Université Libre de Bruxelles Faculté de Médecine
Laboratoire de Médecine Expérimentale
« Identification and characterization of the endoplasmic reticulum (ER)-stress pathways in pancreatic beta-cells. »
Pierre Pirot
Promoter : Décio Laks Eizirik
Co-Promoter : Alessandra Kupper Cardozo
Thèse présentée en vue de l’obtention du Grade de
Docteur en Sciences Biomédicales.
I dedicate this thesis to Guy, Marie-Claire and Félicien,
my beloved parents and brother.
Table of contents
Composition of the thesis jury ……... 4
List of reports constituting this thesis... 5
Summary ... 6
Résumé …... 10
List of abbreviations ... 15
1. Introduction ... 18
1.1 The endoplasmic reticulum ... 18
1.1.1 Physiological role ... 18
1.1.2 Stress of the endoplasmic reticulum ... 22
a) The unfolded protein response (UPR) ... 22
b) Apoptosis ... 25
1.2 Diabetes Mellitus... 34
1.2.1 Definition and epidemiology ... 34
1.2.2. Type 1 diabetes mellitus (T1DM) ... 34
1.2.3.1. Genetics of T1DM ... 34
1.2.2.2. T1DM onset: environmental triggers, recruitment and activation of immune cells ... 37
1.2.2.3. Mediators of beta-cell destruction in T1DM …... 43
1.2.3. Type 2 diabetes mellitus (T2DM) and beta-cell death ... 48
1.3 Endoplasmic reticulum and pancreatic beta-cell ... 50
1.3.1 Physiological role ... 50
1.3.2 ER stress and diabetes mellitus ... 53
1.3.2.1 Lessons from mouse mutants ... 53
a) The Akita mice …... 53
b) Mutations of the PERK-eIF2a pathway ... 54
1.3.2.2. ER stress and human diabetes ... 55
1.3.2.3. Pathophysiological ER stress inducers ... 57
a) ER stress and T1DM: role for cytokines and nitric oxide ... 57
b) ER stress and T2DM: role for free fatty acids and chronic hyperglycemia …. 58 1.4. Chemical ER stress inducers: the SERCA blockers ... 59
1.5. The APOCHIP …... 61
1.5.1. Background ... 61
1.5.2. Microarray procedure ... 61
a) Target preparation and hybridization ... 61
b) Scanning ... 62
c) Data extraction, normalisation and statistical analysis ... 64
2. Aims of the study ... 67
3. Results 3.I. Interferon-g potentiates endoplasmic reticulum stress-induced beta-cell death by reducing beta-cell defense mechanisms ... 68
3.II. Transcriptional regulation of the endoplasmic reticulum (ER) stress gene Chop in pancreatic insulin producing cells ... 77
3.III. Global profiling of genes modified by endoplasmic reticulum (ER) stress in pancreatic beta-cells reveals the early degradation of insulin mRNAs ... 89
4. General discussion and conclusions ... 104
5. Future experiments and perspectives ... 114
6. Thesis annexe summary ... 118
7.Curriculum vitae ... 119
8. Acknowledgments ... 121
9. References ... 123
Composition of the thesis jury:
• Mr. Philippe Lebrun (President) Laboratory of Pharmacology.
Université Libre de Bruxelles (ULB), Faculty of Medicine.
Brussels, Belgium.
• Mr. Decio L. Eizirik (Secretary) Laboratory of Experimental Medicine.
Université Libre de Bruxelles (ULB), Faculty of Medicine.
Brussels, Belgium.
• Mrs. Alexandra K. Cardozo (Co-promoter) Laboratory of Experimental Medicine.
Université Libre de Bruxelles (ULB), Faculty of Medicine.
Brussels, Belgium.
• Mr. Harry Heimberg (Foreign expert) Diabetes Research Center.
Vrije Universiteit Brussel (VUB).
Brussels, Belgium
• Mr. Peter Vandenabeele (Foreign expert) Department for Molecular Biomedical Research.
Flanders Institute for Biotechnology (VIB).
Gent, Belgium.
• Mr. Patrick Robberecht (ULB jury member) Department of Biological Chemistry and Nutrition.
Université Libre de Bruxelles (ULB), Faculty of Medicine.
Brussels, Belgium.
• Mr. Cédric Blanpain (ULB jury member) Interdisciplinary Research Institute (IRIBHM).
Université Libre de Bruxelles (ULB), Faculty of Medicine.
Brussels, Belgium.
• Mr. André Herchuelz (ULB jury member) Laboratory of Pharmacology.
Université Libre de Bruxelles (ULB), Faculty of Medicine.
Brussels, Belgium.
List of reports constituting this thesis:
I. Pirot P, Eizirik DL, Cardozo AK, Interferon-γ potentiates endoplasmic reticulum stress-induced death by reducing pancreatic beta cell defence mechanisms.
Diabetologia. 2006 49:1229-36.
II. Pirot P , Ortis F, Cnop M, Ma Y, Hendershot LM, Eizirik DL, Cardozo AK, Transcriptional regulation of the endoplasmic reticulum (ER) stress gene Chop in pancreatic insulin producing cells. Diabetes. 2007 56:1069-77.
III. Pirot P , Naamane N, Libert F, Magnusson NE, Ørtonft TF, Cardozo AK, Eizirik DL,
Global profiling of genes modified by endoplasmic reticulum stress in pancreatic beta
cells reveals the early degradation of insulin mRNAs. Diabetologia. 2007 50:1006-14.
Summary
The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells.
This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR).
The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6.
The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK.
Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both
type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency
resulting from chronic and progressive destruction of pancreatic beta-cells by the immune
system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the
immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-
inflamatory cytokines interleukin-1β (IL-1β) and interferon−γ (IFN-γ), via nitric oxide (NO)
formation, downregulate expression and function of the ER Ca
2+pump SERCA2. This
depletes beta-cell ER Ca
2+stores, leading to ER stress and apoptosis. Of note, IL-1β alone
triggers ER stress but does not induce beta-cell death, while IFN-γ neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown.
Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis.
In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%).
The first goal of the work was to understand the synergistic effects of IL-1β and IFN-γ
leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray
analysis of primary beta-cells, that IFN-γ down-regulates mRNAs encoding for some ER
chaperones. Against this background, our hypothesis was that IFN-γ aggravates beta-cell ER
stress by decreasing the ability of these cells to mount an adequate UPR. To test this
hypothesis, we investigated whether IFN-γ pre-treatment augments CPA-induced ER stress
and beta cell death. The results obtained indicated that IFN-γ pre-treatment potentiates CPA-
induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-γ, since
neither IL-1β nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-
1E cells. These effects of IFN-γ were mediated via the down regulation of genes involved in
beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as
well as Sec61, a component of the ERAD pathway. This had functional consequences as
evidenced by a decreased basal and CPA-induced activity of a reporter construct for the
unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop.
We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1- β + IFN-γ), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate.
