the role of er stress and translation in cell death
TRANSCRIPT
Randal J. Kaufman, Ph.D.
Departments of Biological Chemistry & Internal MedicineInvestigator, Howard Hughes Medical Institute
University of Michigan Medical Center
Ann Arbor, MI
The Role of ER Stress and Translation in Cell Death
Calcium storage and gated release
Oxidative protein folding
Quality control
ER-associated protein degradation
Core oligosaccharide biosynthesis
Lipid and sterol biosynthesis
The Endoplasmic Reticulum:
Chinese hamster ovary cell
Factor VIII Expression Induces ER Stress
Activation of the Unfolded Protein Response
Calcium depletion thapsigargin, ionophore
Altered glycosylation tunicamycin, castanospermine
Nutrient deprivation (GRP) glucose, hypoxia
Reductive/oxidative stress DTT, homocysteine
Growth arrest / DNA damage (GADD) etoposide, UV
Protein expression wild-type / mutant / subunits
UPR
Activation of the Unfolded Protein Response
Pathological conditions:
Viral infection, tumorigenesis, DNA damage, diabetes,
atherosclerosis, ischemic injury, conformational diseases
Physiological responses:
Glucose regulation of insulin production (β cells)
Response to a misfolded protein (hepatocytes)
ER Lumen
Nucleus
ER Chaperone
The Unfolded Protein Response
ER-Associated Degradation
Proteasome
5’
Translation Attenuation
5’
TranscriptionalActivation
ERSE
ER Lumen
Nucleus
ER Chaperone
The Unfolded Protein Response
ER-Associated Degradation
Proteasome
5’
Translation Attenuation
5’
TranscriptionalActivation
PERKIRE1
ATF6 / CREBH
ERSE
ER Lumen
PERK
Nucleus
IRE1
ATF6 / CREBH
ER Chaperone
The Unfolded Protein Response
BIP
S1P/S2P Golgi
mRNA Splicing
XBP1uXBP1s
P P P P
ER-Associated Degradation
Proteasome
eIF2α eIF2α P
5’ ATF4 Specific
5’ General
mRNATranslation
5’
5’
Anti-ROS AA metabolismApoptosis (CHOP)
ATF4
ERSE
ER Lumen
unfoldedproteins
unfoldedproteins
ER-Associated Degradation
PERK
Splicing
XBP1uXBP1s
Nucleus
IRE1
ATF6 / CREBH Golgi
Processing
5’
eIF2α eIF2α P
5’
5’5’ ATF4 Specific
General
BiP
Proteasome
ER Chaperone
UPR Signaling Responses
P P P P
B lymphocytes /Hepatocytes /Acinar cells Pancreatic β cells
Inflammation
mRNATranslation
ER
Mitochondria
Nucleus
ER Stress-Induced Apoptosis
Casp 9
Cyt-c
Casp 3
APAF1
BH3 only
Apoptosis
Ca+2
Casp 12
pCasp 12
Calpain
IRE1
TRAF2
JNK-P
ER stress
PERK
Chop
Bcl-2
ATF-6
ATF-4
Gadd34
ER perturbation by mild stress
NT 25 ng/ml Tm overnight 25 ng/ml Tm 2 weeks
All stress pathways are activated during
mild stress
ATF6α
PERK
IRE1α
Differential outcomes for UPR pathways during
persistent stress
Selective instability of mRNAs in the PERK-
CHOP axis
Cells pretreated to induce UPR and ActD added to block transcription
hours0 4
Selective instability of proteins in the PERK-CHOP axis
Cells pretreated to induce UPR and CHX added to block translation
BiPGrp94p58IPK
ATF4CHOPGADD34
Long (>8h) half-life
Short (<4h) half-life
5’
UPR-induced Alterations in Gene Expression
adaptive pro-apoptotic
ATF4
eIF2α CHOPPPP
PERK
XBP1PP Chaperones, ERADIRE1
ATF6 Chaperones (BiP)
GADD34
BCL2
T1/2
T1/2
T1/2 T1/2
T1/2
Rutkowski et al. PLOS Biol. (In Press)
eIF2α Kinase
Translation Initiation Response to External Stimuli
PERK HRI
PKRGCN2
eIF2α - PeIF2α
High rate of AUGcodon recognition
Low rate of AUGcodon recognition
eIF2α Kinase
Translation Initiation Response to External Stimuli
PERK
Hemin DeficiencyArsenite
HRI
dsRNA
Amino Acid Starvation
ER Stress
PKR
TNFα
GCN2
Growth Factor Depletion
ER Ca++
Depletion
Virus Infection
eIF2α - PeIF2α
High rate of AUGcodon recognition
Low rate of AUGcodon recognition
eIF2α Kinase
Translation Initiation Response to External Stimuli
PERK
Hemin DeficiencyArsenite
HRI
dsRNA
Amino Acid Starvation
ER Stress
PKR
TNFα
GCN2
Growth Factor Depletion
ER Ca++
Depletion
Virus Infection
eIF2α - PeIF2α
High rate of AUGcodon recognition
Low rate of AUGcodon recognition
Ser51Ala knock-in
1 2 3 4
S/S A/A- + - + Thapsigargin
eIF2α phosphorylation is required for translation attenuation upon ER stress
S/S
A/A
eIF2α A/A mice die within 24 hr after birth
Glucose Rescue of eIF2α A/A Mouse
A/A
S/S
Insulin Glucagon
S/S A/A
A/A islets have reduced β-cells and insulin content
Scheuner et al. 2001 Mol. Cell 7: 1165
Insulin
Glucagon
Translational control is required to prevent ERdistension/stress in pancreatic β cells
A/A
S/S
2 µM 0.5 µM
*
Wolcott-Rallison Syndrome
Rare autosomal recessive disorder
Diabetes mellitus in early infancy
Multiple epiphyseal dysplasia/osteoporosis
Growth retardation
Due to mutations in PERK/PEK
Delépine et al. 2000 Nat. Gen. 25: 406
+
Partial loss of UPR
Diabetes
?
