Tumor Necrosis Factor-alpha -inducible Ikappa Balpha Proteolysis Mediated by Cytosolic m-Calpain
A MECHANISM PARALLEL TO THE UBIQUITIN-PROTEASOME PATHWAY FOR NUCLEAR FACTOR-kappa B ACTIVATION*

Youqi HanDagger , Steven WeinmanDagger §, Istvan Boldogh, Randall K. Walkerparallel , and Allan R. BrasierDagger parallel **

From the Dagger  Department of Internal Medicine, the § Department of Physiology and Biophysics, the  Department of Microbiology & Immunology, and the parallel  Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas 77555-1060

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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, Ikappa Balpha , through the 26 S proteasome. Here, we show a parallel proteasome-independent pathway for cytokine-inducible Ikappa Balpha 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 Ikappa Balpha proteolysis; pretreatment with both is required to completely block Ikappa Balpha proteolysis. Similarly, in transient cotransfection assays, expression of the specific inhibitor, calpastatin, partially blocks TNF-alpha -inducible NF-kappa B-dependent promoter activity and Ikappa Balpha 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 Ikappa Balpha compartment. Addition of catalytically active m-calpain into broken cells was sufficient to produce ligand-independent Ikappa Balpha proteolysis and NF-kappa B translocation. As additional evidence for calpain-dependent Ikappa Balpha 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, Ikappa Balpha proteolysis occurs in a manner sensitive only to calpain inhibitors. Our results demonstrate that TNF-alpha activates cytosolic calpains, a parallel pathway that degrades Ikappa Balpha and activates NF-kappa B activation independently of the ubiquitin-proteasome pathway.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (1-3). 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 (1). 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. 2) and, of special interest to the pathophysiology of inflammatory processes, mechanisms for intracellular signaling produced by the cytokine tumor necrosis factor-alpha (TNF-alpha ).1

Following binding its receptor on the plasma membrane, TNF-alpha initiates de novo transcription of genetic networks, in part, through activating nuclear translocation of the cytoplasmic transcription factor nuclear factor-kappa B (NF-kappa B) (4, 5). NF-kappa B, a multiprotein complex inactivated in the cytoplasm by association with its Ikappa B inhibitor, translocates into the nucleus following dissociation of the NF-kappa B·Ikappa B complex. TNF-alpha modifies NF-kappa B·Ikappa B association through a process initiated by inducible Ikappa Balpha serine phosphorylation on its amino-terminal regulatory domain, a modification coupled to Ikappa B polyubiquitination (Ubn) on adjacent lysine residues (6). NF-kappa B·Ikappa B dissociation requires Ikappa B proteolysis because phosphorylated and ubiquitinated Ikappa B still inactivates NF-kappa B (Ref. 6 and references therein).

Presently, the ubiquitin-proteasome system has been the only pathway identified in mediating cytokine-inducible Ikappa B proteolysis. Pretreatment with cell-permeant proteasome inhibitors blocks TNF-alpha -inducible Ikappa B proteolysis concomitantly with the accumulation of Ubn- and phosphorylated Ikappa B intermediates (6, 8). Independently, inducible Ikappa Balpha proteolysis in cells harboring thermolabile ubiquitin-activating enzymes is markedly slowed at non-permissive temperatures (9).

Several lines of evidence indicate the presence of alternative (nonproteasome-dependent) processing pathways for Ikappa B proteolysis. First, in pre-B lymphocytes, c-Rel:NF-kappa B1 is constitutively nuclear as the consequence of a calcium-dependent proteolytic activity that preferentially affects Ikappa Balpha (rather than Ikappa Bbeta (10)). Second, we have observed a non-proteasome-dependent pathway mediating inducible Ikappa Balpha proteolysis (and NF-kappa B activation) following respiratory syncytial virus infection of human airway epithelial cells (11). However, whether additional nonproteasome-dependent pathways participate in cytokine-inducible NF-kappa B activation have not been explored.

