Characterisation of TGF-beta2 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 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.
Wormstone
I M
IM
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Tamiya
S
S
Eldred
J A
JA
Lazaridis
K
K
Chantry
A
A
Reddan
J R
JR
Anderson
I
I
Duncan
G
G
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D055513:Connective Tissue Growth Factor; D004268:DNA-Binding Proteins / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D051901:Smad4 Protein; D015534:Trans-Activators / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053781:Transforming Growth Factor beta2
D000199:Actins; C526317:CCN2 protein, human; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C509041:TGFB2 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D055513:Connective Tissue Growth Factor