title: Screening for novel lead compounds increasing insulin expression in medullary thymic epithelial cells.
abstract: Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance to pancreatic beta-cells. Thymic insulin expression modulation might be an important drug target for preventing type 1 diabetes. We performed a high-throughput screening to identify compounds with such activity. A reporter plasmid was constructed with the human insulin promoter sequence including a short allele of the upstream variable number tandem repeat (VNTR) sequence (32 repeats), subcloned into the pGL4.17 vector. The plasmid was stably transfected into an insulin-transcribing medullary thymic epithelial cell (mTEC) line. Primary high-throughput screening assays were carried out by stimulating with candidate compounds for 24h, and the activity of luciferase was measured. Positive compounds were further validated by real-time PCR. Of 19,707 compounds, we identified one compound that could enhance mTEC insulin expression, as confirmed by real-time PCR. We also observed that transfection with the autoimmune regulator (AIRE) increased endogenous AIRE expression in mTECs. Our insulin-VNTRI-promoter reporter system is consistent with the insulin expression regulation in mTECs, and one compound that was identified could increase insulin expression in mTECs. A positive feedback effect of AIRE in mTECS was observed. Whether these efforts in murine thymus cells apply to humans remains to be determined.
authors.affiliation: Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China.
authors.forename: Xiu-Ying
authors.initials: XY
authors.lastname: Yang
authors.forename: Dina
authors.initials: D
authors.lastname: Levi
authors.forename: Houria
authors.initials: H
authors.lastname: Ounissi-Benkalha
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Yu
authors.forename: Hui-Qi
authors.initials: HQ
authors.lastname: Qu
authors.forename: Constantin
authors.initials: C
authors.lastname: Polychronakos
authors.forename: Guan-Hua
authors.initials: GH
authors.lastname: Du
mesh_terms: D000818:Animals; D002460:Cell Line; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D057166:High-Throughput Screening Assays; D006801:Humans; D007328:Insulin / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D013950:Thymus Gland / Q000166:cytology*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D000096927:AIRE Protein
chemical_list: D007328:Insulin; D008156:Luciferases; D014157:Transcription Factors; D000096927:AIRE Protein