15-Deoxy-Δ12,14-prostaglandin J2 promotes phosphorylation of eukaryotic initiation factor 2α and activates the integrated stress response - PubMed (original) (raw)
15-Deoxy-Δ12,14-prostaglandin J2 promotes phosphorylation of eukaryotic initiation factor 2α and activates the integrated stress response
Devin Tauber et al. J Biol Chem. 2019.
Abstract
Stress granules (SGs) are cytoplasmic RNA-protein aggregates formed in response to inhibition of translation initiation. SGs contribute to the stress response and are implicated in a variety of diseases, including cancer and some forms of neurodegeneration. Neurodegenerative diseases often involve chronic phosphorylation of eukaryotic initiation factor 2α (eIF2α), with deletions of eIF2α kinases or treatment with eIF2α kinase inhibitors being protective in some animal models of disease. However, how and why the integrated stress response (ISR) is activated in different forms of neurodegeneration remains unclear. Because neuroinflammation is common to many neurodegenerative diseases, we hypothesized that inflammatory factors contribute to ISR activation in a cell-nonautonomous manner. Using fluorescence microscopy and immunoblotting, we show here that the endogenously produced product of inflammation, 15-deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2), triggers eIF2α phosphorylation, thereby activating the ISR, repressing bulk translation, and triggering SG formation. Our findings define a mechanism by which inflammation activates the ISR in a cell-nonautonomous manner and suggest that inhibition of 15-d-PGJ2 production might be a useful therapeutic strategy in some neuroinflammatory contexts.
Keywords: eukaryotic initiation factor 2 (eIF2); neuroinflammation; prostaglandin; proteasome; stress granule; stress response.
© 2019 Tauber and Parker.
Conflict of interest statement
The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health
Figures
Figure 1.
ISRIB disrupts 15-d-PGJ2–induced stress granules, and translational shutoff coincides with eIF2α phosphorylation in GFP-G3BP1 U-2 OS cells. A, U-2 OS cells stably expressing the SG marker GFP-G3BP1 were treated with NaAsO2 (100 μ
m
), PGJ2 (50 μ
m
), or PatA (100 n
m
) for 1 h and imaged (left panels). ISRIB (5 n
m
) was then added, and cells were imaged 5 min later (right panels) (scale bars represent 15 μm). B, SG induction kinetics were assessed by quantifying SG area per cell area over time following stress by NaAsO2 (100 μ
m
), 15-d-PGJ2 (50 μ
m
), or PatA (100 n
m
). Shown is the average (± S.D.) SG area (μm2) of SGs from an entire frame from images collected in live U-2 OS cells every 5 min for 200 min (n = 3). C, quantification of ISRIB addition SG kinetics from A, depicting SG area per cell area per time; 15-d-PGJ2 and NaAsO2 SGs disappear rapidly whereas PatA SGs do not (n = 3). D, immunoblot depicting puromycin incorporation into nascent peptides as a measure of translation in conjunction with P-eIF2α induction over a period of 4 h after treatment with 10 μ
m
15-d-PGJ2. Puromycin incorporation decreases, whereas P-eIF2α increases, after 15-d-PGJ2 addition. E, quantification of D with normalization to the 0-min time point. Translational reduction and P-eIF2α induction display inverse kinetics in relation to each other, with translational reduction plateauing when P-eIF2α levels reach a steady-state maximum after 15-d-PGJ2 stress. *, p < 0.05; unpaired Student's t test; results are displayed as the mean ± S.D.; n = 3. F, correlation plot between P-eIF2α and translation from -fold changes depicted in E. Translation and P-eIF2α negatively correlate after 15-d-PGJ2 addition, suggesting that the two are interrelated (R2 = 0.878).
Figure 2.
ISRIB partially restores translation in 15-d-PGJ2–treated U-2 OS cells. A, immunoblot depicting puromycin incorporation as a marker for translation to examine the effects of ISRIB on translation and P-eIF2α. U-2 OS cells were stressed with NaAsO2 (100 μ
m
) or 15-d-PGJ2 (10 μ
m
) for an hour and subsequently treated with ISRIB (5 n
m
) for 5 min before puromycin pulsing. Notice that P-eIF2α is unchanged, whereas translation is partially restored, by ISRIB for both NaAsO2 and 15-d-PGJ2. B, images depicting α-puro fluorescence intensity in U-2 OS cells exposed to NaAsO2 or 15-d-PGJ2 ± ISRIB addition to confirm the results from A. U-2 OS cells were subjected to the same conditions as in A but fixed and stained for immunofluorescence. α-Puro fluorescence intensity drops an hour after being exposed to NaAsO2 and 15-d-PGJ2 but is partially restored after ISRIB addition. Note the single cell in both NaAsO2 and 15-d-PGJ2 frames displaying high α-puro fluorescence intensity while not containing SGs. Presumably, translation is not shut off in those cells, and therefore they do not contain SGs (scale bars represent 15 μm). C, quantification of fluorescence intensities from B, depicting an increase in translation after ISRIB addition for both NaAsO2 and 15-d-PGJ2. *, p < 0.05; unpaired Student's t test; results are displayed as the mean ± S.D.; n = 3).
