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 2ϩ -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 mitogenactivated 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.Nitric oxide (NO) is an important intracellular and intercellular mediator involved in the modulation of many physiological processes in different tissues, including blood flow regulation, platelet aggregation, smooth muscle relaxation, apoptosis, central and peripheral neurotransmission, and different neuroendocrine responses (Moncada and Higgs, 1993;Nathan and Xie, 1994).NO is synthesized by a family of three distinctive isoforms of nitric-oxide synthase (NOS), named after the tissues in which they were originally described. Neuronal and endothelial NOS [nNOS (or NOS I) and eNOS (or NOS III), respectively] are Ca 2ϩ /calmodulin-dependent enzymes constitutively expressed not only in neuronal and endothelial cells

Recent advances in the immunobiology of ceramide

“…Many studies indicate that low physiological concentrations of NO reduce the generation of ceramide and are implicated in cell survival and differentiation. High levels of NO increase ceramide levels and contribute to cell death (Sciorati et al, 1999;Florio et al, 2003;Huwiler et al, 1999;Takeda et al, 1999).…”

Is Endothelial Nitric Oxide Synthase a Moonlighting Protein Whose Day Job is Cholesterol Sulfate Synthesis? Implications for Cholesterol Transport, Diabetes and Cardiovascular Disease

“…Fibroblasts: Fibroblasts, cells whose sole responsibility is to synthesize extracellular matrix and collagen, contain both eNOS and nNOS [175,3]. Fibroblasts are responsible for synthesizing HS, dermatan sulfate, and chondroitin sulfate, all of which contribute to the extracellular matrix proteins that decorate the exterior of essentially all cells [176].…”

“…It is expressed not only in the endothelial cells lining blood vessel walls, but also in fibroblasts [3], cardiomyocytes [4], platelets [5], and red blood cells (RBCs) [6], among others. It maintains a "resting" state bound to the protein caveolin in the small invaginations in the membrane surface known as caveolae associated with lipid rafts on the plasma membrane [4,7].…”

Sphingosine Kinase–Dependent Activation of Endothelial Nitric Oxide Synthase by Angiotensin II

“…However, studies in several cell types and tissues demonstrate that various stimuli can elicit local ceramide and S1P formation, which then act in an autocrine or paracrine manner. 1,[21][22][23] Therefore, it was the aim of the present study to determine whether known vasoconstrictive compounds may exert their vascular effects at least in part by modulating the ceramide/S1P rheostat. For this purpose, we have used the specific sphingosine kinase inhibitor dimethylsphingosine (DMS) 24 to block S1P formation.…”