title: Biochemical measurements on single erythroid progenitor cells shed light on the combinatorial regulation of red blood cell production.
abstract: Adult bone marrow (BM) erythrocyte colony-forming units (CFU-Es) are important cellular targets for the treatment of anemia and also for the manufacture of red blood cells (RBCs) ex vivo. We obtained quantitative biochemical measurements from single and small numbers of CFU-Es by isolating and analyzing c-Kit(+)CD71(high)Ter119(-) cells from adult mouse BM and this allowed us to identify two mechanisms that can be manipulated to increase RBC production. As expected, maximum RBC output was obtained when CFU-Es were stimulated with a combination of Stem Cell Factor (SCF) and Erythropoietin (EPO) mainly because SCF supports a transient CFU-E expansion and EPO promotes the survival and terminal differentiation of erythroid progenitors. However, we found that one of the main factors limiting the output in RBCs was that EPO induces a downregulation of c-Kit expression which limits the transient expansion of CFU-Es. In the presence of SCF, the EPO-mediated downregulation of c-Kit on CFU-Es is delayed but still significant. Moreover, treatment of CFU-Es with 1-Naphthyl PP1 could partially inhibit the downregulation of c-Kit induced by EPO, suggesting that this process is dependent on a Src family kinase, v-Src and/or c-Fyn. We also found that CFU-E survival and proliferation was dependent on the level of time-integrated extracellular-regulated kinase (ERK) activation in these cells, all of which could be significantly increased when SCF and EPO were combined with mouse fetal liver-derived factors. Taken together, these results suggest two novel molecular strategies to increase RBC production and regeneration.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, 164 College Street, Rm 407, Toronto, ON, Canada M5S 3G9.
authors.forename: Weijia
authors.initials: W
authors.lastname: Wang
authors.forename: Vahe
authors.initials: V
authors.lastname: Akbarian
authors.forename: Julie
authors.initials: J
authors.lastname: Audet
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D001789:Blood Group Antigens / Q000235:genetics / Q000378:metabolism; D002468:Cell Physiological Phenomena / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016000:Cluster Analysis; D056742:Cyclin D2 / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004912:Erythrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology; D005260:Female; D020869:Gene Expression Profiling; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D019089:Stem Cell Factor / Q000494:pharmacology*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D001789:Blood Group Antigens; C068126:CD71 antigen; C486196:Ccnd2 protein, mouse; D056742:Cyclin D2; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011990:Receptors, Transferrin; D019089:Stem Cell Factor; C489122:TER-119 antigen, mouse; D004921:Erythropoietin; D019009:Proto-Oncogene Proteins c-kit; D019061:src-Family Kinases; D020929:Mitogen-Activated Protein Kinase Kinases