Global gene profiling reveals novel glucocorticoid induced changes in gene expression of human lens epithelial cells.
OBJECTIVE
Prolonged use of glucocorticoids can lead to the formation of a cataract, however the mechanism is not known. We recently reported the presence of the functional glucocorticoid receptor in immortalized cultured mammalian lens epithelial cells (LECs), but the biological effect is not known. This study seeks to determine if freshly isolated human LECs respond to glucocorticoid treatment and to examine glucocorticoid induced changes in global gene expression in LECs.


METHODS
Capsulorhexis specimens obtained in surgery from eyes with cataract were cultured. Primary lens cultures were transfected, in triplicate, with pGRE.Luc, which drives the expression of firefly luciferase, and treated with dexamethasone (Dex) or vehicle (Veh). RNA isolated from HLE B-3 cells, treated with Dex or Veh for 4 or 16 h in triplicate, was used to analyze global changes in gene expression by microarray hybridization. Data and cluster analyses were performed using Microarray Suite 5.0, GeneSpring 6.1, EASE, NetAffx, and SAM. Real Time PCR was used to confirm microarray data in RNA isolated from HLE B-3 cells in triplicate and a primary culture of human lens epithelial cells.


RESULTS
Transfected primary cultures of human LECs treated with Dex demonstrated a glucocorticoid response with a greater than 4 fold increase in firefly luciferase activity over controls. Microarray data revealed that 136 genes were modulated with 4 h treatment with Dex. Of the 136 genes, 93 transcripts were upregulated and 43 were downregulated by greater than 1.5 fold. Eighty-six genes were modulated with 16 h Dex treatment. Of the 86 genes, 30 transcripts were upregulated and 56 were downregulated by greater than 1.5 fold. Microarray results were verified by Real Time PCR in both the HLE B-3 and primary cultures of lens epithelial cell.


CONCLUSIONS
The activation of a GRE reporter gene in primary cultures of human LECs demonstrates that the glucocorticoid receptor is functional in non-immortalized human lens cells. Microarray studies at 2 time periods demonstrate that glucocorticoids modulate gene expression in immortalized human LECs, reveal novel changes in gene expression, and confirm an endogenous genomic lens glucocorticoid response. This study demonstrates that primary cultures of lens epithelial cells and microarray technology can be used to determine pathways involved in a lens glucocorticoid response and lead to a better understanding of the formation of a steroid induced cataract.
Gupta
Vanita
V
Department of Biochemistry & Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ 07101-1709, USA.
Galante
Anthony
A
Soteropoulos
Patricia
P
Guo
Suqin
S
Wagner
B J
BJ
D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism; D049409:Luciferases, Firefly / Q000378:metabolism; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015854:Up-Regulation
D005136:Eye Proteins; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D049409:Luciferases, Firefly