Characterisation of TGF-beta2 signalling and function in a human lens cell line

Exp Eye Res. 2004 Mar;78(3):705-14. doi: 10.1016/j.exer.2003.08.006.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / biosynthesis
  • Actins / genetics
  • Cell Division / physiology
  • Cell Line
  • Cell Movement / physiology
  • Connective Tissue Growth Factor
  • DNA-Binding Proteins / physiology
  • Epithelial Cells / metabolism
  • Humans
  • Immediate-Early Proteins / biosynthesis
  • Immediate-Early Proteins / genetics
  • Intercellular Signaling Peptides and Proteins / biosynthesis
  • Intercellular Signaling Peptides and Proteins / genetics
  • Lens, Crystalline / cytology
  • Lens, Crystalline / metabolism*
  • Oligonucleotide Array Sequence Analysis
  • RNA, Messenger / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / physiology*
  • Smad4 Protein
  • Trans-Activators / physiology
  • Transcription, Genetic / physiology
  • Transforming Growth Factor beta / physiology*
  • Transforming Growth Factor beta2

Substances

  • Actins
  • CCN2 protein, human
  • DNA-Binding Proteins
  • Immediate-Early Proteins
  • Intercellular Signaling Peptides and Proteins
  • RNA, Messenger
  • SMAD4 protein, human
  • Smad4 Protein
  • TGFB2 protein, human
  • Trans-Activators
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta2
  • Connective Tissue Growth Factor