LINDA HENDERSHOT MAY 27, 2012

“UPR mechanisms: role in development and disease”

CSSI Workshop on Cell Stress

Pre-meeting Course

NucleusMitochondria

ER

THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT

THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT

THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT

THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT

KDELKDEL

BiP

THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT

THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT

THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT

THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT

Major aspects of the mammalian UPR

• Up-regulation of ER chaperones (i.e., BiP, GRP94, GRP170).- prevents protein aggregation and contributes to

maintaining ER function during stress conditions- promotes refolding when stress subsides

• Transient inhibition of protein translation.- limits the accumulation of unfolded proteins- not restricted to secretory pathway proteins

• Increased degradative capacity of the cell.- reduces load of unfolded proteins

• Arrest of cells in the G1 phase of cell cycle.- ensures cells experiencing stress are not replicated

• Activation of apoptotic pathways if stress persists.- protects organism at the expense of the cell

The Cellular Response to ER Stress or the Unfolded Protein Response

1. The signal – how damage is assessed

2. The transducers – how the signal is relayed

3. The response – characterized by damage control

4. Defeat – protecting the organism

1. Signal?2. Transducers?

3. Downstream Elements?

4. Regulatory Elements?

GRPs

Nucleus

ER

Unfolded proteins that bind to BiP activate the UPR, and over-expression of BiP inhibits activation

CHOP

COS -un

trans

fected

pSG5

- GFP

sorte

d

pSG5-

s G

FP so

rted

BiP

s

Kozutsumi et al, Nature 1988

Morris et al, JBC, 1997

ERp72

The Signal

1. The signal is the accumulation of unfolded

proteins; specifically those that bind to BiP

(the Hsp70 chaperone of the ER)

2. Levels of BiP and not other chaperones are

monitored

Yeast BiP promoter linked to E. coli lacZ gene

No stress

+ stress EMS mutagenesis22 bp

UPRElacZ

Introduce into yeast

Ern1p

Ire1p

Mori et al. Cell, 1993

Cox et al. Cell, 1993

Identification of the UPR signaling machinery

Kohno et al. MCB, 1993

Components of yeast unfolded protein response (UPR)

Ern1p/Ire1p

S/T S/T

P P Rnase L homology domain endonuclease activity

Hac1 pre-mRNA

ER stress ??

tRNA ligase

Hac1p

GRPsUPRE

Mammalian ER stress transducers

Ire1 /

S/T S/T

P P

↑ degradative capacity

PERK

S/TP

S/TP

Inhibition of protein synthesis and growth arrest

Identification of mammalian UPR transducers using one-hybrid screen methodology

Human BiP promoter

XBP1

ATF6

Yoshida et al, JBC, 1998

Tm +-

ER lumen

cytosol

ATF6 is a transmembrane protein with a cytosolicallyoriented transcription factor domain that is liberated upon

UPR activation

Yoshida et al, JBC, 1998

Stress-sensing

Proteolyticcleavage

DTT: 0’ 15’ 30’

Shen et al Dev Cell 2002

BiP levels regulate activation of ER stress signaling molecules

ER stress signal

- changes in ER environment

- accumulation of unfolded proteins

- binding of unfolded proteins to BiP

Ire1/Ire1

PERK

ATF6

P

S/T

P

S/T

P

S/T

P

S/T

Bertolotti et al, Nat. Cell Biology,

Shen et al Dev Cell 2002

The Ire1 pathway

ER stress

Rnase L homology domain endonuclease activity

XBP-1 mRNA

S/T S/T

P

RNA ligase

XBP-1(S)EDEM/ERdj3/ERdj4/p58

UPRE

P

- 26 bases

PERK pathway

S/T S/T

P P

eIF-2P Translation inhibition

ER stress

CHOP/HERPATF composite

Increased degradation

Cell cycle arrest

Cyclin D1

ATF4ORF ORFORF

Protection from oxidative stress

ER stress induces a transient block in general protein synthesis

0 Tg (h):

Commassie

35S label

0.5 1 2 3 4 6 8

Brewer et al, PNAS 1995

ATF6 pathway

Golgi

GRPs / XBP-1 / CHOP

1. ER Stress SignalBiP-associated unfolded proteins

ER

Nucleus

2. Signal TransducersIre1, PERK, ATF6

3. Downstream ElementseIF2- phosphorylationp38 activationATF6 cleavageCHOP inductionNFB activationXBP1 cleavageATF4 induction

eIF-2 P

P

P

PP

Translation inhibitionCell cycle arrestATF4 synthesis

5. DefeatCaspase 12 activationApoptosis

4. Transcriptional Responses

GRPs / XBP-1CHOP

??NFB targets

XBP1 targets

--

UPR and development

1. Some aspects are required for plasma cell differentiation

2. Essential for regulating normal homeostatsis of the pancreas

3. Required for hepatocyte differentiation

4. Plays a critical role in the normal physiology of osteoblasts

5. Contributes to adipocyte differentiation

6. Two ER stress sensing transgenic mice also show evidence of UPR activation in heart, somites, neural tubes, and skeletal muscle

Producing a mouse that expresses GFP in response to ER stress

Iwawaki,T. et al. Nat. Med. 2004.

