title: Effect of increased HoxB4 on human megakaryocytic development.
abstract: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.
authors.affiliation: Department of Pathology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhong
authors.forename: Brent
authors.initials: B
authors.lastname: Sullenbarger
authors.forename: Larry C
authors.initials: LC
authors.lastname: Lasky
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D019938:Cyclin D1 / Q000235:genetics; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism; D016086:Lentivirus; D055016:Megakaryocyte Progenitor Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D017713:Platelet Transfusion; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D034061:Thrombopoiesis / Q000187:drug effects / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*
chemical_list: C078433:FLI1 protein, human; C090460:HOXB4 protein, human; D018398:Homeodomain Proteins; C061248:ITGB3 protein, human; D039661:Integrin beta3; D009687:Nuclear Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D051789:Proto-Oncogene Protein c-fli-1; D020598:Proto-Oncogene Proteins c-myb; D011994:Recombinant Proteins; D014157:Transcription Factors; C498432:ZFPM1 protein, human; D019938:Cyclin D1