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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

Expression of PKD isoforms in mouse embryonic stem cells.(A) mRNA levels of PKD 1/2/3 in undifferentiated A2lox mouse ESCs. (B) Western Blot analysis of PKD1/2/3 in A2lox.cre and CgR8 ESCs. MEFs served as positive control. (C) Immunofluorescence staining for PKD1, PKD2 and PKD3 (green) in undifferentiated A2lox mES cells 6 h and 48 h after plating under pluripotency conditions. Nuclei are stained with DAPI (blue). MEFs served as positive control. (D) PKD2 mRNA expression levels during EB differentiation until day 6 of ESCs. (E) Western blot analysis of PKD1 and PKD2 protein levels during differentiation until day6 in differentiating ES cells. (F) Western Blot of phospho-PKD and PKD2 indicating catalytic activity by phosphorylation within the activation loop at two conserved serine residues. (G,H) Relative band intensity of PKD2 (G) and pPKD2 (H) days 0, 2 and 6. Two experiments of three included into the quantification. (I) Immunoprecipitation of phospho-PKD2 (pPKD2) and PKD2 on Days 0 and 6. (J) Western Blot of PKCδ and phospho-PKCδ on Days 0 and 6. qPCRs were performed n = 3 in replicates. Western Blots are representative for three independent experiments. Scale bars 20 μm.
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f1: Expression of PKD isoforms in mouse embryonic stem cells.(A) mRNA levels of PKD 1/2/3 in undifferentiated A2lox mouse ESCs. (B) Western Blot analysis of PKD1/2/3 in A2lox.cre and CgR8 ESCs. MEFs served as positive control. (C) Immunofluorescence staining for PKD1, PKD2 and PKD3 (green) in undifferentiated A2lox mES cells 6 h and 48 h after plating under pluripotency conditions. Nuclei are stained with DAPI (blue). MEFs served as positive control. (D) PKD2 mRNA expression levels during EB differentiation until day 6 of ESCs. (E) Western blot analysis of PKD1 and PKD2 protein levels during differentiation until day6 in differentiating ES cells. (F) Western Blot of phospho-PKD and PKD2 indicating catalytic activity by phosphorylation within the activation loop at two conserved serine residues. (G,H) Relative band intensity of PKD2 (G) and pPKD2 (H) days 0, 2 and 6. Two experiments of three included into the quantification. (I) Immunoprecipitation of phospho-PKD2 (pPKD2) and PKD2 on Days 0 and 6. (J) Western Blot of PKCδ and phospho-PKCδ on Days 0 and 6. qPCRs were performed n = 3 in replicates. Western Blots are representative for three independent experiments. Scale bars 20 μm.

Mentions: PKDs are significantly expressed in the developing mouse embryo and in particular, PKD2 shows a differentially regulated expression pattern12. In embryonic stem cells, PKCs (the upstream activators of PKDs) drive primitive endoderm formation, while their inhibition facilitates pluripotency45. However, the contribution of PKDs during cell fate determination is not clearly understood. Employing murine ESCs as a tool, we sought to characterize the role of PKDs in early stages of differentiation. First, we investigated the mRNA and protein expression of PKD1, PKD2 and PKD3 in undifferentiated mouse ESCs. As shown in Fig. 1A and B, Pkd2 was highly expressed in ESCs, followed by Pkd3. Pkd1 was barely detectable at mRNA and protein level (Fig. 1A,B). Transcriptome data from a public database confirmed our findings (http://biit.cs.ut.ee/fungenes/). Furthermore, the examination of the abundance of PKD1-3 via immunochemical staining (ICC) indicated predominant cytoplasmic localization of PKD2 in both single ESCs and compact ESC colonies. In line with the mRNA data, PKD1 staining was hardly visible, while PKD3 immunoreactivity confirmed an intermediate expression ranking between PKD1 and PKD2 (Fig. 1C). Accordingly, we next examined the expression of PKD2 during ESC differentiation using embryoid body (EB)-based assays. We observed prominent and significant PKD2 expression peaks at day 2 and day 6 of differentiation on mRNA levels (Fig. 1D). Interestingly, protein levels of PKD2 increased continuously until day 6 (Fig. 1E,G). Of note, the PKD2 antibody used in this study specifically detects PKD2 and does not show cross reactivity with either PKD1 or PKD315. Notably, PKD1 showed moderate protein levels starting at day 4 and day 6 with negligible levels on day 0 and day 2 (Fig. 1E). Next we aimed to assess PKD activity. Western blot analysis with a phospho-specific PKD antibody targeting the activation loop of the kinase demonstrated that undifferentiated ESCs exhibited a moderate basal PKD activity that peaked on day 2 and persisted on lower levels through day 6 of differentiation (Fig. 1F,H). Of note, PKD activity on day 2 mostly reflects PKD2 activity as PKD1 is virtually absent at this stage (Fig. 1B,E). PKD2 specific phosphorylation is further confirmed by immunoprecipitation of the PKD2 protein and subsequent immunoblotting with a phospho-specific PKD antibody (Fig. 1I). PKDs are activated by PKCδ and PKCε, two serine threonine kinases belonging to the novel PKC family45. In mouse embryonic stem cells PKCδ has been shown to be functionally expressed46. Indeed, we found phosphorylated PKCδ coincide with PKD2 activity suggesting that in differentiating ESCs, PKCδ could activate PKD2 (Fig. 1J). Together, these data suggest that PKD2 expression and activity may play a role during ESC differentiation. Based on these observations, we decided to focus on PKD2 in further experiments.


