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Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation.

Bidzhekov K, Hautmann M, Semisch M, Weber C, Engelmann B, Hatzopoulos AK - J. Cell. Mol. Med. (2007 Nov-Dec)

Bottom Line: Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation.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 eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro.

View Article: PubMed Central - PubMed

Affiliation: GSF-National Research Center for Environment and Health, Institute of Clinical Molecular Biology and Tumor Genetics, Munich, Germany.

ABSTRACT
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 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.

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Active C-Raf kinase domain stimulates eEPC growth. eEPCs were transiently transfected with ΔB-Raf:ER*, ΔC-Raf:ER* or an EGFP reporter construct as a control. The cells were left untreated (−) or stimulated (+) with 100 nmol/l tamoxifen (4-HT) and counted 48 hrs later. Activation of the C-Raf kinase domain strongly induces eEPC growth, whereas activation of the corresponding B-Raf domain has only a modest effect (*P<0.01 and **P<0.001 versus control, respectively).
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fig03: Active C-Raf kinase domain stimulates eEPC growth. eEPCs were transiently transfected with ΔB-Raf:ER*, ΔC-Raf:ER* or an EGFP reporter construct as a control. The cells were left untreated (−) or stimulated (+) with 100 nmol/l tamoxifen (4-HT) and counted 48 hrs later. Activation of the C-Raf kinase domain strongly induces eEPC growth, whereas activation of the corresponding B-Raf domain has only a modest effect (*P<0.01 and **P<0.001 versus control, respectively).

Mentions: Raf proteins have been implicated in the regulation of proliferation in different cell types [26, 27]. To identify possible effects of activated Raf members on eEPC growth, we compared ΔB-Raf:ER* and ΔC-Raf:ER* transiently transfected eEPC with or without 4-HT induction of kinase activity. Cells transfected in parallel with an enhanced green fluorescent protein (EGFP) expression construct served as negative control. We induced eEPCs with 4-HT for 8 hrs and counted cell numbers 48 hrs later. As shown in Figure 3, 4-HT treatment led to 2.5-fold higher cell numbers in ΔC-Raf:ER* engineered eEPCs compared to controls, whereas there was only a slight increase in ΔB-Raf:ER* expressing cells. We obtained similar results with stably transfected eEPCs (not shown). Although both B- and C-Raf:ER proteins are capable of phosphorylating MEKs (Fig. 2), it appears that the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than the corresponding B-Raf domain.


Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation.

Bidzhekov K, Hautmann M, Semisch M, Weber C, Engelmann B, Hatzopoulos AK - J. Cell. Mol. Med. (2007 Nov-Dec)

Active C-Raf kinase domain stimulates eEPC growth. eEPCs were transiently transfected with ΔB-Raf:ER*, ΔC-Raf:ER* or an EGFP reporter construct as a control. The cells were left untreated (−) or stimulated (+) with 100 nmol/l tamoxifen (4-HT) and counted 48 hrs later. Activation of the C-Raf kinase domain strongly induces eEPC growth, whereas activation of the corresponding B-Raf domain has only a modest effect (*P<0.01 and **P<0.001 versus control, respectively).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401289&req=5

fig03: Active C-Raf kinase domain stimulates eEPC growth. eEPCs were transiently transfected with ΔB-Raf:ER*, ΔC-Raf:ER* or an EGFP reporter construct as a control. The cells were left untreated (−) or stimulated (+) with 100 nmol/l tamoxifen (4-HT) and counted 48 hrs later. Activation of the C-Raf kinase domain strongly induces eEPC growth, whereas activation of the corresponding B-Raf domain has only a modest effect (*P<0.01 and **P<0.001 versus control, respectively).
Mentions: Raf proteins have been implicated in the regulation of proliferation in different cell types [26, 27]. To identify possible effects of activated Raf members on eEPC growth, we compared ΔB-Raf:ER* and ΔC-Raf:ER* transiently transfected eEPC with or without 4-HT induction of kinase activity. Cells transfected in parallel with an enhanced green fluorescent protein (EGFP) expression construct served as negative control. We induced eEPCs with 4-HT for 8 hrs and counted cell numbers 48 hrs later. As shown in Figure 3, 4-HT treatment led to 2.5-fold higher cell numbers in ΔC-Raf:ER* engineered eEPCs compared to controls, whereas there was only a slight increase in ΔB-Raf:ER* expressing cells. We obtained similar results with stably transfected eEPCs (not shown). Although both B- and C-Raf:ER proteins are capable of phosphorylating MEKs (Fig. 2), it appears that the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than the corresponding B-Raf domain.

Bottom Line: Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation.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 eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro.

View Article: PubMed Central - PubMed

Affiliation: GSF-National Research Center for Environment and Health, Institute of Clinical Molecular Biology and Tumor Genetics, Munich, Germany.

ABSTRACT
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 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.

Show MeSH
Related in: MedlinePlus