The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6+/- mouse model of epithelial wound-healing delay.
BACKGROUND
Congenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells.


RESULTS
Pax6+/- mouse corneal epithelia exhibited a 2-hour delay in their response to wounding, but subsequently the cells migrated normally to repair the wound. Both Pax6+/+ and Pax6+/- epithelia activated immediate wound-induced waves of intracellular calcium signaling. However, the intensity and speed of propagation of the calcium wave, mediated by release from intracellular stores, was reduced in Pax6+/- cells. Initiation and propagation of the calcium wave could be largely decoupled, and both phases of the calcium wave responses were required for wound healing. Wounded cells phosphorylated the extracellular signal-related kinases 1/2 (phospho-ERK1/2). ERK1/2 activation was shown to be required for rapid initiation of wound healing, but had only a minor effect on the rate of cell migration in a healing epithelial sheet. Addition of exogenous epidermal growth factor (EGF) to wounded Pax6+/- cells restored the calcium wave, increased ERK1/2 activation and restored the immediate healing response to wild-type levels.


CONCLUSIONS
The study links Pax6 deficiency to a previously overlooked wound-healing delay. It demonstrates that defective calcium signaling in Pax6+/- cells underlies this delay, and shows that it can be pharmacologically corrected. ERK1/2 phosphorylation is required for the rapid initiation of wound healing. A model is presented whereby minor abrasions, which are quickly healed in normal corneas, transiently persist in aniridic patients, compromising the corneal stroma.
Leiper
Lucy J
LJ
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK. l.leiper@abdn.ac.uk
Walczysko
Petr
P
Kucerova
Romana
R
Ou
Jingxing
J
Shanley
Lynne J
LJ
Lawson
Diane
D
Forrester
John V
JV
McCaig
Colin D
CD
Zhao
Min
M
Collinson
J Martin
JM
D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000293:injuries* / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism*; D010766:Phosphorylation; D012097:Repressor Proteins / Q000378:metabolism*; D014945:Wound Healing / Q000502:physiology*
D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D019950:Mitogen-Activated Protein Kinase 1