ldb 145 geni mutanti_2014-11-19 jamora - dalla ricerca al prodotto 5
TRANSCRIPT
Investigating the role of fibulin-5 in linking tissue stiffness with inflammation
Colin JamoraIFOM-inStem Joint Research Laboratory
Center for Inflammation and Tissue HomeostasisBangalore, India
University of Salento, Nov. 2014
Activated Fibroblasts in Tissue Repair and Disease
Wound healing
Adapted from: Kalluri and Zeisberg 2006
Koontongkaew 2013 Tissue Fibrosis
Hyperactive
Myofib
roblast
Tumor Stroma
CAF
Inflammation Proliferation Remodeling
K14Snail
(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 ARTICLEAssociation of the Autoimmune Disease Scleroderma with an Immunologic Response to CancerChristine 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
Mechanical characterization of the Snail transgenic skin
At higher strain
Avg
. Eta
n m
odul
us (k
Pa)
0
200
400
600
800
1000*
WT Snail Tg
K5/
colla
gen
Collagen:WT Snail Tg 0.0 0.1 0.2 0.3 0.4
0
30
60
90
120
Strain
Str
ess
(kP
a)
0.0 0.1 0.2 0.30
30
60
90
120
Strain
Str
ess (
kP
a)
Stress-Strain curve
WT
Snail Tg
Collaborator: Shyni Varghese (UCSD)
Avg
. Eta
n m
odul
us (k
Pa)
At lower strain
0
30
60
90
120
150
*
WT Snail Tg
Elastic Fibers:
Elastic fiber staining (Verhoeff-Van Gieson Stain)
WT Snail Tg
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
Rel
ativ
e ex
pres
sion
*
WT Snail Tg
Fibu
lin-5
/ K
5
100 µm
Fibulin-5 affects elastic but not collagen fiber formation
WT
Fibulin
-5 K
O
Snail Tg
Snail Tg/
Fibulin
-5 K
O
0
200
400
600
800
1000
*
*E
lastin c
onte
nt (μ
g)
*
Effect on Elastic fibers:
WT
Fibulin
-5 KO
Snail T
g0
20
40
60
80
100 **
Hyd
roxy
prol
ine
cont
ent (μ
g)
Snail T
g/
Fibulin
-5 KO
0
50
100
150
200
250
Snail Tg/
Fibulin
-5 K
O
Snail Tg
At higher strain
Avg
. E
tan m
od
ulu
s (
kP
a)
Effect on Collagen fibers:
Can fibulin-5 regulate tissue inflammation to impact fibrosis?
Depleting fibulin-5: Blocking inflammation:
Fibulin-5 is required for a robust inflammatory response
WT
Fibulin
-5 K
O
Snail Tg
Snail Tg/
Fibulin
-5 K
O
0
20
40
60
80*** *
Ma
c1
-po
sitiv
e c
ells/m
m
K5/M
ac-1
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO
K5/C
D3
0
20
40
60
80
100** *
CD
3-p
ositiv
e c
ells/m
m
WT
Fibulin
-5 K
O
Snail Tg
Snail Tg/
Fibulin
-5 K
O
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO
adapted from Kalluri and Zeisberg 2006
How can fibulin-5 regulate the inflammatory response?
Activated fibroblast/CAF
?
Fibulin-5 accentuates activation of dermal fibroblasts in vivo
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO
α-S
MA
SMA SPARC CTGF
0
2
4
6
8
10
***
##*
**
##
Rel
ativ
e fo
ld e
xpre
ssio
n
WT
Fibulin-5 KO
Snail Tg
Snail Tg/Fibulin-5 KO
adapted from Kalluri and Zeisberg 2006
How can fibulin-5 regulate the inflammatory response?
Activated fibroblast/CAF
?
Fibulin-5 promotes inflammatory chemokine production from dermal fibroblasts in vivo
CXCL1 CXCL5 CXCL10 CXCL160
10
20
30
40WT
Fibulin-5 KO
Snail Tg
Snail Tg/Fibulin-5 KO
***##
Re
lative
fo
ld e
xp
ressio
n
####
### ***
**
***
Activated fibroblast/CAF
Intercellular crosstalk in the tumor stroma
Mindin
adapted from Kalluri and Zeisberg 2006
CXCL1 CXCL5 CXCL10 CXCL160
5
10
15
20
252 kPa WT CM2 kPa Snail CM5 kPa WT CM5 kPa Snail CM
* ***
**
**
Rel
ativ
e fo
ld e
xpre
ssio
nStiffer substrate accentuates the inflammatory response
2 kPa buffer 2 kPa mindin 5 kPa buffer 5 kPa mindin
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
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
Active fibroblasts are not a product of an EMT
Guarino et al., Human Pathology 2009
Figure 2. Lineage tracing of the activated fibroblasts. Epithelial cells in the skin of wild type (WT) or Snail transgenic mice (K14-Snail) were labeled in blue with X-gal staining when these respective mice were mated with ROSA26/K14-Cre mice. Activated dermal fibroblasts were labeled with an antibody recognizing
αSMA (brown).
β-ga
l/αSM
A
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 transgenickeratinocytes
What is in secreted by Snail expressing keratinocytes to activate dermal fibroblasts?
epid
erm
isde
rmis
derm
is
Snail? Collagen contraction activity
Induction of CTGF & αSMA
Concanavalin A sepharose columnCollagen contraction activityInduction of CTGF & αSMA
WT or Snail Tg Conditioned media
Anion exchange chromatography (Mono Q)
MassSpec
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
Image of function of fibulin-5 (and interacting partners??)