XBP-1 mRNA

XBP-1S

XBP-1 mRNA 26 bases

P P

Ire1 kinase

ATG

actin promoter Venus

Venus

ATG

No stress

UPR activation actin promoter

Up-regulation of proteins involved in ER expansion occur in waves during plasma cell differentiation

Van Anken, et. al.,Immunity 18:243-53, 2003

B lymphocyte

Plasma cell

XBP-1 is required for plasma cell differentiation and Ig secretion

Reimold, et. al., Nature 2001

B220

IgM

IgD IgD

BRdUDividing cells

TUNELApoptotic cells

Reimold, et. al., G&D, 2000

And for normal liver development

Whereas PERK is required for exocrine pancreatic function

Harding, et. al., Mol. Cell, 2001

UPR and disease

Cytoprotective Protect, then destroy Cytodestructive

HBV Ischemia Diabetes

HCV glomerular nephritis Neuropathgen. virusMMLV

CMV FrCasE

Simian Virus 5HSV hauntaviruses

Cancers Neurodegen. DiseasesSurvival Alzheimer’sAngiogenesis Parkinson’sChemosensitivity Huntington’s

Lysosomal storage dis.Sandoff-gal deficiencyGaucher’s

Solid tumors experience an altered physiological environment

Oxygen

Nutrients

Wastes, pH

Hypoxic Resp.

Inhib. Glycosyl.

& ATP production

Alter side chain charges

protein folding UPR

High metabolic state

demands on molecular

chaperones

Hypoxic condition: Distance of tumor cell from functional blood

vessel >0.2mm

Inhib. S-S

The UPR and cancer

• Some UPR elements have been shown to be induced in breast, esophageal, prostate, pancreatic, hepatocellular, gastric, and multiple myeloma cancers and in a number of xenograft studies.

Proangiogenicfactors

XBP1(S)ATF4

UPR

Hypoxia

HIF

Tumor growth

Increased Metabolic rate

Proangiogenicfactors

Pereira ER, et al., Endoplasmic Reticulum Stress in Health and Disease, (In press)

Tumors depend on angiogenesis to alleviate stresses caused by an insufficient environment

Rapid cell division Increased Metabolic Rate

UPR ??

1069 probesDifferential expression

185 Angiogenesisassociated 

33 secreted proteinsor transcription factors

↑14/19 positive regulators

↓1 negative regulator

The UPR induces expression of a significant number of proangiogenic factors

Pereira ER, et al., PLoS ONE, 2010

Gene Symbol Angiogenic Effect Fold change

3 hour ThapsigarginFold change

8 hour Thapsigargin

ANG Positive 2.4 8.1

ANGPT2

Positive 2.2

CTGF Positive 2.2 1.4

EPAS1 Positive 1.8 2.6

EREG Positive 2.3 6.3

FGF2 Positive 1.5 3.1

F3 Positive 2.9 1.6

FGF1 Positive 1.1

IL1A Positive 4.4 10.8

IL6 Positive 4.8 7.0

IL8 Positive 54.25 27.9

KLF5 Positive 2.6 3.5

TGFB2 Positive 2.9

VEGFA Positive 2.7

VASH1 Negative -3.0

The UPR induces expression of a significant number of proangiogenic factors

Pereira ER, et al., PLoS ONE, 2010

VEGF human

-5k -4k -3k -2k -1k-6k-7k-8k

TSS

VEGF mouse

-5k -4k -3k -2k -1k-6k-7k-8k

VEGF rat

-5k -4k -3k -2k -1k-6k-7k-8k

XBP1 ATF4 NFB HIF1

+1k

+1k

+1k

Potential binding sites for UPR inducible transcription factors in VEGF promoters

Does the UPR contribute to angiogenesis under normal physiological states? Why should this be a component of

the UPR in normal cells?

Does the UPR contribute to angiogenesis under normal physiological states? Why should this be a component of

the UPR in normal cells?

-Ire1 KO is lethal at 12.5 days

Zhang, K. et al., JCI, 2005

Iwawaki, T., et al., PNAS, 2009

XBP-1 is highly expressed in the placenta

And Ire1 KO mice have decreased expression of VEGF in placenta

Targeted disruption of Ire1 everywhere but in trophoblastsrescued embryonic lethality, so Ire1 expression in trophoblasts

is critical for angiogenesis in mouse placenta!

Iwawaki, T., et al., PNAS, 2009

Summary

1. Disruption of ER homeostasis adversely affects normal protein folding and results in the activation of a cytoprotective signal transduction response known as the unfolded protein response.

2. This response is characterized by increased expression of molecular chaperones, transient inhibition of protein synthesis, up-regulation of ERAD components, and exit from cell cycle. However prolonged UPR activation can result in induction of apoptotic programs.

3. Three ER localized proteins (Ire1, PERK, and ATF6) signal the response and are coordinately activated by the accumulation of unfolded proteins and decreases in levels of free BiP.

4. A number of normal development and differentiation programs are dependent on UPR activation.

5. A growing list of disease states are associated with UPR activation. In some instances, the prosurvival aspects of the UPR lead to disease (i.e., cancer and some viruses), whereas in other cases the proapoptotic elements of the response are responsible for the disease pathology (i.e., neurodegenerative diseases).