Download of control - Dr. Falk Pharma GmbH

INSERM Laboratory U624
Cellular Stress
Campus of Luminy, Marseille
Acute pancreatitis
What is the reaction of the acinar cells at the
cellular and at the molecular level ?
acinar cell of the pancreas
with acute pancreatitis
Gene expression in pancreas with
acute pancreatitis
Experimental
acute pancreatitis
pancreas
RNA extraction
mRNA expression
mRNA expression in pancreas with acute pancreatitis
150
acute phase reaction
% of control
Amylase mRNA
100
50
0
0
0.5
1
2
5
10
Time (days)
mRNA expression in pancreas with acute pancreatitis
150
Elastase mRNA
acute phase reaction
% of control
Amylase mRNA
100
ChTg B mRNA
Tg I mRNA
pCpA mRNA
50
0
0
0.5
1
2
5
10
Time (days)
mRNA expression in pancreas with acute pancreatitis
300
% of control
250
200
150
acute phase reaction
100
Amylase mRNA
Lithostathine mRNA
50
0
0
0.5
1
2
5
10
Time (days)
• Changes in mRNA expression in the acinar
cell of the pancreas with acute pancreatitis
seems to be complex
• Some
genes
became
down-regulated
whereas others were activated
• This is reminder of the response to cellular stress
Stress response
pancreatitis
gene
expression
phenotypic
changes
transcription
factors
second
messengers
cellular resistance
Stress response
pancreatitis
gene
expression
transcription
factors
second
messengers
novel activated genes (new mechanisms !!!)
phenotypic
changes
cellular resistance
Strategy of cloning
mRNA
cDNA
cDNA library
acute pancreatitis
candidate genes
analysis of
expression
analysis of
sequences
Systematic sequencing
(>2000 clones)
over-expressed in
pancreas with pancreatitis
1996
Strategy of cloning
mRNA
cDNA
cDNA library
acute pancreatitis
candidate genes
analysis of
expression
analysis of
sequences
Systematic sequencing
(>2000 clones)
over-expressed in
pancreas with pancreatitis
VMP1
1996
VMP1-EGFP
VMP1
EGFP
VMP1-EGFP
VMP1
EGFP
Autophagy ?
autophagy = autodigestion
Autophagy
Stress-induced autophagy
Benefit for the cell
Cellular damage
(for example chemotherapy)
Removal of damaged organelles
Limitation of infection
Intracellular pathogens
Nutrient/growth factor deprivation
Hypoxia
To produce (in extreme cases) :
- substrates for protein synthesis
- ATP
Autophagy during acute pancreatitis (?)
Autophagy during acute pancreatitis (?)
Electronic microscopy
pre-autophagosome
autophagosome
autophagosome
lysosome
autophagosome
prepre-autophagosome
autophagolysosome
autophagolysosome
VMP1 expression induces LC3 recruitment
pre-autophagosome
LC3
LC3 Immunohistochemistry
control
autophagosome
lysosome
autophagosome
autophagolysosome
pcDNA4-VMP1
VMP1 expression is induced by autophagic
stimuli and is necessary for autophagy
starvation
rapamycin
VMP1 IHC
VMP1
GAPDH
hours
0
2
4
0
2
4
starvation
starvation
rapamycin
VMP1
β−tubulin
hours
0
2
4
0
2
4
rapamycin
VMP1 expression is induced by autophagic
stimuli and is necessary for autophagy
starvation
rapamycin
VMP1 IHC
starvation
VMP1-siRNA
scble-siRNA
VMP1
+
-
rapamycin
+
+
-
VMP1
GAPDH
hours
+
GAPDH
0
2
4
0
2
4
starvation
starvation
VMP1-siRNA
scble-siRNA
+
+
-
rapamycin
+
+
-
VMP1
starvation
rapamycin
β−tubulin
VMP1
β−tubulin
0
2
4
rapamycin
VMP1-siRNA
scble-siRNA
starvation
starvation
VMP1-siRNA
scble-siRNA
rapamycin
rapamycin
VMP1-siRNA
scble-siRNA
control
control
VMP1-siRNA
scble-siRNA
100
90
80
70
60
50
40
30
20
10
0
control
4
rapamycin
2
starvation
0
RFP-LC3 positive cell with
punctate per transfected cells
hours
MCF7
MCF7
MCF7
starvation
100
90
80
70
60
50
40
30
20
10
0
pcDNA4 -VMP1
pcDNA4-VMP1
control
starvation
per transfected cells
control
%ERFP-LC3 positive puncta
VMP1 binds to the beclin1 protein
MCF7
MCF7
MCF7
starvation
100
90
80
70
60
50
40
30
20
10
0
pcDNA4 -VMP1
pcDNA4-VMP1
control
starvation
per transfected cells
control
%ERFP-LC3 positive puncta