The third objective of this work was to obtain a broad picture of the pancreatic beta-cell
molecular responses during and after (recovery period) a severe ER stress. For this purpose,
we utilized an “in home” spotted microarray, the APOCHIP, containing nearly 600 probes
selected for the study of beta-cell apoptosis. Time-dependent gene expression profiles were
measured in INS-1E cells exposed to CPA. CPA-induced ER-stress modified expression of
183 genes in at least one of the time points studied. Most of theses genes returned to control
levels 3h after CPA removal from the culture medium. We observed full beta-cell recovery
and survival, indicating that these cells trigger efficient defenses against ER stress. Beta-cell
recovery is associated with a sustained increase in the expression of ER chaperones and a
rapid decrease of pro-apoptotic mRNAs following CPA removal. Two groups of genes were
particularly affected by CPA, namely those related to the cellular responses to ER stress,
which were mostly up-regulated, and those related to differentiated beta-cell functions, which
were down-regulated. Among this last group, we observed a 40-90% decrease of the mRNAs
for insulin-1 and -2. These findings were confirmed in INS-1E cells exposed to cytokines or thapsigargin (another SERCA blocker), and in primary beta-cells exposed to the same treatments. This decrease in insulin mRNA expression is due to transcript degradation, most probably caused by IRE-1 activation and triggering of its endoribonuclease activity, as recently described in Drosophila cells.
In conclusion, our work enabled a better understanding of the pancreatic beta-cell responses to ER stress:
1.) We identified a sensitizing effect of IFN-γ to ER stress in beta-cells via downregulation of key ER chaperones.
2.) We observed a differential regulation of Chop transcription by different treatments suggesting distinct responses of pancreatic beta-cells to diverse ER stress inducers.
3.) We provided the first global analysis of gene expression modifications in pancreatic beta-cells following ER stress.
4.) We demonstrated a high capacity of beta-cells to cope and recover from a severe ER stress.
5.) We identified a new protective mechanism against ER stress, namely the
degradation of insulin mRNA which limits the load posed on the ER by insulin
synthesis. This, coupled to a marked increase in ER chaperones and a fast
degradation of pro-apoptotic mRNAs, enables beta cells to recover from ER
stress after the causes of this stress are removed.
Résumé.
Le réticulum endoplasmique (RE) est l’organelle cellulaire responsable de la production et du repliement des protéines sécrétées et membranaires. C’est également le lieu de synthèse des lipides et un des stock principaux de calcium de la cellule. La fonction principale de la cellule bêta pancréatique est la production et la sécrétion d’insuline sur demande afin de réguler la concentration sanguine de glucose. Lors d’une augmentation de la glycémie, la production d’insuline par la cellule bêta représente plus de 50% de la synthèse protéique totale. Cette synthèse d’insuline pose une charge énorme sur le RE. Il apparaît donc évident que toute perturbation de la fonction normale du RE, communément appelée « stress du RE », est nocive pour la cellule bêta. En condition de stress du RE, des protéines mal repliées vont s’accumuler dans la lumière du RE. Les cellules déclenchent alors une réponse adaptative, appelée « Unfolded protein response » (UPR) afin de récupérer la fonction normale du RE. L’UPR est transduite par trois protéines transmembranaire située à la surface du RE : PERK, IRE-1 et ATF6. Les mécanismes protecteurs déclenchés en aval de ces trois protéines senseur du stress du RE incluent: a) l’induction de l’expression de protéines chaperonnes et de protéines foldases qui augmentent la capacité de repliement protéique dans le RE ; b) une atténuation de la synthèse protéique limitant l’arrivée de nouvelles protéines au sein du RE; c) une augmentation d’expression des constituants de l’appareil de dégradation protéique du RE (ERAD) afin de diminuer la charge de protéines mal repliées dans le RE.
Lorsque cette réponse adaptative est insuffisante, une réponse pro-apoptotique est déclenchée.
A ce jour, les mécanismes constitutifs au stress du RE provoquant l’apoptose ne sont pas encore bien caractérisés. Cependant, certains médiateurs potentiels de l’apoptose ont été identifiés: les facteurs de transcription Chop et ATF3, des membres de la famille Bcl-2 la caspase 12 ainsi que la protéine kinase JNK.
De plus en plus d’observations suggèrent que le stress du RE semble être impliqué
dans l’induction de l’apoptose des cellules bêta dans le diabète de type 1 et 2. Le diabète de
type 1 est caractérisé par une déficience en insuline secondaire à la destruction des cellules
bêta par le système immunitaire. Lors de cette attaque auto-immune, les cellules bêta sont exposées aux cytokines émises par les cellules immunitaires infiltrant les îlots pancréatiques.
Notre groupe a récemment montré que le traitement de cellules bêta avec les cytokines pro- inflammatoires interferon-γ (IFN-γ) et interleukine-1β (IL-1β), via un mécanisme dépendent de la production de monoxyde d’azote (NO), provoquent une diminution d’expression de la pompe de calcium SERCA2 située à la surface du RE. Cette diminution de SERCA2 va vider le stock de calcium du RE, provoquant un stress du RE et l’apoptose. L’IL-1β seul provoque un stress du RE sans causer la mort des cellules bêta alors que l’IFN-γ seul n’induit quant à lui ni le stress du RE, ni la mort des cellules bêta. Cependant, la combinaison de ces deux cytokines cause l’apoptose des cellules bêta mais les mécanismes synergiques provoquant cet effet restent à ce jour inconnu.
Le diabète de type 2 est caractérisé par une résistance des tissus périphériques à l’insuline et une sécrétion d’insuline déficiente résultant d’une défaillance des cellules bêta.
L’obésité est un facteur de risque du diabète de type 2. En effet, les patients obèses ont un niveau élevé d’acides gras dans le sang et plusieurs études ont montré que l’exposition chronique de cellules bêta à l’acide gras palmitate induit un stress du RE et l’apoptose.
La première étape de notre travail de recherche fut d’établir un modèle expérimental permettant l’activation spécifique du stress du RE dans les cellules bêta. A cette fin, des cellules d’insulinome (INS-1E) ou des cellules bêta primaires de rat ont été traitées avec de l’acide cyclopiazonique (CPA), un bloqueur réversible de la pompe SERCA. Des expériences de dose-réponse et de cinétique d’induction de l’apoptose ont permis de déterminer les meilleures conditions expérimentales induisant un stress marqué du RE sans provoquer un niveau trop élevé d’apoptose (<25%).
Le premier but de notre travail fut de comprendre les effets synergiques de la
combinaison de l’IL-1β et de l’IFN-γ causant l’apoptose de la cellule bêta. Notre groupe a
précédemment observé, lors d’une analyse par puce à ADN de cellules bêta primaires, que
l’IFN-γ diminue l’expression d’ARN messager codant pour diverses protéines chaperonnes.
