with the crude extracts varied depending on the source, indicating species specificity.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors/isolation & purification , Oxidoreductases Acting on CH-NH Group Donors/physiology , Saccharomyces/enzymology , Amino Acid Sequence , Animals , Cross Reactions , Diamines/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Mammals/metabolism , Molecular Sequence Data , Molecular Weight , Neurospora/enzymology , Nucleotides/pharmacology , Oxidoreductases Acting on CH-NH Group Donors/antagonists & inhibitors , Polyamines/pharmacology , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces/chemistry , Sequence Analysis , Species Specificity , TemperatureABSTRACT
Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine, a modification of a specific lysine residue in the precursor of eukaryotic translation initiation factor 5A. We have purified deoxyhypusine synthase from yeast, and cloned and sequenced the corresponding gene encoding a 387-amino acid protein from Saccharomyces cerevisiae. Gene disruption experiments indicated that the deoxyhypusine synthase gene is essential for cell growth in yeast. This gene was shown to be an intron-free, single-copy gene, and its product can catalyze the synthesis of deoxyhypusine equally in two precursor forms of eIF-5A, derived from two distinct genes of yeast.
