lpa triggered signalling in epidermal keratinocyte migration and skin wound healing

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