title: All-trans retinoic acid directly up-regulates thrombopoietin transcription in human bone marrow stromal cells.
abstract: OBJECTIVE
  All-trans retinoic acid (ATRA) has been used as the first-line therapy for patients with acute promyelocytic leukemia (APL). We previously reported that ATRA increased serum thrombopoietin (TPO) levels accompanied by thrombocytosis during ATRA therapy for APL. In this study, we investigated the mechanism of transcriptional regulation of TPO gene by ATRA using human bone marrow stromal cells.
  
  
  METHODS
  Real time reverse transcriptase polymerase chain reaction and Western blotting were performed to quantify TPO mRNA and protein levels in cells from the human bone marrow stromal cell line KM101. Luciferase-based reporter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were performed to identify a retinoic acid responsive element in the promoter region of TPO gene (TPO-RARE).
  
  
  RESULTS
  TPO mRNA expression was up-regulated by approximately 2.9 times 8 hours after stimulation with 10(-6) M ATRA in KM101 cells. In contrast, ATRA did not alter TPO mRNA expression in cells from the human hepatoma cell line HepG2. Protein level of KM101 cells also was increased with 10(-6) M ATRA for 48 hours in KM101 cells. We found the synthesized RARalpha protein bound to [gamma-32P]-labeled TPO-RARE probe and its binding was competed by adding 200x amount of cold TPO-RARE probe by EMSA. In addition, [gamma-32P]-labeled TPO-RARE probe bound to KM101 nuclear protein extract was supershifted by anti-RARalpha antibody and modified by treatment with ATRA. The relative luciferase activity of TPO gene was increased by 2.2x and the histone H4 was acetylated through TPO-RARE after ATRA stimulation in KM101 cells by ChIP assay.
  
  
  CONCLUSIONS
  These data support the direct up-regulation of TPO transcription by ATRA stimulation in human bone marrow stromal cells and propose one of the mechanisms of thrombocytosis during ATRA therapy for APL.
authors.affiliation: Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kinjo
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Miyakawa
authors.forename: Hideo
authors.initials: H
authors.lastname: Uchida
authors.forename: Shigetaka
authors.initials: S
authors.lastname: Kitajima
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kizaki
mesh_terms: D024506:5' Flanking Region; D001854:Bone Marrow Cells / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D017154:Stromal Cells / Q000187:drug effects; D013926:Thrombopoietin / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D014212:Tretinoin; D013926:Thrombopoietin