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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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B-Raf is essential for eEPC differentiation. (A) eEPCs were transfected for 72 hrs with 20 nM siRNAs against B-Raf (lanes 3,4), C-Raf (lanes 5,6), or mock-transfected with oligofectamine as control (lanes 1,2). Duplicates were treated with no (−) or 0.5 μmol/l cAMP (+) for the last 8 hrs of the siRNA treatment. RNA was then isolated and served as template for RT-PCR analysis using gene-specific primers. Silencing of B-Raf completely abolishes the cAMP-induced differentiation of eEPCs, whereas knockdown of C-Raf has no effect—as monitored by Flk-1, P-selectin, Thrombomodulin (TM), vWF and M-CSF expression levels. Aldolase and Tie-2, whose expression does not change during the differentiation process, are not effected by knockdown of B- and C-Raf. The expected band sizes for the various PCR products are indicated on the right. (B) B-Raf eEPCs isolated from B-Raf-/- embryos (KO) were left untreated (−) or induced to differentiate with cAMP (+) for 12 hrs. eEPCs isolated from wild-type siblings (WT) served as controls. The B-Raf  eEPCs do not differentiate after cAMP treatment. The analysed genes are indicated on the right.
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fig07: B-Raf is essential for eEPC differentiation. (A) eEPCs were transfected for 72 hrs with 20 nM siRNAs against B-Raf (lanes 3,4), C-Raf (lanes 5,6), or mock-transfected with oligofectamine as control (lanes 1,2). Duplicates were treated with no (−) or 0.5 μmol/l cAMP (+) for the last 8 hrs of the siRNA treatment. RNA was then isolated and served as template for RT-PCR analysis using gene-specific primers. Silencing of B-Raf completely abolishes the cAMP-induced differentiation of eEPCs, whereas knockdown of C-Raf has no effect—as monitored by Flk-1, P-selectin, Thrombomodulin (TM), vWF and M-CSF expression levels. Aldolase and Tie-2, whose expression does not change during the differentiation process, are not effected by knockdown of B- and C-Raf. The expected band sizes for the various PCR products are indicated on the right. (B) B-Raf eEPCs isolated from B-Raf-/- embryos (KO) were left untreated (−) or induced to differentiate with cAMP (+) for 12 hrs. eEPCs isolated from wild-type siblings (WT) served as controls. The B-Raf eEPCs do not differentiate after cAMP treatment. The analysed genes are indicated on the right.

Mentions: We showed previously that eEPCs differentiate after cAMP treatment leading to induction of genes like Flk-1, vWF, eNOS, Alk-1, Tie-1, M-CSF, TM and P-selectin[4,5]. To investigate a possible link between B- or C-Raf and cAMP-induced differentiation, we tested gene expression in ΔB-Raf:ER and ΔC-Raf:ER expressing eEPCs following activation with estrogen for 8 hrs. We observed induction of Flk-1, Tie-1, P-selectin and M-CSF, which were also stimulated upon cAMP-treatment (Fig. 4). Estrogeninduced gene up-regulation (Fig. 4, lanes 7,11) to a similar extent as cAMP (Fig. 4, lanes 6,10). Mocktransfected cells did not respond to estrogen, but showed a robust induction with cAMP as expected (Fig. 7, lanes 3,4). Of note, we did not observe additive effects between cAMP and estrogen activation (Fig. 4, lane 8,12). Thus, it is likely that the cAMP effects on eEPC differentiation are mediated mostly by activation of Rafs. We tested this idea in cAMP treated eEPCs after knockdown of Raf proteins as described below.


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)

B-Raf is essential for eEPC differentiation. (A) eEPCs were transfected for 72 hrs with 20 nM siRNAs against B-Raf (lanes 3,4), C-Raf (lanes 5,6), or mock-transfected with oligofectamine as control (lanes 1,2). Duplicates were treated with no (−) or 0.5 μmol/l cAMP (+) for the last 8 hrs of the siRNA treatment. RNA was then isolated and served as template for RT-PCR analysis using gene-specific primers. Silencing of B-Raf completely abolishes the cAMP-induced differentiation of eEPCs, whereas knockdown of C-Raf has no effect—as monitored by Flk-1, P-selectin, Thrombomodulin (TM), vWF and M-CSF expression levels. Aldolase and Tie-2, whose expression does not change during the differentiation process, are not effected by knockdown of B- and C-Raf. The expected band sizes for the various PCR products are indicated on the right. (B) B-Raf eEPCs isolated from B-Raf-/- embryos (KO) were left untreated (−) or induced to differentiate with cAMP (+) for 12 hrs. eEPCs isolated from wild-type siblings (WT) served as controls. The B-Raf  eEPCs do not differentiate after cAMP treatment. The analysed genes are indicated on the right.
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Related In: Results  -  Collection

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fig07: B-Raf is essential for eEPC differentiation. (A) eEPCs were transfected for 72 hrs with 20 nM siRNAs against B-Raf (lanes 3,4), C-Raf (lanes 5,6), or mock-transfected with oligofectamine as control (lanes 1,2). Duplicates were treated with no (−) or 0.5 μmol/l cAMP (+) for the last 8 hrs of the siRNA treatment. RNA was then isolated and served as template for RT-PCR analysis using gene-specific primers. Silencing of B-Raf completely abolishes the cAMP-induced differentiation of eEPCs, whereas knockdown of C-Raf has no effect—as monitored by Flk-1, P-selectin, Thrombomodulin (TM), vWF and M-CSF expression levels. Aldolase and Tie-2, whose expression does not change during the differentiation process, are not effected by knockdown of B- and C-Raf. The expected band sizes for the various PCR products are indicated on the right. (B) B-Raf eEPCs isolated from B-Raf-/- embryos (KO) were left untreated (−) or induced to differentiate with cAMP (+) for 12 hrs. eEPCs isolated from wild-type siblings (WT) served as controls. The B-Raf eEPCs do not differentiate after cAMP treatment. The analysed genes are indicated on the right.
Mentions: We showed previously that eEPCs differentiate after cAMP treatment leading to induction of genes like Flk-1, vWF, eNOS, Alk-1, Tie-1, M-CSF, TM and P-selectin[4,5]. To investigate a possible link between B- or C-Raf and cAMP-induced differentiation, we tested gene expression in ΔB-Raf:ER and ΔC-Raf:ER expressing eEPCs following activation with estrogen for 8 hrs. We observed induction of Flk-1, Tie-1, P-selectin and M-CSF, which were also stimulated upon cAMP-treatment (Fig. 4). Estrogeninduced gene up-regulation (Fig. 4, lanes 7,11) to a similar extent as cAMP (Fig. 4, lanes 6,10). Mocktransfected cells did not respond to estrogen, but showed a robust induction with cAMP as expected (Fig. 7, lanes 3,4). Of note, we did not observe additive effects between cAMP and estrogen activation (Fig. 4, lane 8,12). Thus, it is likely that the cAMP effects on eEPC differentiation are mediated mostly by activation of Rafs. We tested this idea in cAMP treated eEPCs after knockdown of Raf proteins as described below.

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