Ldb 145 Geni Mutanti_2014-11-19 Jamora - dalla ricerca al prodotto 5

1. Investigating the role of fibulin-5 in linking
tissue stiffness with inflammation
Colin Jamora
IFOM-inStem Joint Research Laboratory
Center for Inflammation and Tissue Homeostasis
Bangalore, India
University of Salento, Nov. 2014

2. Probing fibroblast biology in the mammalian skin

3. Activated Fibroblasts in Tissue Repair and Disease
Wound healing
Adapted from:
Kalluri and Zeisberg 2006
Koontongkaew 2013
Tissue Fibrosis
Hyperactive
Myofibroblast
Tumor Stroma
CAF
Inflammation
Proliferation
Remodeling

4. Scar formation in wounded Snail transgenic skin
Human skin
Mouse skin

5. Snail is involved in development and disease

6. K14
Snail
(Hoot K. E. et al. J. Clin. Invest 2008)
Human cutaneous SCC
Snail expression in cancer & fibrotic disease
Du et al., Cancer Research 2010
in collaboration with
John Varga (Northwestern)
Figure 5. Expression of Snail in human sclerotic skin. Immunohistochemistry
of normal and sclerotic (Ssc) skin using an antibody specific for human Snail.
scleroderma
RESEARCH ARTICLE
Association of the Autoimmune Disease Scleroderma
with an Immunologic Response to Cancer
Christine G. Joseph1,*, Erika Darrah2,*, Ami A. Shah2,*, Andrew D. Skora1,*,
Livia A. Casciola-Rosen2, Fredrick M. Wigley2, Francesco Boin2, Andrea Fava2,
Chris Thoburn3, Isaac Kinde1, Yuchen Jiao1, Nickolas Papadopoulos1, Kenneth
W. Kinzler1,†, Bert Vogelstein1,†, Antony Rosen2,†
Science 10 January 2014

7. Snail transgenic skin displays features of fibrosis
WT Snail Tg
epi
der
der
epi
WT Snail Tg

8. Mechanical characterization of the Snail transgenic skin
At higher strain
Avg.Etanmodulus(kPa)
0
200
400
600
800
1000
*
WT Snail Tg
K5/collagen
Collagen:
WT Snail Tg
0.0 0.1 0.2 0.3 0.4
0
30
60
90
120
Strain
Stress(kPa)
0.0 0.1 0.2 0.3
0
30
60
90
120
Strain
Stress(kPa)
Stress-Strain curve
WT
Snail Tg
Collaborator: Shyni Varghese (UCSD)
Avg.Etanmodulus(kPa)
At lower strain
0
30
60
90
120
150
*
WT Snail Tg
Elastic Fibers:
Elastic fiber staining
(Verhoeff-Van Gieson Stain)
WT Snail Tg

9. Kielty, Journal of Cell Science, 2002
Fibulin-5 in elastic fiber assembly
2.0
WT Snail Tg
0.0
0.5
1.0
1.5
Relativeexpression
*
WT Snail Tg
Fibulin-5/K5
100 µm

10. Fibulin-5 affects elastic but not collagen fiber formation
W
TFibulin-5
KO
SnailTg
SnailTg/
Fibulin-5
KO
0
200
400
600
800
1000
*
*
Elastincontent(μg)
*
Effect on Elastic fibers:
W
TFibulin-5
KO
SnailTg
0
20
40
60
80
100 **
Hydroxyprolinecontent(μg)
SnailTg/
Fibulin-5
KO
0
50
100
150
200
250
SnailTg/
Fibulin-5
KO
SnailTg
At higher strain
Avg.Etanmodulus(kPa)
Effect on Collagen fibers:

11. Can fibulin-5 regulate tissue inflammation to impact fibrosis?
Depleting fibulin-5: Blocking inflammation:

12. Fibulin-5 is required for a robust inflammatory response
W
T
Fibulin-5
KO
SnailTgSnailTg/
Fibulin-5
KO
0
20
40
60
80
*** *
Mac1-positivecells/mm
K5/Mac-1
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO
K5/CD3
0
20
40
60
80
100
** *
CD3-positivecells/mm
W
T
Fibulin-5
KO
SnailTgSnailTg/
Fibulin-5
KO
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO

13. adapted from Kalluri and Zeisberg 2006
How can fibulin-5 regulate the inflammatory response?
Activated fibroblast/CAF
?

14. Fibulin-5 accentuates activation of dermal fibroblasts in vivo
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO
α-SMA
SMA SPARC CTGF
0
2
4
6
8
10
***
##
*
**
##
Relativefoldexpression
WT
Fibulin-5 KO
Snail Tg
Snail Tg/Fibulin-5 KO

15. adapted from Kalluri and Zeisberg 2006
How can fibulin-5 regulate the inflammatory response?
Activated fibroblast/CAF
?

