liver to pancreas transdifferentiation by pancreatic transcription factors
DESCRIPTION
Liver to pancreas transdifferentiation by pancreatic transcription factors. Shiraz Gefen-Halevi Ph.D Proposal. Dr. Sarah Ferber Prof. Jacob Shoham. The Pancreas. Diabetes Type I. Autoimmune distruction of b cells No Insulin production - PowerPoint PPT PresentationTRANSCRIPT
Liver to pancreas transdifferentiation by
pancreatic transcription factors
Shiraz Gefen-Halevi
Ph.D Proposal
Dr. Sarah Ferber Prof. Jacob Shoham
The Pancreas
Diabetes Type I
Autoimmune distruction of cellsNo Insulin productionHyperglicemia related complications
Pancreas/islets cells transplantation
Achieves good glycemic control
Necessitates life long immunosupression
Shortage of organs
Todays Clinical therapies and complications
Insuin administration Hazards of hypoglycemia Diabetic complications
Number of adult islet allografts done in the last 4 yeats
Cell replacement therapy for diabetic patients will become widely available only when new sources of islets or cells are found.
Liver & Pancreas
• Originate from the primitive Originate from the primitive
foregut endoderm.foregut endoderm.
• Share common characteristics, Share common characteristics,
such as glucose sensing ability, such as glucose sensing ability,
due to GLUT-2 & GK due to GLUT-2 & GK
expression.expression.
• Share the expression of several Share the expression of several
unique transcription factors unique transcription factors
expression.expression.
PDX
-1+
Ad-CMV-PDX-1
PDX-1 induces transdifferentiation of liver to pancreasboth in vivo and in vitro
Pancreatic hormone gene expression
Ameliorates hyperglycemia in diabetic mice
Pancreatic Phenotype
PDX-1
600
450
300
150
00 2 4 6 8 10
Blo
od
Glu
co
se
, m
g/d
l
virus injectionSTZ
-gal
Time, days
PDX-1
600
450
300
150
00 2 4 6 8 10
Blo
od
Glu
co
se
, m
g/d
l
virus injectionSTZ
-gal
Time, days
600
450
300
150
00 2 4 6 8 10
Blo
od
Glu
co
se
, m
g/d
l
virus injectionSTZ
-gal
Time, days
Insulin production and accumulation in liver
In-vitro In-vivo
Gene expressionProtein production Function
*Ferber et al Nature Medicine 2000 & Ber et al J. Biol. Chem 2003
Pancreatic precursor
Endocrine precursor
cell specific
Pancreatic transcription factors cascase in pancreas development cascade
Aims
To use liver tissue as a source of ectopic pancreas
To re-evaluate the functional hierarchy of transcription factors by gain of function study.
To examine whether other pancreatic transcription factors (NGN3 and Nkx6.1) besides PDX-1 can induce liver to pancreas transdifferentiation
To examine whether NGN3 and/or Nkx6.1 augments PDX-1 induced liver to pancreas transdifferentiation.
To examine the sequential effects of the transcription factor ectopic expression
Ectopic expression of pancreatic transcription factors in embryonic hepatocytes
Results
GF
+P
dx-
1+
Ngn
3
beta
- TC
Con
trol
GF
GF
+P
dx-1
GF
+N
kx6.
1
GF
+N
gn3
GF
+P
dx-
1+
Nkx
6.1
GF
+N
gn3+
Nkx
6.1
GF
+P
dx-
1+
Ngn
3+N
kx6.
1
Ins
RT-
Ectopic PDX-1
Ectopic NKX6.1
Endogenic β- ACTIN
Ectopic NGN3
GF=EGF + Nicotinamide
Insulin compared to -actin gene expression 3E+09
0
5000
10000
15000
20000
25000
control GF
GF+Pdx1
GF+Ngn3
GF+Nkx6.1
GF+Pdx1+Ngn3
GF+Pdx1+Nkx6.1
GF+Ngn3+Nkx6.1
GF+Pdx1+Ngn3+Nkx6.1 Ins
Fol
d o
f In
cre
ase
B
Quantitative Insulin gene expression
Somatostatin
GF+
Pdx
-1+
Ngn
3+N
kx6.
1
β-actin
Glucagon
Con
trol
GF
GF+
Pdx
-1
GF+
Ngn
3
GF+
Nkx
6.1
GF+
Pdx
-1+
Ngn
3
GF+
Pdx
- 1+
Nkx
6.1
GF+
Ngn
3+N
kx6
.1
Ins
Insulin
Glucagon and Somatostatin compared to -actin gene expression
0
50
100
150
200
control
GF
GF+Pdx1
GF+Ngn3
GF+Nkx6.1
GF+Pdx1+Ngn3
GF+Pdx1+Nkx6.1
GF+Ngn3+Nkx6.1
GF+Pdx1+Ngn3+Nkx6.1 Ins
Fol
d o
f In
crea
se
Glucagon
Somatostatin
Quantitative Glucagon and Somatostatin gene expression
Somatostatin
GF+
Pdx
-1+
Ngn
3+N
kx6.
