BH(4) (tetrahydrobiopterin)-dependent activation, but not the expression, of inducible NOS (nitric oxide synthase)-2 in proinflammatory cytokine-stimulated, cultured normal human astrocytes is mediated by MEK-ERK kinases.
Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.
Chiarini
Anna
A
Department of Biomedical and Surgical Sciences, University of Verona Medical School, Verona I-37134, Italy.
Dal Pra
Ilaria
I
Gottardo
Rossella
R
Bortolotti
Federica
F
Whitfield
James F
JF
Armato
Ubaldo
U
D001253:Astrocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002070:Butadienes / Q000494:pharmacology; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D018836:Inflammation Mediators / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009570:Nitriles / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002070:Butadienes; D016207:Cytokines; D018836:Inflammation Mediators; D007375:Interleukin-1; D009570:Nitriles; D014409:Tumor Necrosis Factor-alpha; C113580:U 0126; D001708:Biopterins; D009569:Nitric Oxide; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C003402:sapropterin