Reduced expression of laminin-5 in corneal epithelial cells under high glucose condition.
OBJECTIVE
Laminin-5 (alpha3, beta3, gamma2 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.


METHODS
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.


RESULTS
We found that alpha3 chain expression was reduced at the mRNA level in a high glucose condition, whereas beta3 and gamma2 chains showed no change. The high glucose condition induced the inhibition in the synthesis of 190-kd, 160-kd alpha3 chain and 105-kd gamma2 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.


CONCLUSIONS
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.
Lu
WenNan
W
Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan.
Ebihara
Nobuyuki
N
Miyazaki
Kaoru
K
Murakami
Akira
A
D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D018929:Cell Culture Techniques; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D005947:Glucose; D012333:RNA, Messenger; D000097794:Kalinin