Inhibition of PC synthesis results in the expression of pro-apoptotic CHOP/GADD153 and the activation of JNK kinase

Michiel van der Sanden

A phospholipid bilayer

(Alberts et al, Essential cell biology)

Phosphatidylcholine (PC)

(Alberts et al, Essential cell biology)

Choline

PC

Phosphocholine

CDP-choline

Choline kinase

Choline phosphate transferase

ATP

ADP

CTP

PPi

CMP

CTP:phosphocholine cytidylyltransferase (CT)

DAG

De novo synthesis of PC, the Kennedy pathway

Hemicholium-3 (HC-3)

Inhibitors:

Alkyl-lysophospholipids (ALP’s)

Farnesol and geranylgeraniol

• Inhibition of PC synthesis by using inhibitors like Alkyl-LysoPhospholipids (ALPs), as HePC and Edelfosine (ET-18-OCH3).

Disadvantage of using ALPs : Beside inhibition of CT, they affect several other processes in the cell, like formation of pro-apoptotic ceramide, stimulation of SAP/JNK pathway, FAS clustering , PKC activation.

• Inhibition of PC de novo synthesis in a genetic model.

Chinese Hamster Ovary (CHO) cell line, which contains a thermo-sensitive mutation in the rate-limiting enzyme CT• CHO-K1 = Wild-type• CHO-MT58 = Temperature-sensitive mutant

• CHO-MT58-CT = Mutant with re-introduced stable CT

Methods

Regulation of membrane homeostasis

What happens if a cell cannot make

enough membrane lipids?

Effect of the non-permissive temperature on PC biosynthesis in CHO cells

33 °C 40 ° (5 h) 40 °C (24 h)0

20

40

60

80

100W T-K1MT58MT58 + CTα

Inco

rpor

atio

n of

[3H

]cho

line

into

PC

[dpm

/nm

ol p

hosp

hate

]

Temperature ( °C)

PC biosynthesis inhibition leads to reduction in PC pools

hours at 40°C

0 4 8 12 16 20 240

20

40

60

80

WT-K1

MT-58

nm

ol

PC

/mg

pro

tein

Inhibition of cell proliferation and induction of apoptosis in MT58 at 40 ˚C

0 24 48 720

25

50

75

100

125

150

K1 33°C

K1 40°CMT58 33°CMT58 40°C

time (h)

cell

nu

mb

er

(x 1

04

) /d

ish

0

10

20

30

40

50

60

time (h)

% a

po

pto

tic

ce

lls

Rescue from apoptosis, caused by PC depletion, with LysoPC

LPC rescue-CHO-K1MT58

- LPC (0) LPC (16) LPC (24) LPC (30) LPC (48)0

20

40

60

80

100

Cells incubated for 72 h at 40°C

% a

po

pto

tic

cell

s

LPE rescue

LPE (0) LPE (24) LPE (30)

How does PC depletion signals to the apoptotic machinery (= executive caspases etc).

ER stress response, pausing to decide

ER

BiP /GRP78

Unfolded protein response (UPR)

HSPs

PERK

Translational attenuation

Pro-apoptotic targets

CHOP/GADD153Caspase 12

misfolded orunfolded proteins

Glucose starvation

Ca2+

overloadPhospholipid depletion ??

Role of ER stress proteins in the PC depletion induced apoptosis

0

0

8

8

2 4

2 4 2 4

2 3 0

3 0

1 6

1 6

4

4

M T 5 8

C H O - K 1

3 3 4 0

3 3 4 0

A

A c t i n

A c t i n

BiP induction HSP 70 induction

0

0

8

8

24

24 24

2 48

48

30

30

16

16

4

4

MT58

CHO-K1

33 40

33 40

2

2

B

Actin

Actin

Measurement of protein synthesis byincorporation of [35S]methionine

CHO-K1 CHO-MT580

20

40

60

80

100

120

33° (24 h)40° (24 h)Tun (24 h)CHX (3 h)

% n

ewly

syn

thes

ized

pro

tein

,co

mp

ared

to

co

ntr

ol a

t 33

°No Translational attenuation in MT58

CHOP induction in PC depleted MT58 cells

time (h)

time

CHOP

°

CHOP

time

CHOP

°

°

Actin

ActinC H O PC H O P

M T 5 8

3 3 ° t = 0

3 3 ° t = 2 4

4 0 ° t = 2 4

4 0 ° t = 2 4

M T 5 8 + C T α

A c t i n

Caspase 12 is not induced or activated in

MT58

33 °

C t

=24

33 °

C t

=040

° C t

=24

40 °

C t

=24

33 °

C t=0

33 °

C t

=24

40 °

C t=3

0Tu

nica

myc

in

CHO-K1 MT58

Actin

Caspase 12 (60 kD)

No classical ER stress response in PC depleted MT58 cells

- Bip/GRP 78 or HSP 70 induction

- Translational attenuation of proteins

- Induction of caspase 12

+ Induction of CHOP, so what is responsible for the induction of CHOP

Conclusions (1)

• Inhibition of PC synthesis results in a rapid decline of cellular PC content and induces apoptosis within 48 h.

