JNK and p53 cause human and mouse β cell death during excessive unfolded protein response
Rohit B. Sharma, Christine Darko, Ying Wang, Thalia A. Castro, Tara Doma Lama, Brian Gablaski, Andrew Rappa, David Redmond, Jason K. Kim, Amy S. Lee, Laura C. Alonso
Rohit B. Sharma, Christine Darko, Ying Wang, Thalia A. Castro, Tara Doma Lama, Brian Gablaski, Andrew Rappa, David Redmond, Jason K. Kim, Amy S. Lee, Laura C. Alonso
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Research Article Endocrinology Metabolism

JNK and p53 cause human and mouse β cell death during excessive unfolded protein response

Abstract

Endoplasmic reticulum (ER) stress contributes to β cell death in both Type 1 and Type 2 diabetes (T1D and T2D). However, the molecular mechanisms driving β cell death during ER stress remain insufficiently defined, limiting development of protective therapies. GRP78, an ER chaperone, is the master regulator of unfolded protein response (UPR), suppressing UPR initiators during the unstressed state and releasing them to allow UPR activation during stress. To dissect the pathways leading to ER-stress response related β cell decompensation, we engineered mice genetically lacking GRP78 in pancreatic β cells. GRP78 deletion caused acute insulin-deficient diabetes in pups before weaning, with reduced β cell mass due to increased apoptosis. Molecular studies identified deregulated UPR, specifically IRE1 activity, as driving cell death. Unbiased and targeted analyses identified a JNK-p53 axis downstream of IRE1 kinase as a key mediator of β cell death during UPR activation. In vivo JNK inhibition protected against β cell death in 2 distinct ER stress diabetes models. In human β cells, pharmacological inhibition of both JNK and p53 improved β cell survival during GRP78 knockdown–induced UPR. These findings provide insight into mechanisms causing β cell death during ER stress and outline possible therapeutic targets to preserve insulin secretory capacity in diabetes.

Authors

Rohit B. Sharma, Christine Darko, Ying Wang, Thalia A. Castro, Tara Doma Lama, Brian Gablaski, Andrew Rappa, David Redmond, Jason K. Kim, Amy S. Lee, Laura C. Alonso

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Figure 6

JNK inhibition or acute p53 inhibition both rescue β cell death.

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JNK inhibition or acute p53 inhibition both rescue β cell death. All pan...
All panels show experiments on dispersed Grp78fl/fl mouse islet cells cultured for 72 hours in 15 mM glucose. (A) GSEA for hallmark gene sets on RNA-seq of Grp78 knockdown (KD) mouse islet cells with or without JNK inhibition. Only downregulated gene sets are show. Blue, p53-related gene sets. (B) TTRUST transcription factor prediction analysis on apoptosis gene set genes upregulated by Grp78 knockdown. Orange, p53-related; red, JNK-related. (C) Enrichment plot, p53 hallmark genes upregulated after Grp78 knockdown. (D) Heatmap, p53 hallmark genes upregulated after Grp78 knockdown. (E) Venn diagram showing the overlap of p53 genes upregulated by reducing GRP78 and downregulated by JNK inhibition during GRP78 knockdown. (F and G) Islet cells immunostained for insulin (green), phospho-p53 (red), and DAPI (blue) imaged by confocal microscopy, percentage β cells with nuclear phospho-p53 (n = 4). (H and I) qPCR analysis for Grp78 (H) or p53 transcriptional targets Atf3, Bak1, Trib3, Phlda3, Sdc1, and Ier5 (n = 5). (I). (J and K) Islet cells with Grp78 knockdown with or without p53 inhibition were fixed, labeled for insulin (red), TUNEL (green), and DAPI (blue), imaged, and counted (n = 7). (L–O) Islet cells withGrp78 knockdown with or without p53 knockdown underwent qPCR for Grp78, p53, or p53 target genes (L and M), or TUNEL staining and quantification (N and O), n = 3. Scale bars: 50μm. Statistics by paired t test (H and J) or 1-way ANOVA (F and G). *P < 0.05; **P < 0.01.