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)

Expression of PKD isoforms in mouse embryonic stem cells.(A) mRNA levels of PKD 1/2/3 in undifferentiated A2lox mouse ESCs. (B) Western Blot analysis of PKD1/2/3 in A2lox.cre and CgR8 ESCs. MEFs served as positive control. (C) Immunofluorescence staining for PKD1, PKD2 and PKD3 (green) in undifferentiated A2lox mES cells 6 h and 48 h after plating under pluripotency conditions. Nuclei are stained with DAPI (blue). MEFs served as positive control. (D) PKD2 mRNA expression levels during EB differentiation until day 6 of ESCs. (E) Western blot analysis of PKD1 and PKD2 protein levels during differentiation until day6 in differentiating ES cells. (F) Western Blot of phospho-PKD and PKD2 indicating catalytic activity by phosphorylation within the activation loop at two conserved serine residues. (G,H) Relative band intensity of PKD2 (G) and pPKD2 (H) days 0, 2 and 6. Two experiments of three included into the quantification. (I) Immunoprecipitation of phospho-PKD2 (pPKD2) and PKD2 on Days 0 and 6. (J) Western Blot of PKCδ and phospho-PKCδ on Days 0 and 6. qPCRs were performed n = 3 in replicates. Western Blots are representative for three independent experiments. Scale bars 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493579&req=5

f1: Expression of PKD isoforms in mouse embryonic stem cells.(A) mRNA levels of PKD 1/2/3 in undifferentiated A2lox mouse ESCs. (B) Western Blot analysis of PKD1/2/3 in A2lox.cre and CgR8 ESCs. MEFs served as positive control. (C) Immunofluorescence staining for PKD1, PKD2 and PKD3 (green) in undifferentiated A2lox mES cells 6 h and 48 h after plating under pluripotency conditions. Nuclei are stained with DAPI (blue). MEFs served as positive control. (D) PKD2 mRNA expression levels during EB differentiation until day 6 of ESCs. (E) Western blot analysis of PKD1 and PKD2 protein levels during differentiation until day6 in differentiating ES cells. (F) Western Blot of phospho-PKD and PKD2 indicating catalytic activity by phosphorylation within the activation loop at two conserved serine residues. (G,H) Relative band intensity of PKD2 (G) and pPKD2 (H) days 0, 2 and 6. Two experiments of three included into the quantification. (I) Immunoprecipitation of phospho-PKD2 (pPKD2) and PKD2 on Days 0 and 6. (J) Western Blot of PKCδ and phospho-PKCδ on Days 0 and 6. qPCRs were performed n = 3 in replicates. Western Blots are representative for three independent experiments. Scale bars 20 μm.
Mentions: PKDs are significantly expressed in the developing mouse embryo and in particular, PKD2 shows a differentially regulated expression pattern12. In embryonic stem cells, PKCs (the upstream activators of PKDs) drive primitive endoderm formation, while their inhibition facilitates pluripotency45. However, the contribution of PKDs during cell fate determination is not clearly understood. Employing murine ESCs as a tool, we sought to characterize the role of PKDs in early stages of differentiation. First, we investigated the mRNA and protein expression of PKD1, PKD2 and PKD3 in undifferentiated mouse ESCs. As shown in Fig. 1A and B, Pkd2 was highly expressed in ESCs, followed by Pkd3. Pkd1 was barely detectable at mRNA and protein level (Fig. 1A,B). Transcriptome data from a public database confirmed our findings (http://biit.cs.ut.ee/fungenes/). Furthermore, the examination of the abundance of PKD1-3 via immunochemical staining (ICC) indicated predominant cytoplasmic localization of PKD2 in both single ESCs and compact ESC colonies. In line with the mRNA data, PKD1 staining was hardly visible, while PKD3 immunoreactivity confirmed an intermediate expression ranking between PKD1 and PKD2 (Fig. 1C). Accordingly, we next examined the expression of PKD2 during ESC differentiation using embryoid body (EB)-based assays. We observed prominent and significant PKD2 expression peaks at day 2 and day 6 of differentiation on mRNA levels (Fig. 1D). Interestingly, protein levels of PKD2 increased continuously until day 6 (Fig. 1E,G). Of note, the PKD2 antibody used in this study specifically detects PKD2 and does not show cross reactivity with either PKD1 or PKD315. Notably, PKD1 showed moderate protein levels starting at day 4 and day 6 with negligible levels on day 0 and day 2 (Fig. 1E). Next we aimed to assess PKD activity. Western blot analysis with a phospho-specific PKD antibody targeting the activation loop of the kinase demonstrated that undifferentiated ESCs exhibited a moderate basal PKD activity that peaked on day 2 and persisted on lower levels through day 6 of differentiation (Fig. 1F,H). Of note, PKD activity on day 2 mostly reflects PKD2 activity as PKD1 is virtually absent at this stage (Fig. 1B,E). PKD2 specific phosphorylation is further confirmed by immunoprecipitation of the PKD2 protein and subsequent immunoblotting with a phospho-specific PKD antibody (Fig. 1I). PKDs are activated by PKCδ and PKCε, two serine threonine kinases belonging to the novel PKC family45. In mouse embryonic stem cells PKCδ has been shown to be functionally expressed46. Indeed, we found phosphorylated PKCδ coincide with PKD2 activity suggesting that in differentiating ESCs, PKCδ could activate PKD2 (Fig. 1J). Together, these data suggest that PKD2 expression and activity may play a role during ESC differentiation. Based on these observations, we decided to focus on PKD2 in further experiments.

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