Tumor Necrosis Factor-α-inducible IκBα Proteolysis Mediated by Cytosolic m-Calpain

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. 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. Nonlysosomal (cytoplasmic) protease systems have recently been identified as important regulators of intracellular activities including programmed cell death, protein kinase abundance, and cell-cycle progression (1Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (782) Google Scholar, 2Pahl H. Baeuerle P.A. Curr. Opin. Cell Biol. 1996; 8: 340-347Crossref PubMed Scopus (140) Google Scholar, 3Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar). In viable cells, two prominent cytoplasmic protease systems have been identified. These include the ubiquitin-proteasome pathway, mediating targeted turnover of misfolded and unstable proteins, and the calcium-activated neutral protease (calpain)-calpastatin system, initially thought to be important in regulating turnover of protein kinases and key structural proteins in the cell (1Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (782) Google Scholar). More recently, however, inducible proteolysis has also been shown to be important in hormonal control of gene expression by modulating the nuclear abundance of certain transcription factors. These processes include cholesterol-induced cleavage of the sterol-regulated element binding protein (reviewed in Ref. 2Pahl H. Baeuerle P.A. Curr. Opin. Cell Biol. 1996; 8: 340-347Crossref PubMed Scopus (140) Google Scholar) and, of special interest to the pathophysiology of inflammatory processes, mechanisms for intracellular signaling produced by the cytokine tumor necrosis factor-α (TNF-α). 1The abbreviations used are: TNF-α, tumor necrosis factor-α; AMC, 7-amino-4-methylcoumarin; E64, trans-epoxysuccinyl-l-leucylamido-(4-guanido)butane; IκBα, inhibitor of NF-κB; NF-κB, nuclear factor-κB; PMSF, phenylmethylsulfonyl fluoride; Ubn, polyubiquitination; Z, benzyloxycarbonyl; Z-LLF, benzyloxycarbonyl-Leu-Leu-phenylalaninal; Z-LLL, Z-Leu-Leu-Leucinal (Z-LLL); Z-LnL, Z-Leu-norleucinal (calpeptin); Z-LLY, Z-l-leucyl-l-leucyl-l-tyrosine diazomethyl ketone; IL, interleukin; CAT, chloramphenicol acetyltransferase; Boc, t-butoxycarbonyl; CMAC, 7-amino-4-chloromethylcoumarin; FACS, fluorescence-activated cell sorter; Lacta, lactacystin; E1, ubiquitin-activating enzyme; Suc, succinyl. Following binding its receptor on the plasma membrane, TNF-α initiates de novo transcription of genetic networks, in part, through activating nuclear translocation of the cytoplasmic transcription factor nuclear factor-κB (NF-κB) (4Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar, 5Verma I.M. Stevenson J.K. Schwartz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1665) Google Scholar). NF-κB, a multiprotein complex inactivated in the cytoplasm by association with its IκB inhibitor, translocates into the nucleus following dissociation of the NF-κB·IκB complex. TNF-α modifies NF-κB·IκB association through a process initiated by inducible IκBα serine phosphorylation on its amino-terminal regulatory domain, a modification coupled to IκB polyubiquitination (Ubn) on adjacent lysine residues (6DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). NF-κB·IκB dissociation requires IκB proteolysis because phosphorylated and ubiquitinated IκB still inactivates NF-κB (Ref. 6DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar and references therein). Presently, the ubiquitin-proteasome system has been the only pathway identified in mediating cytokine-inducible IκB proteolysis. Pretreatment with cell-permeant proteasome inhibitors blocks TNF-α-inducible IκB proteolysis concomitantly with the accumulation of Ubn- and phosphorylated IκB intermediates (6DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar, 8Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar). Independently, inducible IκBα proteolysis in cells harboring thermolabile ubiquitin-activating enzymes is markedly slowed at non-permissive temperatures (9Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben-Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (395) Google Scholar). Several lines of evidence indicate the presence of alternative (nonproteasome-dependent) processing pathways for IκB proteolysis. First, in pre-B lymphocytes, c-Rel:NF-κB1 is constitutively nuclear as the consequence of a calcium-dependent proteolytic activity that preferentially affects IκBα (rather than IκBβ (10Miyamoto S. Seufzer B.J. Shumway S.D. Mol. Cell. Biol. 1998; 18: 19-29Crossref PubMed Google Scholar)). Second, we have observed a non-proteasome-dependent pathway mediating inducible IκBα proteolysis (and NF-κB activation) following respiratory syncytial virus infection of human airway epithelial cells (11Jamaluddin M. Casola A. Garofalo R.P. Han Y. Elliott T. Ogra P.L. Brasier A.R. J. Virol. 