Characterisation of TGF-beta 2 signalling and function in a human lens cell line.

IM Wormstone, S Tamiya, JA Eldred, K Lazaridis, A Chantry, JR Reddan, I Anderson, G Duncan

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Abstract

There is increasing evidence implicating Transforming growth factor ß (TGF-ß) in pathological states of the lens. However, the underlying signalling mechanisms in human cells have not been fully examined. We have therefore investigated in a human lens cell line, FHL 124, the signalling characteristics of TGF-ß and Smad proteins. Moreover, we have tested the effectiveness of a fully human monoclonal anti-TGF-ß2 antibody, CAT-152, in suppressing TGF-ß2 induced changes in a number of conditions. FHL 124 cells were routinely cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% FCS. Characterisation of the cell line was determined using Affymetrix gene microarrays and compared to native human lens epithelium. Cells were serum starved for 24 hr prior to exposure to TGF-ß2 in the presence and absence of CAT-152. Non-stimulated cells served as controls. Smad 4 localisation was observed by immunocytochemistry. To study Smad-dependent transcriptional activity, cells were transfected with SBE4-luc, an artificial smad-specific reporter, using Fugene-6. Transcriptional activity was determined by luciferase activity. Gene expression was assessed using reverse transcriptase–polymerase chain reaction (RT–PCR). Proliferation was determined by 3H-thymidine DNA incorporation. Growth and contraction were assessed using a scratch and patch assay. Affymettrix gene microarrays identified 99·5% homology between FHL124 cells and the native lens epithelium with respect to expression pattern of the 22 270 genes on the chip. Moreover, FHL124 cells expressed phenotypic markers, aA-crystallin and pax6 along with lens epithelial cell specific marker FoxE3. Immunocytochemical studies revealed the presence of Smad 4 which following TGF-ß2 exposure accumulated in the cell nucleus. Furthermore, Smad-dependent transcriptional activity was also stimulated. TGF-ß2 enhanced the expression of mRNA levels of a smooth muscle actin (aSMA) and connective tissue growth factor (CTGF). Exposure to TGF-ß2 resulted in a relatively small inhibition of 3H-thymidine incorporation of FHL 124 cells. However, a more marked contractile effect was also observed. In serum-supplemented medium, growth rates and TGF-ß induced contraction were enhanced. Treatment with 0·1–10 µg ml-1 CAT-152 dose-dependently inhibited 10 ng ml-1 TGF-ß2 induced effects in the presence and absence of serum. Exposure of FHL 124 cells to TGF-ß therefore induces Smad translocation, transcription, expression of transdifferentiation markers and induces marked contraction. Treatment with CAT-152 can effectively inhibit these responses. TGF-ß2 induced changes can also persist long after the period of exposure and when in the presence of serum TGF-ß induced contraction is enhanced. The work presented therefore demonstrates a platform technology to study TGF-ß2 signalling in human lens epithelial cells and provides evidence to show TGF-ß2 can be a potent factor in the development of posterior capsule opacification following cataract surgery.
Original languageEnglish
Pages (from-to)705-714
Number of pages10
JournalExperimental Eye Research
Volume78
Issue number3
DOIs
Publication statusPublished - Mar 2004

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