Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions. (January 2015)

• Record Type: Journal Article
• Authors: Finotti, Alessia; Bianchi, Nicoletta; Fabbri, Enrica; Borgatti, Monica; Breveglieri, Giulia; Gasparello, Jessica; Gambari, Roberto
• Is Part Of: Pharmacological research. Volume 91(2015:Jan.)
• Page Start: 57
• Page End: 68
• Publication Date: 2015-01
• Subjects: Raptor regulatory associated protein of mTOR -- Rictor rapamycin-insensitive companion of mTOR -- mTOR mammalian target of rapamycin -- mTORC1 mTOR complex 1 -- m-TORC2 mTOR complex 2 -- Sp1 specific protein 1 -- MTH mithramycin -- RAPA rapamycin -- ChIP chromatin immunoprecipitation -- EMSA electrophoretic mobility shift assay -- FBS fetal bovine serum -- PBS phosphate-buffered saline -- TBS tris-buffered saline -- HbF fetal hemoglobin -- ODN oligonucleotide; Mithramycin (PubChem CID: 457831); Raptor -- mTOR -- Sp1 -- Mithramycin -- Erythroid induction -- Fetal hemoglobin; Pharmacology -- Periodicals; Pharmacology -- Periodicals; Research -- Periodicals; Médicaments -- Recherche -- Périodiques; Pharmacologie -- Périodiques; 615.105
• DOI: 10.1016/j.phrs.2014.11.005
• Languages: English
• ISSNs: 1043-6618

Abstract

Graphical abstract: Abstract: Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G + C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.