Oligonucleotide microarray analysis of human lens epithelial cells: TGFbeta regulated gene expression.
OBJECTIVE
Transforming growth factor beta (TGFbeta), a pro-fibrotic cytokine has been proposed a causative factor in the progression of lens pathologies including posterior capsule opacification (PCO), a condition that occurs after cataract surgery. This study employs oligonucleotide microarrays to provide a global profile of gene expression in FHL 124 cells, to identify changes in gene expression following treatment with TGFbeta1 and TGFbeta2, and to enable putative genes relating to TGFbeta regulation and PCO to be identified.


METHODS
Routinely cultured FHL 124 cells maintained in serum free Eagle's Minimum Essential Medium (EMEM) were treated with either TGFbeta1 or TGFbeta2 at 10 ng/ml for 24 h then total RNA extraction was carried out. Total RNA (16 microg) was used to analyze gene expression by spotted oligonucleotide microarray hybridization. The spotted oligonucleotide microarrays employed contained 13,971 oligonucleotide probes, each designed to be specific for an individual gene. Array images were analyzed using GenePix Pro 3.0, followed by raw data import into GeneSpring 7.0 where a cross gene error model (CGEM) filter was applied. Data was subjected to LoWess normalization prior to comparison of the different treatment groups. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to validate the oligonucleotide microarray data, using a select number of genes exhibiting differential expression.


RESULTS
A total of 301 genes were up-regulated by more than 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these up-regulated genes had biological functions relevant to lens epithelial cells including roles in contraction, transdifferentiation and as extracellular matrix (ECM) components. A total of 164 genes were down-regulated by more that 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these down-regulated genes have biological functions including roles in apoptosis, signaling, and as anti-oxidants. Following treatment with TGFbeta1 and TGFbeta2, QRT-PCR successfully validated the differential changes in gene expression detected by oligonucleotide microarrays.


CONCLUSIONS
TGFbeta1 and TGFbeta2 regulate the gene expression of genes that have important roles in human lens epithelial cell biology. Most importantly, TGFbeta induces the gene expression of a number of fibrotic markers which may have a role in promoting the development of PCO such as transdifferentiation markers, contractile factors, and ECM components.
Dawes
L J
LJ
School of Biological Sciences, University of East Anglia, Norwich, UK.
Elliott
R M
RM
Reddan
J R
JR
Wormstone
Y M
YM
Wormstone
I M
IM
D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D016326:Extracellular Matrix Proteins; D006133:Growth Substances; D016023:Integrins; D018994:Myosin Light Chains; D011956:Receptors, Cell Surface; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2