A chemical screen identifies small molecules that regulate hepcidin expression

• DOI: 10.1016/j.bcmd.2014.06.002
• Publication Date: 2014-07-04T03:45:32Z
• Authors: Vera Gaun; Bonnie Patchen; Josephine Volovetz; Aileen W. Zhen; Aleksandr Andreev; Michael P. Pollastri; Paula G. Fraenkel
• Keywords: STAT3 Transcription Factor; 0301 basic medicine; Cell Survival; Recombinant Fusion Proteins; Smad Proteins; Transfection; Small Molecule Libraries; 03 medical and health sciences; Hepcidins; Genes, Reporter; Drug Discovery; Humans; Luciferases; Promoter Regions, Genetic; Molecular Biology; 0303 health sciences; Stat3; Interleukin-6; Cell Biology; Hematology; Hep G2 Cells; High-Throughput Screening Assays; 3. Good health; Gene Expression Regulation; Bone Morphogenetic Proteins; Thalassemia; Molecular Medicine; Hemochromatosis; Bone morphogenic protein; Databases, Chemical; Plasmids; Signal Transduction

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1 standard deviation below the mean of the other chemicals (z-score >3 or <1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.