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 4

Ex vivo GRP78 depletion increased β cell death and transcriptional signatures of apoptosis pathways.

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Ex vivo GRP78 depletion increased β cell death and transcriptional signa...
Dispersed Grp78fl/fl male and female mouse islet cells were transduced with Ad-LacZ (control) or Ad-Cre (knockdown, KD) and cultured for 72 hours in 15 mM glucose for all experiments in this figure. (A and B) Western blots for GRP78, cleaved caspase 3 (cCASP3), CHOP, and Actin (A) were quantitated in B (n ≥ 5). (C and D) Cells were lifted, stained for Annexin V (ANXA5) and propidium iodide (PI), analyzed by flow cytometry (C), and quantitated (D) (n ≥ 5). (E and F) Cells plated on glass coverslips were stained for insulin (red), TUNEL (green) and DAPI (blue), imaged (E), and dying β cells were counted (F) (n ≥ 4). (G and H) qPCR analysis on parallel aliquots of RNA to those sent for library preparation and sequencing were tested for Grp78 (G) or compensatory chaperones Grp94 and Calr (H) (n = 4). (I–L) Bulk RNA sequencing of islet cell cultures prepared as above; n = 4 biological replicates (2 male, 2 female). (I) Scatter plot of differentially expressed genes (P < 0.05, FC ≥ 1.5); red dots are upregulated in Grp78 KD, blue dots are downregulated in Grp78 KD. (J) Log2 fold change with top 10 up- and downregulated genes labeled. (K) Illustration of significantly enriched (FDR < 0.05) upregulated (red, related to cell death) and downregulated (blue) Hallmark gene sets. (L) Enrichment plot and heat map of hallmark apoptosis gene set showing gene expression changes. Scale bars: 50 μm. Statistics by paired t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.