The Role of ER Stress and Translation in Cell Death

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

kaufmanr@umich.edu