HERC5, a HECT E3 ubiquitin ligase tightly regulated in LPS activated endothelial cells

Renate Kroismayr, Ulrike Baranyi, Christian Stehlik, Andrea Dorfleutner, Bernd R. Binder*, Joachim Lipp

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

By differential screening we isolated genes upregulated in inflammatory cytokine-stimulated human skin microvascular endothelial cells. One of these cDNAs encoded RCC1 (regulator of chromosome condensation 1)-like repeats and a HECT (homologous to E6-AP C-terminus) domain, representing a member of the HERC (HECT and RCC1 domain protein) family of ubiquitin ligases. The mRNA level of this member, HERC5, is specifically upregulated in endothelial cells by the proinflammatory cytokines tumor necrosis factor α and interleukin 1β, and by lipopolysaccharide (LPS), but is hardly expressed in other cells of the vascular wall such as primary smooth muscle cells and fibroblasts. Regulation of HERC5 gene expression suggests a critical role for the transcription factor NF-κB. In contrast to mRNA expression HERC5 protein is subject of enhanced degradation upon LPS stimulation of endothelial cells. The time course of LPS-induced changes in HERC5 protein and mRNA levels suggests that the initial drop in HERC5 protein is balanced by increased protein synthesis due to upregulation of HERC5 mRNA. This leads to recovery of HERC5 protein levels within 12 hours of LPS stimulation and points at a tight control of HERC5 protein. To analyze functional activity of this putative member of the ubiquitin-conjugating pathway we performed in vitro assays with different ubiquitin-conjugating enzymes. We found that HERC5 possesses ubiquitin ligase activity and requires the presence of the ubiquitin-conjugating enzyme UbcH5a for its activity. These data show for the first time that a functionally active HECT ubiquitin ligase exhibits a tightly controlled cytosolic level under inflammatory conditions in endothelial cells.

Original languageEnglish (US)
Pages (from-to)4749-4756
Number of pages8
JournalJournal of cell science
Volume117
Issue number20
DOIs
StatePublished - Sep 15 2004

Keywords

  • Endothelial cells
  • Inflammation
  • LPS
  • NF-κ
  • Ubiquitin ligase

ASJC Scopus subject areas

  • Cell Biology

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