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 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 FHL 124 cells and the native lens epithelium with respect to expression pattern of the 22,270 genes on the chip. Moreover, FHL 124 cells expressed phenotypic markers, alphaA-
crystallin and pax6 along with lens epithelial cell specific marker 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 alpha smooth muscle actin (alphaSMA) and
connective tissue growth factor (CTGF). Exposure to
TGF-beta2 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-beta induced contraction were enhanced. Treatment with 0.1-10 microg 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 epithelial cells and provides evidence to show
TGF-beta2 can be a potent factor in the development of posterior
capsule opacification following
cataract surgery.