Lpa triggered signalling in epidermal keratinocyte migration and skin wound healing
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![Epidermal re-epithelialization & LPA
wound edge
During wound healing,
regeneration of skin barrier
epidermis
keratinocyte activation
requires & migration
re-epithelialization
dermis
Signalling poorly understood
Lysophosphatidic acid
water-soluble lipid present
in serum/platelets and LPA-R TGFβR EGFR
released upon wounding
[Ca2+]
Smad3/4 MAPK
mobilization
promotes migration of ???
keratinocytes and enhances gene expression
wound repair in
experimental wounds in
migration
vivo](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)
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Lpa triggered signalling in epidermal keratinocyte migration and skin wound healing
- 1. Investigation of the signalling mechanism by which lysophosphatidic acid promotes epithelial cell migration R. Jans, A.M. Brown, K. Ross & N.J. Reynolds Dermatological Sciences at the Institute of Cellular Medicine Newcastle upon Tyne, UK
- 2. Epidermal re-epithelialization & LPA wound edge During wound healing, regeneration of skin barrier epidermis keratinocyte activation requires & migration re-epithelialization dermis Signalling poorly understood Lysophosphatidic acid water-soluble lipid present in serum/platelets and LPA-R TGFβR EGFR released upon wounding [Ca2+] Smad3/4 MAPK mobilization promotes migration of ??? keratinocytes and enhances gene expression wound repair in experimental wounds in migration vivo
- 3. Agonist-induced Ca2+ Ca2+o-dependent Ca2+ release and influx mobilization by LPA in primary keratinocytes agonist Ca2+ 4 3 Fluo-4 Ft/F0 2 2-way n=215 1 n=183 ] ANOVA: p<0.05 10 µM LPA 0 0 200 400 600 800 Time [s] Ca 2+ LPA added at endoplasmic reticulum 60 µM Ca2+o (basal medium) Ca2+ store 1.2 mM Ca2+o (≈serum levels)
- 4. Ca2+ LPA (2) Orai1 channels? (3) Lipid rafts? (4) Downstream signalling cascade? (1) STIM1 sensor? Ca2+
- 5. STIM1: Effect of RNAi on LPA- induced Ca2+ influx
- 6. Orai1: Overexpression of Lipid rafts: disruption by dominant/negative mutant methyl-β-cyclodextrin Orai1R91W treatment 1.6 5 0 mM Ca 2+ 1.2 mM Ca 2+ 0 mM Ca2+ 1.2 mM Ca2+ 1.4 4 Fura-PE3 340/380nm Empty vector (n=38) untreated (n=23) Orai1 wt (n=30) 1% MβCD (n=31) 1.2 3 Fluo-4 Ft/F0 Orai1R91W (n=28) 1.0 2 0.8 1 LPA LPA 0.6 0 0 200 400 600 0 200 400 600 800 Time [s] Time [s]
- 7. Ca2+ LPA calcineurin A/B Ca2+ P P P P P P NFAT P P P P P P NFAT
- 8. Effect of LPA on NFAT- Effect of LPA on Ca i and 2+ dependent transcriptional NFAT2 activation activity FuraRed NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT NFAT luciferase (inverse probe) NFAT2-GFP LPA added in 60 µM [Ca2+]o LPA added in 1.2 mM [Ca2+]o 12 1.2 mM [Ca2+]o NFAT-luc expression 10 8 6 4 2 n≥3 0 0 4 8 12 16 20 24 15 fps (12 min total) Time [h] n=30
- 9. Ca2+ LPA (1) diethylstilbestrol (2) Orai1 d/n (3) lipid raft disruption calcineurin A/B (4) cyclosporin A Ca2+ P P P P P P NFAT P P P P P P NFAT
- 10. 8 p<0.05 p<0.05 8 vehicle untreated NFAT-luc expression NFAT-luc expression 10 µM DES 1% MβCD 6 6 4 4 2 2 n=3 n=3 0 untreated 10 µM LPA 0 untreated 10 µM LPA 5 p<0.05 p<0.001 p<0.001 NFAT-luc expression 6 NFAT-luc expression 4 vehicle 1 µM CsA (calcineurin inhibitor) 4 3 2 2 1 n=3 0 0 A ed R91W LP EV Orai1 wt Orai1 ate µM tr un 10
- 11. Ca2+ LPA (1) STIM1 calcineurin A/B (2) cyclosporin A siRNA Ca2+ P P P P P (3) NFAT2 P NFAT P P P P P P siRNA NFAT cell migration
- 12. Effect of STIM1 siRNA top 3D chemotactic bottom migration assay overnight migration scrape off Transwell chemotactic non-migrating cells migration assay stain and count migratory cells
- 14. Effect of cyclosporin A t 2D scratch wounding motility assay top 3D chemotactic bottom migration assay overnight migration scrape off Transwell chemotactic non-migrating cells migration assay stain and count migratory cells
- 16. Effect of NFAT2 siRNA top 3D chemotactic bottom migration assay overnight migration scrape off Transwell chemotactic non-migrating cells migration assay stain and count migratory cells
- 18. Conclusion Ca2+ LPA induces: LPA Ca2+ mobilization Orai1 and NFAT/NFAT2 activation in lipid raft keratinocytes requiring STIM1, Orai1 and lipid rafts STIM1 calcineurin A/B keratinocyte Ca2+ P P P P P migration via P NFAT(2) P P P P P P calcineurin & NFAT2 NFAT(2) cell migration
Editor's Notes
