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

Martin Müller,Clair Weidgang,Jana Schröer,Ralf Köhntop,Thomas Seufferlein,Ninel Azoitei,Qiong Lin,Wendy Bergmann,Felicitas Genze,Tim Eiseler,Martin Zenke,Stefan Liebau,Ivan G Costa,Alexander Kleger

doi:10.1038/srep11742

Martin Müller, Clair Weidgang + Show 12 more

Open Access

https://doi.org/10.1038/srep11742

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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.

Highlights

The protein kinase D (PKD) family belongs to the calcium-/calmodulin-dependent protein kinase superfamily[1] and comprises the three evolutionary conserved isoforms, PKD1, − 2 and − 32
PKD1 kinase-dead knock-in mice die in utero around day E9.5 while mice featuring a PKD2 deletion are viable and fertile[40,41]
A detailed phenotypic analysis of this early lethality in PKD1 null remains elusive but primitive haematopoiesis and vasculogenesis start around this time period

Summary

Introduction

The protein kinase D (PKD) family belongs to the calcium-/calmodulin-dependent protein kinase superfamily[1] and comprises the three evolutionary conserved isoforms, PKD1, − 2 and − 32. PKDs regulate protein transport by facilitating the fission of budding vesicles from the trans-Golgi network[6,7,8,9,10] Despite their broad expression in the early embryo, the role of PKD isoforms during development and cell fate choice is largely elusive[11,12]. A recent study identified PKD1 as an anti-differentiate, pro-proliferate signal in the skin tissue[18] This observation is limited to physiological skin formation and to cancer initiation. Other recent studies from our laboratory described PKD1 and − 2 isoform-selective effects on cancer cell invasion and angiogenesis[17,31,32]. Given the high corroboration between embryonic development (in vivo) and pluripotent stem cell differentiation (in vitro), various factors can be investigated in a time- and stage-dependent manner regarding their impact on cell fate determination, differentiation and/or tissue formation[21,38,39,43]

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