1998; 72: 4849-4857Crossref PubMed Google Scholar). However, whether additional nonproteasome-dependent pathways participate in cytokine-inducible NF-κB activation have not been explored. These studies prompted us to examine whether nonproteasome-dependent pathways participate in cytokine-inducible IκBα degradation. Here we investigate the proteolytic mechanism involved in a well characterized model of NF-κB activation in TNF-α-stimulated HepG2 hepatocytes, where NF-κB activation mediates the expression of acute phase reactants (12Han Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 13Brasier A.R. Ron D. Tate J.E. Habener J.F. EMBO J. 1990; 9: 3933-3944Crossref PubMed Scopus (130) Google Scholar). By using calpain and proteasome-selective inhibitors, we demonstrate that inducible IκBα proteolysis is partially blocked following inhibition of either pathway and completely blocked following inhibition of both. By using a specific fluorescent assay in intact cells, we describe for the first time that TNF-α rapidly activates cytosolic calpain proteolytic activity. In subcellular fractionations of TNF-α-stimulated cells, the catalytic m-calpain subunit translocates from the particulate into the cytosolic fraction (the latter containing IκBα) coincidentally with IκBα proteolysis. Moreover, TNF-α-inducible IκBα proteolysis occurs in cells conditionally deficient in the ubiquitin-proteasome pathway, and in cells expressing IκBα mutations deficient in proteasome-dependent processing. Together, these data implicate calpains are a parallel pathway in mediating IκBα proteolysis and NF-κB activation. Purified phosphorylated bovine casein (sodium salt) and rabbit skeletal muscle m-calpain (specific activity 30 units/mg protein, >90% 80- and 30-kDa subunits by SDS-polyacrylamide gel electrophoresis) from and was a of J. was a of Z-Leu-norleucinal and phenylmethylsulfonyl from diazomethyl was from The temperature-sensitive cell and its with enzyme, was from containing to of the human promoter expression of was produced by the of the gene into the of a and the used to the promoter (11Jamaluddin M. Casola A. Garofalo R.P. Han Y. Elliott T. Ogra P.L. Brasier A.R. J. Virol. 1998; 72: 4849-4857Crossref PubMed Google Scholar). by and to to HepG2 cells and with 30 human TNF-α as (12Han Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). protease inhibitor cells for 1 with Z-LLF, Z-LLL, E64, PMSF, or as HepG2 cells into with of human of and of expression with TNF-α for to assay. cells following cotransfection with of the in by and on to rabbit as T. M. PubMed Scopus Google Scholar). In using cells and cells from to for a to the temperature-sensitive ubiquitin-activating Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). with 30 TNF-α at cells as protease inhibitor cells for 1 at or with Z-LnL, E64, Z-LLY, Lacta, and to TNF-α used to IκBα and IκBβ from and m-calpain rabbit to α and to the human proteasome subunit and from of cytosolic or nuclear by SDS-polyacrylamide gel and to (12Han Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Following with in the assay following the proteasome of HepG2 from control or with in 1 of C. K. D. Goldberg A.L. Cell. 1994; 78: Full Text PDF PubMed Scopus Google Scholar) in the presence or of protease inhibitor 30 was by at to are S.D. of activity was on proteasome HepG2 was by 26 or to the proteasome and and in the in the and in the By the m-calpain catalytic subunit in the of the was in the that proteasome activity was measured by this assay. of m-calpain human m-calpain was with phosphorylated bovine casein in the presence or of inhibitors at 30 1 was by assay M. PubMed Scopus Google Scholar). results with rabbit skeletal muscle m-calpain HepG2 cells, calpain activity was measured by the of of the fluorescent AMC, from intracellular J. Biol. Chem. Full Text PDF PubMed Google Scholar). on with at and with a system M. S. M. C. J. M. