Genetics and pathophysiology of ARVC AJ Marian, M.D. Center for - - PowerPoint PPT Presentation

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Genetics and pathophysiology of ARVC AJ Marian, M.D. Center for - - PowerPoint PPT Presentation

Sep 15, 2022 155 likes •704 views

8 th International SADS Foundation Conference: Preventing Unexpected Sudden Death in the Young Jointly Provided by: NYU Post Graduate Medical School (NYU PGMS) And Sudden Arrhythmia Death Syndromes Foundation (SADS) Genetics and

slide-1
SLIDE 1

AJ Marian, M.D. Center for Cardiovascular Genetics

Brown ¡Foundation ¡Institute ¡of ¡Molecular ¡ Medicine ¡

for ¡the ¡Prevention ¡of ¡Human ¡Diseases ¡ Center ¡for ¡Cardiovascular ¡Genetics ¡

8th International SADS Foundation Conference: Preventing Unexpected Sudden Death in the Young Jointly Provided by: NYU Post Graduate Medical School (NYU‐PGMS) And Sudden Arrhythmia Death Syndromes Foundation (SADS)

Genetics and pathophysiology of ARVC

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SLIDE 2

Rare Variant-Rare Disease

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SLIDE 3

Arrhythmogenic ¡Cardiomyopathy ¡(AC) ¡

Normal

AC

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SLIDE 4
slide-5
SLIDE 5

Causes ¡of ¡SCD ¡in ¡young ¡athletes ¡

HCM

HCM ARVC DCM Mitral valve prolapse CCAA CAD Others

ARVC (22%) HCM (46%) ARVC

Corrado D. N Engl J Med 1998 Maron BJ. JAMA 1996

U.S.A. Italy

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SLIDE 6

The ¡Enigma ¡of ¡ARVC ¡

H&E ¡ Masson ¡trichrome ¡

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SLIDE 7

Dionysos, the God of vines and wine, who was born from a thigh of his father Jupiter (parthenogenesis), sits resting on a rock, his leg on the thigh of Ariadne, the daughter of Minos, a pose symbolic of ‘sacred marriage’.(Bronze krater from Derveni, 320–300 BC). Naxos Island

The Island that Solved the Riddle of AC

slide-8
SLIDE 8

Naxos Disease

D

Coonar AS Circulation. 1998

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SLIDE 9

A Truncated Mutation in Junction Plakoglobin (JUP) Causes Naxos Disease

McKoy G et al. The Lancet 2002

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SLIDE 10

Locus Gene Symbol Gene Prevalence

12p11 PKP2 Plakophilin 2 ~25% 18q12 DSG2 Desmoglein 2 ~10-15% 18q12 DSC2 Desmocollin 2 ~10-15% 6p24 DSP1 Desmoplakin <5% 17q21 JUP Plakoglobin Uncommon 3p23 TMEM43 Transmembrane protein 43 Rare 1q21.2

LMNA ¡

Lamin A/C Rare 14q24 TGFB3 ? Transforming growth factor β3 Rare 6p22.1

PLN? ¡

Phospholamban Rare 11p15.5

KCNQ1? ¡

Iks channels Rare

Known Causal Genes for ARVC

slide-11
SLIDE 11
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SLIDE 12
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SLIDE 13

PKP2 PKP2

D S G D S C JUP

β-cat

CHD2 JUP

β-cat

LRP5/6

Dvl

Wnt

PKP2 PKP2

Canonical Wnt Desmosome Adherens junction (AJ)

DSP

Gap junction (GJ)

GJA1

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SLIDE 14

Pathogenesis ¡of ¡ARVC ¡

?

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SLIDE 15

Molecular Remodeling Of IDs in Human AC

JUP PKP2 DSC2 DSG2 GJA1 CDH2 MT Normal AC DSP Human Heart

1 2 1 2 3 4

Normal AC

kD

100 75 100 75 150 150

GAPDH

50 37 50 37 37 100 150 50

GAPDH

250

Human Heart JUP PKP2 DSC2 DSG2 DSP GJA1 CDH2

JUP

*** *** ***

PKP2 DSC2 DSG2 GJA1

* ** *** *

DSP

AC Normal Fold change Fold change

1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0

slide-16
SLIDE 16

Transcriptome ¡in ¡Human ¡AC ¡

Human Heart

Normal AC

1 2 3 5 1 2 3

Wnt9a Prickle1 Sfrp1 Glul Gadd45g Dixdc1 Jun Atm Ddx17 Akr1c3 Myh6 Ndnf Lrrc32 Ndrg2 Serpina3 Lmna Eif5a Ngfr Orc4