Considérant ces précédentes observations, notre hypothèse de départ fut que l’IFN-γ aggrave le stress du RE provoqué par l'IL-1β en diminuant la capacité des cellules bêta à déclencher une UPR adéquate. Afin de vérifier cette hypothèse, nous avons testé si le prétraitement de cellules bêta avec de l’IFN-γ augmente l’intensité du stress du RE et le niveau d’apoptose induit par le CPA dans des cellules bêta. Les résultats obtenus montrent que le prétraitement avec l’IFN-γ intensifie l’apoptose induite par le CPA dans les cellules INS-1E et les cellules bêta primaires de rat. Le prétraitement de cellules bêta avec l’IL-1β ou de faibles doses de CPA n’augmentent pas le niveau d’apoptose induit par une exposition ultérieure au CPA indiquant que les effets de l’IFN-γ sont spécifiques. L’IFN-γ sensibilise les cellules bêta au stress du RE via une diminution de l’expression de gènes impliqués dans les mécanismes de défense contre le stress du RE tels que les chaperonnes du RE BiP, Orp150 et Grp94 ainsi que Sec61, un composant du ERAD. Ces effets ont des conséquences fonctionnelles pour la cellule bêta, notamment une diminution de l’activité d’une construction luciférase sous le contrôle du « unfolded protein response element » (UPRE) au niveau basal et après son activation avec du CPA, ainsi qu’une augmentation de la transcription du gène pro- apoptotique Chop.
Nous avons ensuite étudié les mécanismes moléculaires régulant l’expression du gène pro-apoptotique « C/EBP homologous protein » (Chop) dans des cellules INS-1E en réponse à divers composé induisant un stress du RE tel que les cytokines (IL-1β + IFN-γ), le palmitate et le CPA. Nous avons réalisé une étude détaillée incluant l’utilisation de constructions rapporteur luciférase contenant différentes délétions et mutations du promoteur du gène Chop.
Les résultats obtenus indiquent une régulation distincte du gène Chop en réponse aux trois
différents composés utilisés. Les cytokines (via la production de monoxyde d’azote) et le
palmitate induisent l’expression de Chop par l’intermédiaire de deux éléments régulateurs : le
premier liant les facteurs de transcription de la famille C/EBP et ATF et le second ceux de la
famille AP-1. Le CPA, quant à lui, implique l’utilisation du site liant les membres de la famille de facteurs de transcription C/EBP et ATF et d’un « ER stress response element » (ERSE). Nous avons également démontré par Western blot que les cytokines, le palmitate et le CPA induisent l’expression du facteur de transcription ATF4 et prouvé par gel retard sa liaison au niveau du site de liaison C/EBP-ATF. De plus, nous avons mis en évidence par gel retard et supershift la formation de dimères AP-1 différents en réponse aux cytokines et au palmitate et leur liaison au promoteur Chop.
Le troisième objectif de notre travail de recherche fut d’obtenir un aperçu global de la
modification d’expression génique dans la cellule bêta pancréatique en réponse à un stress du
RE. Dans ce but, nous avons utilisé une puce à ADN élaborée par notre laboratoire,
l’APOCHIP, qui contient près de 600 gènes sélectionnés spécifiquement pour l’étude de
l’apoptose de la cellule bêta pancréatique. Les variations d’expression génique ont été
analysées au cours du temps après induction du stress du RE dans des cellules INS-1E avec
du CPA. Nous avons observé que lorsque le CPA est retiré du milieu de culture, les cellules
récupèrent du stress du RE et survivent, indiquant que ces dernières déclenchent des
mécanismes de défense très efficaces contre le stress RE. L’analyse des puces à ADN nous a
permis d’identifier 183 gènes dont l’expression est modifiée pour au moins une des périodes
de temps étudiées. La plupart de ces gènes retournent à leur niveau d’expression basale 3
heures après avoir stoppé l’induction du stress du RE avec le CPA. Le processus de
récupération des cellules bêta implique entre autre une augmentation soutenue de l’expression
de protéines chaperonnes du RE et une diminution rapide de l’expression de gènes pro-
apoptotiques lorsque le stress du RE est arrêté. Deux groupes de gènes sont particulièrement
affectés par le traitement avec le CPA. D’une part, les gènes impliqués dans la réponse
cellulaire au stress du RE qui sont majoritairement induits, et d’autre part les gènes impliqués
dans la fonction spécifique de la cellules bêta, qui sont eux principalement inhibés. Au sein de
ce dernier groupe, nous avons observé une diminution d’expression de 40 à 90% pour
l’insuline 1 et 2. Cette observation fut confirmée d’une part dans les cellules INS-1E en
utilisant différents agents induisant un stress du RE tels que les cytokines et la thapsigargine (un autre bloqueur de SERCA) et d’autre part dans des cellules bêta primaires exposées aux mêmes traitements. Cette diminution d’expression résulte d’une dégradation de l’ARNm de l’insuline très probablement causée par l’activité endoribonucléase de IRE-1 récemment décrit dans une lignée cellulaire de Drosophile.
En conclusion, notre travail a permis d’obtenir une meilleure compréhension de la façon dont la cellule bêta répond au stress du RE.
1.) Nous avons identifié un effet sensibilisateur de l’IFN-γ au stress du RE via la diminution d’expression de gènes codant pour des protéines chaperonnes du RE.
2.) Nous avons observé des différences dans la régulation du gène Chop en réponse à différents traitements, suggérant que la cellule bêta peut déclencher des types de réponse au stress du RE distinctes en fonction du stimulus déclenchant le stress.
3.) Nous avons également fourni la première analyse globale de modification d’expression génique dans des cellules bêta en réponse à un stress du RE.
4.) Nous avons mis en évidence que les cellules bêta pancréatiques ont une grande capacité de défense et de récupération en cas de stress du RE.
5.) Nous avons identifié un nouveau mécanisme de défense contre le stress du RE à
savoir la dégradation de l’ARN codant pour l’insuline, ce qui limite la charge posée
sur le RE par sa production. Ce mécanisme protecteur couplé à une augmentation
marquée des protéines chaperonnes du RE et une dégradation rapide des ARNs codant
pour des protéines pro-apoptotiques, permettent aux cellules bêta de récupérer du
stress du RE une fois le stress du RE arreté.
List of abbreviations:
AARE: Amino-acid-regulatory element Acyl-CoA: Acyl-Coenzyme A
Ag: Antigen
Akt: Thymoma viral proto-oncogene 1 AP-1: Activator protein 1
APC: Antigen presenting cells
Ask-1: Apoptosis signal-regulating kinase 1 ATF: Activating transcription factor
ATP2C1: ATPase, Ca2+ transporting, type 2C, member 1 Bad: Bcl-associated death promoter
Bak: Bcl2-antagonist/killer
Bap31: B-cell receptor-associated protein 31 Bax: Bcl2-associated X protein
BB rat: Biobreeding rat B-cell: B-lymphocyte
Bcl-2: B-cell leukemia/lymphoma 2 Bid: BH3 interacting domain death agonist BiP: Immunoglobulin heavy chain binding protein bZIP: Basic leucine zipper
C/EBP: CAAT enhancer binding protein cDNA: Complementary DNA
Chop: CCAAT/enhancer-binding protein homologous protein CPA: Cyclopiazonic acid
Crebl-1: cAMP responsive element binding protein-like 1 cRNA: Complementary RNA
CTLA4: Cytotoxic T lymphocyte antigen-4 Cy: Cyanine
DC: Dendritic cell
Dcr2: Phosphatase of IRE-1
Ddit-3: DNA-damage-inducible transcript 3 DNA: Desoxyribonucleic acid
DR-5: Death receptor 5
eEF1A-1: Eukaryotic elongation factor 1A-1 eIF2α: Eukaryotic initiation factor 2 α.