• PC depletion leads to the induction of the pro-apoptotic transcription factor CHOP

• CHOP induction is not observed with the classical ER stress response.

van der Sanden et al. Biochem J. 2003

C/EBP homologous protein (CHOP/GADD 153)

• b-Zip Transcription factor of 27 kD• Binds to a subset of C/EBP promoter sites• Required for stress-activation of genes,

known as DOC’s (downstream of CHOP)• Basal expression of CHOP is very low,

almost undetectable.• CHOP expression is often associated with

the ER-stress response• Activation of CHOP via phosphorylation by

p38 or JNK-kinase

De novo synthesis of proteins (CHOP?) is necessary for apoptosis induced by PC depletion

Rescue from apoptosis, induced by PCdepletion with 20 µ g/ml cycloheximide

(CHX)

- CHX (0) CHX (8) CHX (16) CHX (24)0

20

40

60

80

100 CHO-K1MT58

Cells incubated for 72 h at 40 ° C

% a

popt

otic

cel

ls

Inhibition of CHOP expression by anti- sense mRNA CHOP delays the onset of

apoptosis

0

20

40

60

80

100

t=0 t=16 t=24 t=32 t=40 t=48 t=72

MT58

MT58 + anti senseCHOP 2

MT58 + anti senseCHOP 6

TATAAP1

JNK

ERSE (i)Constitutive-247 till -239 -75 till -71 -62 till -57

C/EBP-ATF

-313 till -295

ATF-2 C-jun

5’ deletion mutans of the CHOP promoter :

TATA luciferase

TATA luciferase

-442

-211

TATA luciferase649

ATF-6ATF-4XBP-1

IRE1PERK

Activation of the CHOP promoter in MT58

-211 -442 -6490

500

1000

1500 K1MT58MT58 + CT

DP

M/n

mo

l ON

PG

Activation of the CHOP promoter in MT58 requires a C/EBP ATF motif

AP-1ATF LUCERSE-442

AP-1ATF LUCERSE-442

AP-1ATF LUCERSE-442

XX

Phosphorylation of transcription factor ATF-2 during PC depletion

33 °

C t

=24

40 °

C t=2

4

40 °

C t

=24

33 °

C t=0

33 °

C t

=24

40 °

C t=8

40 °

C t

=16

CHO-K1 MT58

Actin

ATF2

Phospho-ATF2

Effect of PC depletion on JNK activation33

° C

t=2

4

40

° C t

=24

40 °

C t

=24

33 °

C t=0

33 °

C t

=24

40 °

C t=3

0

40 °

C t

=16

CHO K1 MT-58

JNK

p46 phospho JNK

p54 phospho JNK

JNK activation is necessary for apoptosis induced by PC depletion

Rescue from apoptotis, induced by PCdepletion with JNK inhibitor SP600125

(40 µ M)

- SP (0) SP (8) SP (16) SP (24)0

20

40

60

80

100 CHO-K1MT58

Cells incubated for 72 h at 40 ° C

% a

popt

otic

cel

ls

Inhibition of JNK does not influence CHOP expression

CHO K1 MT-58 +

SP600125 JNK inhibitor -

Actin

CHOP

33 °

C t

=24

40

° C t

=24

40 °

C t

=24

33 °

C t=0

33 °

C t

=24

40 °

C t=2

4

40 °

C t

=16

33 °

C t=0

Conclusions (2)

• CHOP transcriptional activation is likely to be regulated by transcription factor ATF2

• CHOP expression is necessary for a quick apoptotic response to PC depletion

• JNK is involved in the induction of the death of MT58

• JNK is likely not involved in the induction of CHOP expression, but could be responsible for the activation of CHOP by phosphorylation

Hypothesis

CHOP/GADD153

CHOP/GADD153

PC depletion

JNK

?

XATF2

Possible up-stream pathways:

- Ceramides and MLK

- Oxidative stress and ASK

Acknowledgements

A.B.Vaandrager

M. Houweling

H. Meems

W. Klein

Prof. J.B. Helms

Prof L.M.G. van Golde

P. Fafournoux

INRA de TheixChampanelle, France

Department of Biochemistry Veterinary Medicine, University of Utrecht