Translational control of inducible nitric oxide synthase by p38 MAPK in islet β-cells

Abstract

The MAPKs are transducers of extracellular signals such as proinflammatory cytokines. In islet β-cells, cytokines acutely activate expression of the Nos2 gene encoding inducible nitric oxide synthase (iNOS), which ultimately impairs insulin release. Because iNOS production can also be regulated posttranscriptionally, we asked whether MAPKs participate in posttranscriptional regulatory events in β-cells and primary islets in response to cytokine signaling. We show that cytokines acutely reduce cellular oxygen consumption rate and impair aconitase activity. Inhibition of iNOS with l-NMMA or inhibition of Nos2 mRNA translation with GC7 [an inhibitor of eukaryotic translation initiation factor 5A (eIF5A) activity] reversed these defects, as did inhibition of p38 MAPK by PD169316. Although inhibition of p38 had no effect on the nuclear translocation of nuclear factor κB or the abundance of Nos2 transcripts during the immediate period after cytokine exposure, its inhibition or knockdown resulted in significant reduction in iNOS protein, a finding suggestive of a permissive role for p38 in Nos2 translation. Polyribosomal profiling experiments using INS-1 β-cells revealed that Nos2 mRNA remained associated with polyribosomes in the setting of p38 inhibition, in a manner similar to that seen with blockade of translational elongation by cycloheximide. Consistent with a role in translational elongation, p38 activity is required in part for the activation of the translational factor eIF5A by promoting its hypusination. Our results suggest a novel signaling pathway in β-cells in which p38 MAPK promotes translation elongation of Nos2 mRNA via regulation of eIF5A hypusination.

Figures

Fig. 1.

Effects of cytokines and enzyme inhibitors on glucose-induced changes in metabolic flux and aconitase activity in INS-1 (832/13) β-cells. INS-1 β-cells were plated in 24-well plates, and then exposed to cytomix at 2 mm glucose for 4, 24, or 48 h before metabolic flux measurements. At the times indicated by the arrows in each panel, glucose concentration in the medium was increased to 20 mm or 1.5 μm rotenone was added. A, ECAR during a cytomix time course; B, OCR during a cytomix time course, C, OCR after 4 h of cytomix exposure, with or without indicated enzyme inhibitors. D, Actonitase activity in cell extract after 4 h exposure to cytomix, with or without the indicated inhibitors. E, OCR after 4 h of cytomix exposure, without or without addition of 1.5 mm isocitrate or 1.5 mm citrate. F, OCR after 4 h of cytomix exposure, without or without addition of p38 inhibitor. C, Control; DHSi, 100 μm DHS inhibitor GC7; iNOSi, 1 mm iNOS inhibitor l-NMMA; p38i, 10 μm p38 inhibitor PD169316. Data represent the mean ± sem of triplicate determinations from at least three independent experiments. In all cases, *, P < 0.05 for the indicated value(s) in comparison with control by one-way ANOVA.

Fig. 2.

Effects of enzyme inhibitors on cytokine-induced Nos2 gene activation and NFκB nuclear translocation. INS-1 β cells (A[b]), rat islets (B[b]), and human islets (C[b]) were untreated (Control) or exposed to a standard cytomix concentration for 4 h, with or without indicated inhibitors, then cells were harvested for RNA isolation and real-time RT-PCR was performed for Nos2 message. In panels A–C, data are corrected for Actb message levels, and then normalized to Nos2 message levels in the absence of cytomix (Control). D, INS-1 cells were exposed to increasing concentrations of cytomix for 4 h, and then cytoplasmic (“C”) and nuclear (“N”) extracts were isolated and subjected to immunoblotting for NFκB p65 subunit and HDAC1 (nuclear protein control). E, INS-1 cells were exposed to a standard cytomix concentration, with or without indicated inhibitors, and then cytoplasmic (“C”) and nuclear (“N”) extracts were isolated and subjected to immunoblotting for NFκB p65 subunit and histone deacetylase (HDAC)1 (nuclear protein control). Panels A[b]–C represent the mean ± sem]r] of triplicate determinations from at least three independent experiments, and panels D and E are representative of experiments performed on three separate occasions. JNKi, 10 μ[scap]m JNK inhibitor SP600125; p38i and DHSi are as indicated in the legend to Fig. 1.

Fig. 3.

Effects of enzyme inhibitors on cytokine-induced iNOS[b] protein levels and stability. A, INS-1 β-cells were incubated for 4 h in the presence or absence of cytomix and the indicated inhibitors, after which whole-cell extracts were harvested and immunoblots for iNOS, phospho-p38, p38, and actin are shown on the left and quantitation of immunoblots in the presence of cytokines (n = 3) is shown on the right. B, Rat islets were incubated for 4 h in the presence or absence of cytomix and the indicated inhibitors, after whichn extracts were harvested and immunoblots for iNOS and actin are shown on the left, and quantitation of the immunoblots in the presence of cytokines (n = 3) is shown on the right. C, INS-1 β cells were incubated with cytomix for 2 h, then with 50 μg/ml CHX for the indicated times to block new protein synthesis; the panel on the left shows a representative immunoblot, whereas the panel on the right shows quantitation of iNOS protein levels (normalized to actin levels) from the immunoblots (n = 3). *, P < 0.05 for the indicated value(s) in comparison with cytokine-only control by one-way ANOVA.