Elk-1: Ets domain protein ER: Endoplasmic reticulum ERAD: ER-associated degradation ERBB3: Receptor tyrosine kinase ErbB3 ERK: Extracellular signal-regulated kinase ERO-1: ER oxidoreductin 1
ERSE: ER stress response element
FADD: Fas-associated protein with death domain FasL: Fas ligand
FDR: False discovery rate FFA: Free fatty acids
GAD65: Glutamic acid decarboxylase 65
GADD-153: Growth arrest and DNA damage 153 GADD-34: Growth arrest and DNA damage 34
GAS: IFN-γ -activated sequences
GCN2: General control of amino-acid synthesis 2 Gip: glucose-dependent insulinotropic polypeptide Glp-1: Glucagon-like peptide 1
GLUT: glucose transporter
GRP94: Glucose-regulated protein 94 HFD: High fat diet
HLA: Human leukocyte antigen HNF4α: Hepatocyte nuclear factor 4 α HRI: Heme regulated translational inhibitor IAP: Inhibitor of apoptosis protein
IAPP: Islet amyloid polypeptide IFN-R: Interferon receptor IFN-γ: Interferon γ
IL-1R: Interleukin-1 receptor
IL-1RAcP: Interleukin-1 receptor accessory protein IL-1β: Interleukin-1β
IL-2: Interleukin-2
iNOS: Inducible nitric oxide synthase Ins-1 or -2: Insulin-1 or -2
INS-1E: Insulin secreting (rat) cell line
IP-3 receptor: Inositol 1,4,5-trisphosphate receptor IRAK: Interleukin receptor associated kinase IRE-1: ER-to-nucleus signal kinase 1
IRR-1: Insulin receptor-related receptor type 1 IRS-1: Insulin receptor substrate-1
IκB kinase: Inhibitor of NF-κB kinase IκB: Inhibitor of NF-κB
Jak: Janus kinase
JNK: c-Jun N-terminal protein kinase KATP: ATP sensitive potassium channel
LOWESS: Locally weighted scatterplot smoothing LyP: Lymphoid tyrosine phosphatase
MAPK: Mitogen activated protein kinase
MAPKK: Mitogen activated protein kinase kinase
MAPKKK (MAP3K): Mitogen activated protein kinase kinase kinase MCP-1: Macrophage chemoattractant protein-1
MEF: Mouse embryonic fibroblast MEKK-1: MAPK/Erk kinase kinase MHC: Major histocompatibility complex MIN-6: Mouse insulinoma cell
MIP-1α: Macrophage inflammatory protein-1α MPTP: Mitochondrial permeability transition pore MyD88: Myeloid differentiation factor 88
NCX: Na+/Ca2+ exchanger NF-Y: Nuclear factor Y NF-κB: Nuclear factor κ B NO: nitric oxide
NOD mice: Non obese diabetic mice
ORF: Open reading frame
ORP-150: Oxygen-regulated protein 150 P2c: Protein 2C
P58IPK: 58KDa inhibitor of the double-stranded RNA-activatedprotein kinase PARP: Poly(ADP-ribose) polymerase
PDI: Protein disulfide isomerase PEK: Pancreatic eIF2α kinase
PERK: Protein kinase RNA-dependent-like ER kinase PKC: Protein kinase C
PKR: Double-stranded-RNA-dependent protein kinase PLC: Phospholipase C
PLN: Pancreatic lymph node
PMCA: Plasma membrane Ca2+ ATPase PP1C: Protein phosphatase 1C
PPARγ: Peroxisome proliferator-activated receptor γ
PTPN11: Protein tyrosine phosphatase non-receptor type 11 PTPN22 : Protein tyrosine phosphatase non-receptor type 22 QC: Quality control
RER: Rough endoplasmic reticulum Rip: Receptor-interacting protein RNA: Ribonucleic acid
ROS: Reactive oxygen species S1P: Site protease 1
S2P: Site protease 2
SER: Smooth endoplasmic reticulum
SERCA: Sarcoendoplasmic reticulum Ca2+ ATPase SH2B3/LNK: SH2B adaptor protein 3
SREBP: Sterol regulatory element binding protein Stat-1: Signal transducers and activators of transcription T1DM: Type 1 diabetes mellitus
T2DM: Type 2 diabetes mellitus T-cell: T-lymphocyte
TCF7L2: Transcription factor 7-like 2 TCR: T-cell receptor
TNF-R1: Tumor necrosis factor receptor R1 TNF-α: Tumor necrosis factor α
Tollip: Toll-interacting protein
TRADD: TNF receptor-associated death domain protein TRAF: TNF receptor associated factor
TRAF-2: Tumor necrosis factor receptor-associated factor-2 TRAFD1: TRAF-type zinc finger domain containing 1 TRB-3: Tribble-3
UPR: Unfolded protein response
UPRE: Unfolded protein response element VNTR: Variable number of tandem repeat WHO: World Health Organization Xbp-1: X-box binding protein
1. Introduction
1.1 The endoplasmic reticulum 1.1.1 Physiological role
The endoplasmic reticulum (ER) is a membranous network, formed in continuity with the outer membrane of the nuclear envelope. It delimits a cellular compartment with a chemical composition distinct from the cytoplasm [1]. The main functions of this organelle are: a. production, folding and modification of membrane and secreted proteins; b. lipid biosynthesis; c. calcium storage and signaling. These functions are specific for distinct areas of the ER. The ER is composed of two interconnected parts, which have different structure and function: 1) The Rough Endoplasmic Reticulum (RER), which has a stacked cistern structure with a granular aspect due to binding of ribosomes to its external surface. The RER is mainly involved in the synthesis and processing of membrane and secreted proteins. Every cell contains a RER but this structure is more abundant in cells with a high secretory function such as the plasmocytes which produce antibodies, and the pancreatic beta-cells which synthesize and secrete insulin; 2) The Smooth Endoplasmic Reticulum (SER) which has a tubular shape without ribosomes on its external surface. The SER is mainly associated with lipid biosynthesis, and its function and abundance differs depending on the cell type. Cells have also a form of SER called the transitional ER, which is involved in vesicle packing and transport of proteins from the ER to the Golgi. More specific functions of the SER involve detoxification of hydrophobic substances in liver cells, production of steroid hormones in cells of the ovary and testes and calcium uptake and release in the process of muscle contraction in myocytes [1].
