-catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total...

29
Sustained β-catenin activation in dermal fibroblasts promotes fibrosis by regulating cell proliferation and extracellular matrix protein-coding genes by Radhika P. Atit, Ph.D. Case Western Reserve University, Cleveland Ohio Depts. Of Biology, Genetics and Genome Sciences, and Dermatology

Transcript of -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total...

Page 1: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Sustained β-catenin activation in dermal fibroblasts promotes fibrosis by regulating cell proliferation and extracellular matrix

protein-coding genes

by Radhika P. Atit, Ph.D.

Case Western Reserve University, Cleveland Ohio Depts. Of Biology, Genetics and Genome Sciences, and Dermatology

Page 2: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Role of Wnt/b-catenin signaling in fibrosis.

• Well established link Wnt signaling and desmoid tumors.

• What is the actual role of Wnt signaling in the etiology of fibrosis?

• Who are the causative cell types?

Dan Geisler ‘10 Emily Hamburg-Shields, Ph.D

MSTP student

Pooja Sandesara ’09 Gregg DiNuoscio, Intrepid Tech who refuses to be photographed

Page 3: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Fibrotic skin has elevated fibroblast β-catenin activity

Patient skin samples generously provided by Monique Hinchcliff & John Varga (Northwestern) and Kord Honda (UH Dermatology)

Page 4: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Wnt activation in other fibrosing diseases

Dupuytren disease/superficial

fibromatosis

Diabetic nephropathy Liver cirrhosis

Morphea Keloid & hypertrophic scars

Idiopathic pulmonary fibrosis Chronic obstructive pulmonary

disease (COPD)

Post-MI scar

Myelofibrosis

Systemic sclerosis Nephrogenic systemic fibrosis Chronic GVHD Adapted from Fibrosis

Research (Varga et al, 2005)

+Tumor stroma

Page 5: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Hypothesis

Activation of Wnt/b-catenin signaling in resident dermal fibroblasts can cause dermal fibrosis.

Page 6: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Advantages of genetic mouse models • Manipulating a single causative cell type at a time: resident dermal fibroblasts

• Modulating a single signaling/factor at a time: Wnt signaling. • Analyze whole dermis/obtain dermal fibroblasts at various time points after

manipulating Wnt signaling. • Identifying context-specific Wnt signaling targets in dermal fibroblasts. • Wnt signaling senstive biomarkers from specific cell types • Testing therapeutic drug targets in vivo.

Our three genetic models

In vivo dermal fibrosis model: Genetically stabilize b-catenin in dermal fibroblasts. Advantages: in vivo, cell-type restricted manipulation, analyze effect on the whole skin

Disadvantages: Difficult to isolate dermal fibroblasts from adult skin for molecular analysis.

In vitro dermal fibroblasts model from perinatal skin: Genetically stabilize b-catenin in dermal fibroblasts. Advantages: dermal fibroblasts for analysis, molecular/genomics

Disadvantages: in vitro, culture conditions.

Inducible/Deinducible over expression of stabilized b-catenin in dermal fibroblasts Advantages: Identify which aspects of the fibrotic phenotype requires sustained expression of b-catenin.

Disadvantages: Duration of deinduction is not known, difficult to isolate dermal fibroblasts from adult/fibrotic skin for molecular analysis.

Page 7: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Model 1: Genetically stabilize β-catenin activity in resident dermal fibroblasts

Page 8: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Masson’s trichrome (P50)

Sustained stabilized β-catenin activity in resident dermal fibroblasts leads

to dermal fibrosis

Page 9: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Sustained stabilized β-catenin activity in dermal fibroblasts leads to increase in collagen deposition with thickened collagen fibrils

Transmission electron microscopy of cross-sectional collagen fibrils

↓ fibril diameter in fibrotic dermis ↑ fibril diameter in fibrotic hypodermis

fiber=bundle of fibrils

Page 10: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

*

*p<0.001

p=0.03

Increased skin thickness: Increased fibroblast proliferation:

hypodermal

fibrosis

dermis

Sustained stabilized β-catenin activity in dermal fibroblasts leads thickened skin and increase in

Page 11: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

matrisome

}

What is the mechanism of pro-fibrotic β-catenin in dermal fibroblasts? - How does dermal gene expression change in response to expression of stabilized β-catenin? - Which of this β-catenin-responsive genes are also dysregulated in human fibrotic tissues?