Canonical Wnt signaling Human Heart

Normal AC

1 2 3 5 1 2 3

Differentially expressed genes

(FDR0.05)

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SLIDE 17

Plakophilin 2 (PKP2) knock down (KD) HL-1 Myocyte and Mouse Models

Conditional Knockdown of PKP2 in mouse heart

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SLIDE 18

Myof Adamts5 Cyp1b1 Cyr61 Lcp1 Prss23 Nav3 Cav1 Amotl2 Ankrd1 Sema3c Irs1 Cav2 Bmp4 Eomes Smad1 Jun Plk2 Slc2a4 Cenpa Itgb2 Myocd Jak1 Adamts1 Runx1t1 Dusp6 Serpine1 Runx1 Inhba Plac8 Ctgf Ccnd1

−1 1

Row Z−Score

2 1 1

RNA-Seq

HL-1 WT HL-1 Pkp2-shRNA

YAP-TEAD Transcription in HL-1Pkp2shRNA Cells

  • 4
  • 2

4 5 10 15

q<0.05

2

Log2 (Fold change)

  • Log10 (q value)

Transcripts/Million

1 2 3 4

Ccnd1 Bmp4 Runxt1 Ctgf Plac8

5 10 15 20

Runx1 Serpine1 Inhba Adamts1 Cyr61

* ** *

* * ** * *

*

qPCR

(Normalized Relative Ratio)

0.0 0.4 0.8 1.2

***

0.0 0.4 0.8 1.2

***

0.0 0.4 0.8 1.2

***

0.0 0.4 0.8 1.2

***

0.0 0.4 0.8 1.2

*

Ankrd1 Ctgf Cyr61 Ccdn1 Inhba

Transcript copy number

0.0 0.5 1.0

**

TEAD-Luciferase activity

HL-1WT HL-1Pkp2:shRNA

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SLIDE 19

U6

Puro

PKP2:shRNA

cPPT hPGK

Puro P<0.001(N=3)

Normalized Relative Pkp2 mRNA

0.0 0.5 1.0 1.5

P<0.001(N=5)

A

0.0 0.5 1.0 1.5

P=0.04(N=3) P=0.03(N=5) Cells

S c r a m b l e s h R N A P k p 2 : s h R N A 1 P k p 2 : s h R N A 2

PKP2

75 100

C e l l s S c r a m b l e : s h R N A P k p 2 : s h R N A 1 P k p 2 : s h R N A 2

HL-1

C

Cells

S c r a m b l e s h R N A P k p 2 : s h R N A 1 P k p 2 : s h R N A 2

D E

  • 4
  • 2

1 2 3 2 4

  • log (p value)

log2 (fold change) miR-184 F

HL-1Pkp2:shRNA HL-1

Row min Row max

miR-184

  • 15.92

miR-881

  • 8.06

miR-574-3p

  • 5.49

miR-465b-5p

  • 5.35

miR-100

  • 4.34

miR-470

  • 4.08

miR-671-3p

  • 4.01

miR-10a

  • 3.75

miR-741

  • 2.90

miR-34b-3p

  • 2.76

miR-23b

  • 2.74

miR-99a

  • 2.71

miR-196b

  • 2.69

miR-1939

  • 2.15

miR-196a

  • 2.12

miR-338-5P

  • 2.09

miR-1198 2.51 miR-133a 2.67 miR-200a 2.68 miR-351 2.71 miR-20b 2.71 miR-132 2.73 miR-342-3p 2.74 miR-362-5p 3.80 miR-1 4.06 miR-676 5.48 miR-429 5.53 miR-188-5p 6.69 miR-487b 7.39 miR-200b 7.79

G

P<0.001 (N=4) P<0.001 (N=14)

0.0 0.5 1.0 1.5 Cells

S c r a m b l e : s h R N A P k p 2 : s h R N A 1 P k p 2 : s h R N A 2 HL-1 HL-1Pkp2:shRNA Normalized Relative miR-184 Normalized Relative miR-200b Normalized Relative miR-429 HL-1 HL-1Pkp2:shRNA

