Reduced Expression of Laminin-5 in Corneal Epithelial Cells Under High Glucose Condition

Authors:
  • NENobuyuki Ebihara
    Nobuyuki Ebihara
  • KMKaoru Miyazaki
    Kaoru Miyazaki
  • AMAkira Murakami
    Akira Murakami
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Abstract
Laminin-5 (α3, β3, γ2 chains) is a major component of corneal basement membrane and has a crucial role in corneal epithelial cell adhesion. On the other hand, diabetic keratopathy has a varied degree of adhesive disturbance in corneal epithelial cells. Therefore, in this study, we investigated whether a high glucose condition altered the expression of laminin-5 in corneal epithelial cells in vitro. Human corneal epithelial (HCE) cells were cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium for 5 passages before being used in experiments. We first examined the effect of a high glucose condition on the expression of mRNA and proteins for 3 chains of laminin-5 in HCE cells by semiquantitative reverse transcriptase-polymerase chain reaction, Western blotting analysis, and immunofluorescence staining. Second, we tried a cell detachment assay. After 5 days of incubation in high or normal glucose medium, HCE cells were treated with a solution of 0.05% trypsin and EDTA (0.2 mmol/L), pH = 8. The number of detached cells at different times after treatment was determined using a cell count machine. We found that α3 chain expression was reduced at the mRNA level in a high glucose condition, whereas β3 and γ2 chains showed no change. The high glucose condition induced the inhibition in the synthesis of 190-kd, 160-kd α3 chain and 105-kd γ2 chain proteins of laminin-5. The adhesion capacity of HCE cells in the high glucose medium was weaker than that of HCE cells in normal glucose medium. Soluble laminin-5 rescued the detachment of HCE cells in high glucose medium. The loss of homeostatic levels of laminin-5 under a high glucose condition may correlate to weaken epithelial cell adhesion, resulting in the clinical manifestation of diabetic keratopathy.

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