Effect of fibulin-5 on vascular cells
The function of fibulin-5 in adult blood vessels wassuggested by the strong upregulation of Fbln5 in theneointima after balloon withdrawal injury or carotid arteryligation, and in activated endothelial cells of atheroscleroticplaques (Kowal et al. 1999; Spencer et al. 2005). Fbln5-nullSMCs displayed enhanced proliferation and migration inresponse to serum and PDGF. This enhancement wasinhibited by over-expression of Fbln5 (Spencer et al.2005). Forced expression of Fbln5 in primary humanendothelial cells improved cell attachment against contin-uous shear stress and enhanced cell retention on artificialgrafts subjected to pulsatile flow. However, the proliferationof endothelial cells was decreased in Fbln5-overexpressingcells compared to control cells (Preis et al. 2006). Humanprimary SMCs plated on recombinant fibulin-5 exhibitedPDGF receptors with undetectable phosphorylation, andpoorly phosphorylated EGF receptors, compared to SMCsplated on fibronectin (Lomas et al. 2007). Interestingly, thepresence of integrin β1-activating antibody in SMCs,plated on fibulin-5, restored phosphorylation of PDGF αand β receptors. Together with the observation that fibulin-5binds fibronectin receptors (α5β1 and α4β1) but fails toactivate downstream signaling, these data suggest a possi-bility that fibulin-5 may regulate vascular cell behavior byantagonizing fibronectin-mediated signaling. However,further investigation is required to establish the in vivorelevance of the fibulin-5-integrin binding during vesseldevelopment and pathological insults.
Fibulin-5 and angiogenesis
The effect of fibulin-5 on angiogenesis was first describedusing mouse brain microvascular MB114 endothelial cells,in which Fbln5 overexpression or incubation with recom-binant fibulin-5 inhibited sprouting, proliferation, and
invasion in matrigel (Albig and Schiemann 2004). Upre-gulation of thrombospondin-1 expression, as well asantagonization of VEGF165 -mediated signaling, includingactivation of P38 MAPK and ERK1/2, were suggested tobe the underlying causes of the inhibitory effect of fibulin-5. Fibulin-5 was also shown to reduce migration of humanmicrovascular HMEC-1 endothelial cells toward fibronec-tin, and overexpression of FBLN5 in MB114 cells down-regulated MMP-2 expression, as well as enzymatic activityduring tubulogenesis on collagen gels (Albig et al. 2006).Matrigel implantation experiments in the skin of wild-typemice showed that matrigel containing high or low doses offibulin-5 stimulated the invasion of bFGF-induced fibro-blasts, but inhibited vessel formation and angiogenesis,independent of RGD-integrin interactions. Consistent withthese observations, Fbln5-null mice exhibited increasedangiogenesis after physiological wound healing and PVAsponge implantation, without an effect on fibroblastinvasion (Sullivan et al. 2007; Zheng et al. 2006).
Although exogenous and endogenous fibulin-5 wasshown to antagonize angiogenesis, the exact mechanismbehind this effect has remained elusive. We recentlyobserved that fibulin-5 exerts its effect on endothelial cellsand angiogenesis by controlling integrin-induced productionof reactive oxygen species (ROS), which have pro-angiogenic properties (Schluterman et al. 2009). ROS,including O2
- and H2O2, are highly reactive moleculesproduced commonly as by products of aerobic respirationand the mitochondrial transport chain. ROS were originallyidentified as host defense molecules produced by neutrophilsvia NAD(P)H oxidases, and have a critical function ineliciting biological processes required for the initiation ofangiogenesis (Wu 2006). By functioning as signalingmolecules, ROS have been shown to activate pathways suchas proliferation, cell adhesion, motility and invasion inendothelial cells (Ushio-Fukai 2007).
Endothelial cells, treated with H2O2, produced higherlevels of VEGF, thereby increasing their proliferation and
Fig. 3 A model of elastic fiber assembly. a Secretion of tropoelastin(TE) from elastogenic cells (green). Tropoelastin undergoes self-aggregation and fibulin-5 (purple) mediates this process. Tropoealstinbinds both N-terminal (N) and C-terminal tropoelastin-binding
domains (square) of fibulin-5. b Fibulin-5 binds tropoelastin, micro-fibrils (blue), and lysyl oxidase-like enzyme (yellow) to aid in elasticfiber assembly. c Cross-linked insoluble elastin (grey) is polymerizedand organized into functional elastic fibers
Fibulin-5 in development and disease 343
PMC full text: J Cell Commun Signal. Dec 2009; 3(3-4): 337–347.Published online Oct 2, 2009. doi: 10.1007/s12079-009-0065-3Copyright/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 > 1stcbEGF
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 fibronectinfunction
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
Fibulin-5 accentuates the inflammatory response
WT
Fibulin
-5 K
O
Snail Tg
Snail Tg/
Fibulin
-5 K
O
0
20
40
60
80*** *
Ma
c1
-po
sitiv
e c
ells/m
m
K5/M
ac-1
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO
K5/C
D3
0
20
40
60
80
100** *
CD
3-p
ositiv
e c
ells/m
m
WT
Fibulin
-5 K
O
Snail Tg
Snail Tg/
Fibulin
-5 K
O
WT Snail TgFibulin-5 KO Snail Tg/Fibulin-5 KO