Global gene profiling reveals novel glucocorticoid inducedchanges in gene expression of human lens epithelial cells

[摘要] Purpose: Prolonged use of glucocorticoids can lead to the formationof a cataract, however the mechanism is not known. We recently reportedthe presence of the functional glucocorticoid receptor in immortalizedcultured mammalian lens epithelial cells (LECs), but the biologicaleffect is not known. This study seeks to determine if freshly isolatedhuman LECs respond to glucocorticoid treatment and to examineglucocorticoid induced changes in global gene expression in LECs.Methods: Capsulorhexis specimens obtained in surgery from eyes withcataract were cultured. Primary lens cultures were transfected, intriplicate, with pGRE.Luc, which drives the expression of fireflyluciferase, and treated with dexamethasone (Dex) or vehicle (Veh). RNAisolated from HLE B-3 cells, treated with Dex or Veh for 4 or 16 h intriplicate, was used to analyze global changes in gene expression bymicroarray hybridization. Data and cluster analyses were performed usingMicroarray Suite 5.0, GeneSpring 6.1, EASE, NetAffx, and SAM. Real TimePCR was used to confirm microarray data in RNA isolated from HLE B-3cells in triplicate and a primary culture of human lens epithelialcells.Results: Transfected primary cultures of human LECs treated with Dexdemonstrated a glucocorticoid response with a greater than 4 foldincrease in firefly luciferase activity over controls. Microarray datarevealed that 136 genes were modulated with 4 h treatment with Dex. Ofthe 136 genes, 93 transcripts were upregulated and 43 were downregulatedby greater than 1.5 fold. Eighty-six genes were modulated with 16 h Dextreatment. Of the 86 genes, 30 transcripts were upregulated and 56 weredownregulated by greater than 1.5 fold. Microarray results were verifiedby Real Time PCR in both the HLE B-3 and primary cultures of lensepithelial cell.Conclusions: The activation of a GRE reporter gene in primarycultures of human LECs demonstrates that the glucocorticoid receptor isfunctional in non-immortalized human lens cells. Microarray studies at 2time periods demonstrate that glucocorticoids modulate gene expressionin immortalized human LECs, reveal novel changes in gene expression, andconfirm an endogenous genomic lens glucocorticoid response. This studydemonstrates that primary cultures of lens epithelial cells andmicroarray technology can be used to determine pathways involved in alens glucocorticoid response and lead to a better understanding of theformation of a steroid induced cataract.

[学科分类] 生物化学/生物物理