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 5

IRE1-dependent JNK activation, but not CHOP induction, leads to β cell death after GRP78 depletion.

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IRE1-dependent JNK activation, but not CHOP induction, leads to β cell d...
Dispersed Grp78fl/fl male and female mouse islet cells were transduced with Ad-LacZ or Ad-Cre and cultured for 72 hours in 15 mM glucose for all panels. (A and B) Cultures were labeled for TUNEL (green), insulin (red), and DAPI (blue) after treatment with inhibitors of IRE1 (4μ8c), ATF6 (AEBSF), or PERK (GSK2606414) with or without GRP78 knockdown (n ≥ 7). Inhibitors were added for the full 72 hours. See Supplemental Figure 3 for Ad-LacZ data with inhibitors. (C and D) Immunoblot of GRP78, p-JNK, and actin 72 hours after GRP78 knockdown (n ≥ 4). (E) Immunoblot of p-IRE1, p-JNK, and actin after GRP78 knockdown in the absence or presence of IRE1 inhibitors specific for the kinase or nuclease activity. (F and G) qPCR for Grp78 or Chop on GRP78 knockdown islet cultures with or without transduction with Ad-sh-Chop (n = 4). (H and I) GRP78 knockdown islet cell cultures transduced with Ad-sh-Chop and labeled for TUNEL (green), insulin (red) and DAPI (n = 4). For F–I, total virus MOI was held constant for all conditions by adjusting the MOI of Ad-LacZ. (J and K) Immunoblot and band intensity quantitation of GRP78, p-JNK, and actin 72 hours after GRP78 knockdown in the presence or absence of JNK inhibitor (JNK-IN-8) (n = 4). (L and M) GRP78 knockdown islet cell cultures treated with pan-JNK inhibitor labeled for TUNEL (green), insulin (red) and DAPI (n ≥ 8). Scale bars: 50μm. Statistics by paired t test (D) or 1-way ANOVA (B, F, G, I, K, and M). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.