title: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture.
abstract: We have recently demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi-quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. In our cellular model, TRAIL treatment not only increased NO levels but also caused a time-dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co-localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans-cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL-increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub-cellular distribution is mediated by cytoskeleton and Golgi complex modifications.
authors.affiliation: Department of Biomorphology, G. d'Annunzio University, Chieti-Pescara, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Di Pietro
authors.forename: Maria A
authors.initials: MA
authors.lastname: Mariggiò
authors.forename: Simone
authors.initials: S
authors.lastname: Guarnieri
authors.forename: Silvia
authors.initials: S
authors.lastname: Sancilio
authors.forename: Annalisa
authors.initials: A
authors.lastname: Giardinelli
authors.forename: Sara
authors.initials: S
authors.lastname: Di Silvestre
authors.forename: Agostino
authors.initials: A
authors.lastname: Consoli
authors.forename: Giorgio
authors.initials: G
authors.lastname: Zauli
authors.forename: Assunta
authors.initials: A
authors.lastname: Pandolfi
mesh_terms: D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000502:physiology*; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000502:physiology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D015739:Nocodazole; D019331:NG-Nitroarginine Methyl Ester