Tumor necrosis factor-alpha-inducible IkappaBalpha proteolysis mediated by cytosolic m-calpain. A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-kappab 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-kappaB (NF-kappaB) transcription factor. NF-kappaB activation results from sequential phosphorylation and hydrolysis of the cytoplasmic inhibitor, IkappaBalpha, through the 26 S proteasome. Here, we show a parallel proteasome-independent pathway for cytokine-inducible IkappaBalpha 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 IkappaBalpha proteolysis; pretreatment with both is required to completely block IkappaBalpha proteolysis. Similarly, in transient cotransfection assays, expression of the specific inhibitor, calpastatin, partially blocks TNF-alpha-inducible NF-kappaB-dependent promoter activity and IkappaBalpha 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 IkappaBalpha compartment. Addition of catalytically active m-calpain into broken cells was sufficient to produce ligand-independent IkappaBalpha proteolysis and NF-kappaB translocation. As additional evidence for calpain-dependent IkappaBalpha proteolysis and NF-kappaB 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, IkappaBalpha proteolysis occurs in a manner sensitive only to calpain inhibitors. Our results demonstrate that TNF-alpha activates cytosolic calpains, a parallel pathway that degrades IkappaBalpha and activates NF-kappaB activation independently of the ubiquitin-proteasome pathway.
Han
Y
Y
Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1060, USA.
Weinman
S
S
Boldogh
I
I
Walker
R K
RK
Brasier
A R
AR
D001483:Base Sequence; D001692:Biological Transport; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex