title: Activation and inhibition of the erythropoietin receptor by a membrane-anchored erythropoietin.
abstract: OBJECTIVE
  To investigate whether expression of a membrane-anchored form of erythropoietin (MbEpo) results in self-controlled, autocrine proliferation, and differentiation of erythroid cells. This would provide a possible approach to the selective expansion of genetically corrected erythroid cells in gene-therapy protocols.
  
  
  METHODS
  We designed retroviral vectors encoding MbEpo or secreted erythropoietin (Epo) and enhanced green fluorescent protein. Several Epo-dependent cell lines were transduced and their proliferative capacity evaluated. This approach was also assessed in human bone marrow CD34(+) cells and mouse bone marrow transplants.
  
  
  RESULTS
  Retroviral vector-mediated MbEpo expression induced autocrine proliferation of the Epo-dependent cell lines DAE7 and UT7/Epo. However, it blocked the Epo receptor (EpoR)-induced activation of granulocyte macrophage colony-stimulating factor-dependent UT7/GM cells and the erythroid differentiation of both human hematopoietic cells in vitro and of mouse bone marrow cells in transplant experiments. MbEpo was present at the surface of UT7/GM cells. It did not affect the membrane localization of the EpoR, but prevented its normal Epo-dependent phosphorylation and internalization. By contrast to these inhibitory effects, a higher rate of EpoR replenishment in UT7/GM cells before MbEpo production rendered cell proliferation independent of exogenous growth factor.
  
  
  CONCLUSIONS
  Activation of EpoR gene expression before MbEpo-induced EpoR activation is essential for activation or inhibition of growth and differentiation of Epo-dependent cell lines. It will be necessary to delay MbEpo expression in late erythroid progenitors until after EpoR gene activation, for erythroid cell expansion to be achieved in vivo.
authors.affiliation: INSERM, Laboratoire de Thérapie Génique Hématopoïétique, Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Negre
authors.forename: Floriane
authors.initials: F
authors.lastname: Fusil
authors.forename: Annie
authors.initials: A
authors.lastname: Henri
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Villette
authors.forename: Philippe
authors.initials: P
authors.lastname: Leboulch
authors.forename: Yves
authors.initials: Y
authors.lastname: Beuzard
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Payen
mesh_terms: D000818:Animals; D016026:Bone Marrow Transplantation; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin