An efficient and economic high-throughput cell screening model targeting the glucocorticoid receptor.
OBJECTIVE
To discover compounds or proteins that can efficiently bind the glucocorticoid receptor (GR) and trigger the transcription of target genes, resulting in clinical improvement of diseases such as rheumatoid arthritis, asthma, inflammatory bowel disease, a high-throughput drug screening cell model using green fluorescent protein 4 (GFP4) as a marker expressed in response to GR activation has been established and evaluated.


METHODS
Eight repeats of the glucocorticoid response element (GRE) were cloned into the Peak12SxSynGFP4 vector, and the resulting recombinant plasmid Peak12GRE8 x SxSynGFP4 was stably transfected into the 293E cells. The stable and sensitive cell line 293E/GRE8 x /GFP4 was selected by dexamethasone (DEX) using fluorescent microscopy and fluorescence-activated cell sorting. DEX induction and phorbol myristate acetate (PMA) inhibition of the green fluorescence intensity of the cell line were tested.


RESULTS
The expression of GFP4 in the cell line was under the control of GRE, up-regulated by DEX treatment and down-regulated by phorbol myristate acetate (PMA). The up-regulation of the GFP4 expression was DEX concentration-dependent, with an EC(50) at approximately 5 x 10(- 8) M. The down-regulation of the GFP4 expression was phorbol myristate acetate (PMA) concentration-dependent, with an IC(50) at approximately 3 x 10(- 6) gl - 1. The expression of GFP4 was effectively activated when cells were treated with triamcinolone acetonide.


CONCLUSIONS
This drug screening cell line can be used for GR-targeted high-throughput drug screening for the treatment of inflammatory diseases.
Zhang
Yanli
Y
National Laboratory of Medical Molecular Biology, Dept. of Biochemistry & Molecular Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, No. 5 dongdansantiao, Beijing, Peoples Republic of China. zhyanli@gmail.com
Guo
Feng
F
Jiang
Chengyu
C
D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D003907:Dexamethasone / Q000008:administration & dosage / Q000627:therapeutic use; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000191:economics* / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000819:agonists*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014222:Triamcinolone Acetonide / Q000494:pharmacology
D000893:Anti-Inflammatory Agents; D004274:DNA, Recombinant; D011965:Receptors, Glucocorticoid; D049452:Green Fluorescent Proteins; D003907:Dexamethasone; D014222:Triamcinolone Acetonide; D013755:Tetradecanoylphorbol Acetate