Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated activation of murine N9 microglial cell line.
Lipopolysaccharide, the main component of the cell wall of Gram-negative bacteria, is known to activate microglial cells following its interaction with the CD14/Toll-like receptor complex (TLR-4). The activation pathway triggered by lipopolysaccharide in microglia involves enhanced basal levels of intracellular calcium ([Ca2+]i) and terminates with increased generation of cytokines/chemokines and nitric oxide. Here we demonstrate that in lipopolysaccharide-stimulated murine N9 microglial cells, cyclic ADP-ribose, a universal and potent Ca2+ mobiliser generated from NAD+ by ADP-ribosyl cyclases (ADPRC), behaves as a second messenger in the cell activation pathway. Lipopolysaccharide induced phosphorylation, mediated by multiple protein kinases, of the mammalian ADPRC CD38, which resulted in significantly enhanced ADPRC activity and in a 1.7-fold increase in the concentration of intracellular cyclic ADP-ribose. This event was paralleled by doubling of the basal [Ca2+]i levels, which was largely prevented by the cyclic ADP-ribose antagonists 8-Br-cyclic ADP-ribose and ryanodine (by 75% and 88%, respectively). Both antagonists inhibited, although incompletely, functional events downstream of the lipopolysaccharide-induced microglia-activating pathway, i.e. expression of inducible nitric oxide synthase, overproduction and release of nitric oxide and of tumor necrosis factor alpha. The identification of cyclic ADP-ribose as a key signal metabolite in the complex cascade of events triggered by lipopolysaccharide and eventually leading to enhanced generation of pro-inflammatory molecules may suggest a new therapeutic target for treatment of neurodegenerative diseases related to microglia activation.
Franco
Luisa
L
Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy.
Bodrato
Nicoletta
N
Moreschi
Iliana
I
Usai
Cesare
C
Bruzzone
Santina
S
Scarf ì
Sonia
S
Zocchi
Elena
E
De Flora
Antonio
A
D051997:ADP-ribosyl Cyclase 1 / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D036563:Cyclic ADP-Ribose / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D015290:Second Messenger Systems
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D047428:Protein Kinase Inhibitors; D036563:Cyclic ADP-Ribose; D052247:Nitric Oxide Synthase Type II; D011494:Protein Kinases; D051997:ADP-ribosyl Cyclase 1