Protein Kinase C Modulates Wnt Signaling In Colon Tumoral Cell Lines Dra Martha Robles Flores Dra Martha Robles Flores Department of Biochemistry Department of Biochemistry Facultad de Medicina Facultad de Medicina Universidad Nacional Autónoma de México Universidad Nacional Autónoma de México
Tissue anatomy of the colonic TG TGF- β, BM BMP epithelium Differentiation Cell cycle arrest Wnt Stem cell renewal Proliferation
Canonical Wnt Signaling Wnt LRP 5/6 Frizzled P P Dvl P P P P N C β‐ catenin P P P Proteosomal Degradation β ‐ catenin acummulation Nuclei Nuclei HDAC TLE-1 ON LEF/TCF OFF LEF/TCF Lgs/Bcl-9 Pygo
Canonical Wnt Signaling Non-canonical Wnt Signaling (Wnt/ β -catenin) (Wnt/Ca++) Wnt3a Wnt5a RoR Fz Fz LRP5/6 G Protein Dvl GSK3- β / Axin/APC Dvl PLC ? IP3 DAG β -catenin Ca ++ mobilization PKC? TCF/LEF CAMK CAMK Planar cell polarity Wnt gene target transcription Negative modulation of TCF actions
APC MI N mice model PKC Colon cell lines References I soform Cellular Frey 1997, Hizli, 2006, differentiation PKC α Protein levels Black 2000. (IEC-18) Cellular differentiat ion Goldstein, 1995, Assert, PKC β I Protein levels 1999. (SW-480) Protein and mRNA levels Apoptosis Black, 2001, Cerda et PKC δ (CaCo-2) al, 2006. Protein and mRNA levels Cancer cell lines Yu, 2003, Murray, et proliferation and PKC β I I al, 1999 migration Cellular Heider et al, 2004; proliferation, PKC ε Protein levels Perletti, 1998 migration (HT-29)
NON MALIGNANT CELL LINES NON MALIGNANT CELL LINES COLON CARCINOMA CELL COLON CARCINOMA CELL LINES LINES IEC IEC- -18 18 112- 112 -CoN CoN RKO RKO SW SW- -480 480 (rat) (rat) (human) (human) (human) (human) (human) (human) COMPLETE COMPLETE COMPLETE COMPLETE COMPLETE COMPLETE TRUNCATED APC TRUNCATED APC APC APC APC APC APC APC Normal Wnt Normal Wnt Normal Wnt Normal Wnt Normal Wnt Normal Wnt Altered Wnt Altered Wnt signaling signaling signaling signaling signaling signaling signaling signaling (constitutively (constitutively active) active) APC Truncated APC PKC δ
Comparative expression profile of PKC isoforms in colon cultured cell linesde colon Normal Malignant PKC PKC 112 CoN 112 CoN HT- HT -29 29 RKO RKO α 0.2 ± 0.05 1 0.2 0.05 0 β I 0.05 ± 0.04 1 0.05 0.04 0 δ 1 ± 0. 1 ± 0. 1 0. .1 .06 06 0. .1 .02 02 β II 3 ± 0.07 4 ± 0.05 II 1 3 0.07 0.05 ε 2 ± 0.03 5 ± 0.09 1 2 0.03 0.09 η 4 ± 1 1.0 ± 0.02 0 1.0 0.02 ζ 4 ± 0. 2 ± 0. 1 .07 07 .01 01 μ 4 ± 0.1 6 ± 0.2 1 0.1 0.2 UP UP- -REGULATED REGULATED DOWN DOWN- -REGULATED REGULATED
IEC-18 n IEC-18 n RKO RKO PKC- ζ β -cat PKC- ζ GSK-3 Merge Merge IP: β -catenin IP: GSK-3 WB PKC ζ PKC- ζ β -cat GSK-3 HT-29 c RKO c I EC-18 n HT-29 c RKO c I EC-18 n
IP: IP: APC APC WB: WB: APC 250 250 IEC IEC- -18 18 (N) (N) 150 150 HT HT- -29 ( C ) 29 ( C ) PKC α 75 75 PKC α Merge APC 50 50 IP: IP: APC APC APC IEC IEC- -18 (N) 18 (N) 250 250 HT HT- -29 ( C ) 29 ( C ) PKC δ 75 75 50 50 PKC δ APC Merge
GOALS ¿What is the biological meaning of PKC α / δ interaction with APC? ¿What is the biological meaning of β -catenin interaction with PKC ζ ? ¿Can PKC modulate canonical Wnt signaling?
TCF-sensitive Trancriptional 6 TOPFlash (functional TCF sites) 5 FOPFlash (mutated TCF sites) activity (Fold) 4 3 2 1 0 IEC-18 RKO SW480 (Normal) 1.2 RKO 1 0.8 FOP 0.6 TOP 0.4 0.2 0 Control Wnt5a Wnt3a
PKC ζ selective inhibitor blocks β -catenin-mediated transcriptional activity in both RKO and SW480 colon carcinoma cell lines SW480 RKO 35 30 25 20 15 10 5 FOPFLASH TOPFLASH 0 - + + Wnt 3a - - - - + - + PKC ζ i (20 μ M)
PKC ζ knockdown blocks in a dose-dependent manner the β -catenin-mediated transcriptional activity Normalized luciferase activity 1 pSuper.PKCz.RNAi ( μ g) 0 1 2 0.8 WB: 0.6 PKC ζ 0.4 0.2 Actin Psuper PKC RNAi ( μ g) 0 1 2
Effect of PKC ζ knockdown on cell proliferation and Wnt target gene expression (c-myc) SW480 cells 120 WB: 100 (% MTT Reduction) c-myc Proliferatiom 80 actin 60 40 PKC ζ i (20 μ M) 20 PCR 0 c-myc − PKC ζ i (20 μ M) + GAPDH − +
Effect of PKC δ inhibition on β -catenin-mediated transcriptional activity SW- SW -480 480 Normalized Luciferase Activity 4 3.5 3 FOP 2.5 TOP 2 1.5 1 0.5 0 Control Vehicle Rottlerin 3uM
Effect of PKC δ inhibition on β‐ catenin ‐ mediated transcriptional activity 0.12 Normalized Luciferase Activity 0.1 RKO RKO 0.08 FOP 0.06 TOP 0.04 0.02 0 Control Wnt3 Rottlerin 3uM+ Normalized Luciferase Activity 10 Wnt3 9 8 7 6 5 4 3 2 1 0 - - + + + + + + + + Wnt3a Wnt3a Rottlerin Rottlerin - - 3 μ M 4 M 4 μ M 6 M 6 μ M 10 M 10 μ M
SUMMARY PKC isoforms associate in vivo with key Wnt canonical proteins: PKC ζ , • upregulated in malignant cells, interacts with GSK3 β and with β -catenin mainly in cancerous cells. PKC δ , downregulated in malignant cells, interacts with APC in both normal and malignant cells. Pharmacological inhibition of PKC ζ , or its decreased expression blocked in • a dose-dependent manner canonical Wnt activation in cancer cell lines, suggesting that PKC ζ modulates in a positive way canonical Wnt activation. Pharmacological inhibition of PKC δ or its decreased expression, improved • in a dose-dependent way canonical Wnt activation in RKO cancer cells, suggesting that PKC δ modulates in a negative way canonical Wnt activation. Altogether, our results indicate that PKC isoforms play an essential role in the regulation of canonical Wnt pathway.
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