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). was into the and of cells was measured at TNF-α was to the with The of the with to time the intracellular calpain activity J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the was for the of fluorescent as shown by the of cell increase to with that produced by requires only the not for was a increase in the with that hydrolysis and not was calpain in cell of HepG2 cells with and in intracellular was measured to and TNF-α at using a of was measured using a and a of protein was with of human IκBα for in at in a of IκBα was by of rabbit skeletal muscle m-calpain was to a of of HepG2 and 1 (12Han Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) in IκB proteolysis in cytoplasmic and nuclear was by following of (12Han Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). assay was using the from the promoter as (12Han Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). have shown that of TNF-α to HepG2 cells induces rapid IκB and proteolysis and NF-κB following of (12Han Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). examine initially proteasome-independent pathways for IκB proteolysis IκB abundance was by in protease inhibitor cells TNF-α of characterized proteasome Z-LLF, V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar, C. K. D. Goldberg A.L. Cell. 1994; 78: Full Text PDF PubMed Scopus Google Scholar, G. S. 1995; PubMed Scopus Google calpain T. J. M. T. T. PubMed Scopus Google and serine protease inhibitors used In data not pretreatment with these on of either IκB In the of protease inhibitors, TNF-α produced rapid proteolysis of both IκBα and IκBβ 1 Pretreatment with Z-LLF, or blocked IκBβ processing with Z-LnL, E64, and These are with a of the proteasome pathway mediating IκBβ proteolysis (6DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). In IκBα proteolysis was partially blocked by the proteasome inhibitors and 1 and the proteasome-selective inhibitors, only was a completely inhibitor of IκBα proteolysis. the calpain inhibitors and also partially blocked IκBα where IκBβ proteolysis was and These data a parallel of in TNF-α-inducible IκBα the of proteasome-independent pathways mediating IκBα IκBα in cells was in cells containing with that in cells proteasome activity 1 In cells not with protease inhibitors, IκBα proteolysis is rapid of min), coincidentally with the of phosphorylated IκBα intermediates 1 In cells with the proteasome inhibitor IκBα proteolysis occurs with a of and is with the of a 1 whether pathway than the for IκBα the of the specific calpain inhibitor M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and 1 completely IκBα proteolysis. In the presence of both inhibitor IκBα proteolysis was completely blocked with accumulation of and phosphorylated IκBα intermediates that was at the time in the presence of proteasome inhibitors not in calpain inhibitors activity of calpains are by the of calpain inhibitor, As additional evidence for the of calpains in NF-κB activation, the of was on NF-κB-dependent activity in transient cotransfection assay M. S. M. C. J. M. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, 1995; Google Scholar). have shown that the human promoter is TNF-α-inducible in a manner on a NF-κB A.R. M. Casola A. Garofalo J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of expression not activity blocked TNF-α-inducible activity 1 As a the of on IκBα was measured in transient HepG2 cells with receptor expression in the or presence of of with Following of cells, a was to in IκBα in cytosolic In the presence of of IκBα proteolysis was by these data a parallel of the calpain system in TNF-α-inducible proteolysis of IκBα and NF-κB activation. The and of protease inhibitors for proteasome and calpain activities measured in of the was used to proteasome activity in cell C. K. D. Goldberg A.L. Cell. 1994; 78: Full Text PDF PubMed Scopus Google Scholar, G. S. 1995; PubMed Scopus Google Scholar). As following proteasome of the activity was that the assay is proteasome activity. As shown in in both control and cells, activity was and than inhibition of proteasome activity was following Z-LLF, and these inhibitors Z-LnL, E64, and proteasome activity. was for either control or TNF-α-stimulated activity of m-calpain was measured in the presence of the inhibitors the proteasome inhibitors and Z-LLL, as well as the calpain inhibitors Z-LnL, E64, and Z-LLY, inhibitors of was to calpain from proteasome and Z-LnL, E64, and Z-LLY, specific for activity of on the proteasome. of in intracellular calpain activity in broken cells has been to the presence of inhibitor that rapidly with active calpains following cell However, the of a specific fluorescent assay using a cell-permeant calpain to in calpain proteolytic activity has the for broken cell J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the into cells, is with to a calpain Following its the fluorescent where its of accumulation is a of intracellular calpain activity J. Biol. Chem. Full