Figure 3.
15-d-PGJ2 requires eIF2α phosphorylation for SG induction and is dependent on an eIF2α kinase. A, MEFsWT/WT or MEFsS51A/S51A were stressed with NaAsO2 (100 μ
m
), 15-d-PGJ2 (50 μ
m
), or PatA (100 n
m
) for an hour and fixed and stained for G3BP immunofluorescence. SGs form under all conditions in MEFsWT/WT, but only PatA SGs form in MEFsS51A/S51A, indicating that P-eIF2α is required for SGs driven by 15-d-PGJ2 (scale bars represent 15 μm). B, U-2 OS cells expressing GFP-G3BP were preincubated with 1 μ
m
PERKi or PKRi for 15 min and stressed with NaAsO2 (100 μ
m
), 15-d-PGJ2 (50 μ
m
), TG (500 n
m
), or PatA (100 n
m
) for 1 h and fixed. Quantification of SG area per cell area was performed to examine how PERKi or PKRi preincubation affects SG formation for the various stressors. Although inconclusive regarding a responsible eIF2αK for 15-d-PGJ2–induced SGs, both inhibitors prevent 15-d-PGJ2 SGs, indicating that an eIF2αK is required. *, p < 0.05; unpaired Student's t test; results are displayed as the mean ± S.D.; n = 3). C, differential graph depicting magnitudes of SG inhibition normalized to conditions without eIF2αK inhibitors present. PERKi prevents SG formation by all stressors except PatA and is a pan-ISR inhibitor. *, p < 0.05; unpaired Student's t test; results are displayed as the mean ± S.D.; n = 3). D, U-2 OS cells were treated under the same conditions outlined in B but were lysed for immunoblotting to examine how PERKi affected 15-d-PGJ2–induced P-eIF2α. eIF2α phosphorylation is prevented by PERKi in both NaAsO2- and 15-d-PGJ2–treated cells, consistent with P-eIF2α being required for 15-d-PGJ2 SG formation.
Figure 4.
Loss of a single eIF2αK does not prevent 15-d-PGJ2–driven P-eIF2α. A, HAP1 cells that were either WT or contained a single eIF2αK deletion were stressed with 15-d-PGJ2 (10 μ
m
) for 1 h, pulsed with puromycin for 5 min, and lysed for immunoblotting to examine how eIF2αK deletions affected 15-d-PGJ2 eIF2α phosphorylation and translation. No single eIF2αK deletion prevents translational shutoff or P-eIF2α; however, ΔHRI has a modest effect on preventing translational shutoff and eIF2α phosphorylation, similar to MG132 (
Fig. S3_A_
). B, quantification of P-eIF2α from A. No single kinase deletion had a significant effect on decreasing P-eIF2α, but ΔHRI had less P-eIF2α than the rest. *, p < 0.05; unpaired Student's t test; results are displayed as the mean ± S.D.; n = 3; NS, not significant. C, quantification of translation from A. No single eIF2α kinase prevents translational shutoff or significant changes from the WT, but ΔHRI had less of a decrease. *, p < 0.05; unpaired Student's t test; results are displayed as the mean ± S.D.; n = 3.
Figure 5.
Proteasomal inhibition precedes P-eIF2α for MG132 but coincides with P-eIF2α for 15-d-PGJ2, suggesting different mechanisms of eIF2α phosphorylation. A, U-2 OS cells were stressed with MG132 (10 μ
m
), 15-d-PGJ2 (10 μ
m
), or PatA (100 n
m
) over a period of 4 h and lysed for immunoblotting to examine the kinetic relationship of K48 poly-Ub accumulation (26S inhibition) and P-eIF2α between stressors. B, quantification of 26S inhibition kinetics. Both MG132 and 15-d-PGJ2 display similar curves. *, p < 0.05; unpaired Student's t test; results are displayed as the mean ± S.D.; n = 3. C, quantification of P-eIF2α kinetics. 15-d-PGJ2 displays faster P-eIF2α induction than MG132, suggesting that 26S inhibition precedes proteasomal induction of P-eIF2α for 15-d-PGJ2 and that 15-d-PGJ2–induced P-eIF2α is not solely explained by 26S inhibition but could be a combinatorial effect from the proteins outlined in
Fig. S2_A_
. *, p < 0.05; unpaired Student's t test; results are displayed as the mean ± S.D.; n = 3. D, correlation plot between MG132- and 15-d-PGJ2–induced poly-Ub and P-eIF2α. Although poly-Ub correlates between stressors (R2 = 0.705), P-eIF2α does not (R2 = 0.166).
References
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