H

P<0.0001 (N=3) P<0.0001 (N=3) 50

TUBA1B

Fold Change

Fold Change

PKP2

B I

Differentially Expressed miRNAs, miRNA:mRNA Pathway Analysis

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SLIDE 20

The Pathway(s) From PKP2 of Desmosomes to YAP/TEAD of the Hippo Pathway

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SLIDE 21

Inactivation of YAP by Phosphorylation in AC: HL-1Pkp2:shRNA Myocytes

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SLIDE 22

Inactivation of YAP by Phosphorylation in AC: Mouse Models

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SLIDE 23

Inactivation of YAP by Phosphorylation in AC: Human Hearts

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SLIDE 24

Activation of The Hippo Pathway in AC: HL-1Pkp2:shRNA Myocytes

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SLIDE 25

Activation of The Hippo Pathway in AC: Mouse Models

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SLIDE 26

Activation of The Hippo Pathway in AC: Human Heart

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SLIDE 27

PKCα as a link between PKP2 and the Hippo Pathway

Intercalated Intercalated Discs Discs

TEAD Hippo

Active Active Active Active Inactive

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SLIDE 28

pPKCα

75 50 75

PKP2

HL-1

WT Pkp2:shRNA TUBA1B

kD

HL-1WT HL-1Pkp2:shRNA PKP2 PKP2 pPKC-α pPKC-α Overlay Overlay pPKC-α

Localization and Level of pPKCα in AC: HL-1Pkp2:shRNA Myocytes

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SLIDE 29

Localization and pPKCα Level in AC: Mouse Models

Fold change Fold change

1.2 0.8 0.6 0.4 0.2 0.0 1.0 1.2 0.8 0.6 0.4 0.2 0.0 1.0

Nkx2.5:DspW/F Myh6:Jup NTG

Mouse pPKC-α

p=0.002 (N=2) p=0.003 (N=2)

NTG

kD

N T G N T G M y h 6 : J u p N k x 2 . 5 : D s p W / F

75

pPKCα

50

TUBA1B

pPKC-α Overlay pPKC-α Overlay

Mouse models of AC NTG Myh6:Jup Nkx2.5:DspW/F

PKP2 pPKC-α Overlay PKP2 PKP2 PKP2

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SLIDE 30

Normal AC Human Heart

PKP2 PKP2 pPKC-α pPKC-α Overlay Overlay

kD

75

pPKCα

50 37

GAPDH

1 2 1 2 3 4

Normal AC

*

AC Normal Fold change

Human pPKC-α

1.2 0.8 0.6 0.4 0.2 0.0 1.0

p=0.007 (N=2-4)

Localization and Level of pPKCα in AC: Human Hearts

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SLIDE 31

The Hippo Pathway

Intercalated Intercalated Discs Discs

TEAD Hippo

Active Active Active Active Inactive

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SLIDE 32

Relative luciferase activity TCF-Luciferase Reporter Assay

HL-1 WT HL-1Pkp2:shRNA

*

0.0 0.4 0.8 1.2

Fzd2 Gsk3b Rarb Akt3 Tgfb3 LRP5 Ppard Dvl3 Jun Ccnd1 Myc

1

  • 1

Color Key

1 2 1 2

Row Z−Score

HL-1WT HL-1Pkp2:shRNA

Csnk1e Tab1 Wnt5b Wnt11 Sox12 Csnk2a1 Tle1 Ppp2r3a Tle4 Nlk Sfrp5 Wif1

Normalized Relative Ccnd1 mRNA

Ccnd1

0.0 0.4 0.8 1.2

Ctgf

***

c-Jun

  • 0. 0
  • 0. 4
  • 0. 8
  • 1. 2

Bves

***

0.0 0.4 0.8 1.2

Sox2

***

0. 0. 0. 1. 4 8 2

Snai1

*** ***

0.0 0.4 0.8 1.2 0.0 0.4 0.8 1.2

***

HL-1WT HL-1Pkp2:shRNA

Suppression of the Canonical Wnt Signaling in the HL-1Pkp2:shRNA Myocytes: Transcriptional Activity

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SLIDE 33

HL-1WT HL-1Pkp2:shRNA CTNNB1 CTNNB1 CTNNB1:DAPI CTNNB1:DAPI pCTNNB1 pCTNNB1:DAPI pCTNNB1:DAPI pCTNNB1

HL-1WT HL-1Pkp2:shRNA

kD 100 50 100 50

pCTNNB1 CTNNB1 GSK3B AXIN1 TUBA1B pCTNNB1

100 100

HL-1 WT Pkp2:shRNA Phos-Tag

Suppression of the Canonical Wnt Signaling in AC: pβ-Catenin Localization and Levels in the HL-1Pkp2:shRNA Myocytes

