The molecular mechanisms of endothelial differentiation into a functional vascular network are incompletely understood. To identify novel factors in endothelial development we employed a microarray screen using differentiating embryonic stem (ES) cells that identified the previously uncharacterized Ankyrin Repeat and SOCS Box Protein 4 (ASB4) as the most highly differentially expressed gene in the vascular lineage during early differentiation. Like other SOCS box-containing proteins, ASB4 acts as the substrate recognition molecule of an Elongin-B/Elongin-C/Cullin/Roc ubiquitin ligase complex that mediates the ubiquitination and degradation of substrate protein(s). We show here that high levels of ASB4 expression in the embryonic vasculature coincide with drastic increases in oxygen tension as placental blood flow is initiated. However, as vessels mature and oxygen levels stabilize, ASB4 expression is quickly downregulated, suggesting that ASB4 may function to modulate an endothelial-specific response to increasing oxygen tension. Consistent with the hypothesis that ASB4 function is regulated by oxygen concentration, ASB4 directly interacts with Factor Inhibiting HIF1-alpha (FIH), and is a substrate for FIH-mediated hydroxylation via an oxygen-dependent mechanism. Additionally, overexpression of ASB4 in ES cells promotes differentiation into the endothelial lineage in an oxygen-dependent manner. We propose that hydroxylation of ASB4 in normoxia promotes binding to and degradation of substrate protein(s) to modulate vascular differentiation.
Ankyrin Repeat and SOCS Box Protein 2