FOXO3 Modulates Endothelial Gene Expression and Function by Classical and Alternative Mechanisms

We demonstrate that FOXO3 controls expression of vascular remodeling genes in an forkhead-responsive elements -dependent manner

Tobias Czymai; Dorothee Viemann; Carsten Sticht; Grietje Molema; Matthias Goebeler; Marc Schmidt


Scholarcy highlights

  • The FOXOs represent an evolutionarily conserved subfamily of forkhead transcription factors unified by the presence of a highly homologous DNA-binding domain termed the “forkhead box.” In contrast to invertebrates, which only express a single FOXO gene mammals contain four distinct isoforms: FOXO1, FOXO3, FOXO4 and the atypical FOXO6, which shows divergent modes of regulation and expression
  • We generated an forkhead-responsive elements-binding mutant of FOXO3 by site-directed mutagenesis of histidine 212 in the third helix of the forkhead box to arginine
  • This loss of functionality was further supported by Chromatin immunoprecipitation experiments, which confirmed its failure to bind to an established FREcontaining region of the IGFBP1 promoter in vivo
  • 4OHT stimulation of FOXO3.A3.ER.H212R-infected human umbilical vein endothelial cells had no effect on the levels of IGFBP1 mRNA or protein expression of p27KIP1, whereas both genes were strongly induced by conditional activation of FOXO.3.A3.ER
  • Apoptosis induction was less pronounced with the FRE-binding mutant as evident from statistical analysis of multiple experiments, prolonged activation of both mutants showed no major differences in colony formation assays and quantitative cytotoxicity assays, indicating that FOXO3 can affect endothelial cells proliferation and viability in the absence of a functional FRE-binding domain
  • Colony Formation Assays—Control cells and cells expressing various transgenes were cultured until control cells reached confluence and fixed by methanol and subsequently stained by 0.1% crystal violet as described
  • Our bioinformatical analysis of the functional gene clusters regulated by FOXO3.A3.ER and its corresponding FRE-binding mutant revealed several overrepresented clusters regulated by classical and alternative mechanisms
  • This appears to be at odds with the finding that FOXO-induced cell cycle arrest does not require forkhead-responsive elements-dependent gene expression

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