Presence of a transcriptionally active glucocorticoid receptor alpha in lens epithelial cells.
OBJECTIVE
The purpose of this study was to determine whether lens epithelial cells (LECs) contain a glucocorticoid receptor (GR) that is transcriptionally active and that is able to induce production of known glucocorticoid-inducible proteins.


METHODS
Protein and mRNA were obtained from human, rabbit, and bovine lens epithelia and from cultured human lens epithelial cells (B3, hLECs) and rabbit lens epithelial cells (N/N1003A, rLECs). Paraffin-embedded sections were prepared from human lenses for immunohistochemical localization of GR. RT-PCR was performed to amplify portions of GR, and the products were sequenced. Protein samples were analyzed by Western blot. hLECs and rLECs were transfected with pTAT3-luc and assayed for luciferase activity after treatment with dexamethasone (Dex) and/or RU486. Dex-treated LECs were also analyzed by quantitative real-time PCR and by Western blot for expression of specific mRNA and proteins.


RESULTS
By PCR and sequencing, products consistent with GR sequences were obtained from human, rabbit, and bovine lenses and from hLECs and rLECs. The complete GRalpha sequence was obtained from rLECs and was found to be 89% identical with human GR. A 1757-bp 3' fragment of bovine GRalpha cDNA was also amplified from bovine lens. By Western blot, bands of approximately 94 kDa, the expected size of GR, were identified from human, rabbit, and bovine lens samples and from hLECs and rLECs, using anti-GR antibodies. Anti-GR antisera localized GR to both the cytosol of anterior and bow region LECs and to the nuclei of epithelial and early-differentiating lens fiber cells. Luciferase expression was induced in pTAT3-luc-transfected rLECs and hLECs by Dex treatment and this expression was partially (rLECs) or completely (hLECs) blocked by pretreatment with RU486. mRNA levels for type-1 glucocorticoid-induced target genes and also mRNA and protein levels for type-2 genes were upregulated after Dex exposure.


CONCLUSIONS
The data confirm the existence of GR in hLECs, indicate that GR is present in rLECs, and resolve the controversy over the presence of GR in bovine lens. The GRalpha in hLECs and rLECs was shown to be transcriptionally active and the expression levels in hLECs of mRNAs and proteins known to be regulated by glucocorticoids were modified in these cells by glucocorticoid treatment.
James
Eric R
ER
Department of Ophthalmology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA. jameser@musc.edu
Robertson
Lorie
L
Ehlert
Erich
E
Fitzgerald
Patrick
P
Droin
Nathalie
N
Green
Douglas R
DR
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D015735:Mifepristone / Q000494:pharmacology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; C411470:glucocorticoid receptor alpha; D015735:Mifepristone; D003907:Dexamethasone