1
β-actin
Glucagon
Con
trol
GF
GF+
Pdx
-1
GF+
Ngn
3
GF+
Nkx
6.1
GF+
Pdx
-1+
Ngn
3
GF+
Pdx
- 1+
Nkx
6.1
GF+
Ngn
3+N
k6.1
Ins
Insulin
Insulin secretion from human embryonic hepatocytes during 72 hours
0
5
10
15
20
25
30
35
40
45
Contro
l (n=
4)
GF
(n=4)
GF+
Pdx
1 (n
=4)
GF+
Ngn
3 (n
=4)
GF+
Nkx
6.1
(n=4
)
GF+
Pdx1+
Ngn
3 (n=4)
GF+
Pdx
1+ N
kx6.
1 (n=4)
GF+
Ngn
3+ N
kx6.1
(n=2
)
GF+
Pdx
1+ N
gn3+
Nkx6.
1 (n
=4)
Ins
(n=4
)
Insu
lin (
ng
/ml/m
g p
rote
in)
Insulin secretion
Only PDX-1 alone can induce Insulin production and secretionCombinations with other transcription factors didn’t increase Insulin secretion
Somatostatin
GF+
Pdx
-1+
Ngn
3+N
kx6.
1
Glucagon
Con
trol
GF
GF+
Pdx
-1
GF+
Ngn
3
GF+
Nkx
6.1
GF+
Pdx
-1+
Ngn
3
GF+
Pdx
- 1+
Nkx
6.1
GF+
Ngn
3+N
k6.1
Ins
Insulin Elastase
Quantitative exocrine gene expression
Summary:
1. PDX-1 induces a pancreatic phenotype in human embryonic liver cells
2. Other pancreatic transcription factors (NGN3 and Nkx6.1) can not induce a pancreatic phenotype in liver cells as does PDX-1
3. Nkx6.1 supports PDX-1 in the transdifferentiation process by increasing Insulin, Glucagon and Somatostatin gene expression
4. NGN3 subtracts from PDX-1 induced liver to pancreas transdifferentiation by decreasing Insulin, Glucagon and Somatostatin gene expression
5. Co expression of PDX-1 and Nkx6.1 seems to increase Elastase gene expression
GF
+P
dx-1
+N
gn3
+N
kx
6.1
Nkx2.2
Isl-1
Co
ntr
ol
GF
GF
+P
dx-1
GF
+N
gn
3
GF
+N
kx
6.1
GF
+P
dx-1
+N
gn3
GF
+P
dx-1
+N
kx6
.1
GF
+N
gn
3+
Nkx
6.1
Ins
Neuro-D
Nkx6.1
-actin
Quantitative gene expression of pancreatic transcription factors
NKX2.2 is not expresseed by embryonic liver cells PDX-1 increases NKX2.2 expression NGN3 and NKX6.1 do not increase NKX2.2 expression
Isl1
GF
+P
dx-1
+N
gn3
+N
kx
6.1
Nkx2.2
Isl-1
Co
ntr
ol
GF
GF
+P
dx-1
GF
+N
gn
3
GF
+N
kx
6.1
GF
+P
dx-1
+N
gn3
GF
+P
dx-1
+N
kx6
.1
GF
+N
gn
3+
Nkx
6.1
Ins
Neuro-D
Nkx6.1
-actin
Quantitative gene expression of pancreatic transcription factors
Isl1
ISL1 is expressed by embryonic liver cellsPDX-1 increases ISL1 gene expression by 2 foldsNGN3 and NKX6.1 hardly increase ISL1 expressionNKX6.1 + PDX-1 increases ISL1 expression by 3 folds
GF
+P
dx-1
+N
gn3
+N
kx
6.1
Nkx2.2
Isl-1
Co
ntr
ol
GF
GF
+P
dx-1
GF
+N
gn
3
GF
+N
kx
6.1
GF
+P
dx-1
+N
gn3
GF
+P
dx-1
+N
kx6
.1
GF
+N
gn
3+
Nkx
6.1
Ins
Neuro-D
Nkx6.1
-actin
Quantitative gene expression of pancreatic transcription factors
Neuro D in not expressed by embryonic liver cellsNGN3 is the main inducer of Neuro D gene expression
Neuro D
GF
+P
dx-1
+N
gn3
+N
kx
6.1
Nkx2.2
Isl-1
Co
ntr
ol
GF
GF
+P
dx-1
GF
+N
gn
3
GF
+N
kx
6.1
GF
+P
dx-1
+N
gn3
GF
+P
dx-1
+N
kx6
.1
GF
+N
gn
3+
Nkx
6.1
Ins
Neuro-D
Nkx6.1
-actin
Quantitative gene expression of pancreatic transcription factors
NKx6.1 is not expressed by embryonic liver cells Pdx-1 increases Nkx6.1 gene expression Ectopic Nkx6.1 abolishes its own endogenic expression
Summary:
1. Nkx2.2, Isl1 and Nkx6.1 endogenic expression seem to be involved inthe pancreatic phenotype induced in the liver
2 .Neuro D seems not to be involved in the pancreatic phenotype induced in the liver
3 .NGN3 induces Neuro D gene expression
4 .PDX-1 induces Nkx2.2, Isl1 and Nkx6.1 gene expression
5 .Nkx6.1 seems to increase Isl1 gene expression induced by PDX-1 and to decrease NKX2.2 and endogenic Nkx6.1 gene expression induced by PDX-1
Ngn3
Neuro D
Pdx-1 Nkx6.1
Nkx2.2
Nkx6.1
אינסולין
Isl-1
?