The composition of the intra-ER milieu is oxidative and it has a calcium concentration
hundred to thousand times higher than in the cytoplasm [2]. The ER intraluminal free calcium
concentration is maintained by: 1) Calcium flux between the ER and the cytoplasm, mediated
mainly by the sarcoendoplasmic reticulum pump Ca
2+ATPase (SERCA) (entrance of Ca
2+)
and the IP-3 and ryanodine receptor (exit of Ca
2+); 2) ER resident protein Ca
2+buffers,
including calreticulin, calbindin and calnexin that bind calcium and release it upon demand
(Figure 1A). Several of these ER residents Ca
2+binding proteins are also chaperones. This
specific environmental conditions, together with the presence of ER resident chaperones, is
crucial for the correct folding of proteins inside the ER [2]. Indeed, the luminal ER calcium
interacts and modulates the activity of Ca
2+sensitive ER resident chaperones such as BiP,
GRP94, ORP150, calnexin and calreticulin, among others. Several Ca
2+binding chaperones
also regulate the function of other chaperones. For example, the protein disulfide isomerase
(PDI), which is involved in disulfides bond formation, is regulated via a Ca
2+dependent
binding with calreticulin [2,3]. The oxidative condition of the ER milieu is mainly maintained
by the low ratio between reduced/oxidized glutathione, which is around 1:1 to 1:3 in the ER
as compared to >50:1 in the cytosol [2,4]. This oxidative environment enables cysteine
oxidation in nascent proteins, an important step for the formation of disulfide bonds [2]. In
addition to disulfide bond formation, another common post-translation modification made in
the ER is the N-glycosilation of proteins on their asparagines, when included in a specific
amino-acid consensus sequence (i.e. asparagine-X-threonine/serine, where X can be any
amino acid residue except for proline). Besides its role in protein folding, ER resident
chaperones are also involved in a quality control (QC) system that targets misfolded proteins
to the protein ER associated degradation (ERAD) pathway [5] (Figure 1B). The QC system
involves two main kinds of chaperone complexes: one containing BiP and the other
containing calnexin and calreticulin, which are more specific for the glycoproteins. It remains
to be clarified, however, how the QC “decides” that a protein cannot be folded correctly and
must be degraded via the ERAD.
S1P
EDEM
ERAD (pr oteasome)
ATF6
ATF6 ATF6ATF6
XBP-1 (unspliced) XBP-1 (spliced)
AAAAAAAAAAAAAA PstI P PPERK
PERK
ATF6ATF6
GOLGI
Bip APOPTOSISS2P
CYT OPLASM
Ca2+ER STRESS
BipPERK
Bip
Calbind in
Calreticulin Calnexin
Ca2+ [1mM]
Ca2+ [100nM]
P P
Bip
ORP-150
Sec61Grp94
IRE-1
CASPASE-12 CASPASE-12 TRAF-2
Bip Bip
XBP-1
Translational attenuation
APOPTOSIS Caspase 9Caspase 3,7
Ask1
JNK IRE-1 ATF6 éER ChaperonesXBP-1
ATF4eIF2α
eIF2αP
PERK
éChop/GADD-153ATF6 ATF4ATF6 éERAD ComponentsXBP-1 éXbp-1ATF6 XBP-1
NUCLEUS
é [Ca
2+] cyt.
Calcineurin Calpain
Bcl-2 Bcl-XL SERCA
IP3 rec.
ryanodine Bak Bax
BAP-31
IRE-1
IRE-1
BipBipBip
BaxBak Cyt. CApoptosome
p58
MIT O. A C B
F E D
G H
GADD-34PP1CI
Pro-caspase-8LCaspase-8
Bid
BAP-20 BAP-20 BAP-29
J
Figure 1 : Scheme of the unfolded protein response (UPR) cytoprotective and pro-apoptotic mechanisms triggered in response to ER stress.(A) Maintenance of ER calcium homeostasis is a dynamic process relying on calcium uptake from the cytosol by Serca pumps, release of calcium by the IP3 and ryanodine receptors and the buffer capacity of several chaperones such as calreticulin, calbindin and calnexin. (B) Protein folding is a finely tuned process involving ER resident chaperones, which may both help proteins to fold or contribute to their degradation by the ERAD in case of wrong conformation. In response to ER stress, cells trigger a cytoprotective mechanism called the UPRs which is transduced by three trans-membrane proteins, namely PERK, IRE-1 and ATF6. (C) PERK dimerization and activation leads to phosphorylation of eIF2α, which inhibits general protein translation and activates ATF4. (D) Subsequent dimerization and activation of IRE-1 leads to XBP-1 mRNA unconventional splicing, translation of the spliced mRNA, and activation of XBP-1-regulated genes including ERAD components, ER resident chaperones and XBP-1 itself. (E) Bip dissociation from ATF6 allows its transport to the Golgi where cleavage by S1P and S2P facilitates release of the cytoplasmic part of ATF6 which then migrates to the nucleus where it regulates the expression of target genes such as ERAD components, ER resident chaperones, XBP-1 and Chop.Failure of the UPR to restore a normal ER function leads to activation of an apoptotic response that may involve: (F) an ER calcium release via Bap-20, Bak/Bax and ryanodine and IP3-receptor provoking a Bcl-2 family mediated release of cytochrome-C from the mitochondria, (G) the Caspase-8 and the mitochondria death pathway, (H) the JNK pathway (I) the transcription factor Chop and (J) the Caspase-12 although the importance its function in ER stress mediated cell death remain highly controversial. See text for further description, references and definitions of abbreviations.
1.1.2 Stress of the endoplasmic reticulum:
a.) The Unfolded Protein Response (UPR):
Maintenance of ER homeostasis and protein folding are tightly regulated and interdependent processes. Disturbances in one of these processes will profoundly influence the other. For instance, perturbation of intra-ER calcium concentration can affect protein folding while accumulation of misfolded protein can disturb calcium homeostasis [2].
Pathological events provoking serious perturbation of the ER homeostasis, excessive protein translation exceeding the folding capacity of the ER and mutations affecting the correct folding of proteins lead to accumulation and aggregation of misfolded proteins in the ER lumen provoking an ER stress. The cells respond to this kind of stress by triggering a cytoprotective response called the unfolded protein response (UPR), which aims to restore normal ER function [6,7] (Figure 1). The UPR is mediated by three trans-membrane ER proteins, namely protein kinase RNA-dependent-like ER kinase (PERK), inositol requiring ER-to-nucleus signal kinase 1 (IRE-1) and activating transcription factor 6 (ATF6). Under physiological conditions these proteins are maintained in an inactive state due to binding of their ER luminal regulatory domain to the chaperone BiP [8,9]. Upon ER stress, BiP migrates to the lumen of the ER to bind misfolded proteins; release of Bip leads to activation of transmembrane proteins as described below.
