A chemical screen identifies small molecules that regulate hepcidin expression

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
DOI: 10.1016/j.bcmd.2014.06.002 Publication Date: 2014-07-04T03:45:32Z
ABSTRACT
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.
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