Protein Kinase C M odulates Wnt Signaling In Colon Tumoral ... · Protein Kinase C M odulates Wnt...
Transcript of Protein Kinase C M odulates Wnt Signaling In Colon Tumoral ... · Protein Kinase C M odulates Wnt...
Dra Martha Robles FloresDra Martha Robles FloresDepartment of BiochemistryDepartment of BiochemistryFacultad de MedicinaFacultad de MedicinaUniversidad Nacional Autónoma de MéxicoUniversidad Nacional Autónoma de México
Protein Kinase C Modulates Wnt Signaling In Colon Tumoral Cell Lines
Wnt
DifferentiationCell cycle arrest
TGF-β, BMP
Stem cell renewalProliferation
Tissue anatomy of the colonic epithelium
β‐cateninC
ProteosomalDegradation
OFFLEF/TCF
TLE-1
Wnt
LRP 5/6 Frizzled
N
PP P
PPP
Dvl
PPP
Nuclei
LEF/TCF
ON
HDAC
Nuclei
β‐catenin acummulation
Lgs/Bcl-9 Pygo
Canonical Wnt Signaling
PP
LRP5/6FzWnt3a
GSK3-β / Axin/APC
β-catenin
TCF/LEF
Wnt gene target transcription
FzWnt5a
Dvl
G Protein
Ca++ mobilization
PKC?
Canonical Wnt Signaling(Wnt/ β -catenin)
Non-canonical Wnt Signaling(Wnt/Ca++)
Planar cell polarityNegative modulation of TCF actions
Dvl PLC
IP3DAG
?
CAMKCAMK
RoR
Heider et al, 2004;
Perletti, 1998
Yu, 2003, Murray, et
al, 1999
Black, 2001, Cerda et
al, 2006.
Goldstein, 1995, Assert,1999.
Frey 1997, Hizli, 2006,
Black 2000.
References
Cancer cell lines proliferation and
migration
Protein and mRNA levels
PKCβ II
Cellular proliferation,
migration (HT-29)Protein levelsPKCε
Apoptosis (CaCo-2)
Protein and mRNA levels
PKCδ
Cellular differentiation
(SW-480)Protein levelsPKCβ I
Cellular differentiation
(IEC-18) Protein levelsPKCα
Colon cell linesAPC MIN mice modelPKC Isoform
NON MALIGNANT CELL LINESNON MALIGNANT CELL LINES COLON CARCINOMA CELL COLON CARCINOMA CELL LINESLINES
IECIEC--1818(rat)(rat)
112112--CoNCoN(human)(human)
RKORKO(human)(human)
SWSW--480480(human)(human)
COMPLETE COMPLETE APCAPC
COMPLETE COMPLETE APCAPC
COMPLETE COMPLETE APCAPC
TRUNCATED APCTRUNCATED APC
Normal Wnt Normal Wnt signalingsignaling
Normal Wnt Normal Wnt signalingsignaling
Normal Wnt Normal Wnt signalingsignaling
Altered Wnt Altered Wnt signaling signaling (constitutively (constitutively active)active)
APC
PKCδ
Truncated APC
Comparative expression profile of PKC isoforms in colon cultured cell linesde colon
66±±0.20.244±±0.10.111μμ
22±± 00..010144±±00..070711ζζ
1.0 1.0 ±±0.020.0244±±1100ηη
55±± 0.090.092 2 ±±0.030.0311εε
44±±0.050.053 3 ±±0.070.0711ββIIII
00..11±±00..020200..1 1 ±±00..060611δδ
000.05 0.05 ±±0.040.0411ββ II
000.2 0.2 ±±0.050.0511αα
RKORKOHTHT--2929112 CoN112 CoNPKCPKC
DOWNDOWN--REGULATEDREGULATED UPUP--REGULATEDREGULATED
Normal Malignant
RKO
GSK-3 PKC-ζ Merge
IEC-18n
β-cat PKC-ζ Merge
IP: GSK-3
PKC-ζ
GSK-3
HT-29c RKOc IEC-18n
WB
IEC-18n
RKO
IP: β-catenin
PKC ζ
β-cat
HT-29c RKOc IEC-18n
APC
IP: APCIP: APC
PKCδ Merge
IECIEC--18 (N) 18 (N)
HTHT--29 ( C ) 29 ( C )
250250
150150
7575
5050
PKCα
APC
IP: APCIP: APCWB:WB:
PKCδ
APC
APC
PKCα Merge
250250
7575
5050
IECIEC--18 18 (N) (N)
HTHT--29 ( C ) 29 ( C )
GOALS
¿What is the biological meaning of PKCα/δinteraction with APC?