- Reepithelialization is required to regenerate an efficient skin barrier during wound healing. However, the physiology of this process is complex and incompletely understood, which limits the amount of treatments that actively enhance it. We were therefore interested in further characterizing the signalling mechanisms that are recruited upon the activation of keratinocyte migration. Lysophosphatidic acid or LPA is a bioactive lipid found in platelets and serum and released upon wounding. LPA promotes the migration of keratinocytes and enhances wound repair in experimental skin wounds in rats and mice. The mechanism by which LPA does so is partially known – LPA activates TGFbeta and EGFR signalling leading to enhanced migration. LPA binding to its own receptors triggers calcium mobilization but its effect on migration remains unknown.
- In several primary cell types, agonist activation of G protein-coupled receptors evokes IP3-dependent store release which leads to calcium entry. Such calcium mobilization can then lead to activation of the calcineurin/NFAT pathway, in which calcineurin dephosphorylates cytoplasmic NFAT transcription factors, which then translocate into the nucleus to modulate gene expression. To determine if LPA can induce calcium fluxes liable to activate the NFAT pathway, we investigated the effect of stimulating primary keratinocytes with LPA in their basal culture medium at a calcium concentration of 60 µM, or in medium containing physiologically relevant millimolar levels. As you can see in blue, LPA triggers only what appears to be store release in basal medium, whereas in red, stimulation in high calcium medium evokes store release followed by a sustained increase suggesting the occurrence of calcium entry.
- We then took interest into the mechanism by which LPA induces calcium entry in keratinocytes. We first verified using the inhibitor diethylstilbestrol (DES) that entry was actually occurring and then investigated the involvement of Orai1 or CRAC channel proteins and of lipid rafts, because these components are of known relevance both in the control of keratinocyte physiology and of cellular motility. The effect of DES was studied used an addback technique in which we first induced release of calcium from stores by stimulating the cells with LPA in calcium-free medium, and then induced entry by adding back extracellular calcium Addition of DES during the entry period efficiently impairs LPA-induced entry and concurrently, the presence of DES significantly impairs NFAT activation during LPA stimulation. We examined the involvement of Orai1 by overexpressing Orai1 R91W, a dominant negative mutant of Orai1, which was first detected in human SCID patients. Using the addback technique, we show that cells expressing mutant Orai1 show impaired calcium entry compared to cells expressing the empty vector or wild-type Orai1. Downstream NFAT activation is also impaired in mutant Orai1 expressing cells The involvement of lipid rafts was studied by examining the effect of disrupting lipid rafts using methyl beta cyclodextrin, which is an established method that we have used in previous studies. This treatment has a slight inhibitory effect on LPA-induced store release, which hints at a weak involvement of rafts in regulating store release. Lipid rafts disruption potently impairs calcium entry and subsequent NFAT activation.
- We then took interest into the mechanism by which LPA induces calcium entry in keratinocytes. We first verified using the inhibitor diethylstilbestrol (DES) that entry was actually occurring and then investigated the involvement of Orai1 or CRAC channel proteins and of lipid rafts, because these components are of known relevance both in the control of keratinocyte physiology and of cellular motility. The effect of DES was studied used an addback technique in which we first induced release of calcium from stores by stimulating the cells with LPA in calcium-free medium, and then induced entry by adding back extracellular calcium Addition of DES during the entry period efficiently impairs LPA-induced entry and concurrently, the presence of DES significantly impairs NFAT activation during LPA stimulation. We examined the involvement of Orai1 by overexpressing Orai1 R91W, a dominant negative mutant of Orai1, which was first detected in human SCID patients. Using the addback technique, we show that cells expressing mutant Orai1 show impaired calcium entry compared to cells expressing the empty vector or wild-type Orai1. Downstream NFAT activation is also impaired in mutant Orai1 expressing cells The involvement of lipid rafts was studied by examining the effect of disrupting lipid rafts using methyl beta cyclodextrin, which is an established method that we have used in previous studies. This treatment has a slight inhibitory effect on LPA-induced store release, which hints at a weak involvement of rafts in regulating store release. Lipid rafts disruption potently impairs calcium entry and subsequent NFAT activation.