16. Fibulin-5 promotes inflammatory chemokine
production from dermal fibroblasts in vivo
CXCL1 CXCL5 CXCL10 CXCL16
0
10
20
30
40 WT
Fibulin-5 KO
Snail Tg
Snail Tg/Fibulin-5 KO
***
##
Relativefoldexpression
####
### ***
**
***

17. Activated fibroblast/CAF
Intercellular crosstalk in the tumor stroma
Mindin
adapted from Kalluri and Zeisberg 2006

18. CXCL1 CXCL5 CXCL10 CXCL16
0
5
10
15
20
25
2 kPa WT CM
2 kPa Snail CM
5 kPa WT CM
5 kPa Snail CM
* ***
**
**
Relativefoldexpression
Stiffer substrate accentuates the inflammatory response
2 kPa buffer
2 kPa mindin
5 kPa buffer
5 kPa mindin

19. Summary
• Snail transgenic skin is a model of tissue/tumor fibrosis
• Elastin binding protein (Fibulin-5) is elevated in
transgenic dermis
• Deletion of Fibulin-5 decreases elastin (collagen remains
elevated), dermal thickness, and corresponding tissue
stiffness
• Deletion of Fibulin-5 decreases inflammation in the fibrotic
skin
• Small increase in substrate rigidity is able to enhance a
proinflammatory response in dermal fibroblasts

20. Acknowledgements
Manando Nakasaki MD, PhD
Tuan-Lin Tan, PhD
Subhasri Ghosh, PhD
Tanay Bhatt
Sunny Kataria
Neha Pincha
Federica Centonze
Syed Abrar Rizvi
Edries Hajam
Isha Rana
Bhavya Byantri
Ajai Pulianmackal
Prof. Tomoyuki Nakamura (Kansai Medical Univ)
Prof. Steven Briggs (UCSD)
Prof. Shyni Varghese (UCSD)
Yongsung (Joshua) Hwang, PhD

21. Active fibroblasts are not a product of an EMT
Guarino et al., Human Pathology 2009
β-gal/αSMA

22. Primary Dermal fibroblasts
Control CM
Snail CM
WT CM
Snail CM
Assay to test for secreted factor capable
of activating dermal fibroblasts
WT keratinocytes
Snail transgenic
keratinocytes

23. What is in secreted by Snail expressing
keratinocytes to activate dermal fibroblasts?
epidermis
dermis
dermis
Snail
?
Collagen contraction activity
Induction of CTGF & αSMA
Concanavalin A sepharose column
Collagen contraction activity
Induction of CTGF & αSMA
WT or Snail Tg Conditioned media
Anion exchange chromatography (Mono Q)
Mass
Spec

24. Candidate proteins mediating
epithelial-mesenchymal crosstalk

25. Fibulin 5 on activated fibroblasts

26. Background on spondin-2 (aka mindin)
What is spondin-2/mindin?
• Member of the F-spondin family
• Secreted, associates with ECM
• Component of basal lamina (zebrafish)
• Ligand for integrins
• PRR – production of inflammatory cytokines
• Promotes outgrowth of hippocampal neurons
Wynn & Ramalingam 2012

27. Image of function of fibulin-5 (and interacting partners??)
bulin-5 on vascular cells
on of fibulin-5 in adult blood vessels was
invasion in matrigel (Albig and Schiemann 2004
gulation of thrombospondin-1 expression, as
antagonization of VEGF165 -mediated signaling, in
del of elastic fiber assembly. a Secretion of tropoelastin
astogenic cells (green). Tropoelastin undergoes self-
nd fibulin-5 (purple) mediates this process. Tropoealstin
N-terminal (N) and C-terminal tropoelastin-binding
domains (square) of fibulin-5. b Fibulin-5 binds tropoelasti
fibrils (blue), and lysyl oxidase-like enzyme (yellow) to aid
fiber assembly. c Cross-linked insoluble elastin (grey) is po
and organized into functional elastic fibers
evelopment and disease

28. PMC full text: J Cell Commun Signal. Dec 2009; 3(3-4): 337–347.
Published online Oct 2, 2009. doi: 10.1007/s12079-009-0065-3
Copyright/License ► Request permission to reuse
Table 1
Interacting partners of Fibulin-5
Interacting
proteins
Binding site(s) within Fibulin-5 Cellular and/or Biological functions Reference
Fibulin-5 ND Unknown Zheng et al. 2007
Tropoelastin cbEGF domains Jones et al. 2009
N-terminal cbEGF domains Elastic fiber assembly Zheng et al. 2007
C-terminal EB domain
Loxl-1 a. C-terminal fibulin domain > 1st
cbEGF
Elastic fiber assembly a. Hirai et al. 2007a
b. C-terminal region (245-448) b. Liu et al. 2004
Loxl-2, 4 C-terminal fibulin domain Elastic fiber assembly Hirai et al. 2007a
Emilin-1 ND Elastic fiber assembly Zanetti et al. 2004
LTBP-2 6th cbEGF Elastic fiber assembly Hirai et al. 2007b
Fibrillin-1 ND Elastic fiber assembly Freeman et al. 2005
SOD3 C-terminal region (320-448) Superoxide scavenge Nguyen et al. 2004
α5β1, α4β1 N-terminal half containing RGD Cell attachment, Antagonize fibronectin
function
Lomas et al. 2007
αvβ3, αvβ5 N-terminal half containing RGD Cell attachment Nakamura et al. 2002
α9β1 ND Cell attachment Nakamura et al. 2002
Lipoprotein(a) C-terminal domain (350-448) Unknown Kapetanopoulos et al.
2002
ND not determined

29. Fibulin-5 accentuates the inflammatory response
W
T
Fibulin-5
KO
SnailTgSnailTg/
Fibulin-5
KO
0
20
40
60
80
*** *
Mac1-positivecells/mm
K5/Mac-1
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO
K5/CD3
0
20
40
60
80
100
** *
CD3-positivecells/mm
W
T
Fibulin-5
KO
SnailTgSnailTg/
Fibulin-5
KO
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO