Abstract
We have recently shown that the crosstalk between mild endoplasmic reticulum (ER) stress and low concentrations of the pro-inflammatory cytokine interleukin (IL)-1β exacerbates beta cell inflammatory responses via the IRE1α/XBP1 pathway. We presently investigated whether mild ER stress also sensitizes beta cells to cytokine-induced apoptosis. Cyclopiazonic acid (CPA)-induced ER stress enhanced the IL-1β apoptosis in INS-1E and primary rat beta cells. This was not prevented by XBP1 knockdown (KD), indicating the dissociation between the pathways leading to inflammation and cell death. Analysis of the role of pro- and anti-apoptotic proteins in cytokine-induced apoptosis indicated a central role for the pro-apoptotic BH3 (Bcl-2 homology 3)-only protein Bim (Bcl-2-interacting mediator of cell death), which was counteracted by four anti-apoptotic Bcl-2 (B-cell lymphoma-2) proteins, namely Bcl-2, Bcl-XL, Mcl-1 and A1. CPA+IL-1β-induced beta cell apoptosis was accompanied by increased expression of Bim, particularly the most pro-apoptotic variant, small isoform of Bim (BimS), and decreased expression of A1. Bim silencing protected against CPA+IL-1β-induced apoptosis, whereas A1 KD aggravated cell death. Bim inhibition protected against cell death caused by A1 silencing under all conditions studied. In conclusion, mild ER stress predisposes beta cells to the pro-apoptotic effects of IL-1β by disrupting the balance between pro- and anti-apoptotic Bcl-2 proteins. These findings link ER stress to exacerbated apoptosis during islet inflammation and provide potential mechanistic targets for beta cell protection, namely downregulation of Bim and upregulation of A1.
Highlights
When faced with compromised endoplasmic reticulum (ER) homeostasis and ER stress, cells trigger the unfolded protein response (UPR) through activation of three ER transmembrane proteins: activating transcription factor-6 (ATF-6), inositol-requiring enzyme 1a (IRE1a) and RNA-activated protein kinase-like eukaryotic initiation factor 2a kinase (PERK).[7,9,10,11] The UPR attenuates the protein load on the ER by decreasing global protein synthesis in parallel to increasing translation of ER chaperones and foldases involved in correct protein maturation; terminally misfolded proteins are degraded
A dose–response analysis of ER stress in cyclopiazonic acid (CPA)-treated INS-1E cells indicated that 6.25 mM clearly induces the expression of X-box-binding protein-1 (XBP1) and C/EBP homologous protein (CHOP), but not degradation of Ins-2 mRNA, which is an indicator of severe ER stress[30] (Supplementary Figure 1); similar observations were obtained at later time points[19,29] and
We presently describe that mild ER stress sensitizes pancreatic beta cells to the pro-apoptotic effects of the cytokine IL-1b by altering the balance between the antiapoptotic Bcl-2 protein A1 and the pro-apoptotic BH3-only protein Bcl-2-interacting mediator of cell death (Bim)
Summary
When faced with compromised ER homeostasis and ER stress, cells trigger the unfolded protein response (UPR) through activation of three ER transmembrane proteins: activating transcription factor-6 (ATF-6), inositol-requiring enzyme 1a (IRE1a) and RNA-activated protein kinase-like eukaryotic initiation factor 2a kinase (PERK).[7,9,10,11] The UPR attenuates the protein load on the ER by decreasing global protein synthesis in parallel to increasing translation of ER chaperones and foldases involved in correct protein maturation; terminally misfolded proteins are degraded. Protracted or excessive ER stress and pro-inflammatory cytokines trigger beta cell death through the ‘mitochondrial’ or intrinsic pathway of apoptosis. This pathway is regulated by the Bcl-2 (B-cell lymphoma-2) protein family, which includes pro- (such as the Bcl-2 homology 3 (BH3)-only proteins) and anti-apoptotic proteins.[12,20,21] Recent studies suggest that death protein 5 (DP5, known as Harakiri),[22] p53-upregulated modulator of apoptosis (PUMA)[23] and Bcl-2-interacting mediator of cell death (Bim)[21,24,25,26] are the key pro-apoptotic BH3-only proteins in cytokine-treated beta cells, whereas relevant anti-apoptotic proteins include Bcl-2, Bcl-XL, myeloid cell leukemia sequence-1 (Mcl-1) and A1 ( known as Bcl-2A1).[23,25,27,28] It remains to be defined, how the balance between these proteins regulates the activation of Bcl-2-associated X protein (Bax) and Bcl-2 antagonist/killer (Bak), the consequent activation of caspases 9 and 3, and the execution of beta cell apoptosis.[12]
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