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

The cytokine tumor necrosis factor alpha (TNF-alpha) induces expression of inflammatory gene networks by activating cytoplasmic to nuclear translocation of the nuclear factor-kappa B (NF-kappa B) transcription factor. NF-kappa B activation results from sequential phosphorylation and hydrolysis of the cytoplasmic inhibitor, I kappa B alpha, through the 26 S proteasome, Here, we show a parallel proteasome-independent pathway for cytokine-inducible I kappa B alpha 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-alpha-inducible I kappa B alpha proteolysis; pretreatment with both is required to completely block I kappa B alpha proteolysis. Similarly, in transient cotransfection assays, expression of the specific inhibitor, calpastatin, partially blocks TNF-alpha-inducible NF-kappa B-dependent promoter activity and I kappa B alpha proteolysis. In TNF-alpha-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 I kappa Ba alpha compartment, Addition of catalytically active m-calpain into broken cells was sufficient to produce ligand-independent I kappa B alpha proteolysis and NF-kappa B translocation. As additional evidence for calpain-dependent I kappa B alpha proteolysis and NF-kappa B 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, I kappa B alpha proteolysis occurs in a manner sensitive only to calpain inhibitors. Our results demonstrate that TNF-alpha activates cytosolic calpains, a parallel pathway that degrades I kappa B alpha and activates NF-kappa B activation independently of the ubiquitin-proteasome pathway.