VMP1 binds to the beclin1 protein
antiprebeclin1 immune
pull-down
Ni-agarose
beclin1
VMP1-V5
beclin1
TCL
VMP1-V5
VMP1-V5
VMP1-V5
TCL
beclin1
pcDNA4-VMP1
+
-
pcDNA4-empty
-
+
pcDNA4-VMP1
+
+
antiprebeclin1 immune
anti- preVMP1 immune
beclin1
VMP1
IP
VMP1
IgG
rapamycin
IP
beclin1
IP
beclin1
IgG
+
+
rapamycin
+
+
VMP1 binds to the beclin1 protein
MCF7
MCF7
MCF7
starvation
100
90
80
70
60
50
40
30
20
10
0
pcDNA4 -VMP1
pcDNA4-VMP1
control
starvation
per transfected cells
control
%ERFP-LC3 positive puncta
D
B
TCL
VMP1-V5
beclin1
VMP1-V5
pcDNA4-VMP1
+
-
pcDNA4-empty
-
+
pcDNA4-VMP1
+
+
antiprebeclin1 immune
anti- preVMP1 immune
beclin1
VMP1
IP
VMP1
IgG
rapamycin
TCL
beclin1
IP
beclin1
IgG
+
+
rapamycin
+
+
GST
beclin1
IP
GST
VMP1-V5
VMP1-V5
GST
pull-down
Ni-agarose
GST
antiprebeclin1 immune
beclin1
C
A
A
B
C D
+
His6- beclin1
pull-down
VMP1 binds to the beclin1 protein
MCF7
MCF7
MCF7
starvation
100
90
80
70
60
50
40
30
20
10
0
pcDNA4 -VMP1
pcDNA4-VMP1
control
starvation
per transfected cells
control
%ERFP-LC3 positive puncta
D
B
TCL
VMP1-V5
IP
+
-
pcDNA4-empty
-
+
B
C D
pull-down
VMP1-V5
TCL
beclin1
pcDNA4-VMP1
GST
beclin1
A
+
His6- beclin1
VMP1-V5
beclin1
GST
VMP1-V5
GST
pull-down
Ni-agarose
GST
antiprebeclin1 immune
beclin1
C
A
pcDNA4-VMP1
+
GST-VMP1A
His6- beclin1
+
-
+
GST-VMP1B
-
+
GST-VMP1C GST-VMP1D
-
+
IB : anti-beclin1
antiprebeclin1 immune
anti- preVMP1 immune
beclin1
VMP1
IP
IgG
rapamycin
IP
beclin1
IgG
+
+
+
IB : anti-GST
beclin1
VMP1
-
rapamycin
beclin1
+
+
VMP1 binds to the beclin1 protein
MCF7
MCF7
MCF7
starvation
100
90
80
70
60
50
40
30
20
10
0
pcDNA4 -VMP1
pcDNA4-VMP1
control
starvation
per transfected cells
control
%ERFP-LC3 positive puncta
D
B
TCL
VMP1-V5
IP
+
-
pcDNA4-empty
-
+
B
C D
pull-down
VMP1-V5
TCL
beclin1
pcDNA4-VMP1
GST
beclin1
A
+
His6- beclin1
VMP1-V5
beclin1
GST
VMP1-V5
GST
pull-down
Ni-agarose
GST
antiprebeclin1 immune
beclin1
C
A
pcDNA4-VMP1
+
GST-VMP1A
His6- beclin1
+
-
+
GST-VMP1B
-
+
GST-VMP1C GST-VMP1D
-
+
-
IB : anti-GST
IB : anti-beclin1
beclin1
antiprebeclin1 immune
anti- preVMP1 immune
beclin1
VMP1
IP
VMP1
IgG
rapamycin
IP
beclin1
IgG
+
+
+
rapamycin
PI3k
beclin1
+
+
VMP1-beclin1 interaction is necessary for autophagy
VMP1ΔD/V5
beclin1
?
beclin1
pull-down
Ni-agarose
V5
beclin1
+
-
-
pcDNA4-VMP1ΔD/V5
-
+
-
pcDNA4-empty
-
-
+
VMP1ΔD/V5
Triton
treatment
pH11.0
treatment
1.5M NaCl
treatment
No
treatment
VMP1ΔD/V5
No
treatment
pcDNA4-VMP1/V5
empty/V5
TCL
V5
VMP1-beclin1 interaction is necessary for autophagy
VMP1ΔD/V5
beclin1
?
beclin1
pull-down
Ni-agarose
V5
beclin1
+
-
-
pcDNA4-VMP1ΔD/V5
-
+
-
pcDNA4-empty
-
-
+
Triton
treatment
pH11.0
treatment
1.5M NaCl
treatment
No
treatment
VMP1ΔD/V5
No
treatment
pcDNA4-VMP1/V5
empty/V5
TCL
V5
VMP1ΔD/V5
VMP1/EGFP
VMP1ΔD/EGFP
VMP1 expression in vivo
VMP1
LC3
Merge
VMP1
beclin1
Merge
0
3
6
12
control
hours
experimental
pancreatitis
β−tubulin
control
VMP1
experimental
pancreatitis
experimental
pancreatitis
VMP1 transgenic mice
DNA construct
Promotor-Elastase
VMP1
EGFP
Anti-EGFP
?
Anti-EGFP
Hypothesis
acute pancreatitis
PI3k
VMP1 mRNA
VMP1 gene
autophagolysosome
beclin1
ER
PI3k
autophagosome
beclin1
ER
lysosome
LC3
PI3k
autophagosome
beclin1
ER
pre-autophagosome
Conclusion I
Expression
of
VMP1
necessary and sufficient
induce autophagy
is
to
Conclusion II
During the acute pancreatitis,
acinar cells respond to the
cellular stress induced by this
disease, by changing their gene
expression profile in a very
complex manner but with the aim
to survive…..
Conclusion III
Starting autophagy, during the acute
phase of the pancreatitis, could be a
protective mechanism for the acinar cell
because
:
1/ it could provide energy substrates…..
2/ it could remove damaged organelles…..
Calanques of Luminy, Marseille
at 5 min walking from the lab…