Michiel van der Sanden

1. Inhibition of PC synthesis results in the
expression of pro-apoptotic CHOP/GADD153
and the activation of JNK kinase
Michiel van der Sanden

2. A phospholipid bilayer
(Alberts et al, Essential cell biology)

3. Phosphatidylcholine (PC)
(Alberts et al, Essential cell biology)

4. De novo synthesis of PC, the Kennedy pathway
Choline Inhibitors:
ATP Hemicholium-3
Choline kinase
ADP (HC-3)
Phosphocholine
CTP:phosphocholine CTP Alkyl-lysophospholipids
cytidylyltransferase (CT) PPi (ALP’s)
CDP-choline
Choline phosphate DAG Farnesol and
transferase CMP geranylgeraniol
PC

5. Methods
• 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

6. Regulation of membrane homeostasis
What happens if a
cell cannot make
enough membrane
lipids?

7. Effect of the non-permissive temperature
on PC biosynthesis in CHO cells
W T-K1
Incorporation of [3H]choline into PC
100 MT58
MT58 + CT α
[dpm/nmol phosphate]
80
60
40
20
0
°
33 °C 40 ° (5 h) 40 C (24 h)
Temperature ( °C)

8. PC biosynthesis inhibition leads to
reduction in PC pools
80
nmol PC/mg protein
60
WT-K1
40
MT-58
20
0
0 4 8 12 16 20 24
hours at 40 °C

9. Inhibition of cell proliferation and induction
of apoptosis in MT58 at 40 ˚C
60
cell number (x 10 4 ) /dish
150
% a p optotic cells
50
125
100 40
75 30
K1 33°C
50 20
K1 40°C
25 MT58 33°C 10
MT58 40°C
0 0
0 24 48 72
time (h) time (h)

10. Rescue from apoptosis, caused by PC
depletion, with LysoPC
- LPC rescue LPE rescue
100
CHO-K1
% apoptotic cells
MT58
80
60
40
20
0
- LPC (0) LPC (16) LPC (24) LPC (30) LPC (48) LPE (0) LPE (24) LPE (30)
Cells incubated for 72 h at 40 ° C

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

12. ER stress response, pausing to decide
misfolded or Ca2+ Glucose Phospholipid
unfolded proteins overload starvation depletion ??
Unfolded protein
response (UPR)
BiP / PERK
GRP78
ER
HSPs
Translational
attenuation
Caspase 12 CHOP/GADD153
Pro-apoptotic targets

13. Role of ER stress proteins in the PC
depletion induced apoptosis
A BiP induction B HSP 70 induction
C H O -K 1 CHO-K1
33 40 33 40
2 0 4 8 16 24 30 2 0 2 4 8 16 24 30 48
A c tin
Actin
M T 58 MT58
33 40 33 40
24 0 4 8 16 24 30 24 0 2 4 8 16 24 30 48
A c tin Actin

14. No Translational attenuation in MT58
Measurement of protein synthesis by
incorporation of [35S]methionine
% newly synthesized protein,
120
°
compared to control at 33
100
33° (24 h)
80 40° (24 h)
Tun (24 h)
60
CHX (3 h)
40
20
0
CHO-K1 CHO-MT58

15. CHOP induction in PC depleted MT58 cells
° °
time
0 24 4 24
CHOP
t = t= t= 2 t =
3 3° 33° 40° 4 0°
Actin
CH O P
A c tin
°
M T58 + C Tα M T58
time (h)
CHOP
Actin

16. Caspase 12 is not induced or activated in
MT58
in
4
4
0
4
4
yc
t=2
t=2
t=2
t=3
t=0
t=2
t=0
am
C
C
C
C
°C
C
°C
nic
°
°
°
°
°
Tu
33
40
33
40
33
33
40
Caspase 12 (60 kD)
Actin
CHO-K1 MT58

17. 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

18. 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

19. 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

20. De novo synthesis of proteins (CHOP?) is
necessary for apoptosis induced by PC depletion
Rescue from apoptosis, induced by PC
depletion with 20 µ g/ml cycloheximide
(CHX)
100 CHO-K1
MT58
80
% apoptotic cells
60
40
20
0
- CHX (0) CHX (8) CHX (16) CHX (24)
Cells incubated for 72 h at 40 ° C

21. Inhibition of CHOP expression by anti-
sense mRNA CHOP delays the onset of
apoptosis
100
80
MT58
60
MT58 + anti sense
CHOP 2
40
MT58 + anti sense
CHOP 6
20
0
t=0 t=16 t=24 t=32 t=40 t=48 t=72

22. JNK IRE1
ATF-6 PERK
ATF-4
ATF-2 C-jun XBP-1
C/EBP-ATF AP1 Constitutive ERSE (i) TATA
-313 till -295 -247 till -239 -75 till -71 -62 till -57
5’ deletion mutans of the CHOP promoter :
649 TATA luciferase
-442 TATA luciferase
-211 TATA luciferase

23. Activation of the CHOP promoter in MT58
1500 K1
DPM/nmol ONPG
MT58
MT58 + CT
1000
500
0
-211 -442 -649

24. Activation of the CHOP promoter in
MT58 requires a C/EBP ATF motif
-442 ATF AP-1 ERSE LUC
-442 ATF
X
AP-1 ERSE LUC
-442
X
ATF AP-1 ERSE LUC

25. Phosphorylation of transcription factor
ATF-2 during PC depletion
4
4
6
4
4
t=2
t=1
t=2
t=2
t=2
t=8
t=0
C
°C
C
C
C
C
°C
°
°
°
°
°
40
40
33
40
33
40
33
Phospho-ATF2
ATF2
Actin
CHO-K1 MT58

26. Effect of PC depletion on JNK activation
4
6
4
0
4
4
t=1
t=2
t=2
t=2
t=3
t=2
t=0
°C
C
C
C
C
C
C
°
°
°
°
°
°
40
33
40
33
40
33
40
p54 phospho JNK
p46 phospho JNK
JNK
CHO K1 MT-58

27. JNK activation is necessary for apoptosis
induced by PC depletion
Rescue from apoptotis, induced by PC
depletion with JNK inhibitor SP600125
(40 µ M)
100 CHO-K1
MT58
% apoptotic cells
80
60
40
20
0
- SP (0) SP (8) SP (16) SP (24)
Cells incubated for 72 h at 40 ° C

28. Inhibition of JNK does not influence
CHOP expression
4
6
4
4
4
24
t=1
t=2
t=2
t=0
t=2
t=0
t=2
t=
°C
C
C
C
°C
C
C
C
°
°
°
°
°
°
40
33
40
33
33
40
33
40 -
SP600125 JNK inhibitor
CHOP
Actin
CHO K1 MT-58 +

29. 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

30. Hypothesis
Possible up-stream pathways:
PC depletion - Ceramides and MLK
- Oxidative stress and ASK
ATF2
JNK
X
CHOP/GADD153
?
CHOP/GADD153

31. Acknowledgements
Department of Biochemistry INRA de Theix
Veterinary Medicine, Champanelle, France
University of Utrecht
A.B.Vaandrager P. Fafournoux
M. Houweling
H. Meems
W. Klein
Prof. J.B. Helms
Prof L.M.G. van Golde