Does the UPR play a role in the etiology of type II diabetes?
DIET ENVIRONMENTAL STRESS GENETIC FACTORS
DIET ENVIRONMENTAL STRESS GENETIC FACTORS
High-Fat or db/db
+
Partial loss of UPR (eIF2α S/A)
Diabetes
?
Does the UPR play a role in the etiology of type II diabetes?
15 WksTime (wks)
S/A HF
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1520
25
30
35
40
45
50
55
Bo
dy
Wei
gh
t (g
)
HF
S/A
LF
Heterozygous eIF2α S/A mice become obese upon high-fat diet
S/S
S/S HF
S/A HF
S/A
0 40 80 120
Time (min)
0
200
400
600
800
1000
1200
0 50 100 150 200 250
Time (min)
Blo
od
Glu
cose
(mg
/dL
)
S/S
0
0.5
1
1.5
2
Insu
lin (
ng
/ml)
0 40 80 120
Time (min)
Heterozygous eIF2α S/A mice are glucose intolerantwith impaired insulin secretion in vivo
S/S S/A
Scheuner et al. 2005 Nat. Med. 11: 757
500 nm
High-fat diet induces ER stress in beta cells of eIF2α S/A mice
HF S/S HF S/A LF S/A
Genotype: S/S S/A S/S S/A
LF HF
BiP
Proinsulin
Diet:
Antibody: α BiP
Increased Association of Proinsulin with BiPin HF-Fed S/A Mice
α PC2
Genotype: S/S S/A S/S S/A
LF HF
BiP
Proinsulin
Diet:
Increased Association of Proinsulin with BiPin HF-Fed S/A Mice
HFLF
Insulin
Antibody: α BiP α Insulin
α PC2
S/S S/A S/S S/A
Scheuner et al. 2005 Nat. Med. 11: 757
The UPR is essential for beta cell compensation
Fatty acid
Insulin resistance
Insulin secretion / transcription / translation
Increased ER load
UPR activation
ER integrity preserved
(Beta cell compensation)
The UPR is essential for beta cell compensation
Fatty acid
Insulin resistance
Insulin secretion / transcription / translation
Increased ER load
UPR activation
ER integrity preserved
(Beta cell compensation)
+ eIF2α S/A (Partial loss of UPR)
ER dilation/ER stress
Beta cell decompensation
Glucose intolerance
The UPR is essential for beta cell compensation
Fatty acid
Insulin resistance
Insulin secretion / transcription / translation
Increased ER load
UPR activation
ER integrity preserved
(Beta cell compensation)
+ eIF2α S/A (Partial loss of UPR)
ER dilation/ER stress
Beta cell decompensation
Glucose intolerance
CHOP
Apoptosis
?
