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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

Effects of PKD2 over expression after day 4 of EB development.(A) Scheme illustrating treatment regimen of iPKD2 ES cells. (B,C) mRNA levels of late cardiac markers Myh6 and Myl2a. (D) Immunostaining of α-actinin at day 14 of differentiation. (E–G) mRNA levels of vascular markers CD31, CD34 and von Willebrand factor (vWF). (H) Immunostaining of the vascular protein CD31 in Dox− an Dox 4–14 conditions. Time points and treatment regimen as indicated in the figure. All experiments were performed n = 3 in replicates. Scale bars 20 μm. Significances were calculated using R. Raw p values were adjusted using Bonferroni correction (§p < 0.05; §§p < 0.01; §§§p < 0.001). Adjusted p-values are listed in Suppl. Table 2.
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f4: Effects of PKD2 over expression after day 4 of EB development.(A) Scheme illustrating treatment regimen of iPKD2 ES cells. (B,C) mRNA levels of late cardiac markers Myh6 and Myl2a. (D) Immunostaining of α-actinin at day 14 of differentiation. (E–G) mRNA levels of vascular markers CD31, CD34 and von Willebrand factor (vWF). (H) Immunostaining of the vascular protein CD31 in Dox− an Dox 4–14 conditions. Time points and treatment regimen as indicated in the figure. All experiments were performed n = 3 in replicates. Scale bars 20 μm. Significances were calculated using R. Raw p values were adjusted using Bonferroni correction (§p < 0.05; §§p < 0.01; §§§p < 0.001). Adjusted p-values are listed in Suppl. Table 2.

Mentions: The primary vascular plexus in the EB is remodelled by vessel sprouting starting at day 6 and later, thus marking angiogenesis54. Given our primary aim to define a PKD2-responsive time-window during vascular development, we triggered the overexpression of PKD2 via Dox-exposure starting at day 4 of differentiation and maintained doxycycline in the culture medium until day 14 of differentiation (day 4 to day 14). Continuous Dox-treatment (day 0 to day 14) was administered to distinguish between an early and late PKD2 responsive window (Fig. 4A). Given the close association between the developmental course of the vascular and cardiac lineage, we next sought to investigate the cardiac differentiation potential. Quantification of beating clusters in differentiating EBs showed no significant difference in cardiac differentiation (data not shown). This observation was further mirrored by the Myh6, Myl2a gene expression analyses at days 6, 9 and 14 of EB differentiation (Fig. 4B,C) and immunostaining of α-actinin (Fig. 4D). Consistent with an early inhibitory effect of PKD2, when activated from day 0 to day 4, continuous activation led to reduced expression of cardiac markers (Fig. 4B,C). Next, we assessed the development of our primary lineage of interest, namely endothelial cell differentiation. qPCR analysis revealed a significant upregulation of vascular marker genes (vWF, CD34, CD31) in Dox-treated compared to untreated cultures particularly at days 6, 9 and also 14 though less pronounced (Fig. 4E–G). These findings were corroborated with immunostaining for CD31 (Fig. 4H). Of note, Tubb3 expression was reduced upon later PKD2 induction (data not shown).


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)

Effects of PKD2 over expression after day 4 of EB development.(A) Scheme illustrating treatment regimen of iPKD2 ES cells. (B,C) mRNA levels of late cardiac markers Myh6 and Myl2a. (D) Immunostaining of α-actinin at day 14 of differentiation. (E–G) mRNA levels of vascular markers CD31, CD34 and von Willebrand factor (vWF). (H) Immunostaining of the vascular protein CD31 in Dox− an Dox 4–14 conditions. Time points and treatment regimen as indicated in the figure. All experiments were performed n = 3 in replicates. Scale bars 20 μm. Significances were calculated using R. Raw p values were adjusted using Bonferroni correction (§p < 0.05; §§p < 0.01; §§§p < 0.001). Adjusted p-values are listed in Suppl. Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Effects of PKD2 over expression after day 4 of EB development.(A) Scheme illustrating treatment regimen of iPKD2 ES cells. (B,C) mRNA levels of late cardiac markers Myh6 and Myl2a. (D) Immunostaining of α-actinin at day 14 of differentiation. (E–G) mRNA levels of vascular markers CD31, CD34 and von Willebrand factor (vWF). (H) Immunostaining of the vascular protein CD31 in Dox− an Dox 4–14 conditions. Time points and treatment regimen as indicated in the figure. All experiments were performed n = 3 in replicates. Scale bars 20 μm. Significances were calculated using R. Raw p values were adjusted using Bonferroni correction (§p < 0.05; §§p < 0.01; §§§p < 0.001). Adjusted p-values are listed in Suppl. Table 2.
Mentions: The primary vascular plexus in the EB is remodelled by vessel sprouting starting at day 6 and later, thus marking angiogenesis54. Given our primary aim to define a PKD2-responsive time-window during vascular development, we triggered the overexpression of PKD2 via Dox-exposure starting at day 4 of differentiation and maintained doxycycline in the culture medium until day 14 of differentiation (day 4 to day 14). Continuous Dox-treatment (day 0 to day 14) was administered to distinguish between an early and late PKD2 responsive window (Fig. 4A). Given the close association between the developmental course of the vascular and cardiac lineage, we next sought to investigate the cardiac differentiation potential. Quantification of beating clusters in differentiating EBs showed no significant difference in cardiac differentiation (data not shown). This observation was further mirrored by the Myh6, Myl2a gene expression analyses at days 6, 9 and 14 of EB differentiation (Fig. 4B,C) and immunostaining of α-actinin (Fig. 4D). Consistent with an early inhibitory effect of PKD2, when activated from day 0 to day 4, continuous activation led to reduced expression of cardiac markers (Fig. 4B,C). Next, we assessed the development of our primary lineage of interest, namely endothelial cell differentiation. qPCR analysis revealed a significant upregulation of vascular marker genes (vWF, CD34, CD31) in Dox-treated compared to untreated cultures particularly at days 6, 9 and also 14 though less pronounced (Fig. 4E–G). These findings were corroborated with immunostaining for CD31 (Fig. 4H). Of note, Tubb3 expression was reduced upon later PKD2 induction (data not shown).

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