BH₄ (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

Abstract

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.

Roles of Ca2+ and the Ca2+‐sensing receptor (CASR) in the expression of inducible NOS (nitric oxide synthase)‐2 and its BH4 (tetrahydrobiopterin)‐dependent activation in cytokine‐stimulated adult human astrocytes

“…The inhibitor was first dissolved in dimethylsulfoxide (DMSO) to make a 100 mM stock solution which was finally diluted to 100 nM in the culture medium. In one set of experiments, NPS 89636 (like the U0126 MEK1/MEK2 inhibitor used in our previous study [Chiarini et al, 2005]) was added for 30 min and then washed out just before the cytokines were added, and then added again at 24 h and washed out at 24.5 h. To do this, the cell-conditioned cytokine-free medium was carefully removed and medium containing 100 nM NPS 89636 was put on the cultures for 30 min after which it was removed by washout and the original cell-conditioned NPS 89636-free, but now cytokine-containing medium was put back on the cultures and the experiment began. This was repeated, washout included, for another 30 min at 24 h after adding the cytokines.…”

“…As described in more detail by Chiarini et al [2005], the cells in the cortical sample were released by mild treatment with 0.25% w/v trypsin (Sigma-Aldrich, Italy) and trituration in Hanks' Balanced Salt Solution (BSS; Eurobio, France) at 188C.…”

“…But the large amount of NO made by proinflammatory cytokine-activated astrocytes is made by NOS-2 which, unlike the other two enzymes, is not directly controlled by Ca 2þ or Ca 2þ Á calmodulin because it carries its own ''built-in'' calmodulin Baltrons and Garcia, 2001]. Also unlike the other two enzymes, NOS-2 is not expressed by the astrocytes in a normal brain or by proliferatively quiescent astrocytes in culture, but it is induced when the cells are activated by the signals from proinflammatory cytokine receptors [Suzuki et al, 1995;Grzybicki et al, 1998;Wada et al, 1998;Zamora et al, 2000;Baltrons and Garcia, 2001;Chiarini et al, 2005].…”

“…We have been explicating the mechanisms responsible for the build-up of NOS-2 in freshly isolated proliferatively quiescent, phenotypically stable, adult human cerebral astrocytes exposed to the three proinflammatory cytokines (IL-1b, IFN-g, TNF-a) that are most often found in injured or diseased brains [Chiarini et al, 2005]. We have so far found that this cytokine triad turns on two mechanisms that together induce the appearance, accumulation, and activation of NOS-2.…”

“…We have so far found that this cytokine triad turns on two mechanisms that together induce the appearance, accumulation, and activation of NOS-2. One mechanism stimulates the build-up of inactive NOS-2 protein and needs p38 MAPK activation to do so [Neufeld and Liu, 2003a,b;Chiarini et al, 2005]. The second mechanism, which operates between 24 and 24.5 h after adding the cytokines to the culture medium, stimulates the production of BH 4 (tetrahydrobiopterin) needed to activate the accumulating NOS-2 proteins and requires MEK1/MEK2 activities to do so [Chiarini et al, 2005].…”