¿What is the biological meaning of β-catenininteraction with PKCζ?
¿Can PKC modulate canonical Wnt signaling?
IEC-18(Normal)
RKO SW4800
1
2
3
4
5
6TOPFlash (functional TCF sites)FOPFlash (mutated TCF sites)
TCF-
sens
itive
Tra
ncri
ptio
nal
activ
ity (F
old)
0
0.2
0.4
0.6
0.8
1
1.2
FOPTOP
Control Wnt5a Wnt3a
RKO
0
5
10
15
20
25
30
35
FOPFLASHTOPFLASH
Wnt 3a
PKCζ i (20μM)
-
-
-
+
- + +
- - +
SW480 RKO
PKCζ selective inhibitor blocks β-catenin-mediated transcriptional activityin both RKO and SW480 colon carcinoma cell lines
Nor
mal
ized
luci
fera
se a
ctiv
ity
Psuper PKC RNAi (μg) 0 1 2
1
0.8
0.6
0.4
0.2
PKCζ
0 1 2
Actin
pSuper.PKCz.RNAi (μg)
PKCζ knockdown blocks in a dose-dependent manner theβ-catenin-mediated transcriptional activity
WB:
0
20
40
60
80
100
120
PKCζ i (20 μM) +−
Prol
ifera
tiom
(%
MTT
Red
uctio
n)
SW480 cellsWB:
c-myc
c-myc
GAPDH
PKCζi (20 μM)
+−
PCR
Effect of PKCζ knockdown on cell proliferation and Wnt targetgene expression (c-myc)
actin
0
0.5
1
1.5
2
2.5
3
3.5
4
Control Vehicle Rottlerin 3uM
Nor
mal
ized
Luc
ifera
se A
ctiv
ity
FOP
TOP
SWSW--480480
Effect of PKCδ inhibition on β-catenin-mediated transcriptional activity
0
0.02
0.04
0.06
0.08
0.1
0.12
Control Wnt3 Rottlerin 3uM+Wnt3
Nor
mal
ized
Luc
ifera
se A
ctiv
ity
FOPTOP
RKORKO
Effect of PKCδ inhibition on β‐catenin‐mediated transcriptional activity
0123456789
10
Nor
mal
ized
Luc
ifera
se A
ctiv
ity
Wnt3aWnt3aRottlerinRottlerin
-- + + + ++ + + +
-- 33μμM 4M 4μμM 6M 6μμM 10M 10μμMM
SUMMARY
• PKC isoforms associate in vivo with key Wnt canonical proteins: PKCζ,upregulated in malignant cells, interacts with GSK3β and with β-cateninmainly in cancerous cells. PKCδ, downregulated in malignant cells,interacts with APC in both normal and malignant cells.
• Pharmacological inhibition of PKCζ, or its decreased expression blocked ina dose-dependent manner canonical Wnt activation in cancer cell lines,suggesting that PKCζ modulates in a positive way canonical Wnt activation.
• Pharmacological inhibition of PKCδ or its decreased expression, improvedin a dose-dependent way canonical Wnt activation in RKO cancer cells,suggesting that PKCδ modulates in a negative way canonical Wntactivation.
Altogether, our results indicate that PKC isoforms play an essential role in the regulation of canonical
Wnt pathway.