Tumor necrosis factor, α-inducible IκBα proteolysis mediated by cytosolic M-calpain: A mechanism parallel to tile ubiquitin-proteasome pathway for nuclear factor-κB activation

Youqi Han, Steven Weinman, Istvan Boldogh, Randall K. Walker, Allan R. Brasier

Research output: Contribution to journalArticle

207 Scopus citations

Abstract

The cytokine tumor necrosis factor α (TNF-α) induces expression of inflammatory gene networks by activating cytoplasmic to nuclear translocation of the nuclear factor-κB (NF-κB) transcription factor. NF-κB activation results from sequential phosphorylation and hydrolysis of the cytoplasmic inhibitor, IκBα, through the 26 S proteasome. Here, we show a parallel proteasome-independent pathway for cytokine-inducible IκBα proteolysis in HepG2 liver cells mediated by cytosolic calcium-activated neutral protease (calpains). Pretreatment with either calpain- or proteasome-selective inhibitors partially blocks up to 50% of TNF-α-inducible IκBα proteolysis; pretreatment with both is required to completely block IκBα proteolysis. Similarly, in transient cotransfection assays, expression of the specific inhibitor, calpastatin, partially blocks TNF-α-inducible NF-κB-dependent promoter activity and IκBα proteolysis. In TNF-α-stimulated cells, a rapid (within 1 min), 2.2-fold increase in cytosolic calpain proteolytic activity is measured using a specific fluorescent assay. Inducible calpain proteolytic activity occurs coincidentally with the particulate-to-cytosol redistribution of the catalytic m-calpain subunit into the Iκbα compartment. Addition of catalytically active m-calpain into broken cells was sufficient to produce ligand-independent IκBα proteolysis and NF-κB translocation. As additional evidence for calpain-dependent IκBα proteolysis and NF-κB activation, we demonstrate that this process occurs in a cell line (ts20b) deficient in the ubiquitin-proteasome pathway. Following inactivation of the temperature- sensitive ubiquitin-activating enzyme, IκBα proteolysis occurs in a manner sensitive only to calpain inhibitors. Our results demonstrate that TNF-α activates cytosolic calpains, a parallel pathway that degrades IκBα and activates NF-κB activation independently of the ubiquitin-proteasome pathway.

Original languageEnglish (US)
Pages (from-to)787-794
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number2
DOIs
StatePublished - Jan 8 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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