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Wormstone, I. M. 
ORCID: https://orcid.org/0000-0002-6423-7766, Tamiya, S., Eldred, J. A., Lazaridis, K., Chantry, A., Reddan, J. R., Anderson, I. and Duncan, G.
(2004)
Characterisation of TGF-beta 2 signalling and function in a human lens cell line.
Experimental Eye Research, 78 (3).
pp. 705-714.
ISSN 1096-0007
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.
| Item Type: | Article |
|---|---|
| Faculty \ School: | Faculty of Science > School of Biological Sciences |
| UEA Research Groups: | Faculty of Science > Research Groups > Cells and Tissues |
| Depositing User: | EPrints Services |
| Date Deposited: | 01 Oct 2010 13:37 |
| Last Modified: | 24 Sep 2024 10:02 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/755 |
| DOI: | 10.1016/j.exer.2003.08.006 |
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