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 9

Inhibition of either JNK or p53 reduces human β cell death after GRP78 depletion.

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Inhibition of either JNK or p53 reduces human β cell death after GRP78 d...
Panels A–F contain data from human pancreatic sections, and panels G–M contain data from ex vivo culture of dispersed human islet cells transduced with Ad-LacZ or Ad-sh-GRP78 and cultured for 72 hours. (A–C) Sections from individuals with T2D or controls (n = 5 of each) were immunostained for insulin (green), p-JNK (red), and DAPI (blue), imaged with confocal microscopy (A), and p-JNK staining intensity over the insulin+ area quantified using ImageJ either in absolute (B) or normalized to insulin staining intensity (C). (D–F) Sections from individuals with T2D or controls (n = 5 each) were immunostained for insulin (green), GRP78 (red), and DAPI (blue), imaged with confocal microscopy (D), and GRP78 staining intensity over the insulin+ area quantified using ImageJ either in absolute (E) or normalized to insulin staining intensity (F). (G) Dispersed human islet cells transduced with Ad-LacZ or Ad-sh-GRP78 were assessed for GRP78 or GRP94 abundance by qPCR (n = 5). (H and I) Dispersed human islet cells transduced with Ad-LacZ or Ad-sh-GRP78 and plated on glass coverslips (n = 6) were assessed for apoptosis by labeling for insulin (red), TUNEL (green), and DAPI (blue); imaged (H); and quantified for the percentage of β cells labeling for TUNEL (I). (J) Human islet cells treated as in G were assessed by qPCR for abundance of cJUN target genes (n = 3). (K–M) Dispersed human islet cells transduced with Ad-LacZ or Ad-sh-GRP78 were treated with vehicle (DMSO), JNK-IN-8 inhibitor (n = 11), or p53 inhibitor (n = 10) for 72 hours; labeled for insulin (red), TUNEL (green), and DAPI (blue); imaged (K); and quantified for the percentage of β cells labeling for TUNEL (L and M). Statistics by t test (B, C, E–G, I, and J) or 1-way ANOVA (L and M). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.