Hypothesis:

Page 12: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

(1) Isolate whole dermis RNA (all transcribed genes) (2) RNA-sequencing & alignment of transcribed genes (3) Determine differentially expressed genes

Measuring differentially expressed genes in stabilized β-catenin After 3 weeks in whole dermis in vivo

Page 13: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Stabilized β-catenin results in increased expression of 175 genes

Page 14: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

β-catenin-responsive matrisome genes are putative targets of TCF binding

16 matrisome genes have ↑ expression & Tcf/Lef binding sites:

Page 15: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

(1) Isolate dermal fibroblasts, culture 3 days, AdCre infect, verify recombination

(2) Mouse dermal fibroblasts RNA (all transcribed genes, lncRNAs)

(2) RNA-sequencing & alignment of transcribed genes (3) Determine differentially expressed genes

Model #2: Early responders of Wnt/β-catenin signaling in dermal fibroblasts

after 4 days in vitro

+AdCre +AdCre

Nathaniel Mullin ‘16

Page 16: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

• Input/output considered: Upregulated genes (FC > 1.5, p< 0.05) from B-cat. Stab P4 ventral dermal fibroblasts (in vitro)

Representative annotation terms

Enrichment Score

% of total genes present in cluster

1 Glycoprotein 10.10

2 Glycoprotein/ Membrane

8.01

3 Vessel development 6.52

4 Pleckstrin Homology 5.52

5 Lipoprotein 4.70

Functional annotation clustering of Genes FC>1.5 GOF P4 Ventral Dermal Fibroblasts

-20 0 20 40 60

50

100

150

Z Score

Fis

he

r s

co

re

TFBS P4 B-Cat. Upreg. (>1.5FC) Genes

Foxj1_1

Lef1_1Lef1_2

Smad3_1

Smad3_2

Sox12_1Sox18_1

Sox30_1

Tbp_1

Tcf7l2_1

Tcf7l2_2

Early target genes of Wnt/β-catenin signaling in dermal fibroblasts are

enriched for matrisome genes

Page 17: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

NAME SIZE NES NOM p-val. FDR q-val. FWER p-val. REACTOME NEUROTRANSMITTER RECEPTOR BINDING AND DOWNSTREAM TRANSMISSION IN THE POSTSYNAPTIC CELL

26 2.876609 0 0 0

BENPORATH PRC2 TARGETS 119 2.6450212 0 0 0

BENPORATH ES WITH H3K27ME3 213 2.6280246 0 0 0

MEISSNER NPC HCP WITH H3K4ME2 AND H3K27ME3 61 2.6235106 0 0 0

MIKKELSEN MEF HCP WITH H3K27ME3 41 2.6077304 0 0 0

BENPORATH SUZ12 TARGETS 212 2.5702548 0 0 0

MIKKELSEN MCV6 HCP WITH H3K27ME3 56 2.5578966 0 0 0

REACTOME NEURONAL SYSTEM 47 2.552818 0 0 0

REACTOME TRANSMISSION ACROSS CHEMICAL SYNAPSES

33 2.5163605 0 0 0

REACTOME INTEGRATION OF ENERGY METABOLISM 23 2.3828208 0 6.58E-04 0.008

JAEGER METASTASIS DN 58 2.3701718 0 5.99E-04 0.008

ONDER CDH1 TARGETS 2 DN 128 2.3566086 0 0.00110515

5 0.016

MIKKELSEN NPC HCP WITH H3K4ME3 AND H3K27ME3 46 2.3337145 0 0.00108390

8 0.017

REACTOME REGULATION OF INSULIN SECRETION 17 2.306346 0 0.00136384

2 0.023

BENPORATH EED TARGETS 235 2.2810423 0 0.00177367

6 0.031

RICKMAN HEAD AND NECK CANCER C 19 2.262737 0 0.00197635

1 0.037

Gene sets most enriched in P4 B-Cat. GOF Ventral Dermal Fibroblasts. Selected by GSEA from c2 Curated Gene Sets. FDR < 0.05, FWER p Value < 0.05, Nom. p Value < 0.001

Early responders to Wnt/β-catenin signaling in dermal fibroblasts are targets of Epigenetic repression and poised for regulation by Wnt signaling

• Input data: All DE genes (p<0.05) from B-cat. Stab P4 ventral dermal fibroblasts (in vitro) • MSigDB Gene Set selected: Ben-Porath PRC2 targets (ranked #2 of all C2 gene sets)

– (NES = 2.640442, p<.001, FDR = 2.79E-5 ) – human embryonic stem cells

Representative annotation terms

Enrichment Score

1 Signal/ disulfide bond 4.59

2 Glycoprotein/ Membrane 2.50

3 Wnt Signaling 2.42

4 Growth Factor/ TGFB 2.39

5 Regulation of signal transduction

1.85

Functional annotation clustering of BENPORATH PRC2 Targets enriched in GOF P4 Ventral Dermal Fibroblasts

Page 18: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

LATE (P21)

LILRB3

CD53

BGN

TPSB2

CD48

HCLS1

LUM

LHFPL2

LSAMP

DOCK2

PIK3CG

CRIP1

MYOC

THBS2

CSF2RA

MMRN1

MMP16

QPCT

THY1

PAPPA

COL7A1

WISP2

TWIST1

EPHB2

G0S2

CD14

THBS4

GEM

EARLY B-cat Stab. (P4) (1535)

LATE B-cat Stab. (P21) (171)

Both EARLY and LATE

Desmoid Tumor (1556) (Bacac et al.)

