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W. Li, H. He, Y.-T. Chen, Y. Hayashida, D. Y. Tseng, M. Alonso, S.-Y. Chen, X. Xi, S. C. G. Tseng; Abnormal Epidermal Differentiation of Human Limbal Epithelial Cells Can Be Induced by Air Exposure Through p38 MAPK Signaling Pathway. Invest. Ophthalmol. Vis. Sci. 2007;48(13):475.
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The mechanism of squamous metaplasia, a pathological process frequently occurring in non-keratinized stratified ocular surface epithelia, is largely unknown.
Human limbal explants were cultured in an airlift manner with or without p38 MAPK inhibitor SB203580, or in a submerged manner for different durations. Epithelial proliferation, differentiation, and stem cell status were studied by immunostaining to Ki67, p63, K12, K10, Filaggrin, Pax6, ABCG-2, and Musashi-1. Expression of phospho-p38 and its down stream transcription factors C/EBPα and C/EBPß were also studied under different culture conditions. Clonal growth and differentiation of epithelial cells harvested from explants after 2 weeks cultured under different manners on 3T3 feeder layers for 12 days were investigated by double-staining to K12 and K10.
In airlift, but not submerged, cultures, there were dramatic stratification and digital intrastromal invasion of the limbal, but not corneal, epithelium and a marked increase of Ki67 staining in the limbal epithelium from day 4 to 6 and gradually decreased till day 14. p63 staining was expanded to superficial epithelial cells at day 4 but regressed to basal and suprabasal cell layers after day 6. ABCG-2 and Musashi-1 expression was temporarily activated around day 4 to 6 and gradually decreased to the normal level at day 14. Pax6 expression was maintained in limbal epithelium throughout. Intriguingly, limbal suprabasal epithelial cells started to express K10 at day 4 and Filaggrin at day 6 with a gradual increase toward day 14. Addition of p38 inhibitor SB203580 abolished expression of K10 and Filaggrin in airlift cultures without affecting stratification. Epithelial clones generated on 3T3 feeder layers from different cultures were still K12 positive but K10 negative. Phospho-p38 expression induced by airlift was suppressed by SB203580, a pattern also observed for expression of C/EBP α and C/EBPß more so in airlift than submerged cultures.
Exposure to air (dryness) activates limbal, but not corneal, progenitor cell proliferation, which is coupled with abnormal epidermal differentiation but without intrinsic alteration of stem cells via p38 MAPK signaling. Therefore, abnormal epidermal terminal differentiation can be added as another manifesto of limbal progenitor cells. p38 inhibitor SB203580 may be used to direct normal differentiation and potentially reverse the pathological process of squamous metaplasia.
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