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SLIDE 34

CTNNB1 TUBA1 pCTNNB1

100 100

NTG NTG Myh6:Jup Nkx2.5: DspW/F

kD

50

pCTNNB1

Fold Change Fold Change

Nkx2.5:DspW/F Myh6:Jup NTG

CTNNB1

8.0 6.0 4.0 0.0

*

2.0

***

5.5 3.5 1.5 0.0 2.5 2.0 1.5 1.0 0.5 0.0

*

1.2 1.0 0.8 0.6 0.4 0.2 0.0

NTG Myh6:Jup (PG) Nkx2.5:DspW/F

pCTNNB1:DAPI CTNNB1:DAPI pCTNNB1:DAPI CTNNB1:DAPI

Suppression of the Canonical Wnt Signaling in AC: pβ-Catenin Localization and Levels in the Mouse Models

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SLIDE 35

Suppression of the Canonical Wnt Signaling in AC: pβ-Catenin Localization and Levels in the Human Hearts

CTNNB1 GAPDH pCTNNB1 1 2 3 4

Normal AC

kD

Normal AC

pCTNNB1:DAPI

37 100 100 Fold Change

CTNNB1:DAPI

1 2 3 4

Normal AC

pCTNNB1 *

2.5 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0

CTNNB1

Fold Change

Normal AC

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SLIDE 36

Hippo/Wnt Interactions

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SLIDE 37

Amino ¡acid ¡sequence ¡iden;ty ¡= ¡88% ¡ β-­‑catenin ¡

TCF7L2

Plakoglobin ¡ (JUP) ¡

Shared Origin of JUP (Plakoglobin, γ-Catenin) and β-Catenin Through Gene Duplication

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SLIDE 38

Wild Type

siDSP β-Catenin JUP β-Catenin JUP DAPI DAPI Overlay Overlay Control siDSP-1102

JUP

Cytoplasmic Nuclear Control siDSP-1102

β-Catenin Tubulin

Nuclear Localization of JUP Upon Knock Down of DSP in HL1 Cells

slide-39
SLIDE 39

B

50 37

75 ¡ Lamin A JUPTR Specific Pan JUP Tubulin Flag (JUPWT)

Nucleus Cytosol Membrane

100 100 75 75 100 50 75

Antibodies Cx 43

A

NTG JUPWT JUPTR

DNA:Pan-JUP DNA:Pan-JUP DNA:Pan-JUP DNA:JUPTR DNA:Flag (JUPWT) DNA:Flag (JUPWT) DNA:Flag (JUPWT) DNA:JUPTR DNA:JUPTR

Nuclear Localization of Transgene JUP

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SLIDE 40

IB:Pan-JUP

kDa 100 75

IB:Flag (JUPWT) IB:JUPTR - Specific

100 75 100 75

Input: TCF7L2-4

75

IP: TCF7L2

Nuclear Localization and Co-IP of JUP and TCF7L2 Transcription Factor

HL-1WT HL-1Pkp2:shRNA JUP JUP JUP:DAPI JUP:DAPI

slide-41
SLIDE 41

5 10 15 20 25

F=48.7, p<0.001 * p<0.001

* * * * * * FOP FOP FOP FOP TOP TOP TOP TOP Control GFP siRNA Dsp siRNA-799 Dsp siRNA-1102

Fold Change Suppression of The Canonical Wnt/β-Catenin Signaling

slide-42
SLIDE 42

100 75 100 75

IgG IB IP: YAP

HL-1 Cells

YAP CTNNB1

100 75

YAP

75

JUP Input

75 100 75

IP: YAP CTNNB1 JUP IgG Normal Normal ARVC ARVC Input IB

Human Heart

A B

WT Pkp2: shRNA WT Pkp2: shRNA

Hippo:Canonical Wnt Interactions: Co-IP of YAP , β-Catenin (CTNNB1) and JUP

slide-43
SLIDE 43

HL-1WT HL-1Pkp2:shRNA 40x 40x 20x 100x 63x 100x 63x 20x

A

B

Phenotypic Consequences of Activation of the Hippo Pathway in AC: Cell shape, size, and morphology

slide-44
SLIDE 44

HL-1WT HL-1Pkp2:shRNA

Oil Red O Oil Red O

PPARG PPARG:DAPI PPARG:DAPI % ORO+ cells

10 20 30 40 50 60 70

**

HL-1WT HL-1Pkp2:shRNA

% PPARG+ cells

HL-1WT HL-1Pkp2:shRNA

10 5

Phenotypic Consequences of Activation of the Hippo Pathway in AC: Enhanced Adipogenesis

