Activation and inhibition of the erythropoietin receptor by a membrane-anchored erythropoietin.
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.
Negre
Olivier
O
INSERM, Laboratoire de Thérapie Génique Hématopoïétique, Paris, France.
Fusil
Floriane
F
Henri
Annie
A
Villette
Jean-Marie
JM
Leboulch
Philippe
P
Beuzard
Yves
Y
Payen
Emmanuel
E
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
D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin