title: Basic fibroblast growth factor activates endothelial nitric-oxide synthase in CHO-K1 cells via the activation of ceramide synthesis.
abstract: In this study, we analyzed the intracellular mechanisms leading to basic fibroblast growth factor (bFGF)-dependent production of NO in Chinese hamster ovary (CHO)-K1 cells and a possible physiological role for such an effect. bFGF induces NO production through the activation of the endothelial form of NO synthase (eNOS), causing a subsequent increase in the cGMP levels. In these cells, the activation of eNOS by bFGF is Ca(2+)- and mitogen-activated protein kinase-independent. The translocation of the enzyme from the plasma membrane, where it is located in caveolae bound to caveolin 1, to the cytosol is the crucial step for the synthesis of NO through the eNOS isoform. We demonstrate that bFGF activates a sphingomyelinase to synthesize ceramide, which, in turn, allows the dissociation of eNOS from caveolin 1 and its translocation to the cytosol in the active form, where it catalyzes the synthesis of NO. In fact, drugs interfering with sphingomyelinase activity blocked bFGF activation of eNOS, and an increase in ceramide content was detected after bFGF treatment. Moreover, in fibroblasts derived from patients with Niemann-Pick disease, in which the enzyme is genetically inactive, bFGF is unable to elicit eNOS activation. The NO produced after bFGF treatment, through the activation of guanylyl cyclase and protein kinase G, mediates a mitogen-activated protein kinase-independent cell proliferation. In conclusion, our data show that, in CHO-K1 cells, bFGF regulates the activity of eNOS through a novel intracellular pathway, involving the induction of ceramide synthesis and that the NO released participates in bFGF proliferative activity.
authors.affiliation: Pharmacology and Neurosciences, National Institute for Cancer Research c/o Advanced Biotechnology Center, Genova, Italy. florio@cba.unige.it
authors.forename: Tullio
authors.initials: T
authors.lastname: Florio
authors.forename: Sara
authors.initials: S
authors.lastname: Arena
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pattarozzi
authors.forename: Stefano
authors.initials: S
authors.lastname: Thellung
authors.forename: Alessandro
authors.initials: A
authors.lastname: Corsaro
authors.forename: Valentina
authors.initials: V
authors.lastname: Villa
authors.forename: Alessandro
authors.initials: A
authors.lastname: Massa
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Diana
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Spoto
authors.forename: Sabrina
authors.initials: S
authors.lastname: Forcella
authors.forename: Gianluca
authors.initials: G
authors.lastname: Damonte
authors.forename: Mirella
authors.initials: M
authors.lastname: Filocamo
authors.forename: Umberto
authors.initials: U
authors.lastname: Benatti
authors.forename: Gennaro
authors.initials: G
authors.lastname: Schettini
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002518:Ceramides / Q000096:biosynthesis*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D008246:Lysophospholipids; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D002518:Ceramides; D008246:Lysophospholipids; D016222:Fibroblast Growth Factor 2; C060506:sphingosine 1-phosphate; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine; D002118:Calcium