Smad4 Is Critical for TGFß Induced Transdifferentiation but Does Not Mediate Matrix Contraction by Human Lens Epithelial Cells

Abstract

Purpose:: Smad4, a key component in the TGFß/Smad signalling pathway, is proposed to regulate transdifferentiation of lens epithelial cells to myofibroblasts and matrix contraction. These processes contribute to posterior capsule opacification (PCO) formation following cataract surgery. The primary objective of the study was to determine the importance of Smad4 in TGFß2 induced transdifferentiation and matrix contraction by human lens epithelial cells.

Methods:: The human lens cell line FHL 124 was employed. Smad4 knockdown was achieved using siRNA technology and efficiency tested at the message and protein level using Real-time PCR and western blots respectively. Gene expression of transdifferentiation markers, alpha smooth muscle actin and fibronectin was determined using Real-time PCR. Contraction was assessed using a patch growth assay, whereby all areas covered by cells were measured using imaging techniques. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. To determine Smad4 distribution immunocytochemistry was employed.

Results:: SiRNA targeted against Smad4, successfully reduced expression of Smad4 message (79.73 ± 1.8% inhibition) and protein (92.77 ± 2.6% inhibition) 24 and 48 hours following the initial transfection period respectively. These findings were confirmed by immunocytochemistry for Smad4, which showed a significant reduction in nuclear Smad4 following siSmad4 treatment relative to -ve control siRNA in both the presence and absence of 10ng/ml TGFß2. Real-time PCR analysis showed that 10ng/ml TGFß2 significantly increased expression of mαSMA (197.7 ± 6.2%) and mfibronectin (197.4 ± 20.4%) in Smad4 expressing cells (-ve control siRNA group). These inductions were not evident in Smad4 knockdown cells. Smad4 expressing cells did not exhibit a contractile response following 24 hours culture in the presence or absence of 10ng/ml TGFß2. However, Smad4 knockdown cells did demonstrate a significant reduction in patch area i.e. contraction, following TGFß2 treatment. No significant differences in cell population between any groups were observed.

Conclusions:: Smad4 is critical for the expression of transdifferentiation markers, αSMA and fibronectin. However, Smad4 does not appear to be essential for matrix contraction to occur. TGFß2 regulation of transdifferentiation and matrix contraction therefore appears to be regulated by distinct pathways.

Keywords: posterior capsular opacification (PCO) • EMT (epithelial mesenchymal transition) • signal transduction