PERK, initially called pancreatic eIF2α kinase (PEK), is a type 1 ER-transmembrane
protein with a cytoplasmic serine/threonine kinase activity [10] (Figure 1C). In response to
ER stress, BiP dissociates from the PERK ER-luminal domain, promoting its activation by
oligomerization and trans-phosphorylation [9]. The principal substrate of PERK is the
eukaryotic initiation factor 2α (eIF2α), a protein involved in the formation of the translational
initiation complex. Phosphorylation of eIF2α by PERK provokes its inactivation, leading to
inhibition of global protein synthesis [11]. This is a protective mechanism, limiting the burden
posed on the stressed ER by the arrival of newly formed proteins [11]. Prolonged translational inhibition, however, becomes deleterious for the cell [12,13]. Thus, extended translational block causes the disappearance of proteins with short half life, such as cyclin D1 [13] (leading to cell cycle arrest) and anti-apoptotic proteins from the Inhibitor of Apoptosis Proteins (IAPs) family [14]. The duration of the translational block depends on a tightly regulated activation of the PERK/eIF2α pathway. This is mediated by two protein phosphatases, namely p58
IPKthat dephosphorylates PERK [15] and the GADD-34/PP1C complex that dephosphorylates eIF2α [16], both contributing to reactivation of protein translation. Of note, the phosphorylated form of eIF2α decreases general protein translation, while allowing preferential translation of the activating transcription factor 4 (ATF4) and of other key proteins involved in the UPR [17]. The fact that the translation of ATF4 escapes the general inhibition of protein synthesis is due to the particular structure of its transcript, which contains several micro-open reading frames (µORF) in front of the main one [17]. The ATF4 targets include genes involved in amino-acid metabolism, resistance to oxidative stress and the pro- apoptotic transcription factor Chop (this pro-apoptotic function is described below in 1.1.2.b) [18-20]. Translational block is not a specific feature of ER stress, since eIF2α can be phosphorylated by other protein kinases, namely double-stranded-RNA-dependent protein kinase (PKR), general control of amino-acid synthesis 2 (GCN2), and heme regulated translational inhibitor (HRI) which are activated by ER independent mechanisms. These mechanisms include viral infection for PKR, amino-acid starvation for GCN2 and heme deficiency for HRI [19].
IRE-1 is a type 1 ER-transmembrane protein which contains both a serine/threonine kinase
and cytoplasmic RNase domains [21] (Figure 1D). Activation of IRE-1 in response to ER
stress occurs by release of BiP from its regulatory domains leading to dimerisation and trans-
phophorylation and provoking the subsequent activation of its RNase activity [9]. IRE-1
mediates the unconventional splicing of the mRNA encoding the transcription factor X-box binding protein-1 (Xbp-1). The processing of Xbp-1 transcript by IRE-1 involves the removal of a 26 bp intron leading to an ORF frame shift and translation of the functional form of Xbp- 1 [22]. Xbp-1-target genes include ER resident chaperones and components of the ER associated degradation pathway (ERAD) [23-25]. Altough Xbp-1 was previously reported to bind to ERSE sequence such as the one present in the promoter pro-apoptotic transcription factor Chop [20,26], overexpression of the spliced form of XBP-1 in MEF is not able to induce Chop while a chemical ER stress inducer is able to stimulates its expression [23].
Spliced Xbp-1 also regulates its own promoter thus increasing total Xbp-1 expression [27]. It was recently shown that Xbp-1 splicing is not the only target of IRE-1 nuclease activity. Thus, it was observed in Drosophila cells under ER stress conditions that IRE-1 mediates the degradation of mRNAs encoding for secreted and membrane proteins [28]. A similar phenomenon was recently reported in Hela cells, causing the degradation of the CD59 mRNA upon ER stress [29]. These findings suggest a new protective mechanism for the UPR downstream of IRE-1, namely degradation of mRNAs encoding for ER targeted proteins; this would complement the translational repression mediated by eIF2α phosphorylation, further reducing the load placed by newly synthesized proteins on the stressed ER [30]. Evidence for a similar phenomenon in beta-cells was provided by the present work (See 3.III)
ATF-6 is a type II ER-transmembrane protein with a basic leucine zipper (bZIP) DNA
binding domain and a transcriptional activation domain in its cytoplasmic part [31] (Figure
1E). ER stress-induced activation of ATF6 occurs via release of BiP from its N-terminal
domain domain unveiling a Golgi localization signal [8]. This leads to ATF6 translocation to
the Golgi apparatus and subsequent sequential cleavage by the site 1 and site 2 proteases (S1P
and S2P) resulting in the release of its cytoplasmic part and further translocation into the
nucleus. ATF-6 target genes include ER resident chaperones, components of the ER
associated degradation pathway (ERAD), the pro-apoptotic transcription factor Chop, the phosphatase p58
IPKand Xbp-1 [20,27,32,33].
In conclusion, the UPR has three main mechanisms to correct the ER stress: 1) induction of ER chaperones in order to increase the folding capacity of the ER; 2) attenuation of total protein biosynthesis to avoid overloading the stressed ER with new proteins; 3) upregulation of ERAD components, which decreases the unfolded protein load of the ER.
Failure of the UPR to restore a normal ER function leads to activation of an apoptotic response by the mechanisms described in the section below.
b.) Apoptosis:
The process of apoptosis initiated by the UPR aims to eliminate cells which reached an irreversible state of ER stress [6,7,34]. It is not yet clearly understood how the irreversible state of ER stress is sensed and when exactly is apoptosis triggered by the ER stress. However several putative pathways mediating and regulating apoptosis in response to ER stress were identified and are described below.
Caspases are a well characterized group of cysteine proteases involved in the integration of death signals and the further execution of the apoptosis [35]. The caspase family is divided in several groups including the initiator caspases -2, -8, -9, -10 and -12, and the effector caspases -3, -6 and -7. A recent study of mouse embryonic fibroblasts deficient for the caspases-3, -7 and -9, alone or in combination, demonstrate a requirement for caspases -3 and -9 to induce apoptosis under ER-stress conditions [36]. Caspase activation requires the proteolitical cleavage of a domain of its inactive pro-caspase form. This is usually mediated by another caspase, but initiator caspase activation can also occur via a caspase independent cleavage.
Although mainly regulated by proteolytic cleavage, caspases can also be regulated by other
mechanisms, including for instance phosphorylation by Akt which inhibits activity of caspase
9 [37] and ubiquitinilation by inhibitor of apoptosis proteins (IAPs) which cause caspase
inhibition [38]. Caspases mediate their pro-apoptotic effects via proteolitic cleavage of specific targets containing a particular amino-acid consensus sequence. Caspase targets involves caspases themselves, regulators of apoptosis (e.g. protein kinases; Bcl-2 family members) as well as proteins concluding the apoptotic cascade and truly provoking cell death, such as cell cycle regulators, factors involved in cellular repair, proteins involved in essential cellular functions and structural proteins [35]. ER stress induces apoptosis by triggering several mechanisms that lead to activation and regulation of the caspase cascade [39].
Two initiator caspases, namely caspase-8 and -12 , are proposed to trigger cell-death signaling cascades upon ER-stress [39]. The caspase-8 (Figure 1G) is activated in response to extrinsic death signal downstream of death receptors of the TNF-α family localized at the plasma membrane (See 1.2.2.3). It was also shown in human epithelial cells that procaspase-8L, an N-terminus extended isoform of procaspase-8, can interact with a proteic complex containing Bap-31 and -29 at the cytosolic face of the ER membrane [40]. Although the activation of caspase-8 in ER stress remains elusive, it could involve the processed form of Bap31, namely, Bap20 [40,41]. Interestingly, caspase-8 deficiency delays the onset of apoptosis in mouse embryonal carcinoma (P19 cells) upon ER stress [42]. Substrates of caspase-8 includes Bap31 and Bid [41-43]. Bap31 is an integral membrane protein which mediates the transport of certain membrane proteins from the ER to the Golgi [41,44]. Cleavage of Bap-31 into Bap-20 stimulates Ca
2+release from the ER and favors the release of cytochrome c from the mitochondria (See below and [41]). The cleaved form of Bid (tBid) favors the oligomerization of Bak, thus leading to release of cytochrome c from the mitochondria (See below and [45]).
