Characterisation of TGF-β2 signalling and function in a human lens cell line

There is increasing evidence implicating Transforming growth factor beta (TGF-beta) 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-beta and Smad proteins. Moreover, we have tested the effectiveness of a fully human monoclonal anti-TGF-beta2 antibody, CAT-152, in suppressing TGF-beta2 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-beta2 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 striad-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 3 H-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, alphaA-crystallin and pax6 along with lens epithelial cell specific market FoxE3. Immunocytochemical studies revealed the presence of Smad 4 which following TGF-beta2 exposure accumulated in the cell nucleus. Furthermore, Smad-dependent transcriptional activity was also stimulated. TGF-beta2 enhanced the expression of mRNA levels of a smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Exposure to TGF-beta2 resulted in a relatively small inhibition of H-3-thymidine incorporation of FHL 124 cells. However, a more marked contractile effect was also observed. In serum-supplemented medium, growth rates and TGF-beta induced contraction were enhanced. Treatment with 0.1 - 10 mug ml(-1) CAT-152 dose-dependently inhibited 10 ng ml(-1) TGF-beta2 induced effects in the presence and absence of serum. Exposure of FHL 124 cells to TGF-beta therefore induces Smad translocation, transcription, expression of transdifferentiation markers and induces marked contraction. Treatment with CAT-152 can effectively inhibit these responses. TGF-beta2 induced changes can also persist long after the period of exposure and when in the presence of serum TGF-beta induced contraction is enhanced. The work presented therefore demonstrates a platform technology to Study TGF-beta2 signalling in human lens epithelia] cells and provides evidence to show TGF-beta2 can he a potent factor in the development of posterior capsule opacification following cataract surgery. (C) 2003 Elsevier Ltd. All rights reserved.