Abstract
Purpose: :
The tropomyosin (Tm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. The epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) is a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs as model systems, we demonstrated overexpression of Tm1α/2β and suggested the involvement of these molecules in remodeling the actin cytoskeleton during EMT. We analyzed the effect of overexpression and inhibition of Tm expression on lens differentiation in rat cataractous LECs. We also examined the expression of Tm in rat and human cataract.
Methods: :
A construct of full-length cDNA from human Tm1α/2β gene was prepared in eukaryotic expression vector (pGFP-C). SiRNA of Tm1α/2β were purchased from Invitrogen. GFP- Tm1α/2β and Tm1α/2β-siRNAs were transfected into rat and human LECs. MTS and TUNEL assays were used to detect apoptotic cell viability. Expressions of several genes related with lens EMT were examined using real-time PCR and Western analysis, respectively. Western analysis was used to monitor Tm1α/2β expression in lenses of Shumiya Cataract Rats (SCRs). Immunohistochemial staining of Tm1α/2β and α-SMA were performed for human dislocated lens capsules with LECs.
Results: :
In SCRs Tm expression was elevated more in cataractous lenses than in clear lenses. After Tm1α/2β overexpression in LECs, cell viability and expression of Prdx6 were decreased and expression of α-SMA and TGF-β were increased. In contrast, with inhibition of Tm1α/2 by siRNAs, cataractous LECs showed phenotypic changes in cell culture that resembled differentiated LECs. In human lens samples, Tm1α/2β was strongly expressed in differentiated LECs.
Conclusions: :
Tm1α/2β overexpression in LECs was correlated with remodeling of actin filaments, possibly leading to EMT/PCO and anterior subcapsular cataract. The findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, contributing to development of Tm-based inhibitors for postponing PCO and cataractogenesis.
Keywords: cataract • EMT (epithelial mesenchymal transition) • differentiation