In agreement with these data, it was shown that caspase-8 deficient P19 cells are resistant to
ER-stress-induced cytochrome c release [42]. In addition, it was also observed that the
specific caspase-8 inhibitor Z-IETD-FMK suppresses the cleavage of BAP-31 in response to
ER stress triggered by bile acids in human hepatoma (HepG2 cells) or by virus infection in
murine astrocytes [44,46]. Caspase-12 (Figure 1J) is an ER transmembrane protein that was suggested to act as a specific mediator of ER-stress-induced apoptosis in rodent cells [39,47].
Pro-caspase-12 is recruited to IRE-1 via the tumor necrosis factor receptor-associated factor-2 (TRAF2) thus leading to its activation by clustering and autoprocessing. A possible alternative activation mechanism involves caspase-12 cleavage by calpain, a Ca
2+sensitive cytoplasmic protease, which is activated by release of ER calcium [48]. The caspase cascade initiated by caspase-12 leads to caspase-9 activation, which in turn activates the downstream caspases 3 and 7. The role of caspase-12 as an important mechanism of ER-stress induced cell death remains, however, unclear. The human orthologue of caspase-12 acquired several mutations during evolution causing a loss of function [39,49] and caspase-12 deletion in rodent models generates contradictory results, depending on the model. Thus: a. loss of caspase-12 in mouse embryonal carcinoma cells (P19) only weakly modifies apoptosis in response to the ER-stress inducer tunicamycin [42]; b. depletion of caspase-12 in murine embryonic telencephalic cells via RNA interference does not prevent caspase-7 and -3 activation upon ER stress triggered by tunicamycin [50]; c. lack of caspase-12 does not protect murine pro B-cell line (FL5.12) cells against ER stress induced cell death [51]; d. in contradiction with these observations, mouse embryonic fibroblasts, kidney and neuronal cells from caspase-12 knock mice are more resistant to ER stress-induced cell death than wild-type cells [52]. These observations were recently re-evaluated in embryonic fibroblasts from another caspase-12 deficient mice, and it was shown that these cells do not have any protection against cell death triggered by several chemical ER stress inducers [53].
Another important ER-stress induced mechanism leading to apoptosis involves the Bcl-2 family members and the mitochondria [54-56]. The Bcl-2 family is subdivided in pro- apoptotic (i.e. Bad, Bax, Bak, Bim etc) and anti-apoptotic (i.e. Bcl-2, Bcl-XL etc) members.
The pro-apoptotic subgroup is further subdivided in two categories. The first contains proteins
affecting membrane permabilisation, such as Bak and Bax. The second includes sensors of death signals which have a regulatory effect on the other members of the Bcl-2 family, the so- called BH3-only proteins including Bim, Bik, Bad, Bid, Puma, Noxa and Hrk [45]. The balance between pro- and anti-apoptotic Bcl-2 members is important to their function since heterodimerization between pro- and anti-apoptotic proteins inhibit the biological activity of their respective partners. The main mechanism of action of Bcl-2-related proteins involve permeabilization of cellular membranes via Bak/Bax pore formation. More particularly, modification of mitochondrial outer membrane permeability leads to release of cytochrome C from the mitochondria matrix. Once factors in the cytoplasm, namely Apaf-1 and pro-caspase-9, forming the apoptosome which trigger the caspase cascade via proteolytic activation of caspase-9. Although there is solid evidence for the involvement of Bcl-2 proteins in ER stress-induced cell death, how are they regulated in response to ER stress is not very well understood [55] (Figure 1F). A possible mechanism involves the rise in cytoplasmic calcium secondary to ER stress-induced ER Ca
2+leak. The mechanisms provoking calcium release in response to ER stress remain to be
clarified, but potential mediators include the physiological ER channels IP3 and ryanodine
receptors, pore formation by Bax and Bak and Bap-20, the cleaved form of Bap-31
[41,57,58]. An increase in cytoplasmic Ca
2+leads to activation of two Ca
2+-dependent
enzymes, calcineurin and calpain. Calcineurin is a serine/threonine phosphatase which
dephosphorylates the BH3-only member Bad enabling its binding to Bcl-XL and consequent
inhibition of Bcl-XL’s anti-apoptotic effects [59]. Calpain is a cysteine protease involved in
the Ca
2+dependent cleavage of cytoplasmic Bax, leading to conformational changes and
translocation to the mitochondria [60]. It was recently shown in human leukemic cells
exposed to the ER stress inducer thapsigargin that Bax cooperates with the mitochondrial
permeability transition pore (MPTP), inducing apoptosis via release of cytochrome-C [61].
Although the MPTP is required for Bax pro-apoptotic effects, blocking Bax does not prevent MPTP pore formation and induction of caspase-independent death mechanisms [61]. In addition to modulation by Ca
2+, the activity of Bcl-2 members can also be regulated downstream of ER stress via Chop and JNK. Chop downregulates Bcl-2 expression and promotes the translocation of Bax and Bak to the mitochondria while JNK affects activity of Bcl-2 members activity via phosphorylation (see below) [62,63].
C/EBP homologous protein (Chop), also known as Growth Arrest and DNA Damage
153 (GADD-153) or DNA-damage-inducible transcript 3 (Ddit-3) is an important ER stress-
induced transcription factor (Figure 1I). It provokes apoptosis by aggravating cellular stress
conditions, including ER stress itself, and by affecting the expression and/or function of
factors involved in the caspase cascade regulation [20]. Chop was first identified in a large
scale screening aiming to discover genes induced in response to DNA damage caused by UV
irradiation [64]. Its expression was also observed in rat beta-cells exposed to alkylating agents
[65]. Chop was initially considered as a growth arrest and DNA-damage inducible gene, but
further studies showed that several of the stimuli used to induce it also provoked ER stress
and that known ER stress inducers lead to a high induction of this gene [66]. The Chop
transcript is expressed at low levels under normal conditions, but it is robustly induced in
response to stimuli that provoke cellular stress. Several regulatory binding sites were
previously reported in the Chop promoter, including an AP-1 site, a C/EBP-ATF composite
site, two amino-acid-regulatory elements (AARE) and two overlapping ER stress response
elements (ERSE) [20]. AARE and the two ERSE bind transcription factors activated
downstream of the three UPR pathways, namely ATF6, Xbp-1 and ATF4. In addition to these
transcriptional enhancers, Chop expression can be inhibited by the transcriptional repressor
ATF3 that is induced downstream of the PERK and IRE-1 pathways [67]. Different
combinations of transcription factors were previously reported to bind to the Chop promoter
depending of the cell type and the inducing stimuli [33,68-71]. In addition to its transcriptional regulation, the Chop expression can also be regulated by stabilization of its mRNA [69,72]. Chop is a transcription factor of the CAAT Enhancer Binding Protein (C/EBP) family. It has a dual role, acting either as a dominant negative inhibitor of C/EBPs or as a gene activator. Chop forms heterodimers with other C/EBP family members hampering their binding to the consensus C/EBP binding sequence [73]. The Chop-C/EBP heterodimer, however, is able to bind to another consensus binding sequence and activate its own target genes [74]. Chop enhances the transcriptional action of AP-1 by tethering to the AP-1 complex without direct binding to DNA [75]. Chop activity can be regulated at a post translational level: phosphorylation of Chop by p38 MAPK enhances its transcriptional activity, while phosphorylation by casein kinase II has the opposite effect [76,77]. p38 MAPK is a substrate of Ask-1, which is recruited to the IRE-1/TRAF2 complex upon ER stress [78].
