Abstract 46: RSK4 Is a Novel Negative Regulator of Endothelial Differentiation via Direct Suppression of Protein Kinase A Activity

Endothelial cells (ECs) differentiation is strictly regulated to generate functional blood vessels. Previously, we demonstrated that Flk1+ cells derived from embryonic stem (ES) cells serve as vascular progenitors and can constructively reproduce the early differentiation of both ECs and Mural cells (MCs; vascular smooth muscle cells and pericytes). We recently reported that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced EC differentiation from Flk1+ vascular progenitors via induction of vascular endothelial growth factor (VEGF) receptors expression. To identify a novel molecule involved in EC differentiation, we performed microarray analyses at various differentiation stages from ES cells to vascular cells; undifferentiated ES cells, Flk1+ vascular progenitors, ECs and MCs. Then, we found that mRNA of p90 ribosomal protein S6 kinase 4 (RSK4) was highly expressed in Flk1+ vascular progenitors and almost diminished during EC differentiation, whereas it remained being expressed in MCs. The addition of a broad-spectrum RSK inhibitor, SL0101, to Flk1+ vascular progenitors enhanced VEGF receptor expression and EC differentiation specifically in the presence of the simultaneous cAMP signal activation. SL0101-elicited EC differentiation was abolished by PKA catalytic subunit alpha (PKAc) knockdown. Specific RSK4 knockdown in Flk1+ vascular progenitors reproduced the effects of SL0101 on EC differentiation via increasing Flk1 expression. RSK4 had potential to bind with PKAc and inhibited PKA activity. Furthermore, an ex vivo whole embryo culture assay showed that SL0101 treatment drastically resulted in increased CD31+ vasculature area, enlarged vessel diameter and defected vascular remodeling in yolk sac. Taken together, these results indicate that RSK4 acts as a novel negative regulator for EC differentiation via inhibition of PKA function. Fine tuning of PKA and Flk1 signaling by RSK4 is critical to normal vascular development. Elucidation of the new-mode cell fate determination by RSK4 would provide novel insights in developmental biology, stem cell biology, and regenerative medicine.