ASB4 Is a Hydroxylation Substrate of FIH and Promotes Vascular Differentiation via an Oxygen-Dependent Mechanism

Abstract

The molecular mechanisms of endothelial differentiation into a functional vascular network are incompletely understood. To identify novel factors in endothelial development, we used a microarray screen with differentiating embryonic stem (ES) cells that identified the gene for 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 is 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). 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 endothelium-specific response to increasing oxygen tension. Consistent with the hypothesis that ASB4 function is regulated by oxygen concentration, ASB4 interacts with the factor inhibiting HIF1α (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 vascular lineage in an oxygen-dependent manner. We postulate that hydroxylation of ASB4 in normoxia promotes binding to and degradation of substrate protein(s) to modulate vascular differentiation.

SUPPLEMENTAL MATERIAL

Thanks go to W. Alexander for ASB1/ASB4 pEF1 constructs; Y. Xiong for Cul2, Cul5, and Roc1 antibodies; C. Scarlett for mass spectrometric expertise; B. Temple for structural modeling; and M. Willis, R. Kelley, J. Schisler, and P. Charles for manuscript critiques.

C.P. is an Established Investigator of the American Heart Association and a Burroughs Wellcome Fund Clinician Scientist in Translational Research. This work was supported by American Heart Association predoctoral fellowship 0515350U to J.F. and National Institutes of Health grants HL 61656, HL 03658, and HL 072347 to C.P.