The role of Hsp70 and Hsp90 in TGF-beta-induced epithelial-to-mesenchymal transition in rat lens epithelial explants.
OBJECTIVE
This study investigates the effects of heat shock treatment and the role of Hsp70 and Hsp90 on tranforming growth factor beta 2 (TGF-beta2)-induced epithelial-to-mesenchymal transition (EMT) in rat lens epithelial explants.


METHODS
Rat lens epithelial explants from 7 to 10 day-old Wistar rats were dissected and incubated for 24 h before treatment. The explants were divided into eight treatment groups: control (culture medium), fibroblast growth factor-2 (FGF-2), TGF-beta2, and TGF-beta2+FGF-2 under normal culture conditions and heat shocked conditions. The explants were heat shocked at 45 degrees C before treatment with the respective media. H&E staining was performed on whole-mount epithelial explants from each group. Immunofluorescence staining for alpha-smooth muscle actin (alpha-SMA), F-actin, and E-cadherin was also used to determine EMT and fibrotic plaque formation in the lens epithelial explants. Apoptotic cell death was determined using the TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) assay. Confocal microscopy was used to visualize immunoreactivity in the whole-mount epithelial explants. Western blot analysis of alpha-SMA, E-cadherin, Hsp70, and Hsp90 were also performed.


RESULTS
TGF-beta2-induced EMT and plaque formation in the lens epithelial explants. The simultaneous treatment of epithelial explants with TGF-beta2+FGF-2-induced the most significant morphological changes and EMT. Heat shock treatment of lens epithelial explants before TGF-beta2 treatment did not inhibit plaque formation, but there was significant reduction of alpha-SMA expression and greater E-cadherin expression when compared to the non-heat shocked TGF-beta2-treated explants. Interestingly, TGF-beta-induced apoptotic cell death was significantly lower in the heat shocked explants compared to the non-heat shock lens explants. Heat-induced accumulation of Hsp70 and Hsp90 expression was reduced in the heat shocked groups at day 4 of treatment.


CONCLUSIONS
TGF-beta2-induced EMT was significantly reduced in the heat shocked TGF-beta2 lens epithelial explants. After four days of culture, there is a reduction in expression of Hsp70 and Hsp90 in the heat-shocked groups, indicating that the lens epithelial cells are under a less stressful condition than the non-heat shocked groups. In conclusion, molecular chaperones can play a protective role against TGF-beta2-induced EMT and enhance cell survival.
Banh
Alice
A
School of Optometry, University of Waterloo, Waterloo, ON, Canada. abanh@scimail.uwaterloo.ca
Deschamps
Paula A
PA
Vijayan
Mathilakath M
MM
Sivak
Jacob G
JG
West-Mays
Judith A
JA
D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015820:Cadherins / Q000378:metabolism; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008648:Mesoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*
D000199:Actins; D015820:Cadherins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D053781:Transforming Growth Factor beta2