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

There is increasing evidence implicating Transforming growth factor β (TGF-β) 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-β and Smad proteins. Moreover, we have tested the effectiveness of a fully human monoclonal anti-TGF-β2 antibody, CAT-152, in suppressing TGF-β2 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 24hr prior to exposure to TGF-β2 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, αA-crystallin and pax6 along with lens epithelial cell specific marker FoxE3. Immunocytochemical studies revealed the presence of Smad 4 which following TGF-β2 exposure accumulated in the cell nucleus. Furthermore, Smad-dependent transcriptional activity was also stimulated. TGF-β2 enhanced the expression of mRNA levels of α smooth muscle actin (αSMA) and connective tissue growth factor (CTGF). Exposure to TGF-β2 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-β induced contraction were enhanced. Treatment with 0.1-10 μg ml^-1 CAT-152 dose-dependently inhibited 10 ng ml^-1 TGF-β2 induced effects in the presence and absence of serum. Exposure of FHL 124 cells to TGF-β therefore induces Smad translocation, transcription, expression of transdifferentiation markers and induces marked contraction. Treatment with CAT-152 can effectively inhibit these responses. TGF-β2 induced changes can also persist long after the period of exposure and when in the presence of serum TGF-β induced contraction is enhanced. The work presented therefore demonstrates a platform technology to study TGF-β2 signalling in human lens epithelial cells and provides evidence to show TGF-β2 can be a potent factor in the development of posterior capsule opacification following cataract surgery.