May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
Dexamethasone Induces Changes in Glucocorticoid Receptor Phosphorylation and Gene Expression in Human Lens Epithelial Cells
Author Affiliations & Notes
  • V.K. Gupta
    Biochem & Mol Bio, UMDNJ–Graduate School of Biomedical Sciences, Newark, NJ
  • B.J. Wagner
    Ophthal and Biochem & Mol Bio, UMDNJ– NJMS & GSBS, Newark, NJ
  • Footnotes
    Commercial Relationships  V.K. Gupta, None; B.J. Wagner, None.
  • Footnotes
    Support  NIH EY02299
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1890. doi:
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    • Get Citation

      V.K. Gupta, B.J. Wagner; Dexamethasone Induces Changes in Glucocorticoid Receptor Phosphorylation and Gene Expression in Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1890.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Prolonged use of glucocorticoid (GC) can lead to steroid cataract. We recently reported the presence of the functional glucocorticoid receptor (GR) in mammalian lens epithelial cells (LEC), but the biological effect is not known. Here we tested the specificity of the GC response and analyzed miroarray data to elucidate a pathway of GC action. Methods: Primary LEC and HLE B–3 (B–3) cultures were transfected with pGRE.Luc, which drives the expression of firefly luciferase, and co–treated with dexamethasone (DEX) +/– GR antagonist, RU–486, or mineralocorticoid receptor (MR) antagonist, spironolactone. GR phosphorylation was visualized by western blotting of cell extracts with specific phospho–GR and total–GR antibodies. Microarray was performed on Affymetrix chips. Cluster and pathway analyses were performed using Microarray Suite 5.0, Genespring 6.1 and Pathway Assist 2.0. Pairs of whole lenses from human donor eyes were incubated in DMEM without serum and treated with DEX. Epithelial cell RNA was used for RT– Real Time PCR. Protein expression in B–3 cells was visualized by western blotting. Results: Transfected LECs demonstrated that RU–486, not the MR antagonist, spironolactone, inhibited DEX induced luciferase activity. DEX–dependent phosphorylation of S211 in the GR was observed in DEX treated LECs, but not with DEX + RU–486. Total GR expression was similar in both DEX and RU–486 treated samples. Analysis of microarray data through literature mining and manual construction of regulatory networks revealed an alteration in MAP Kinase (MAPK) regulators upon DEX treatment. RT–Real Time PCR of RNA from B–3 and primary cultures verified microarray results. Western blotting demonstrated DEX induced changes in MAPK regulators in B–3 cells. Conclusions: Dexamethasone specifically activates the human LEC GR to induce changes in gene expression and GR phosphorylation. Manual analysis of individual genes, identified by microarray and verified by Real Time PCR and western blotting, revealed changes in expression of MAPK regulators but not in MAPK expression. The difficulty in elucidating GC actions in the lens may be due to an indirect action of GCs on MAPK activity. Modulating regulators may lead to a change in MAPK activity which can affect a wide array of biological, molecular, and cellular functions. These changes over time may be involved in the formation of a steroid induced cataract.

Keywords: corticosteroids • receptors • gene/expression 

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