Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation.
Mouse embryonic endothelial progenitor cells (eEPCs) acquire a mature phenotype after treatment with cyclic adenosine monophosphate (cAMP), suggesting an involvement of Raf serine/threonine kinases in the differentiation process. To test this idea, we investigated the role of B-Raf and C-Raf in proliferation and differentiation of eEPCs by expressing fusion proteins consisting of the kinase domains from Raf molecules and the hormone binding site of the estrogen receptor (ER), or its variant, the tamoxifen receptor. Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation. In contrast, the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than B-Raf. In a complimentary approach, we used siRNA silencing to knockdown endogenously expressed B-Raf and C-Raf in eEPCs. In this experimental setting, we found that eEPCs lacking B-Raf failed to differentiate, whereas loss-of C-Raf function primarily slowed cell growth without impairing cAMP-induced differentiation. These findings were further corroborated in B-Raf null eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro. Thus, gain- and loss-of-function experiments point to distinct roles of B-Raf and C-Raf in regulating growth and differentiation of endothelial progenitor cells, which may harbour therapeutic implications.
Bidzhekov
Kiril
K
GSF-National Research Center for Environment and Health, Institute of Clinical Molecular Biology and Tumor Genetics, Munich, Germany.
Hautmann
Martina
M
Semisch
Matthias
M
Weber
Christian
C
Engelmann
Bernd
B
Hatzopoulos
Antonis K
AK
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D003001:Cloning, Molecular; D004622:Embryo, Mammalian / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004967:Estrogens / Q000494:pharmacology; D005818:Genetic Engineering; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D034622:RNA Interference / Q000187:drug effects; D011960:Receptors, Estrogen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology
D004967:Estrogens; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf