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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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Genetic engineering of eEPCs with inducible Raf kinases. (A) Structure of DNA constructs carrying the kinase domains of B- and C-Raf fused to the hormone binding site (hbs) domain of the estrogen (ER) and tamoxifen (ER*) receptors under the PGK promoter (PGK Pr); pA: polyadenylation region of the bovine growth hormone gene. The first and last five amino acids of the B- and C-Raf kinase domains and the hormone binding sites of the estrogen/tamoxifen receptors are provided on top of the diagrams. (B). PCR genotyping of G418-selected eEPC clones transfected with the expression constructs depicted in A. (−) mock-transfected cells; (+) cells transfected with ΔB- or ΔC-Raf:ER expression vectors (ΔB and ΔC). M: DNA size marker; sizes given in base pairs (bp). Lane C represents a positive control using ΔC-Raf:ER plasmid as template. (C). Genetically engineered eEPCs express high levels of ΔB- and ΔC-Raf:ER RNAs. (D). Engineered eEPCs express high levels of ΔB- and ΔC-Raf:ER proteins. The ΔB- and ΔC-Raf:ER fusion proteins were detected by immunoprecipitation (IP) with an anti-ER antibody followed by Western blotting (WB) with antibodies that recognize specifically the kinase domain of B- or C-Raf. In complementary fashion, the same proteins were detected by IP with the B- or C-Raf antibodies followed by Western blotting with anti-ER. Protein sizes are indicated in kDaltons (kD). In C,D: (−) mock transfected cells; (+) cells transfected with ΔB- and ΔC-Raf:ER expression vectors.
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fig01: Genetic engineering of eEPCs with inducible Raf kinases. (A) Structure of DNA constructs carrying the kinase domains of B- and C-Raf fused to the hormone binding site (hbs) domain of the estrogen (ER) and tamoxifen (ER*) receptors under the PGK promoter (PGK Pr); pA: polyadenylation region of the bovine growth hormone gene. The first and last five amino acids of the B- and C-Raf kinase domains and the hormone binding sites of the estrogen/tamoxifen receptors are provided on top of the diagrams. (B). PCR genotyping of G418-selected eEPC clones transfected with the expression constructs depicted in A. (−) mock-transfected cells; (+) cells transfected with ΔB- or ΔC-Raf:ER expression vectors (ΔB and ΔC). M: DNA size marker; sizes given in base pairs (bp). Lane C represents a positive control using ΔC-Raf:ER plasmid as template. (C). Genetically engineered eEPCs express high levels of ΔB- and ΔC-Raf:ER RNAs. (D). Engineered eEPCs express high levels of ΔB- and ΔC-Raf:ER proteins. The ΔB- and ΔC-Raf:ER fusion proteins were detected by immunoprecipitation (IP) with an anti-ER antibody followed by Western blotting (WB) with antibodies that recognize specifically the kinase domain of B- or C-Raf. In complementary fashion, the same proteins were detected by IP with the B- or C-Raf antibodies followed by Western blotting with anti-ER. Protein sizes are indicated in kDaltons (kD). In C,D: (−) mock transfected cells; (+) cells transfected with ΔB- and ΔC-Raf:ER expression vectors.

Mentions: To study the role of B- and C-Raf in eEPCs, we established cell lines expressing conditionally active forms of B- and C-Raf proteins. For this purpose, we constructed fusions of the Raf kinase domains with the hormone-binding part of the human ER α[24]. In this setting, Raf kinase activity can be induced by hormone addition to the culture medium, which liberates fusion proteins from bound heat shock proteins that mask their activity. For efficient expression levels, we cloned ΔB-Raf:ER and ΔC-Raf:ER behind the phosphoglycerate kinase (PGK) promoter (Fig. 1A), which is highly active in embryonic cells.


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)

Genetic engineering of eEPCs with inducible Raf kinases. (A) Structure of DNA constructs carrying the kinase domains of B- and C-Raf fused to the hormone binding site (hbs) domain of the estrogen (ER) and tamoxifen (ER*) receptors under the PGK promoter (PGK Pr); pA: polyadenylation region of the bovine growth hormone gene. The first and last five amino acids of the B- and C-Raf kinase domains and the hormone binding sites of the estrogen/tamoxifen receptors are provided on top of the diagrams. (B). PCR genotyping of G418-selected eEPC clones transfected with the expression constructs depicted in A. (−) mock-transfected cells; (+) cells transfected with ΔB- or ΔC-Raf:ER expression vectors (ΔB and ΔC). M: DNA size marker; sizes given in base pairs (bp). Lane C represents a positive control using ΔC-Raf:ER plasmid as template. (C). Genetically engineered eEPCs express high levels of ΔB- and ΔC-Raf:ER RNAs. (D). Engineered eEPCs express high levels of ΔB- and ΔC-Raf:ER proteins. The ΔB- and ΔC-Raf:ER fusion proteins were detected by immunoprecipitation (IP) with an anti-ER antibody followed by Western blotting (WB) with antibodies that recognize specifically the kinase domain of B- or C-Raf. In complementary fashion, the same proteins were detected by IP with the B- or C-Raf antibodies followed by Western blotting with anti-ER. Protein sizes are indicated in kDaltons (kD). In C,D: (−) mock transfected cells; (+) cells transfected with ΔB- and ΔC-Raf:ER expression vectors.
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Related In: Results  -  Collection

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fig01: Genetic engineering of eEPCs with inducible Raf kinases. (A) Structure of DNA constructs carrying the kinase domains of B- and C-Raf fused to the hormone binding site (hbs) domain of the estrogen (ER) and tamoxifen (ER*) receptors under the PGK promoter (PGK Pr); pA: polyadenylation region of the bovine growth hormone gene. The first and last five amino acids of the B- and C-Raf kinase domains and the hormone binding sites of the estrogen/tamoxifen receptors are provided on top of the diagrams. (B). PCR genotyping of G418-selected eEPC clones transfected with the expression constructs depicted in A. (−) mock-transfected cells; (+) cells transfected with ΔB- or ΔC-Raf:ER expression vectors (ΔB and ΔC). M: DNA size marker; sizes given in base pairs (bp). Lane C represents a positive control using ΔC-Raf:ER plasmid as template. (C). Genetically engineered eEPCs express high levels of ΔB- and ΔC-Raf:ER RNAs. (D). Engineered eEPCs express high levels of ΔB- and ΔC-Raf:ER proteins. The ΔB- and ΔC-Raf:ER fusion proteins were detected by immunoprecipitation (IP) with an anti-ER antibody followed by Western blotting (WB) with antibodies that recognize specifically the kinase domain of B- or C-Raf. In complementary fashion, the same proteins were detected by IP with the B- or C-Raf antibodies followed by Western blotting with anti-ER. Protein sizes are indicated in kDaltons (kD). In C,D: (−) mock transfected cells; (+) cells transfected with ΔB- and ΔC-Raf:ER expression vectors.
Mentions: To study the role of B- and C-Raf in eEPCs, we established cell lines expressing conditionally active forms of B- and C-Raf proteins. For this purpose, we constructed fusions of the Raf kinase domains with the hormone-binding part of the human ER α[24]. In this setting, Raf kinase activity can be induced by hormone addition to the culture medium, which liberates fusion proteins from bound heat shock proteins that mask their activity. For efficient expression levels, we cloned ΔB-Raf:ER and ΔC-Raf:ER behind the phosphoglycerate kinase (PGK) promoter (Fig. 1A), which is highly active in embryonic cells.

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