CHOP deletion increases HF-induced obesity in eIF2αS/A mice
wk0
5
10
15
20
25
0 5 10 15
CHOP(wt)-eIF2α S/S
CHOP(wt)-eIF2α S/A
CHOP(KO)-eIF2α S/S
CHOP(KO)-eIF2α S/A
To
tal w
eig
ht
gai
n (
g)
CHOP(KO)-S/A
CHOP(WT)-S/A
wt-S/A vs ko-S/A ∗ P<0.05; ∗∗ P<0.01
∗∗ ∗
∗∗∗∗
∗ ∗
5 wkHF diet
12 wk
17 wk 32 wk
Blo
od
glu
cose
leve
ls (
mg
/dL
)
Time post glucose injection (min)
CHOP deletion prevents HF diet-induced glucose intolerance in eIF2αS/A mice
0
200
400
600
800
1000
1200
0 40 80 120 0 40 80 120
0
200
400
600
800
1000
1200
0 40 80 120 0 40 80 120
CHOP(wt)-S/S
CHOP(wt)-S/A
CHOP(ko)-S/S
CHOP(ko)-S/A
wt-S/A vs ko-S/A∗ P<0.05∗∗ P<0.01 ∗∗∗ P<0.001
∗∗
∗
∗∗ ∗∗
∗∗∗∗
∗
∗
∗∗∗∗
∗∗∗
CHOP deletion promotes islet hyperplasia in high fat eIF2αS/A mice
wt-S/S wt-S/A ko-S/S ko-S/A
400µm
100µm
50µm
Insulin Glucagon
Chop+/+ S/S Chop-/- S/AChop+/+ S/A
CHOP deletion restores insulin granulesand secretion in HF-fed eIF2αS/A mice
Chop+/+ S/S Chop-/- S/AChop+/+ S/A
CHOP deletion restores insulin granulesand secretion in HF-fed eIF2αS/A mice
02468
10
Insu
linse
cret
ion
(% c
onte
nt) 3mM glucose
15 min17mM glucose30 min
17mM glucose60 min
Does expression of a malfolded protein induce the UPR, oxidative stress, and
apoptosis in vivo?
Does expression of a malfolded protein induce the UPR, oxidative stress, and
apoptosis in vivo?
Clotting factor VIII inefficiently secreted due tomisfolding.
Does factor VIII expression activate the UPR and apoptosis in vivo?
FVIII expression after hydrodynamic DNA injection
C1 C2B A3A1 A2
A1 A2 C1 C2A3B
A1 A2 C1 C2A3
FVIII
226/N6*
BDD
* Miao et al., 2004 Blood 103:3412
0
500
1000
1500
2000
2500
3000
3500
Vect FVIII BDD 226/N6
mU
/ml A
ctiv
ity
Vector
FVIII
FVIII expression after hydrodynamic DNA injection
C1 C2B A3A1 A2
A1 A2 C1 C2A3B
A1 A2 C1 C2A3
FVIII
226/N6*
BDD
* Miao et al., 2004 Blood 103:3412
Vect
FVIII
BDD
226/N6
FVIII Expression Induces Apoptosis in Mouse Liver
TUNEL Nomarski` KDEL Csp-12 Merge`
Vect FVIII BDD 226/N6
Vect
FVIII
BDD
226/N6
0
20
40
60
80
100
DNAse Vect FVIII BDD 226/N6
% A
po
pto
sis
Chop-/-CHOP is required for FVIII induced apoptosis
DNAse
TUNEL Nomarski
Chop-/-
0
5
10
15
20
25
30
35
*
*
*
*p<0.05(n=5)
Chop+/+
% A
po
pto
sis
p-JNK
JNK1
CHOP
p-eIF2α
BiP
Actin
Vect FVIII BDD 226/N6
FVIII Expression Induces ER Stress in Liver
Spliced XBP1mRNA
ER
Mitochondria
Nucleus
ER Stress-Induced Oxidative Stress
IRE1
PERK
Chop
Bcl-2
ATF6
Gadd34
ER
Mitochondria
Nucleus
ER Stress-Induced Oxidative Stress
IRE1
ER stress
PERK
Chop
Bcl-2
ATF6
Gadd34
ER
Mitochondria
Nucleus
ER Stress-Induced Oxidative Stress
IRE1
ER stress
PERK
Chop
Bcl-2
ATF6
Gadd34
ROS
ER
Mitochondria
Nucleus
ER Stress-Induced Apoptosis
BH3 only
Ca+2
IRE1
ER stress
PERK
Chop
Bcl-2
ATF6
Gadd34
ROS
ROS
TRAF2
JNK-P
Casp 12
Casp 9
Casp 3
ER
Mitochondria
ER Stress-Induced Apoptosis
ER stress
BH3 only
Ca+2
ROS
ROS
IRE1
TRAF2
JNK-P
Casp 12
Casp 9
Casp 3Nucleus
PERK
Chop
Bcl-2
ATF6
Gadd34
ATF-4
ACKNOWLEDGEMENTS
Kaufman Laboratory, U of M
Donalyn Scheuner
Benbo Song Kezhong Zhang Robert ClarkSung-hoon Back Kenji SakakiJyoti MalhotraTom RutkowskiMark RibickJun Wu
Past:Stacey ArnoldMartin Schroder Xiaohua ShenEdward McEwen Kyungho Lee Chuan Yin LiuWitoon Tirasophon
Ajith Wehlinda
Dept. of Pediatrics, U of MSteven PipeHonghi Miao
UM Transgenic Animal Core
Patrick Gillespie
Thom SandersLinda Samuelson
Joslin Diabetes Center
Ross Laybutt
Susan Bonner-Weir
Vrije Universiteit BrusselDiabetes Research Center
Daisy Flamez
Frans Schuit