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A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.

Müller M, Schröer J, Azoitei N, Eiseler T, Bergmann W, Köhntop R, Lin Q, Costa IG, Zenke M, Genze F, Weidgang C, Seufferlein T, Liebau S, Kleger A - Sci Rep (2015)

Bottom Line: In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer.Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs.Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, Ulm University, Ulm, Germany.

ABSTRACT
The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

No MeSH data available.


Related in: MedlinePlus

Generation of a conditional PKD2 allele in embryonic stem cells.(A) Schematic display of the strategy to generate a dose-dependent Dox-inducible PKD2 ESC line. (B) PCR band at 450 bp illustrates the correct recombination of the HPRT locus upon homologous cassette exchange. (C) mRNA levels of PKD2 upon Dox-stimulation of iPKD2 ES cells vs. control mES cells (A2lox.cre cell line) (D) Western blot of PKD2 upon Dox-stimulation of iPKD2 ES cells. (E) Immunostaining of SSEA1 upon Dox induced PKD2 overexpression. Note unaltered expression of SSEA1. qPCRs were performed n = 3 in replicates. Western Blots and immunostaining experiments were performed three times. Scale bars 20 μm.
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f2: Generation of a conditional PKD2 allele in embryonic stem cells.(A) Schematic display of the strategy to generate a dose-dependent Dox-inducible PKD2 ESC line. (B) PCR band at 450 bp illustrates the correct recombination of the HPRT locus upon homologous cassette exchange. (C) mRNA levels of PKD2 upon Dox-stimulation of iPKD2 ES cells vs. control mES cells (A2lox.cre cell line) (D) Western blot of PKD2 upon Dox-stimulation of iPKD2 ES cells. (E) Immunostaining of SSEA1 upon Dox induced PKD2 overexpression. Note unaltered expression of SSEA1. qPCRs were performed n = 3 in replicates. Western Blots and immunostaining experiments were performed three times. Scale bars 20 μm.

Mentions: In order to dissect the role of PKD2 in ESC differentiation, we targeted an inducible PKD2-knock-in allele in ESCs to allow the temporally regulated and dose-dependent expression of PKD2 (iPKD2 ESCs; dose-dependence not shown, Fig. 2A). We used a rapid and efficient recombination system in the HPRT locus, where a doxycycline (Dox)-inducible promoter regulates expression of the PKD2 mRNA. This overexpression system has been extensively applied in a series of studies to delineate the function of a particular gene during differentiation2139474849505152. Phosphorylation at three critical serine residues of PKD2 has been reported to solely achieve maximum activation (Ser 706, 710 and 244) and not via gene expression alone4. Consequently, we targeted a constitutively active PKD2 mutant (PKD2 S244/706/710E, PKD2-3SE) that mimics phosphorylation4. The correct gene recombination in the HPRT locus is shown in Fig. 2B. The PKD2 allele was effectively induced upon Dox-exposure as assessed by mRNA and western blot (Fig. 2C,D). Notably, PKD2 overexpression did not affect the expression levels of the SSEA1 protein indicating a negligible role of PKD2 for the pluripotency circuitry (Fig. 2E).


A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.

Müller M, Schröer J, Azoitei N, Eiseler T, Bergmann W, Köhntop R, Lin Q, Costa IG, Zenke M, Genze F, Weidgang C, Seufferlein T, Liebau S, Kleger A - Sci Rep (2015)

Generation of a conditional PKD2 allele in embryonic stem cells.(A) Schematic display of the strategy to generate a dose-dependent Dox-inducible PKD2 ESC line. (B) PCR band at 450 bp illustrates the correct recombination of the HPRT locus upon homologous cassette exchange. (C) mRNA levels of PKD2 upon Dox-stimulation of iPKD2 ES cells vs. control mES cells (A2lox.cre cell line) (D) Western blot of PKD2 upon Dox-stimulation of iPKD2 ES cells. (E) Immunostaining of SSEA1 upon Dox induced PKD2 overexpression. Note unaltered expression of SSEA1. qPCRs were performed n = 3 in replicates. Western Blots and immunostaining experiments were performed three times. Scale bars 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493579&req=5

f2: Generation of a conditional PKD2 allele in embryonic stem cells.(A) Schematic display of the strategy to generate a dose-dependent Dox-inducible PKD2 ESC line. (B) PCR band at 450 bp illustrates the correct recombination of the HPRT locus upon homologous cassette exchange. (C) mRNA levels of PKD2 upon Dox-stimulation of iPKD2 ES cells vs. control mES cells (A2lox.cre cell line) (D) Western blot of PKD2 upon Dox-stimulation of iPKD2 ES cells. (E) Immunostaining of SSEA1 upon Dox induced PKD2 overexpression. Note unaltered expression of SSEA1. qPCRs were performed n = 3 in replicates. Western Blots and immunostaining experiments were performed three times. Scale bars 20 μm.
Mentions: In order to dissect the role of PKD2 in ESC differentiation, we targeted an inducible PKD2-knock-in allele in ESCs to allow the temporally regulated and dose-dependent expression of PKD2 (iPKD2 ESCs; dose-dependence not shown, Fig. 2A). We used a rapid and efficient recombination system in the HPRT locus, where a doxycycline (Dox)-inducible promoter regulates expression of the PKD2 mRNA. This overexpression system has been extensively applied in a series of studies to delineate the function of a particular gene during differentiation2139474849505152. Phosphorylation at three critical serine residues of PKD2 has been reported to solely achieve maximum activation (Ser 706, 710 and 244) and not via gene expression alone4. Consequently, we targeted a constitutively active PKD2 mutant (PKD2 S244/706/710E, PKD2-3SE) that mimics phosphorylation4. The correct gene recombination in the HPRT locus is shown in Fig. 2B. The PKD2 allele was effectively induced upon Dox-exposure as assessed by mRNA and western blot (Fig. 2C,D). Notably, PKD2 overexpression did not affect the expression levels of the SSEA1 protein indicating a negligible role of PKD2 for the pluripotency circuitry (Fig. 2E).

Bottom Line: In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer.Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs.Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, Ulm University, Ulm, Germany.

ABSTRACT
The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

No MeSH data available.


Related in: MedlinePlus