Abstract
Purpose:
Transforming growth factor beta (TGFβ)-induced epithelial-mesenchymal transition (EMT) is involved in the formation of anterior subcapsular cataracts as well as secondary cataract. Our previous work suggests that myocardin related transcription factor A (MRTF-A) is an important mediator of TGFβ-induced EMT in lens epithelial cells (LECs). The following study investigated whether MRTF-A nuclear localization is downstream of TGFβ-induced Rho signaling, which is involved in cytoskeletal regulation and subsequent EMT.
Methods:
Rat LEC explants, with epithelia attached to their native lens capsule, were isolated and maintained in culture. Confluent LEC explants were treated with TGFβ for 48 hours, in the presence or absence of the Rho kinase (ROCK) inhibitor Y27632. Lens explants were immunostained with MRTF-A to determine localization of MRTF-A. αSMA expression was detected with immunofluoresence to determine the state of EMT.
Results:
In the ex vivo untreated rat LEC explants, MRTF-A was predominantly localized in the cytoplasm with negligible αSMA expression detected. Treatment with TGFβ (4ng/ml) triggered a transformation of LECs from a tightly packed cuboidal monolayer into an elongated mesenchymal phenotype, which included nuclear MRTF-A localization and increased expression of filamentous αSMA. In contrast, LEC explants treated with TGFβ in the presence of Y27632 (20μM) exhibited cytoplasmic MRTF-A staining with negligible αSMA expression and resembled control explants.
Conclusions:
The current study demonstrated that the inhibition of the Rho-ROCK signaling pathway was able to prevent TGFβ-induced nuclear MRTF-A migration in conjunction with the suppression of αSMA expression. Overall, our data suggests that MRTF-A is a downstream target of the Rho signaling pathway in TGFβ-induced EMT of LECs.
Keywords: 445 cataract •
512 EMT (epithelial mesenchymal transition) •
493 cytoskeleton