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SLIDE 45

Transcription Switch To Adipogenesis in DSP- KD HL-1 Cells

slide-46
SLIDE 46

Untreated Dex + Ins + IBMX

Control

Suppressed Canonical Wnt Higher Magnification

Suppression of the Canonical Wnt Signaling and Enhanced Adipogenesis

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SLIDE 47

A

Normalized relative Lats1 mRNA

Lats1

No target-shRNA Lats1-shRNA#39 Lats2-shRNA#87

  • Lats2-shRNA#706

+

  • +
  • +

+

  • +
  • +
  • +
  • +

+

  • +

HL-1WT HL-1Pkp2:shRNA

0.0 0.5 1.0 1.5

** ** *

B Lats2

  • +
  • +
  • +

+

  • +
  • +
  • +
  • +

+

  • +

Normalized relative Lats2 mRNA No target-shRNA Lats1-shRNA#39 Lats2-shRNA#87 Lats2-shRNA#706 HL-1WT HL-1Pkp2:shRNA

0.0 0.4 0.8 1.2

*** *** ** **

kD 150 75 75 100 100 50

No target-shRNA Lats1-shRNA #39 Lats1-shRNA #42 Lats2-shRNA #87 Lats2-shRNA #706

  • +
  • +
  • +

+

  • +

+ + + +

  • +
  • +
  • +

+

  • +

+ + + +

  • HL-1WT

HL-1Pkp2:shRNA

pYAP TotalYAP LATS1 pCTNNB1 CTNNB1 TUBA1B

C

Phenotypic Rescue Upon Knock Down of Hippo (Lat1 and Lats2) in the HL-1Pkp2:shRNA Myocytes

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SLIDE 48

Cells only HL-1WT HL-1Pkp2:shRNA

ORO ORO ORO PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI

#39-87 LATS1/2-shRNAs No Target shRNA #39-706

ORO ORO ORO ORO ORO

10 20 30

% PPARG+ Cells

  • +
  • +
  • +

+

  • +
  • +
  • +
  • +

+

  • +

HL-1WT

HL-1Pkp2:shRNA No target-shRNA Lats1-shRNA#39 Lats2-shRNA#87 Lats2-shRNA#706 ** ** *** *** ### % ORO+ Cells

  • +
  • +
  • +

+

  • +
  • +
  • +
  • +

+

  • +

HL-1WT

HL-1Pkp2:shRNA No target-shRNA Lats1-shRNA#39 Lats2-shRNA#87 Lats2-shRNA#706

10 20 30 40 50

### *** ***

50 50

HL-1WT HL-1Pkp2:shRNA

Cells only #39-87 LATS1/2

  • shRNAs

LATS1/2

  • shRNAs

No Target shRNA #39-706 Cells only #39-87 No Target shRNA #39-706

PPARG TUBA1B

Phenotypic Rescue of Adipogenesis Upon Knock Down of Hippo (Lat1 and Lats2) in the HL-1Pkp2:shRNA Myocytes

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SLIDE 49

36.4 ¡ 13.5 ¡ 9.9 ¡ 8.3 ¡ 10 20 30 40 50 *p<0.001 vs. no BIO No BIO BIO-2µM BIO-5µM BIO-10µM # of Oil Red O + cells/field Bio (µM)

37 ¡ 25 ¡ 16 ¡ 1 ¡

10 20 30 40 50 2 5 10

* * *

*p<0.001 vs. no BIO C/EBPα:DAPI C/EBPα:DAPI C/EBPα:DAPI C/EBPα:DAPI % C/EBPα + cells (N=200) 2 5 10 Bio (µM)

* * *

ORO ORO ORO ORO

Activation of Canonical Wnt Signaling Rescues Adipogenesis

slide-50
SLIDE 50

PG+/+ PG-/-

A

c-kit APC

Lin-V450 Flk-1 PE Cy7

Cell Count

B

Lin-V450 Flk-1 PE Cy7

c-kit APC Cell Count

SSCXFSC SSCXFSC

PG+/+ PG-/-

1 22 5 10 15 20 25 1 2

0 ¡ 0.5 ¡ 1 ¡ 1.5 ¡ 2 ¡ 2.5 ¡

PG+/+ PG-/- Fold Change 1 48 10 20 30 40 50 60

Ctgf c-myc

1 3

0 ¡ 1 ¡ 2 ¡ 3 ¡ 4 ¡ Cycin D1

1 7

0 ¡ 2 ¡ 4 ¡ 6 ¡ 8 ¡ Cyclin E1

1 4

0 ¡ 1 ¡ 2 ¡ 3 ¡ 4 ¡ 5 ¡ Sox2

PG+/+ PG-/- PG+/+ PG-/- PG+/+ PG-/- PG+/+ PG-/- p< 0.001 p< 0.001 p< 0.001 p< 0.001 p< 0.001

