Translational control of inducible nitric oxide synthase expression by arginine can explain the arginine paradox

Abstract

L-Arginine is the only endogenous nitrogen-containing substrate of NO synthase (NOS), and it thus governs the production of NO during nervous system development as well as in disease states such as stroke, multiple sclerosis, Parkinson's disease, and HIV dementia. The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine. Herein, we report that decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of inducible NOS (iNOS) protein expression. However, activity of the promoter of the iNOS gene, induction of iNOS mRNA, and stability of iNOS protein were not inhibited under these conditions. Our results indicate that inhibition of iNOS activity by arginine depletion in stimulated astrocyte cultures occurs via inhibition of translation of iNOS mRNA. After stimulation by cytokines, uptake of L-arginine negatively regulates the phosphorylation status of the eukaryotic initiation factor (eIF2 alpha), which, in turn, regulates translation of iNOS mRNA. eIF2 alpha phosphorylation correlates with phosphorylation of the mammalian homolog of yeast GCN2 eIF2 alpha kinase. As the kinase activity of GCN2 is activated by phosphorylation, these findings suggest that GCN2 activity represents a proximal step in the iNOS translational regulation by availability of l-arginine. These results provide an explanation for the arginine paradox for iNOS and define a distinct mechanism by which a substrate can regulate the activity of its associated enzyme.

Figures

Figure 1

Regulation of iNOS and arginase I in primary cultured rat astrocytes. (A) Immunoblot analyses of iNOS and arginase I expression. Rat astrocytes were treated with cAMP (1 mM), IFN-γ (100 units/ml), and cAMP plus IFN-γ for 24 h. Whole-cell lysates were subjected to immunoblotting with antibodies specific for iNOS or arginase I. (B) cAMP and IFN-γ induce iNOS but not arginase I in astrocytes. Rat astrocytes were treated with vehicle (control) or cAMP plus IFN-γ for 24 h. Cells were fixed with 4% paraformaldehyde and incubated with mouse anti-GFAP (a, d, g, and j), rabbit anti-iNOS (b and e), or rabbit anti-arginase I (h and k) antibody. Fluorescein-conjugated secondary antibodies were incubated, and indirect fluorescence was detected by confocal microscopy.

Figure 2

(A) Increased expression of arginase I in astrocytes by using an adenoviral vector. Rat astrocytes were infected with an adenoviral vector containing arginase I (Ad-Arg I) or vector control (Ad-GFP) for 48 h. Whole-cell lysates were subjected to immunoblotting with antibodies specific for arginase I, β-actin, or GFP. (B) Increased arginase activity in virally transfected astrocytes. Rat astrocytes were infected with the indicated moi of Ad-Arg I or Ad-GFP for 48 h. Arginase activity reflects percent conversion of [14C]arginine added to the culture medium to 14C urea. Results are mean ± SD of triplicate experiments. (C) Overexpression of arginase I in astrocytes can decrease cytokine-induced NO levels in astrocytes. Rat astrocytes were infected with the indicated moi of Ad-Arg I or Ad-GFP for 24 h and then further treated with cAMP (1 mM) and IFN-γ (100 units/ml) to induce NO product. After 16 h, NO levels were quantified by measuring the accumulation of nitrite in the culture medium of astrocytes with the Griess reagent. (D) Overexpression of arginase I in astrocytes can decrease cytokine-induced iNOS protein levels. Rat astrocytes were infected with Ad-Arg I or Ad-GFP (vector control) for 24 h and subsequently treated with cAMP (1 mM) and IFN-γ (100 units/ml) for 16 h. Immunoblot analysis of iNOS in whole-cell lysates is shown. (E) For confocal microscopy cells were fixed with 4% paraformaldehyde and incubated with rabbit anti-iNOS (b, e, h, and k) or rabbit anti-arginase I (b′, e′, h′, and k′) antibody. The adenovirus transfer vector used in these studies has a double expression cassette, and GFP has been subcloned into Ad-GFP (a, a′, d, and d′) or Ad-Arg I (g, g′, j, and j′) to monitor vector-derived expression.

