Genomic locus proteomic screening identifies the NF-κB signaling pathway components NFκB1 and IKBKG as transcriptional regulators of Ripk3 in endothelial cells

PLoS One. 2021 Jun 21;16(6):e0253519. doi: 10.1371/journal.pone.0253519. eCollection 2021.

Abstract

The receptor-interacting protein kinase 3 (RIPK3) is a multi-functional protein best known for facilitating cellular necroptosis and inflammation. Recent evidence from our lab indicates that RIPK3 expression must be tightly regulated in endothelial cells to promote angiogenesis, to maintain vascular integrity during embryogenesis, and to provide protection from postnatal atherosclerosis. RIPK3 activity and stability are regulated by post-translational modifications and caspase-dependent cleavage. However, less is known about the transcriptional regulation of Ripk3. Here we utilized an unbiased CRISPR-based technology called genomic locus proteomics (GLoPro) to screen transcription factors and coregulatory proteins associated with the Ripk3 locus in a murine endothelial cell line. We found that 41 nuclear proteins are specifically enriched at the Ripk3 locus, including the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway components NFκB1 and IKBKG. We further verified that NFκB1 and IKBKG directly bind the Ripk3 promoter and prevent TNFα-induced Ripk3 transcription in cultured human primary endothelial cells. Moreover, NFκB1 prevents RIPK3-mediated death of primary endothelial cells. These data provide new insights into NF-κB signaling and Ripk3 transcriptional regulation in endothelial cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Chromatin Immunoprecipitation
  • Endothelial Cells / metabolism*
  • Gene Expression Regulation
  • Gene Knockout Techniques
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mice
  • NF-kappa B / metabolism*
  • NF-kappa B p50 Subunit / genetics
  • NF-kappa B p50 Subunit / metabolism*
  • Proteomics / methods
  • Real-Time Polymerase Chain Reaction
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*
  • Signal Transduction* / genetics
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Intracellular Signaling Peptides and Proteins
  • NEMO protein, mouse
  • NF-kappa B
  • NF-kappa B p50 Subunit
  • Transcription Factors
  • Nfkb1 protein, mouse
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk3 protein, mouse