- This result led us to analyze the effect of LPA on NFAT activity. Firstly, we performed a live-cell dual imaging assay in which intracellular calcium fluctuations were assessed using the inverse probe FuraRed and NFAT activation by monitoring the nuclear translocation of GFP-tagged NFAT2 LPA was added in millimolar calcium medium. In the FuraRed movie, you see the cells lose fluorescence after LPA addition, which indicates sustained calcium mobilization Regarding NFAT2, it is predominantly cytoplasmic in resting cells, in which the nucleus is mostly devoid of NFAT2 and black. Upon LPA stimulation, a noticeable proportion of NFAT2 slowly translocates and becomes clearly nuclear within 10 minutes Next, we studied the effect of LPA on NFAT-dependent transcriptional activity using an NFAT-regulated luciferase construct. While millimolar calcium, in grey, and LPA in basal medium, in blue, slightly upregulated NFAT activity over 24h, LPA added in millimolar calcium conditions, in red, induced a 10-fold upregulation of NFAT activity within 4h of treatment and sustained over 24h. This upregulation was calcineurin-dependent because it was impaired when the cells had been treated with the calcineurin inhibitor cyclosporin A before LPA stimulation.
- We then took interest into the mechanism by which LPA induces calcium entry in keratinocytes. We first verified using the inhibitor diethylstilbestrol (DES) that entry was actually occurring and then investigated the involvement of Orai1 or CRAC channel proteins and of lipid rafts, because these components are of known relevance both in the control of keratinocyte physiology and of cellular motility. The effect of DES was studied used an addback technique in which we first induced release of calcium from stores by stimulating the cells with LPA in calcium-free medium, and then induced entry by adding back extracellular calcium Addition of DES during the entry period efficiently impairs LPA-induced entry and concurrently, the presence of DES significantly impairs NFAT activation during LPA stimulation. We examined the involvement of Orai1 by overexpressing Orai1 R91W, a dominant negative mutant of Orai1, which was first detected in human SCID patients. Using the addback technique, we show that cells expressing mutant Orai1 show impaired calcium entry compared to cells expressing the empty vector or wild-type Orai1. Downstream NFAT activation is also impaired in mutant Orai1 expressing cells The involvement of lipid rafts was studied by examining the effect of disrupting lipid rafts using methyl beta cyclodextrin, which is an established method that we have used in previous studies. This treatment has a slight inhibitory effect on LPA-induced store release, which hints at a weak involvement of rafts in regulating store release. Lipid rafts disruption potently impairs calcium entry and subsequent NFAT activation.
- We then inquired if the calcineurin/NFAT pathway was involved in mediating LPA-induced keratinocyte motility by analyzing the effect of cyclosporin A on LPA-induced migration. We monitored cell motility over 14h using two assays: a two dimensional scratch wounding assay and a three dimensional chemotactic migration assay. In both assays performed in millimolar calcium medium, LPA promoted keratinocyte migration as expected. Treatment with cyclosporin A before LPA stimulation significantly impaired this effect, which confirms our hypothesis that calcineurin activity is required for LPA-induced migration.
- In a second approach, we examined the specific involvement of NFAT2 in LPA-induced migration since we’ve found NFAT2 to be activated by LPA and also because NFAT2 has previously been shown to regulate cell migration in other contexts. So we analyzed the effect of knocking down the expression of NFAT2 by transfecting specific siRNA into keratinocytes before LPA challenge. Using the chemotactic migration assay, NFAT2 knockdown is found to significantly impair LPA-induced cell motility.
- To conclude, our data demonstrate that in physiological Ca2+ conditions, LPA induces Ca2+ entry and downstream activation of the NFAT pathway and we have shown that this event requires functional Orai1 and intact lipid rafts. Downstream, the modulation of gene expression that leads to the induction of migration probably involves NFAT2, which we have shown to be activated. Finally, we have shown that LPA-induced migration requires calcineurin activity. We have therefore identified the calcineurin/NFAT pathway as a novel signal transducer within an experimental setting relevant to wound healing.