Text PDF PubMed Google Scholar). of this assay for calpain has been by its inhibition by the specific calpain inhibitor, Z-LLY, and from or J. Biol. Chem. Full Text PDF PubMed Google Scholar). of of was observed in cells the assay by the of inhibitors on proteolytic activity. of the fluorescent proteolysis is calcium-dependent to the of the intracellular and is by the calpain inhibitors, inhibition at and at and inhibition at and not by the proteasome inhibitor, inhibition at In cells, TNF-α calpain activity the 1 of The of calpain activity is blocked by the calpain inhibitor not parallel of inhibitor for intracellular calpain activity as for m-calpain in and assay was also by to the of HepG2 As shown in in HepG2 as of calpain activation, of in than of cells following TNF-α for of in also the inhibitor as shown in the cell assay m-calpain proteolytic activity to be for IκBα we to the subcellular of m-calpain in control and TNF-α-stimulated particulate and cytosolic at following TNF-α by at and for both m-calpain catalytic subunit and IκBα by m-calpain be in both cytosolic and particulate fraction of protein, the specific activity of m-calpain was in the particulate In the cytosolic m-calpain abundance following TNF-α in the cytosolic In both however, m-calpain abundance time with we that the cytosolic fraction IκBα and that in m-calpain subunit concomitantly with IκBα proteolysis and with calpain proteolytic activity control of in the was used to protein whether cytosolic from cells containing m-calpain catalytic subunit by also IκBα proteolytic in protease assay was In this human IκBα was to TNF-α-stimulated cytosolic and the on proteolysis was by observed a proteolysis of a proteolysis that was blocked either by or the of calpain inhibitors, or not or whether m-calpain IκB the NF-κB·IκB complex and produce ligand-independent NF-κB activation, m-calpain was to broken cell produced a of IκBα IκBα was into intermediates of also in TNF-α-stimulated cells proteasome activity The of m-calpain was and required m-calpain proteolytic activity Inducible phosphorylation is not required for IκBα proteolysis because the IκBα is and IκBα in is whether calpains in NF-κB activation, on and nuclear proteins that m-calpain a and increase in binding activity as complex a characterized by assay (12Han Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar)). demonstrate NF-κB in nuclear abundance was measured by where a increase in in the m-calpain was that ligand-independent IκBα proteolysis and NF-κB activation be by As additional evidence for proteasome-independent pathway for IκB proteolysis and NF-κB activation, we the of TNF-α in cells conditionally in the ubiquitin-proteasome cells a temperature-sensitive for in the Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar, M. S. M. C. J. M. Mol. Cell. Biol. 1997; PubMed Scopus Google control cells are with the Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). of calpain activity was observed in cells hydrolysis of the cell-permeant calpain with TNF-α In cells, calpain activity from to TNF-α inactivation by cells at the IκBα proteolysis was still at and By cells a rapid IκBα proteolysis with a at by its on the TNF-α signaling pathway. In both cell binding activity of the was in parallel to IκBα proteolysis IκBα proteolysis in cells is blocked by calpain E64, and and not by proteasome inhibitors the of IκBα proteolysis in the cells are to of HepG2 cells 1 Together, these data indicate that IκBα turnover is than both proteolytic systems are are intracellular calcium-dependent subunits include and these have and are in subcellular and M. F. I. M. S. 1996; PubMed Scopus Google Scholar, T. Google Scholar). Here we show for the first time that the system is a parallel pathway for TNF-α-inducible IκB proteolysis and NF-κB activation. TNF-α, activates NF-κB through the of two cytoplasmic protease systems as the proteasome pathway, where IκBα proteolysis is by its modification and the inducible system, where protease activity is by In the the of calpains and the proteasome in intracellular regulatory processes has been because inhibitors of the two cytoplasmic protease systems identified. indicate that that be used to the parallel of these protease systems in cytokine for the pathway mediating NF-κB activation is on the of the following Inducible IκBα proteolysis is only be partially blocked by either or proteasome-selective inhibitors and completely blocked by both. In TNF-α-stimulated cells, a rapid (within 1 min), 2.2-fold increase in cytosolic calpain proteolytic activity in intact cells is