pPAC-CMVIPF-IRES6.1
12059 bp
SV40pA
Ad 5
Amp
Ad 5
NotI 11344
NcoI 11881 EcoRI 1
KpnI 10
BamHI 1580
SalI 3310
HindIII 3320
NotI 3750
NcoI 4086
XhoI 6000
IRES-nkx6.1
IPF-PA
CMV
Construction of an Ad-CMV-IPF-1-IRES-NKX6.1
pCMV-IPF-1-IRES-NKX6.1 expression in cells
m-ACTIN
haNKX6 .1
PDX-1
293 Transfected
293
β-TC
RNA
Mouse pancrease
aML+ Ad-PDX1 293
Transfected 293 pAC-PDX1
Transfected 293 pAC -PDX1- NKX6.1
PDX-1
Nkx6.1
293
Transfected 293 pAC-PDX1 - NKX6.1 ML
aML+ Ad-NKX6 .1
Human Hepatocytes
Human Hepatocytes
+Ad - NGN3 +Ad - NKX6.1
Mouse pancrease
Protein
The best pancreatic phenotype will be determined following these questions:
1. Is there an endocrine pancreatic phenotype? And to what extent?RIP Promoter activation (Ad Rip-GFP) Hormone gene expression (quantitative RT-PCR)Hormone secretion (RIA)Hormone content (RIA)
2. Is there an exocrine pancreatic phenotype? And to what extent?Exocrine enzyme gene expression (quantitative RT-PCR)Exocrine enzyme protein (western/Immunofluorescense)
3. Are the cells functional (in vitro and in vivo)?Glucose regulated insulin secretionCell transplantation into SCID-NOD mice
What to do next…..?
4 .What is the distribution of the hormones in the cells?Hormone cell distribution (Immunohistochemistry)
5 .Can the bi-gene adenovirus cure diabetic mice better than PDX-1 or Nkx6.1 alone?
Injection of the virus to diabetic mice
6 .What are the downstream pancreatic transcription factors activated or delayed by the different treatments?
Pancreatic transcriptin factors gene expression (quantitative RT-PCR)DNA microarray
Thank you for your attention…
TaqmanQuantative RT-PCR
Glucose regulated Insulin secretion
0
10
20
30
40
50
60
70
80
90
Insu
lin (
ng
/ml/
mg
pro
tein
)Control (n=2)
GF+Pdx-1 (n=2)
GF+Pdx-1+Ngn3 (n=2)
GF+Pdx-1+Nkx6.1 (n=2)
GF+Pdx-1+Ngn3+Nkx6.1 (n=2)
5.5mM G 2.5mM G 25mM G (45 min)
25mM G (90 min)
25mM G (180 min)
25mM G (12 hours)
Glucose regulated Insulin secretion
Schematic representation
Pdx-1
+
_
Somatostatin
Nkx6.1
Ngn3
Insulin
Pdx-1
Nkx6.1 +
Ngn3 _
Glucagon
Nkx6.1 +
Ngn3 _
Pdx-1
TaqmanReal Time PCR
Pancreatic Transcription factors cascade in pancreas development
Nkx6.1
Nkx2.2
Diabetes mellitus Diabetes is a disease in which the body does not produce or properly use insulin
The cause of diabetes continues to be a mystery, although both genetics and environmental factors appear to play roles
Type 1 Diabetes (5-10%)
Is usually diagnosed in children and young adults
The body does not produce insulin
The insulin-producing cells in the pancreas have been destroyed
Type 2 Diabetes (90-95%)
The most common form of diabetes
Either the pancreas does not produce enough insulin or the cells fail to properly use insulin (insulin resistance)
'maturity onset' diabetes because it usually appears in middle-aged or elderly people
