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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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ΔB- and ΔC-Raf:ER phosphorylate MEK after estrogen stimulation. (A, B) Western blotting with antibodies recognizing MEK1/2 (top panels) and antibodies recognizing phosphorylated MEK1/2 (p-MEK1/2) in serines 217 and 221 (lower panels). eEPCs were grown in starvation medium for 12 hrs with Nutridoma as nutritional supplement, and then induced for 1 hr with either estrogen, or serum as positive control. MEK phosphorylation is evident in ΔB-Raf:ER (A) and ΔCRaf:ER (B) expressing cells, but not in mock-transfected cells after estrogen treatment (compare lanes 3–7). Serum addition leads to MEK phosphorylation in both mock- and ΔRaf:ER- expressing cells (lanes 1,2 and 5,6).
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fig02: ΔB- and ΔC-Raf:ER phosphorylate MEK after estrogen stimulation. (A, B) Western blotting with antibodies recognizing MEK1/2 (top panels) and antibodies recognizing phosphorylated MEK1/2 (p-MEK1/2) in serines 217 and 221 (lower panels). eEPCs were grown in starvation medium for 12 hrs with Nutridoma as nutritional supplement, and then induced for 1 hr with either estrogen, or serum as positive control. MEK phosphorylation is evident in ΔB-Raf:ER (A) and ΔCRaf:ER (B) expressing cells, but not in mock-transfected cells after estrogen treatment (compare lanes 3–7). Serum addition leads to MEK phosphorylation in both mock- and ΔRaf:ER- expressing cells (lanes 1,2 and 5,6).

Mentions: To check if the kinase domains of ΔB-Raf:ER and ΔC-Raf:ER proteins are functional, we investigated the phosphorylation of Raf downstream targets after estrogen activation. Immediate partners in the signalling pathway of Raf proteins are the MAP/extracellular signal-regulated kinases (ERK) kinases MEK1 and MEK2, which in turn phosphorylate the ERKs [25]. To test MEK phosphorylation after estrogen stimulation, ΔB-Raf:ER or ΔC-Raf:ER transfected stable eEPC lines were grown in serum-free medium to reduce basal levels of MEK phosphorylation and then induced with estrogen (Fig. 2). Using MEK-recognizing antibodies, we found comparable MEK protein levels in all samples (Fig. 2A and B upper panels). In contrast, using antibodies recognizing specifically phospho-MEK, we detected phosphorylation only in estrogen-induced cells transfected with the ΔB-Raf:ER and ΔC-Raf:ER constructs (Fig. 2A and B lower panels). Starved, or estrogen-induced mocktransfected cells showed no phosphorylated MEK, demonstrating that the Raf:ER proteins were responsible for MEK activation after estrogen treatment (Fig. 2A and B comparing lanes 3 and 7). We obtained comparable results using ΔB-Raf:ER* and ΔC-Raf:ER* after 4-HT stimulation (not shown).


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)

ΔB- and ΔC-Raf:ER phosphorylate MEK after estrogen stimulation. (A, B) Western blotting with antibodies recognizing MEK1/2 (top panels) and antibodies recognizing phosphorylated MEK1/2 (p-MEK1/2) in serines 217 and 221 (lower panels). eEPCs were grown in starvation medium for 12 hrs with Nutridoma as nutritional supplement, and then induced for 1 hr with either estrogen, or serum as positive control. MEK phosphorylation is evident in ΔB-Raf:ER (A) and ΔCRaf:ER (B) expressing cells, but not in mock-transfected cells after estrogen treatment (compare lanes 3–7). Serum addition leads to MEK phosphorylation in both mock- and ΔRaf:ER- expressing cells (lanes 1,2 and 5,6).
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Related In: Results  -  Collection

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

fig02: ΔB- and ΔC-Raf:ER phosphorylate MEK after estrogen stimulation. (A, B) Western blotting with antibodies recognizing MEK1/2 (top panels) and antibodies recognizing phosphorylated MEK1/2 (p-MEK1/2) in serines 217 and 221 (lower panels). eEPCs were grown in starvation medium for 12 hrs with Nutridoma as nutritional supplement, and then induced for 1 hr with either estrogen, or serum as positive control. MEK phosphorylation is evident in ΔB-Raf:ER (A) and ΔCRaf:ER (B) expressing cells, but not in mock-transfected cells after estrogen treatment (compare lanes 3–7). Serum addition leads to MEK phosphorylation in both mock- and ΔRaf:ER- expressing cells (lanes 1,2 and 5,6).
Mentions: To check if the kinase domains of ΔB-Raf:ER and ΔC-Raf:ER proteins are functional, we investigated the phosphorylation of Raf downstream targets after estrogen activation. Immediate partners in the signalling pathway of Raf proteins are the MAP/extracellular signal-regulated kinases (ERK) kinases MEK1 and MEK2, which in turn phosphorylate the ERKs [25]. To test MEK phosphorylation after estrogen stimulation, ΔB-Raf:ER or ΔC-Raf:ER transfected stable eEPC lines were grown in serum-free medium to reduce basal levels of MEK phosphorylation and then induced with estrogen (Fig. 2). Using MEK-recognizing antibodies, we found comparable MEK protein levels in all samples (Fig. 2A and B upper panels). In contrast, using antibodies recognizing specifically phospho-MEK, we detected phosphorylation only in estrogen-induced cells transfected with the ΔB-Raf:ER and ΔC-Raf:ER constructs (Fig. 2A and B lower panels). Starved, or estrogen-induced mocktransfected cells showed no phosphorylated MEK, demonstrating that the Raf:ER proteins were responsible for MEK activation after estrogen treatment (Fig. 2A and B comparing lanes 3 and 7). We obtained comparable results using ΔB-Raf:ER* and ΔC-Raf:ER* after 4-HT stimulation (not shown).

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