TGF-induced contraction is not promoted by fibronectin-fibronectin receptor interaction, or SMA expression

Lucy Jean Dawes, Julie Ann Eldred, Ian Kevin Anderson, Matthew Sleeman, John R. Reddan, George Duncan, Ian Michael Wormstone

Research output: Contribution to journalArticlepeer-review

35 Citations (Scopus)

Abstract

Purpose: Transforming growth factor (TGF)-β 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 TGFβ-mediated 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 μM RGDS peptide was used. Suppression of TGFβ-induced αSMA expression was mediated by siRNA technology.

Results: Real-time PCR analysis showed 10 ng/mL TGF-β1 or -β2 significantly increased expression of αSMA, fibronectin, and α5β1 integrin (fibronectin receptor components) in FHL 124 cells and human lens epithelia. Cultures maintained in TGFβ and RGDS showed a marked increase in the rate of contraction relative to TGF-β alone. RGDS alone did not differ significantly from the control. Real-time PCR and Western blots showed reduced levels of message and αSMA protein when transfected with siRNA. αSMA knockdown did not prevent TGFβ-induced contraction.

Conclusions: A targeted inhibition approach demonstrated that key elements associated with transdifferentiation are not critical for TGFβ-induced matrix contraction.
Original languageEnglish
Pages (from-to)650-661
Number of pages12
JournalInvestigative Ophthalmology & Visual Science
Volume49
Issue number2
DOIs
Publication statusPublished - Feb 2008

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