The balance between adaptive and apoptotic unfolded protein responses regulates β-cell death under ER stress conditions through XBP1, CHOP and JNK
Introduction
The loss of β-cell mass is critical to the development of both type 1 and type 2 diabetes (Matveyenko and Butler, 2008). However, the triggering stimuli and the cellular mechanisms involved are poorly understood. Elevated plasma levels of pro-inflammatory cytokines and free fatty acids have been observed in type 1 and type 2 diabetes, and have been proposed as key contributors to β-cell apoptosis (Biden et al., 2014, Cnop et al., 2005, Donath and Shoelson, 2011). Indeed, exposure of β-cells to cytokines or fatty acids in vitro induces apoptosis along with the activation of multiple signalling networks, including endoplasmic reticulum (ER) stress (Cardozo et al., 2005, Cunha et al., 2008, Laybutt et al., 2007).
β-cells depend heavily on an efficient ER function due to their high rate of insulin synthesis and secretion. Cytokines and fatty acids have been shown to impair ER homeostasis by inducing depletion of ER Ca2+ and alteration of chaperone function, proinsulin processing and ER-Golgi trafficking, triggering accumulation of misfolded proteins and ER stress (Baldwin et al., 2012, Cardozo et al., 2005, Hara et al., 2014, Jeffrey et al., 2008, Oyadomari et al., 2001, Preston et al., 2009). ER stress and its ensuing unfolded protein response (UPR) have been implicated in β-cell pathophysiology in both forms of diabetes (Laybutt et al., 2007, Marhfour et al., 2012). However, the UPR can also serve an adaptive function to restore ER homeostasis via the ER stress sensors PKR-like ER kinase (PERK), inositol requiring kinase 1 (IRE1), and activating transcription factor (ATF) 6 (Fonseca et al., 2011, Scheuner and Kaufman, 2008). Activation of PERK and subsequent phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) leads to a rapid and transient reduction of global protein translation to reduce ER load, while increasing translation of the transcription factor ATF4. Activation of the endoribonuclease IRE1 results in the unconventional splicing of Xbp1 mRNA and increased translation of active XBP1 transcription factor. ATF6 is activated by proteolytic cleavage in the Golgi to release an active transcription factor. These transcription factors stimulate the expression of chaperones, protein folding enzymes and ER-associated degradation (ERAD) machinery to enhance ER folding capacity and clearance of misfolded proteins. Under prolonged or irresolvable ER stress, the UPR switches from an adaptive to an apoptotic role. The most widely implicated pro-apoptotic UPR effector is the transcription factor C/EBP Homologous Protein (CHOP) (Akerfeldt et al., 2008, Allagnat et al., 2012, Cardozo et al., 2005, Karaskov et al., 2006, Laybutt et al., 2007, Song et al., 2008). However, the mechanisms regulating the transition from adaptive to apoptotic UPR remain to be clarified.
Here, we used in vitro physiological inducers of ER stress (cytokines and saturated fatty acid, palmitate) and in vivo models of type 1 [Nonobese diabetic (NOD) mice] and type 2 diabetes (C57BL/KsJ db/db mice) as well as β-cell compensation (C57BL/6J ob/ob mice) to show that the balance between XBP1-mediated adaptive UPR and CHOP is critically important in the regulation of β-cell survival during ER stress. Furthermore, our studies reveal that JNK activation is a key factor that regulates the transition from adaptive to apoptotic UPR in β-cells. These findings uncover novel interrelationships between XBP1, CHOP and JNK that are crucial for β-cell survival in type 1 and type 2 diabetes.
Section snippets
Cell culture
MIN6 cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) containing 25 mM glucose, 10 mM HEPES, 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. Either 2 × 105 or 8 × 105 cells were seeded per well in a 24-well or 6-well plate, respectively. Cells were treated with 100 U/ml IL-1β, 250 U/ml IFN-γ and 100 U/ml TNF-α (R&D Systems, Minneapolis, MN, USA); 0.92% BSA or 0.4 mM palmitate coupled to 0.92% BSA. 4μ8c (30 μM; Tocris Biosciences, Bristol, UK), and JNK inhibitor II (JNKi,
IRE1/XBP1 inhibition potentiates cytokine-induced β-cell death
We first investigated the influence of IRE1/XBP1 signalling on the ER stress response induced by cytokines. In MIN6 cells, exposure to the combination of cytokines (IL-1β + TNF-α + IFN-γ) for 24 h led to an atypical pattern of UPR activation. Expression of adaptive UPR genes including Xbp1 (Fig. 1A), Bip (Fig. 1C), Erp72 (Fig. 1D), Fkbp11 (Fig. 1E), Grp94 (Fig. 1F) and Edem1 (Fig. 1G) were reduced after cytokine exposure, whereas expression of pro-apoptotic UPR genes, Chop (Fig. 1H) and Trib3 (
Discussion
ER stress has been implicated in β-cell death in type 1 (Marhfour et al., 2012, Tersey et al., 2012) and type 2 diabetes (Laybutt et al., 2007, Oyadomari et al., 2002, Scheuner and Kaufman, 2008, Song et al., 2008). However, an intact UPR is also required for physiological maintenance of β-cell mass (Fonseca et al., 2011, Scheuner and Kaufman, 2008). Our study examines relationships between XBP1, CHOP and JNK in the transition from adaptive to apoptotic UPR during ER stress in models of type 1
Conclusion
Our studies show for the first time that: 1) β-cell survival during cytokine-mediated and lipotoxic ER stress is regulated by the balance between XBP1 and CHOP; 2) the IRE1/XBP1-mediated adaptive UPR protects against hallmarks of β-cell failure associated with type 1 and type 2 diabetes; and 3) JNK is a crucial regulator of the transition from adaptive to apoptotic UPR during ER stress. These observations reveal important molecular mechanisms governing β-cell survival during ER stress and
Acknowledgements
This work was supported by a grant from the National Health and Medical Research Council (NHMRC) of Australia. D.R.L. is supported by an Australian Research Council (ARC) Future Fellowship.
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