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R. Jans, A.M. Brown, K. Ross & N.J. ReynoldsDermatological Sciences at the Institute of Cellular Medicine
Newcastle upon Tyne, UK
Investigation of the signalling mechanism by which lysophosphatidic acid promotes epithelial cell migration
Epidermal re-epithelialization & LPA
During wound healing, regeneration of skin barrier requiresre-epithelialization
Signalling poorly understood
wound edge
keratinocyte activation& migration
ep
ide
rmis
de
rmis
??????
LPA-R EGFR
[Ca2+]mobilization
gene expression
migration
TGFβR
Smad3/4 MAPK
Lysophosphatidic acid
water-soluble lipid present in serum/platelets and released upon wounding
promotes migration of keratinocytes and enhances wound repair in experimental wounds in vivo
Agonist-induced Ca2+ release and influx
Ca2+o-dependent Ca2+
mobilization by LPAin primary keratinocytes
10 µM LPA
0 200 400 600 800
0
1
2
3
4
Time [s]
Flu
o-4
Ft/
F0
LPA added at
60 µM Ca2+o (basal medium)
1.2 mM Ca2+o (≈serum levels)
2-wayANOVA:p<0.05n=183
n=215 ]
agonistCa2+
Ca2+
endoplasmic reticulumCa2+ store
LPA
Ca2+
Ca2+
(4) Downstream signalling cascade?
(3) Lipid rafts?
(2) Orai1 channels?
(1) STIM1 sensor?
STIM1: Effect of RNAi on LPA-
induced Ca2+ influx
LPA
0 mM Ca2+
0 200 400 600 8000
1
2
3
4
5
untreated (n=23)1% MβCD (n=31)
Time [s]
Flu
o-4
Ft/
F0
1.2 mM Ca2+
LPA
0 mM Ca2+
0 200 400 6000.6
0.8
1.0
1.2
1.4
1.6
Empty vector (n=38)Orai1 wt (n=30)
Orai1R91W (n=28)
Time [s]
Fu
ra-P
E3
340/
380n
m
1.2 mM Ca2+
Orai1: Overexpression of dominant/negative mutant
Orai1R91W
Lipid rafts: disruption by methyl-β-cyclodextrin
treatment
LPA
Ca2+
Ca2+
NFATPP P
PPPP P P
PP P
NFAT
calcineurin A/B
n=30
FuraRed(inverse probe) NFAT2-GFP
Effect of LPA on Ca2+i and
NFAT2 activation
Effect of LPA on NFAT-dependent transcriptional
activity
luciferaseNFATNFATNFATNFATNFATNFATNFATNFATNFAT
n≥3
LPA added in 60 µM [Ca2+]o
LPA added in 1.2 mM [Ca2+]o
1.2 mM [Ca2+]o
0 4 8 12 16 20 240
2
4
6
8
10
12
Time [h]N
FA
T-l
uc
ex
pre
ss
ion
15 fps (12 min total)
LPA
Ca2+
Ca2+
(1) diethylstilbestrol
NFATPP P
PPPP P P
PP P
NFAT
calcineurin A/B (4) cyclosporin A
(2) Orai1 d/n
(3) lipid raft disruption
p<0.05
n=3
untreated 10 µM LPA0
2
4
6
8vehicle
10 µM DES
NF
AT
-lu
c ex
pre
ssio
n
p<0.05
n=3
EV Orai1 wtR91W
Orai10
1
2
3
4
5
NF
AT
-lu
c ex
pre
ssio
n
p<0.05
n=3
untreated 10 µM LPA0
2
4
6
8untreated1% MβCD
NF
AT
-lu
c ex
pre
ssio
n
p<0.001 p<0.001
untreat
ed
10 µ
M L
PA0
2
4
6vehicle1 µM CsA (calcineurin inhibitor)
NF
AT
-lu
c ex
pre
ssio
n
LPA
Ca2+
Ca2+
NFATPP P
PPPP P P
PP P
NFAT
calcineurin A/B (2) cyclosporin A(1) STIM1siRNA
(3) NFAT2siRNA
cell migration
3D chemotactic migration assay
Transwell chemotacticmigration assay
top
bottom
overnight migration
scrape offnon-migrating cells
stain and count migratory cells
Effect of STIM1 siRNA
t
2D scratch wounding motility assay
3D chemotactic migration assay
Transwell chemotacticmigration assay
top
bottom
overnight migration
scrape offnon-migrating cells
stain and count migratory cells
Effect of cyclosporin A
Effect of NFAT2 siRNA
3D chemotactic migration assay
Transwell chemotacticmigration assay
top
bottom
overnight migration
scrape offnon-migrating cells
stain and count migratory cells
cell migration
Conclusion
LPA induces:
Ca2+ mobilization and NFAT/NFAT2 activation in keratinocytes requiring STIM1, Orai1 and lipid rafts
keratinocyte migration via calcineurin & NFAT2
LPA
Ca2+
Ca2+
lipid raft
Orai1
NFAT(2)PP P
PPPP P P
PP P
NFAT(2)
calcineurin A/BSTIM1
Acknowledgements
Dermatology staff