proteolytic activity occurs with the particulate to redistribution of the catalytic m-calpain IκBα proteolysis occurs coincidentally with in m-calpain abundance in the of catalytically active m-calpain is sufficient to produce ligand-independent NF-κB activation. IκBα proteolysis is in cells proteasome activity The mechanism for activation of calpains in intact cells is calpains to of activity through of its subunits (1Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (782) Google Scholar). In intact cells, evidence for activation or activation following in intracellular is In calpains are to be proteins with of this not be with protease J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Our data that TNF-α-stimulated calpain activity occurs in the of because are not at in protease activity be Moreover, calpain activation in the of in intracellular has been in D.E. Y. J. Cell. Physiol. 1996; PubMed Scopus Google Scholar). In data not we have not observed in in HepG2 intracellular is required for calpain activity in intact cells, because intracellular block calpain activity and IκBα proteolysis data not mechanism for calpain activation include in subcellular are not the In cell m-calpain is in a in the association with Cell PubMed Scopus Google and in the system, m-calpain is S. J. 1996; PubMed Scopus Google Scholar). In HepG2 cells, we that m-calpain into the cytoplasmic a fraction containing IκBα, following TNF-α redistribution is the mechanism for m-calpain activation additional m-calpain activity in the fraction was thought to be important for proteolysis of protein kinase S. M. J. Biol. Chem. Full Text PDF PubMed Google cytosolic calpain activity to be important in turnover of the M. S. M. C. J. M. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). on subcellular the intracellular of proteolysis of IκBα also occurs in the activity is inducible following activation of including the receptor J. Biol. Chem. Full Text PDF PubMed Google and the receptor A. Y. T. K. S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google a for have shown that including in TNF-α calpain catalytic activity through a mechanism that binding to the 30-kDa regulatory subunit S. J. 1996; PubMed Scopus Google Scholar, J. 1997; PubMed Scopus Google Scholar, C. 1996; PubMed Scopus Google Scholar). These to a S. J. 1996; PubMed Scopus Google Scholar). be important intermediates for calpain activity for First, TNF-α is to increase through its on activity in K. S. T. D. M. Cell. 1994; 78: Full Text PDF PubMed Scopus Google this has been to NF-κB activation S. K. T. D. K. M. Cell. 71: Full Text PDF PubMed Scopus Google Scholar). Second, intracellular calpain activity in cells J. 1997; PubMed Scopus Google Scholar). are to be important regulators of intracellular signaling in turnover of protein kinase and proteins (Ref. D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (782) Google Scholar and references calpains have recently been in mediating turnover of the transcription factor S. M. I. F. M. J. 1996; PubMed Scopus Google S. H. M. K. 1991; PubMed Scopus Google Scholar) and the tumor gene M. S. M. C. J. M. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). both and initially to be proteasome Our data IκBα to the of key regulatory proteins by a parallel pathway. to IκBα, is a for calpain proteolysis M. S. M. C. J. M. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The TNF-α-inducible calpain pathway mediating IκBα proteolysis is from the two nonproteasome-dependent IκBα proteolytic pathways (10Miyamoto S. Seufzer B.J. Shumway S.D. Mol. Cell. Biol. 1998; 18: 19-29Crossref PubMed Google M. Casola A. Garofalo R.P. Han Y. Elliott T. Ogra P.L. Brasier A.R. J. Virol. 1998; 72: 4849-4857Crossref PubMed Google Scholar). In the first IκBα turnover in pre-B cell line was not by the calpain inhibitors, calpain inhibitors and or Z-LLF, that with IκBα proteolysis in system Ref. Y. Brasier A.R. J. Biol. Chem. 1997; 272: 9825-9832Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Second, calpain inhibitors and not have on IκBα proteolysis in respiratory syncytial epithelial cells (11Jamaluddin M. Casola A. Garofalo R.P. Han Y. Elliott T. Ogra P.L. Brasier A.R. J. Virol. 1998; 72: 4849-4857Crossref PubMed Google Scholar). The of these pathways to pathway, In we implicate the and proteasome pathways are parallel mechanisms mediating inducible IκBα proteolysis by the cytokine These data indicate that calpains to rapid IκBα proteolysis through a mechanism in cytosolic calpain proteolytic activity. J. for lactacystin; for for and for J. for and A. and for on the