Interplay Between Endoplasmic Reticulum

Published Date: 02 Nov 2017

Introduction

Type 2 diabetes consists of an array of metabolic dysfunctions that coincide with hyperglycemia and result from the progressive failure of beta cells to secrete insulin at adequate levels [1]. Recent studies suggest that p21 represses β cell – duplication rate and facilitates the recovery of mice from hyperglycemia caused by streptozotocin- induced diabetes [2]. Interference with the induction of apoptosis during diabetes, such as by genetic deletion of chop, rescues β cells from ER stress – related apoptosis [3]. A fundamental question regarding the decision for β-cell survival versus apoptosis is which UPR sub-pathways play a role in this transition and how the latter is being regulated. To that end it is of particular importance to understand which the key sensors of these responses are and how these responses are integrated into the cellular machinery to become protective or disadvantageous to β-cell survival upon short-term or long-term ER stress.

Interplay between Endoplasmic reticulum –homeostasis and protein quality control

The endoplasmic reticulum (ER) is a highly dynamic organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, intracellular calcium homeostasis and lipid biosynthesis. The ER responds to genetic and environmental stimuli and provides a unique folding environment for approximately one third of all proteins. All eukaryotic cells have evolved specific mechanisms to preserve ER functions under conditions of stress [4]. Growing evidence shows that endoplasmic reticulum (ER) stress is an important mechanism linking obesity, insulin resistance and glucose intolerance [5].

The maintenance of ER homeostasis in insulin-secreting beta-cells are extremely important, because when homeostasis is disrupted, misfolded or unfolded proteins may accumulate in the ER, a condition referred to as ER stress [6]. Numerous perturbations in the normal functions of the ER such as hypoxia, alterations in calcium, nutrient availability, mutations in the ER-chaperones, inhibition of protein glycosylation, reduced disulfide bond formation and viral infections, may initiate an evolutionarily conserved cellular response that is designated as the unfolded protein response (UPR). This biochemical response initially aims to restore cellular homeostasis but can eventually promote cell death if ER dysfunction is acute or prolonged [7].

The UPR is comprised of three distinct biochemical branches linked to three intracellular receptors that upon activation initiate specific biochemical events: (1) Activated PERK (endoplasmic reticulum kinase) that causes translational attenuation by phosphorylating, eIF2. However, some specific mRNAs, including ATF4, are translated under these conditions. ATF4 induces transcription of genes encoding adaptive functions including the glucose-regulated proteins. [7]; (2) IRE1a (ER-protein kinase), that induces the alternative splicing, and thus activation of the transcription factor XBP1 mRNA and produces XBP1-spliced (XBP1s). The activated transcription factor upregulates many ER chaperones and genes involved in ERAD, various UPR ‘stress genes’, as well as enzymes involved in membrane biogenesis [7]; (3) ATF6 (activating transcription factor 6) that translocates to the Golgi, where it is processed to proteolytic cleavage that renders the activated form of the protein. ATF6 induces genes involved in ER homeostasis and membrane biogenesis [8];

There is an array of four types of specific cellular responses that are induced during the earlier phases of the UPR and aim to overcome stress:

1. Translational attenuation that occurs in order to overcome the load of ER and is triggered by the PERK-dependent phosphorylation of eIF2.

2. Induction of UPR-related genes, primarily chaperones such as Bip/GRP78 and GRP74 in order to prevent further accumulation of unfolded/misfolded proteins.

3. Enzymes including protein disulfide isomerase (PDI) and SERCA2 (sarcoplasmic ER Ca+2 –ATPase2) that increase the capacity of endoplasmic reticulum for protein folding. In addition during UPR the transcriptional induction of genes involved in the biosynthesis of amino acids occurs, as well as genes implicated in glutathione biosynthesis that protects against oxidative stress.

4. Stimulation of NFkB activity that is a transcription factor acting as a mediator of immune and anti-apoptotic response [9].

If these responses are not sufficient for the re-establishment of the cellular homeostasis, ERAD (ER-associated protein degradation) components are induced in order to eliminate the misfolded proteins [9]. Finally, if cellular damage is deemed irreversible and the pro-survival activity of the UPR is not sufficient for the retention of cellular homeostasis, apoptosis is induced by stimulation of the CCAAT/enhancer-binding homologous protein (CHOP) and activation of the JNK kinase and caspase-12 [9].

Apparently, there is a continuous interplay between survival and death decisions during ER stress that determine the transition from the prosurvival towards the proapoptotic state of UPR [10, 11]. We have provided evidence that chop regulates the expression of the cell cycle regulator p21 during ER stress. Chop suppresses p21 and prompt cells into a pro-apoptotic program. Our findings indicate that CHOP relieves the anti-apoptotic activity of p21 during ER stress. Thus, p21 is implicated in the regulation of the UPR by inhibiting the induction of apoptosis [12].

ER stress- related mechanism of glucose-induced beta cell dysfunction

Prolonged in vitro exposure of beta cell lines or islets to glucose increases ER stress markers in the majority of studies. This is related to the fact that increased glucose levels over-stimulate insulin production that exhausts the insulin producing and secreting activity of the b cells leading to ER stress. The homeostatic consequences of the resulting UPR become apparent by the observation that overproduction of the ER chaperone 78kDa glucose- regulated protein (GRP78) partially prevents glucose-induced beta cell dysfunction in vitro in INS-1 cells (a rat cell line that secretes insulin in response to glucose concentrations in the physiological range) [13].

Evidence for a crosstalk between the generation of ROS and ER stress response

Several experimental results suggest that the production of reactive oxygen species (ROS) results in misfolded protein- mediated cell death. Oxidative protein folding takes place in the ER implicating various oxidoreductases, including protein disulphideisomerase (PDI), PDIR, PDIp, P5 ERp57 and ERp72. In vitro formation, isomerization and reduction of disulphide bonds are catalyzed by PDI. During disulphide bond formation, cysteine residues within the PDI active site accept two electrons from thiol residues in the polypeptide chain substrate resulting in the oxidation of the protein, the reduction of the PDI active site and finally in the production of hydrogen peroxide. Thus, reactive oxygen species (ROS) are produced in a cell during synthesis of disulphide bonds in the ER during oxidative protein folding which may elicit DNA and protein damage [14]. Furthermore, glutathione is involved in the reduction of mispaired disulfide bonds that may destroy the cellular glutathione pool which is crucial for the neutralization of the reactive oxygen species (ROS) and the blockade of the oxidation of the cytosolic proteins. Because oxidative protein folding occurs in the ER and disturbances in protein folding can have damaging consequences, alterations in redox status or the generation of ROS could directly or indirectly (or both) affect ER homeostasis and protein folding. Consequently, cells with a powerful biosynthetic load, deficient UPR, or defective ER-associated protein degradation (ERAD) are more sensitive to oxidative stress [15].

Considering that insulin biosynthesis involves disulphide bond formation oxidative stress plays a causal role in glucose-induced beta cell dysfunction both in vivo and in vitro. Analysis of clinical specimens indicates that both, ER stress and oxidative stress are increased in the islets of individuals with type 2 diabetes while recent studies demonstrate a close interrelationship between oxidative stress and ER-stress in beta cells [15].