Pcna

PG+/+ PG-/- p< 0.001

C

Activation of Canonical Wnt Signaling Suppresses Adipogenesis

slide-51
SLIDE 51

B

7.4

0.5

0 ¡ 2 ¡ 4 ¡ 6 ¡ 8 ¡ 10 ¡ 12 ¡ 14 ¡

p< 0.001 PG+/+ PG-/- # of ORO+ cells/ field PG+/+ PG-/-

9.6 0.6

2 4 6 8 10 12 14 16 18 20

p< 0.001 % C/EBPα + cells(N=400)

A

C-kit+:PG+/+

40x ¡ C/EBPα:DAPI ORO

TPM1

C-kit+: PG-/-

C/EBPα:DAPI ORO

Activation of Canonical Wnt Signaling Rescues Adipogenesis and Enhances Myogenesis

slide-52
SLIDE 52

No target-shRNA Jup-shRNA#21

  • +
  • +

+

  • Jup-shRNA#18

HL-1WT

  • +
  • +

+

  • HL-1Pkp2:shRNA
  • pCTNNB1

100 75 100 50

A

  • +
  • +
  • +
  • Jup
  • +
  • +
  • +
  • 0.0

0.5 1.0 1.5 HL-1WT HL-1Pkp2:shRNA

B

No target-shRNA Jup-shRNA#18 Jup-shRNA#21

HL-1WT HL-1Pkp2:shRNA

  • +
  • +
  • +
  • +
  • +
  • +
  • No target-shRNA

Jup-shRNA#21 Jup-shRNA#18 HL-1WT HL-1Pkp2:shRNA

Normalized relative Luciferase activity

TCF- Canonical Wnt Reporter Assay

  • +
  • +
  • +
  • +
  • +
  • +
  • No target-shRNA

Jup-shRNA#21 Jup-shRNA#18

Jup-shRNA#21 Cells No target-shRNA Jup-shRNA#18 HL-1WT HL-1Pkp2:shRNA

PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI PPARG:DAPI

Oil Red O Oil Red O Oil Red O Oil Red O Oil Red O Oil Red O Oil Red O Oil Red O

C D E F

Normalized Relative Jup mRNA level

% PPARG+ Cells Ccnd1

0.00 0.02 0.04 0.06 0.5 1.0 1.5 2.0

##

Normalized Relative mRNA level

  • +
  • +
  • +
  • +
  • +
  • +
  • HL-1WT

HL-1Pkp2:shRNA

No target-shRNA Jup-shRNA#18 Jup-shRNA#21

Ctgf

1 2 3

#

  • +
  • +
  • +
  • +
  • +
  • +
  • HL-1WT

HL-1Pkp2:shRNA

#

0.5 1.0 1.5 2.0 2.5

Inhba

  • +
  • +
  • +
  • +
  • +
  • +
  • HL-1WT

HL-1Pkp2:shRNA

#

Cyr61

1 2 3 4 5

  • +
  • +
  • +
  • +
  • +
  • +
  • HL-1WT

HL-1Pkp2:shRNA

1 2

Ankrd1

3

  • +
  • +
  • +
  • +
  • +
  • +
  • HL-1WT

HL-1Pkp2:shRNA

0.0 0.5 1.0 1.5 2.0 10 20 30 40 50

75

pYAP TUBA1B JUP

*** *** ******

###

*

slide-53
SLIDE 53

DSG

D S C

JUP

β

  • c

a t

JUP

β

  • c

a t

LRP5/6

Axin APC

Dvl

Wnt

β-cat

P P P P

GSK3β

CKI

PKCα

NF2 (Merlin)

MST1/2

P

LATS1/2

P

YAP1

P

PKP2 Canonical Wnt β

  • c

a t

YAP1

TCF7L2 TEAD

YAP1

β

  • c

a t H i p p

  • P

a t h w a y JUP

Intercalated Discs

β-cat

P P P P

JUP Desmosome Adherens junction (AJ)

DSP UPS BDC CHD

PKP2

PKCα

Activation of the Hippo and Suppression of the Canonical Wnt Pathways in the Pathogenesis of AC

slide-54
SLIDE 54