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Carlos Alonso-Ron, Fernandez-Gonzalez Ana, Miguel Coca-Prados, Ignacio Alcalde, Guilherme Ferrara, Manuel Chacon, Jesus Merayo-Lloves, Carlos Belmonte; Hyaluronan Receptors Expression And Effects In Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):280.
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© ARVO (1962-2015); The Authors (2016-present)
To describe the distribution and the isotypes of hyaluronan (HA) receptors (CD44 and RHAMM) in the corneal epithelium and the effects of HA in cell proliferation and migration.
Total RNA from healthy whole human corneas and SV40 immortalized human corneal epithelial cells (HCEsv) was purified. Thereafter, the whole-genome expression profiling was examined using the Illumina platform. For RT-PCR, total RNA from HCEsv cells was extracted, DNase I digested, and reverse-transcribed. Fragments CD44 and RHAMM transcript variants were amplified by PCR. For immunocytochemistry (ICC) HCEsv cells were growth till 70% of confluence, starvated overnight, fixed in paraformaldehyde and incubated overnight with CD44 or RHAMM antibodies. For immunohistochemistry (IHC), microtome sections of whole C57/BL/6J mice’s eye were fixed in formaline, included in parafine, deparafinated, rehidratated and subsequently incubated with primary antibodies. In both ICC and IHC, receptors were visualized by indirect immunofluorescence. For proliferation and migration assays HCEsv cells were used. To measure proliferation, Wst-1 based commercial kits was used; for migration, we employ Boyden Chamber and ‘scratch-wound’ kits. The protocols followed were those described by the manufacturer.
Microarray analysis of human corneas and HCEsv cells showed that they express three of the 5 CD44’s and two of the 4 RHAMM’s transcripts variants. RT-PCR analysis confirmed these results. Immunolabeling of mice corneas with CD44 and RHAMM antibodies indicated that both receptors co-localized along the corneal epithelium cells of the basal layer, CD44’s labeling being somewhat more intensive than RHAMM’s. HCEsv cells under steady-state conditions were also immuno-stained. HCEsv showed a concentration-dependent positive effect of HA in migration and proliferation.
HA has a trophic effect in HCEsv cells, probably mediated by one or more of the 3 identified isotypes of CD44’s and 2 RHAMM’s. In a mouse model, those receptors are expressed in the basal layer of the healthy corneal epithelium.
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