TGF -Induced Contraction Is Not Promoted by Fibronectin-Fibronectin Receptor Interaction, or SMA Expression

PURPOSE. Transforming growth factor (TGF)- is a potent inducer of both transdifferentiation and contraction, which are regarded as critical processes that underpin tissue fibrosis. Consequently, transdifferentiation is believed to drive TGFmediated contraction. This study was conducted to determine the relationship between transdifferentiation of human lens epithelial cells and matrix contraction. METHODS. Real-time PCR was used to investigate gene expression of transdifferentiation markers in the human lens cell line FHL 124 and native lens epithelia. Contraction was assessed with a patch-contraction assay, whereby all areas covered by cells were measured with imaging techniques after fixation and cell staining with Coomassie blue. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. To prevent fibronectin‐fibronectin receptor interaction 100 M RGDS peptide was used. Suppression of TGF-induced SMA expression was mediated by siRNA technology. RESULTS. Real-time PCR analysis showed 10 ng/mL TGF- 1o r -2 significantly increased expression of SMA, fibronectin, and 51 integrin (fibronectin receptor components) in FHL 124 cells and human lens epithelia. Cultures maintained in TGF and RGDS showed a marked increase in the rate of contraction relative to TGF- alone. RGDS alone did not differ significantly from the control. Real-time PCR and Western blots showed reduced levels of message and SMA protein when transfected with siRNA. SMA knockdown did not prevent TGF-induced contraction. CONCLUSIONS. A targeted inhibition approach demonstrated that key elements associated with transdifferentiation are not critical for TGF-induced matrix contraction. (Invest Ophthalmol Vis Sci. 2008;49:650‐661) DOI:10.1167/iovs.07-0586