Fig. 4.

Effect of enzyme inhibition or siRNA on cytokine-induced iNOS protein production. p38α+/+ mouse embryonic fibroblasts (MEFs) (A[b]) or p38−/− MEFs (B[b]) were incubated for 4 h in the presence or absence of cytomix and the indicated enzyme inhibitors, after which whole-cell extracts were harvested and immunoblotted for iNOS, tubulin, and p38. C[b], INS-1 β cells were treated with siRNAs indicated for 72 h, and then exposed to cytomix for 4 h, after which extracts were subjected to immunoblot analysis for p38 and actin. D[b], INS-1 extracts subjected to RT-PCR for Nos2 mRNA. E, INS-1 extracts subjected to immunoblot for iNOS and actin. In panels C and E, the top panels show two representative immunoblots and the bottom panels show quantitation of the immunoblots from three experiments. *, P < 0.05 for the indicated value(s) in comparison with si-Control by Student's t test.

Fig. 5.

PRP of INS-1 β-cells. INS-1 β-cells were exposed to cytomix for 4 h with or without inhibitors, and then subjected to PRP followed by real-time RT-PCR as detailed in Materials and Methods. In each panel, the solid line indicates the absorbance at 254 nm, whereas the dashed line with shading indicates the percent of total Nos2 message appearing in the indicated fraction. A[b], Cytomix alone. B[b], Cytomix plus Tg. C[b], Cytomix plus CHX. D[b], Cytomix plus p38 inhibitor PD169316. E[b], Cytomix plus JNK inhibitor SP600125. F[b], Cytomix plus DHS inhibitor GC7. Data shown are representative profiles obtained from experiments performed on three to four independent occasions. Tg, 1 μm Tg; CHX, 50 μg/ml CHX; p38i, JNKi, and DHSi are as indicated in the legends to Figs. 1 and 2. Panels indicate the positions of RNAs occupied by 40S, 60S, 80S ribosomal subunits, and the positions where RNA species are occupied by 2, 3, 4, 5, and more than 5 ribosomes (polyribosomes).

Fig. 6.

PRP of p38α+/+ and p38α-/- mouse[b] embryonic fibroblasts. A, p38α+/+ MEFs (left panel) and p38α-/- MEFs (right panel) were exposed to cytomix for 4 h and then subjected to PRP as detailed in Materials and Methods. B, p38α+/+ MEFs (left panel) and p38α-/- MEFs (right panel) were exposed to cytomix plus 1 μm Tg for 4 h, and then subjected to PRP. Panels indicate the positions of RNAs occupied by 40S, 60S, 80S ribosomal subunits, and the shaded region indicates the polyribosome region. P/M indicates the polyribosome/monoribosome RNA ratio, as determined by area under the curve (AUC) analysis. The P/M values are statistically different in panel B by Student's t test.

Fig. 7.

Effects of enzyme inhibitors or siRNA on eIF5A[b] hypusination. INS-1 β cells (A[b]), rat islets (B[b]), and human islets (C[b]) were exposed to [3H]spermidine for 4 h in the presence or absence of cytokines and the indicated enzyme inhibitors, after which whole-cell extracts were harvested and subjected to PAGE and fluorography for hypusinated eIF5A (3H-eIF5AHyp) or immunoblotting for total eIF5A and actin. D, INS-1 β-cells were treated with the siRNAs, then exposed to cytomix for 4 h, and extracts were subjected to PAGE and fluorography for 3H-eIF5AHyp or immunoblotting for total eIF5A. The panels on the left show representative fluorography and immunoblots, whereas the panels on the right show quantitation of [3H]eIF5AHyp protein levels (normalized to actin levels) from three independent experiments. *, P < 0.05 for the indicated value(s) in comparison with cytokine-only control by one-way ANOVA or Student's t test. C, control (no inhibitors); p38i, JNKi, and DHSi are as indicated in the legends to Figs. 1 and 2.

Fig. 8.

Proposed model of p38 MAPK regulation of mRNA translation. The figure depicts pathways by which p38 in the setting of cytokine signaling has been previously shown to affect gene transcription (leftmost pathway) and mRNA translation (middle two pathways) by way of direct phosphorylation of targets (transcription factors, Mnk1/2, and eEF2K). With this study, we propose a new pathway (rightmost pathway in bold), whereby p38 regulates the activity of hypusinating enzymes (DHS and/or DHH), which subsequently control the action of eIF5A. The figure is not meant to indicate that regulation of the hypusination enzyme(s) by p38 is necessarily direct (i.e. by a phosphorylation event), because the mechanism of this regulation remains to be elucidated. eIF4E, eukaryotic translation initiation factor 4E; eEF2K, eukaryotic translation elongation factor 2 kinase.