title: Induction of fetal/embryonic globin gene expression depends on intact cell signaling in definitive erythroid cells.
abstract: OBJECTIVE
  The induction of fetal hemoglobin during definitive erythropoiesis is a major therapeutic goal in β-globin gene disorders. Butyrate induces fetal hemoglobin, and p38 phosphorylation has been implicated in this process. We studied p38 and the effect of its inhibitors in a physiologic primary cell model of fetal/embryonic globin gene induction during definitive erythropoiesis.
  
  
  METHODS
  p38 phosphorylation was evaluated in a short-term culture of definitive erythroid precursor (EryD) cells following butyrate induction, absent prolonged exposure to cytokines. The impact of p38 inhibitors on embryonic/fetal globin gene induction by butyrate and on normal erythroid processes, including proliferation, differentiation, cell cycle occupancy, and RNA transcription, was also examined.
  
  
  RESULTS
  p38 phosphorylation, maximal at harvest of murine fetal liver-derived EryD (FL EryD), when minimal embryonic/fetal globin gene expression is seen, is suppressed by EPO (as reported by others). Butyrate initially delays EPO-mediated suppression of p38 phosphorylation, but p38 phosphorylation thereafter, at 30 minutes to 48 hours, is equivalent and at low levels in EPO-treated FL EryD, with or without butyrate. Inhibitors of p38, at 10-50 μM, prevent butyrate-mediated induction of embryonic/fetal globin gene expression. We found that p38 inhibitors, which also disrupt non-p38 signaling pathways, perturb cell division, erythroid differentiation, transit through the cell cycle, and RNA transcription in primary EryD.
  
  
  CONCLUSIONS
  p38 inhibitors interrupt normal erythropoiesis and the capacity for embryonic/fetal globin gene induction. However, p38 signaling is maximal in primary EryD at harvest, when embryonic globin genes are minimally expressed, and diminishes thereafter. We conclude that p38 inhibitors disrupt cellular pathways that are essential to butyrate-induced embryonic/fetal globin gene expression. However, levels of p38 phosphorylation are not coordinate with embryonic/fetal globin gene expression in EryD, and increased signaling through p38 may not be the sine qua non for embryonic/fetal globin gene induction.
authors.affiliation: Hematology, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Amrita
authors.initials: A
authors.lastname: Dutta
authors.forename: Shay
authors.initials: S
authors.lastname: Karkashon
authors.forename: Radha
authors.initials: R
authors.lastname: Raghupathy
authors.forename: Himanshu
authors.initials: H
authors.lastname: Bhatia
authors.forename: Lydia
authors.initials: L
authors.lastname: Tesfa
authors.forename: Jane A
authors.initials: JA
authors.lastname: Little
mesh_terms: D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000378:metabolism; D041905:Erythroid Cells / Q000187:drug effects / Q000378:metabolism; D004920:Erythropoiesis / Q000187:drug effects / Q000235:genetics*; D005319:Fetal Hemoglobin / Q000096:biosynthesis / Q000235:genetics*; D005333:Fetus / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006453:Hemoglobinopathies / Q000628:therapy; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D002087:Butyrates; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D005914:Globins; D005319:Fetal Hemoglobin; D048051:p38 Mitogen-Activated Protein Kinases