Figure 3

Regulation of iNOS expression by extracellular arginine. (A) Extracellular arginine regulates intracellular production of NO by iNOS. Astrocytes were cultured in L-arginine-free media containing the designated concentration of L-arginine supplementation. Parallel cultures were also treated with cAMP/IFN-γ to induce iNOS expression, and NO levels were quantitated as described. (B) Extracellular levels of L-arginine regulate intracellular iNOS expression induced by cAMP and IFN-γ in astrocytes. (C) Manipulating extracellular arginine does not affect iNOS promoter activity. iNOS promoter (firefly luciferase) activity was normalized to Renilla luciferase activity. The data are mean ± SE obtained from three separate experiments. (D) Manipulating extracellular arginine does not affect iNOS mRNA levels. Northern blot analysis data were obtained from seven separate experiments. (E) Manipulation of extracellular arginine using recombinant arginase I diminishes de novo synthesis of iNOS. Rat astrocytes were treated with cAMP (1 mM) and IFN-γ (100 units/ml) for 16 h before cell harvest. Recombinant arginase I (1 μg/ml) was added 6 or 16 h before cell harvest. Forty minutes before cell harvest, cells were placed in methionine/cysteine-free medium. [35S]methionine/cysteine was added for 15 min before harvest. Harvested cell lysates were immunoprecipitated with an anti-iNOS antibody. Immunoprecipitated samples were resolved by SDS/PAGE, and radiolabeled proteins were detected by autoradiography. (F) Extracellular manipulation of arginine does not trigger the degradation of iNOS protein. Rat astrocytes were treated with cAMP (1 mM) and IFN-γ (100 units/ml) for 16 h before the chase with nonradiolabeled amino acids. Subsequently, cells were placed in methionine/cysteine-free medium 40 min before the chase addition; [35S]methionine/cysteine was added for 15 min before the chase addition. Cells were chased in L-arginine-depleted medium or L-arginine-supplemented medium (control) for the indicated time period. At the designated times, cell lysates were harvested, and levels of radiolabeled iNOS were detected as described above.

Figure 4

Extracellular or intracellular manipulation of intracellular arginine decreases iNOS expression by translational control mechanisms involving eIF2α phosphorylation. (A) Arginase I-induced decreases in iNOS protein levels are associated with the phosphorylation of eIF2α. Rat astrocytes were infected with Ad-Arg I or Ad-GFP for 24 h. Cells were then stimulated by cAMP (1 mM) and IFN-γ (100 units/ml) for 16 h. Whole-cell lysates were subjected to immunoblotting with antibodies specific for the form of eIF2α that is phosphorylated at serine 51. (B) An inverse correlation is observed between the level of iNOS protein and the amount of phospho-eIF2α protein. The densitometric values were calculated from iNOS and phospho-eIF2α immunoblots shown in A and Fig. 2D. (C) Increased levels of extracellular arginine are correlated with a decrease in the level of intracellular phospho-eIF2α. (D) The densitometric value (mean ± SD) of phospho-eIF2α from three separate experiments. Rat astrocytes were cultured in l-arginine-depleted medium or l-arginine-supplemented medium for 24 h. Cells were then stimulated by cAMP (1 mM) and IFN-γ (100 units/ml) for 16 h. (E) Rat astrocytes were infected with Ad-GFP (vector control), Ad-S51A (serine to alanine, nonphosphorylatable mutant), or Ad-S51D (serine to aspartate, phosphomimetic mutant) at an moi of 5 for 24 h and subsequently treated with cAMP (1 mM) and IFN-γ (100 units/ml) for 16 h. Immunoblot analysis of iNOS in whole-cell lysates is shown. (F) For immunofluorescence microscopy, cells were fixed with 4% paraformaldehyde and incubated with rabbit anti-iNOS antibody (b, f, j, n, r, and v) or the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI) (c, g, k, o, s, and w). The adenovirus transfer vector used in these studies has a double expression cassette, and GFP has been subcloned into Ad-GFP (a and e), Ad-S51A (i and m), or Ad-S51D (q and u) to monitor vector-derived expression.

Figure 5

(A) Increased extracellular arginine levels are also associated with decreased phosphorylation of the GCN2 kinase. Cell lysates were immunoprecipitated with GCN2 antibody and immunoblotted with a phospho-GCN2 or a GCN2 antibody. (B) A model for regulation of iNOS activity by arginine. Cytokine stimulation of astrocytes leads to increased transcription of iNOS message. The translation of this message into iNOS protein is governed, in part, by the transport of extracellular arginine into the cell. Increases in intracellular arginine lead to charging of tRNAs, decreased GCN2 phosphorylation and activity, and subsequent decreased phosphorylation of eIF2α. Decreased eIF2α phosphorylation results in increased translational initiation of iNOS. This model resolves the arginine paradox for iNOS and suggests that extracellular arginine regulates the activity of intracellular iNOS by regulating its translation.