162 51 8

Desmoid Tumor and b-Catenin ChiP-seq (Watanabe et al. 2014)

MYO1F AXIN2 WNT11 HYAL1 SYK SMAD7

--

Upreg in P21, P4 and Desmoid Tumor

Cd53

Cadps

Ahr

Qpct

Twist1

Rarres1

Col7a1

Nfatc4

Genes Upregulated in desmoid tumor & In b-catenin stabilized dermal fibroblasts

Page 19: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

On our way to targeting the Wnt pathway

• Test whether Wnt/b-catenin signaling activation needs to be maintained to continue the fibrotic state.

• Wnt is a well-appreciated therapeutic target. . . – Cross talk with other pathways – There are many mesenchymal derivatives that are implicated in

various human and animal models – How to measure the therapeutic efficacy of systemic Wnt

inhibitors on fibroblast-specific effects in vivo? – Wnt responsive biomarkers in specific cell types. – Develop guides for our expectation to evaluate the effect(s) of

Wnt/b-catenin inhibitors (antiproliferative or ECM modulator).

Page 20: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Model#3: New genetic model of Inducible/deinducible b-catenin in mouse dermal fibroblasts

A

Engrailed1 Cre

R26-rtTAneopApApApA

GFP

Cre recombinase

T1 T1

rtTA-IRES-EGFP-pArtTA

Tet-O

my

c

DOX

my

c

Hfα-Myc tagHf

Hf

Hf

Primary-only Control

Miarassa Steele ‘16 Emily Hamburg-Shields Alicia Vignozzi, MSTP student

Page 21: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Deinducing b-catenin in dermal fibroblasts does not amelioration of matrix in 5 weeks

Page 22: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Deinducing b-catenin leads to recovery of cell number and may be adipose tissue

0

100

200

300

400

500

600

Nu

mb

er

of

Cell

s in

a F

ixed

Are

a

Female Controls

Female Mutants

Male Controls

Male Mutants

1week

Induction

3 week

Induction

3 week Induction

5 week Deinduction

P=0.847 P=0.077 P=0.096

Page 23: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

• Increased dermal fibroblasts in fibrotic human skin express nuclear β-catenin.

• Expression of stabilized β-catenin in mouse dermal fibroblasts is sufficient for skin fibrosis.

• Stabilized β-catenin in dermal fibroblasts produces fibrotic extracellular matrix by altering expression of matrisome genes that may be targets of epigenetic regulation.

• Sustained expression of stabilized b-catenin is not required for matrix turnover, but for fibroproliferative response.

Summary

Page 24: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Inpired by my father, Oct 24, 1941-2014

Page 25: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Thanks so much

Atit lab Present/Past members: Thu Tran, Ph.D., Peggy Myung M.D./Ph.D,

Henry Goodnough, M.D./Ph.D., Demeng, Ph.D., James Ferguson

Jennifer Ohtola, Adrie Welsh, Ozimba Anyangawe,

Nikhil Mallipeddi, Mahima Devarajan

Mice: Alex Joyner (Sloan-Kettering), Mark Taketo (Japan)

Susan Mackem (NCI),

RNA-seq: Case Genomics Sequencing core

Human tissues: John Varga & Monique Hinchcliff,

(Northwestern), Robert Lafyatis (Boston University, SSC core),

Shawn Cowper and Erica Herzog (Yale)

FUNDING:

National Institute of Health (NIDCR) R01, F30, F31

March of Dimes, Scleroderma Research Foundation,

Global Fibrosis Foundation,

Skin Disease Research Center (CWRU).

Page 26: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Active Wnt/β-catenin signaling might be due to increased expression of Wnt ligands

Beyer et al. (2012)

↑ Wnt1 protein in SSc skin ↑ WNT4, WNT10A, and WNT10B in SSc skin

↑AXIN2 in SSc skin

AXIN2 = Wnt/β-catenin target gene

Page 27: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Scleroderma biopsy & fibroblasts Tumor stroma IPF lung biopsy & fibroblasts Dupuytren contracture fibroblasts Keloid fibroblasts

Publicly available microarrays (10 total)

β-catenin-responsive genes are also up-regulated in human fibrotic tissues

↑ in a private scleroderma dataset

Nathaniel Mullin ‘16

Page 28: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

Differentially expressed genes

Genes poised for β-catenin regulation

* Which of these genes encode

matrix proteins?

*

Are the differentially upregulated matrisome genes potential transcriptional targets of Wnt/beta-catenin signaling?

Page 29: -catenin activation in dermal fibroblasts promotes ...annotation terms Enrichment Score % of total genes present in cluster 1 Glycoprotein 10.10 2 Glycoprotein/ Membrane 8.01 3 Vessel

β-catenin-responsive matrisome genes are putative targets of TCF binding

proportion of background genes that include that TFBS

oPOSSUM: 2 statistical measures of over-represented transcription factor binding sites (TFBS)

Fisher score ~

proportion of the gene set that includes a TFBS

Z-score ~

rate of occurrence of a TFBS in gene set

rate of occurrence of that TFBS in background genes