Dataset Information

ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning

ABSTRACT: Precise vascular patterning is critical for normal growth and development. The ERG transcription factor drives Delta like ligand 4 (DLL4)/Notch signalling and is thought to act as pivotal regulators of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC specific Focal Adhesion Kinase (FAK)-knockout (KO) and point-mutant FAK-knockin mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins identified that endothelial nuclear-FAK interacts with the de-ubiquitinase USP9x and the ubiquitin ligase TRIM25 enzymes. Further in silico analysis corroborates that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell Of Umbilical Vein

SUBMITTER: Alex von kriegsheim

LAB HEAD: Alex von Kriegsheim

PROVIDER: PXD033447 | Pride | 2022-07-05

REPOSITORIES: Pride

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ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning.

D'Amico Gabriela G Fernandez Isabelle I Gómez-Escudero Jesús J Kim Hyojin H Maniati Eleni E Azman Muhammad Syahmi MS Mardakheh Faraz K FK Serrels Bryan B Serrels Alan A Parsons Maddy M Squire Anthony A Birdsey Graeme M GM Randi Anna M AM Bolado-Carrancio Alfonso A Gangeswaran Rathi R Reynolds Louise E LE Bodrug Natalia N Wang Yaohe Y Wang Jun J Meier Pascal P Hodivala-Dilke Kairbaan M KM

Development (Cambridge, England) 20220712 13

Precise vascular patterning is crucial for normal growth and development. The ERG transcription factor drives Delta-like ligand 4 (DLL4)/Notch signalling and is thought to act as a pivotal regulator of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC-specific focal adhesion kinase (FAK)-knockout (KO) and point-mutant FAK-knock-in mice, we show that loss of ECFAK, its kinase activity or phosphorylati ...[more]

PMID: 35723257

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Project description:Background: Endothelial cells (EC) sit at the forefront of dramatic physiologic changes occurring in the pulmonary circulation during late embryonic and early postnatal life. First, as the lung moves from the hypoxic fetal environment to oxygen-rich postnatal environment, marked changes in pulmonary EC structure and function facilitate a marked increase in blood flow from the placenta to the lungs. Subsequently, pulmonary angiogenesis expands the microvasculature to drive exponential distal lung growth during early postnatal life. Yet, how these marked physiologic changes alter distinct EC subtypes to facilitate the transition of the pulmonary circulation and regulate vascular growth and remodeling remains incompletely understood. Methods: In this report, we employed single cell RNA-transcriptomics and in situ RNA imaging to profile pulmonary EC in the developing mouse lung from just before birth through this period of rapid postnatal growth. Results: Multiple, transcriptionally distinct macro- and microvascular EC were identified in the late embryonic and early postnatal lung, with gene expression profiles distinct from their adult EC counterparts. A novel arterial subtype, unique to the developing lung localized to the distal parenchyma and expressed genes that regulate vascular growth and patterning. Birth particularly heightened microvascular diversity, inducing dramatic shifts in the transcriptome of distinct microvascular subtypes in pathways related to proliferation, migration and antigen presentation. Two distinct waves of EC proliferation were identified, including one just prior to birth, and a second during early alveolarization, a time of exponential pulmonary angiogenesis. Chronic hyperoxia, an injury that impairs parenchymal and vascular growth, induced a common gene signature among all pulmonary EC, unique alterations to distinct microvascular EC subtypes, and disrupted EC-EC and EC-immune cell cross talk. Conclusions: Taken together, these data reveal tremendous diversity of pulmonary EC during a critical window of postnatal vascular growth, and provide a detailed molecular map that can be used to inform both normal vascular development and alterations in EC diversity upon injury. These data have important implications for lung diseases marked by dysregulated angiogenesis and pathologic pulmonary vascular remodeling.

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