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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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Loss of C-Raf function impairs proliferation of eEPCs. eEPCs were transfected with 20 nM siRNA against B-Raf or CRaf for 72 hrs. Oligofectamine alone treatment served as a negative control. Each transfection was done in triplicates. At the end of the 72-hrs period, cells were counted twice using an automatic Coulter counter (*P<0.001 versus control).
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fig06: Loss of C-Raf function impairs proliferation of eEPCs. eEPCs were transfected with 20 nM siRNA against B-Raf or CRaf for 72 hrs. Oligofectamine alone treatment served as a negative control. Each transfection was done in triplicates. At the end of the 72-hrs period, cells were counted twice using an automatic Coulter counter (*P<0.001 versus control).

Mentions: Following the specific and efficient knockdown of B and C-Raf proteins, we analysed the effects of Raf loss-of-function on eEPC proliferation and differentiation. We found that silencing of B-Raf or C-Raf impaired the growth of eEPCs at variable degrees. Specifically, the effect was more pronounced in cells transfected with siRNAs against C-Raf than B-Raf (Fig. 6). This result is in accordance with the gain-of-function experiments, where ΔC-Raf:ER* was more efficient in inducing eEPC proliferation than ΔBRaf: ER* after 4-HT stimulation (Fig. 3).


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)

Loss of C-Raf function impairs proliferation of eEPCs. eEPCs were transfected with 20 nM siRNA against B-Raf or CRaf for 72 hrs. Oligofectamine alone treatment served as a negative control. Each transfection was done in triplicates. At the end of the 72-hrs period, cells were counted twice using an automatic Coulter counter (*P<0.001 versus control).
© Copyright Policy
Related In: Results  -  Collection

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

fig06: Loss of C-Raf function impairs proliferation of eEPCs. eEPCs were transfected with 20 nM siRNA against B-Raf or CRaf for 72 hrs. Oligofectamine alone treatment served as a negative control. Each transfection was done in triplicates. At the end of the 72-hrs period, cells were counted twice using an automatic Coulter counter (*P<0.001 versus control).
Mentions: Following the specific and efficient knockdown of B and C-Raf proteins, we analysed the effects of Raf loss-of-function on eEPC proliferation and differentiation. We found that silencing of B-Raf or C-Raf impaired the growth of eEPCs at variable degrees. Specifically, the effect was more pronounced in cells transfected with siRNAs against C-Raf than B-Raf (Fig. 6). This result is in accordance with the gain-of-function experiments, where ΔC-Raf:ER* was more efficient in inducing eEPC proliferation than ΔBRaf: ER* after 4-HT stimulation (Fig. 3).

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