Possible mechanisms for Chop-induced apoptosis include: 1) Induction of TRB3, a member of the signaling regulator scaffold-protein family Tribbles [79,80]. TRB3 contributes to apoptosis via inhibition of Akt by binding to its activation domain and preventing its phosphorylation [80]. Akt is a serine/threonine kinase that inhibits caspase-9 by phosphorylation [37]. TRB3 can also act in a feedback regulatory loop by directly interacting with Chop and/or ATF4 by inhibiting its transcriptional activity [79,81]; 2) Inhibition of Bcl-2 and depletion of cellular glutathione. This disturbs the cellular redox status, leading to exaggerated production of reactive oxygen species and induction of cell death by oxidative injury [62]; 3) Induction of the GADD-34, a member of the GADD-34/PP1C eIF2α protein phosphatase complexe, that reactivates protein biosynthesis initially blocked by the UPR [82].
This may aggravates the ER stress by the arrival of new proteins. GADD-34 also acts as a
feedback regulator of Chop since ATF4 induction is under the control of the posphorylated
form of eIF2α [17]; 4) Increased expression of the carbonic anhydrase VI, an enzyme
catalyzing the reversible hydratation of CO
2to H
2CO
3[83]. This results in acidification of the intracellular milieu (because of the dissociation of H
2CO
3into H
+and HCO
3-) affecting many cellular process and provoking apoptosis via enhancement of Bax activity at low pH and/or increase in pro-caspase-9 cleavage by the apoptosome, which is more efficient under acidic conditions [83,84]; 5) Upregulation of the death receptor 5 (DR5), with activation of the caspase cascade by the extrinsic pathway via caspase-8 [85,86]; 6) Stimulation of the expression of ER oxidoreductin 1 (ERO1) which promotes oxidizing conditions in the ER, leading to accumulation of reactive oxygen species that contribute to cell death [82]. A scheme summarizing Chop transcriptional and post translational regulation as well as its target genes and their effects is presented in Figure 2.
Another ER stress-induced pro-apoptotic pathway involves the c-Jun N-terminal
protein kinase (JNK) activated downstream of the IRE-1 branch of the UPR (Figure 1H). JNK
is a member of the mitogen activated protein kinase (MAPK) family which has either pro- or
anti-apoptotic functions depending of the cell type, nature of the triggering stimuli and
activation of other signaling pathways modulating its function [63,87]. JNK is activated
downstream of a cascade of phosphorylation involving at least three protein kinases, namely
an initiator MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and the
terminal MAPK (for instance JNK). In response to ER stress, IRE-1 recruits the adaptator
protein TNF receptor-associated factor 2 (TRAF-2) and the MAPKK Apoptosis signal-
regulating kinase 1 (Ask-1) forming a complex which triggers the phosphorylation cascade
leading to JNK activation [88,89]. The mechanism of action of JNK occurs via serine
phosphorylation of target proteins including transcriptions factor such as c-Jun, ATF2, Elk-1,
p53, Myc as well as non-transcription factors such as members of the Bcl-2 family, provoking
either activation or inhibition [63]. A scheme describing JNK target genes and the effects of
JNK-induced phosphorylation is shown is Figure 3.
TRB3Bcl-2ERO-1Death receptor 5GADD34Carbonic anhydrase VI
APOPT OSIS
Reactivation of protein biosynthesis
Inhibition of Akt activity Perturbation of cellular redox status Acidification of intracellular
milieu
Formation of reactive
oxygen species
Promote extrinsic pathway
Chop
p38 MAPK Casein kinase IIPost-translational regulators
IRE-1 Xbp-1
ATF6 ATF3ATF4eIF2α
PERK
GCN2 PKR HRI
TRAF2/Ask1 JNK
AP-1
ER stress dependent pathwaysER str
ess independents pathways
Figure 2: Schematic representation of Chop transcriptional and post translational regulation, and presentation of Chop target genes and their downstream effects. Further information in provided in the text. .
Figure 3: JNK target genes and the effect of JNK-induced phosphorylation on their activity. *The divergent effects of JNK on Bad activity have been attributed to the ability of JNK to phosphorylate distinct sites namely serine (Ser) 128 or threonine (Thr) 201. (Adapted from [57]).
1.2 Diabetes mellitus
1.2.1. Definition and Epidemiology.
According to the World Health Organization (WHO) diagnosis criteria, diabetes mellitus includes a set of metabolic diseases characterized by a fasting blood glucose ≥7.0 mmol/l and/or an elevated blood glucose level (>11.1 mmol/l) measured 2h after ingestion of 75g of glucose by a fasting adult [90]. It was estimated that 2.8% of the worldwide population was diabetic in 2000 and predicted that the prevalence of this disease will reach 4.4%, namely 336 millions of individuals, in 2030 (Figure 4). We are thus dealing with a growing diabetes epidemic, with devastating effects on human health. There are two major types of diabetes mellitus, namely type 1 and type 2. Type 1 diabetes mellitus (T1DM), previously called insulin dependent diabetes mellitus (IDDM), accounts for about 10-15 % of all cases of diabetes [91]. T1DM results from an autoimmune destruction of the pancreatic beta-cells leading to severe or absolute insulin deficiency. Type 2 diabetes mellitus (T2DM), previousely called non insulin dependent diabetes mellitus (NIDDM), is characterized by a resistance of the peripheral tissues to insulin and a defective insulin secretion secondary to progressive beta-cell failure [92].
1.2.2. Type 1 diabetes mellitus (T1DM).
T1DM is a multifactorial disease where a genetic predisposition combines with environmental trigger(s) to induce the activation of a specific autoimmune destruction of beta- cells [91,93-95].
1.2.2.1. Genetics of T1DM.
Several loci giving a risk to develop T1DM were identified [94,96,97]. Among them,
the human leukocyte antigen (HLA) locus is by far the most common predisposing
polymorphism [94,96]. Other well documented predisposing loci include the insulin locus, the
cytotoxic T-lymphocyte antigen 4 (CTLA4) locus and the phosphatase non-receptor type 22
The Americas 2000: 33 millions 2030: 66.8 millions
Africa
2000: 7 millions 2030: 18.2 millions
Europe
2000: 33.3 millions 2030: 48 millions
Middle East 2000: 15.2 millions 2030: 42.6 millions
Asia and Oceania 2000: 82.7 millions 2030: 190.5 millions
<3%
3-5%
6-8%
>8%
Prevalence of Diabetes
Figure 4: Overview of the world diabetes prevalence by regions.
(Adapted from http://www.who.int/entity/diabetes/actionnow/en/mapdiabprev.pdf )
