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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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RNA interference-mediated silencing of B- and C-Raf in eEPCs. (A) eEPCs were transfected with 20 nM siRNAs against B- or C-Raf using oligofectamine alone as control (Co). Total RNA was isolated 48 hrs later and assayed for levels of endogenous B- and C-Raf mRNAs using RT-PCR. cAMP treatment did not interfere with the silencing process. siRNAs against B-raf do not affect C-Raf mRNA levels, and vice versa, indicating that the RNAi tools are specific for the corresponding targeted Raf isoform. (B) eEPCs were transfected for 72 hrs with 20 nM siRNAs against B-Raf or C-Raf. Western blotting shows that siRNA treatment effectively abolishes endogenous B- and C Raf proteins. As for mRNA in A, there is no cross reactivity between the knockdown tools since anti-B-Raf siRNAs do not affect C-Raf protein and vice versa. The unspecific bands in the B-Raf blot (asterisks) served as loading controls.
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fig05: RNA interference-mediated silencing of B- and C-Raf in eEPCs. (A) eEPCs were transfected with 20 nM siRNAs against B- or C-Raf using oligofectamine alone as control (Co). Total RNA was isolated 48 hrs later and assayed for levels of endogenous B- and C-Raf mRNAs using RT-PCR. cAMP treatment did not interfere with the silencing process. siRNAs against B-raf do not affect C-Raf mRNA levels, and vice versa, indicating that the RNAi tools are specific for the corresponding targeted Raf isoform. (B) eEPCs were transfected for 72 hrs with 20 nM siRNAs against B-Raf or C-Raf. Western blotting shows that siRNA treatment effectively abolishes endogenous B- and C Raf proteins. As for mRNA in A, there is no cross reactivity between the knockdown tools since anti-B-Raf siRNAs do not affect C-Raf protein and vice versa. The unspecific bands in the B-Raf blot (asterisks) served as loading controls.

Mentions: For the silencing experiments, we transfected eEPCs with siRNA cocktails against B- and C-Raf for 48 hrs. The siRNAs were first tested for correct and efficient targeting by RT-PCR using gene-specific primer pairs that distinguish between B- and C-Raf transcripts. The results showed that siRNAs against either Raf isoform reduced the corresponding mRNA levels in a specific manner, that is, without affecting the expression levels of the other Raf gene (Fig. 5A). We also observed that cAMP treatment did not interfere with the silencing process (Fig. 5A). Quantification of expression levels demonstrated a reduction of 3-fold in B-Raf and around 2-fold in C-Raf mRNAs after siRNA-mediated knockdown.


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)

RNA interference-mediated silencing of B- and C-Raf in eEPCs. (A) eEPCs were transfected with 20 nM siRNAs against B- or C-Raf using oligofectamine alone as control (Co). Total RNA was isolated 48 hrs later and assayed for levels of endogenous B- and C-Raf mRNAs using RT-PCR. cAMP treatment did not interfere with the silencing process. siRNAs against B-raf do not affect C-Raf mRNA levels, and vice versa, indicating that the RNAi tools are specific for the corresponding targeted Raf isoform. (B) eEPCs were transfected for 72 hrs with 20 nM siRNAs against B-Raf or C-Raf. Western blotting shows that siRNA treatment effectively abolishes endogenous B- and C Raf proteins. As for mRNA in A, there is no cross reactivity between the knockdown tools since anti-B-Raf siRNAs do not affect C-Raf protein and vice versa. The unspecific bands in the B-Raf blot (asterisks) served as loading controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401289&req=5

fig05: RNA interference-mediated silencing of B- and C-Raf in eEPCs. (A) eEPCs were transfected with 20 nM siRNAs against B- or C-Raf using oligofectamine alone as control (Co). Total RNA was isolated 48 hrs later and assayed for levels of endogenous B- and C-Raf mRNAs using RT-PCR. cAMP treatment did not interfere with the silencing process. siRNAs against B-raf do not affect C-Raf mRNA levels, and vice versa, indicating that the RNAi tools are specific for the corresponding targeted Raf isoform. (B) eEPCs were transfected for 72 hrs with 20 nM siRNAs against B-Raf or C-Raf. Western blotting shows that siRNA treatment effectively abolishes endogenous B- and C Raf proteins. As for mRNA in A, there is no cross reactivity between the knockdown tools since anti-B-Raf siRNAs do not affect C-Raf protein and vice versa. The unspecific bands in the B-Raf blot (asterisks) served as loading controls.
Mentions: For the silencing experiments, we transfected eEPCs with siRNA cocktails against B- and C-Raf for 48 hrs. The siRNAs were first tested for correct and efficient targeting by RT-PCR using gene-specific primer pairs that distinguish between B- and C-Raf transcripts. The results showed that siRNAs against either Raf isoform reduced the corresponding mRNA levels in a specific manner, that is, without affecting the expression levels of the other Raf gene (Fig. 5A). We also observed that cAMP treatment did not interfere with the silencing process (Fig. 5A). Quantification of expression levels demonstrated a reduction of 3-fold in B-Raf and around 2-fold in C-Raf mRNAs after siRNA-mediated knockdown.

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