These studies prompted us to examine whether nonproteasome-dependent pathways participate in cytokine-inducible Ikappa Balpha degradation. Here we investigate the proteolytic mechanism involved in a well characterized model of NF-kappa B activation in TNF-alpha -stimulated HepG2 hepatocytes, where NF-kappa B activation mediates the expression of acute phase reactants (12, 13). By using calpain and proteasome-selective inhibitors, we demonstrate that inducible Ikappa Balpha 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-alpha rapidly activates cytosolic calpain proteolytic activity. In subcellular fractionations of TNF-alpha -stimulated cells, the catalytic m-calpain subunit translocates from the particulate into the cytosolic fraction (the latter containing Ikappa Balpha ) coincidentally with Ikappa Balpha proteolysis. Moreover, TNF-alpha -inducible Ikappa Balpha proteolysis occurs in cells conditionally deficient in the ubiquitin-proteasome pathway, and in cells expressing Ikappa Balpha mutations deficient in proteasome-dependent processing. Together, these data implicate calpains are a parallel pathway in mediating Ikappa Balpha proteolysis and NF-kappa B activation.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
Discussion
References

Materials-- 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) were from Sigma and Aldrich. Lactacystin (Lacta), was a generous gift of E. J. Corey (Harvard University). Benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF) was a gift of Mark Suto (Signal Pharmaceuticals, San Diego, CA). Z-Leu-Leu-leucinal (MG132, Z-LLL), Z-Leu-norleucinal (calpeptin, Z-LnL), trans-epoxysuccinyl-L-leucylamido-(4-guanido)butane (E64), and phenylmethylsulfonyl fluoride (PMSF) were from Calbiochem (San Diego, CA). CBZ-L-leucyl-L-leucyl-L-tyrosine diazomethyl ketone (Z-LLY) was from Molecular Probes. The temperature-sensitive Balb/c 3T3 cell line, ts20b, and its corrected version transfected with wild-type E1 enzyme, H38-5, was gift from Harvey Ozer (UMDNJ-New Jersey Medical School).

Plasmid Construction-- A reporter plasmid containing -162 to +44 bp of the human IL-8 promoter driving expression of CAT was produced by subcloning the BamHI/HindIII restricted polymerase chain reaction product of the IL-8 gene into the same sites of a pGEMCAT plasmid. For this, an upstream primer 5'-ACTTGGATCCACTCCGTATTTGATAAGG-3' (BamHI site underlined) and the downstream primer 5'-AGAAGCTTGTGTGCTCTGCTGTCTCTGAA-3' were used to polymerase chain reaction amplify the IL-8 promoter (11). Plasmids were purified by ion exchange chromatography (Qiagen) and sequenced to verify authenticity prior to transfection.

Cell Culture and Transfection-- Human HepG2 cells were cultured and stimulated with 30 ng/ml recombinant human TNF-alpha as described (12). For protease inhibitor experiments, cells were pretreated for 1 h with 10 µM lactacystin, 10 µM Z-LLF, 25 µM Z-LLL, 10 µM Z-LnL, 50 µM E64, 100 µM PMSF, or 100 µM Z-LLY as indicated. HepG2 cells were transfected using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate:DNA liposomes into triplicate 60-mm plates with 20 µg of -162/+44 human IL-8/CAT reporter, 5 µg of SV40-driven alkaline phosphatase internal control, and 4 µg of pcDNA I expression plasmid (InVitrogen). Cells were stimulated with TNF-alpha for 12 h prior to reporter assay. Where indicated, transiently transfected cells were isolated following cotransfection with 5 µg of plasmid CMV.IL2R encoding the IL-2 receptor. Transient transfectants in 100-mm plates (108 cells) were purified by adding anti-human CD25 (Caltag Laboratories) and captured on magnetic beads conjugated to rabbit anti-mouse IgG (Dynabeads, Dynal Inc.) as described (7). In experiments using ubiquitin pathway-defective ts20b cells and their controls, H38-5, cells were transferred from 32 °C (permissive temperature) to 39 °C (restrictive temperature) for a 6-h period to inactivate the temperature-sensitive E1 ubiquitin-activating enzyme (14). Cells were stimulated with 30 ng/ml TNF-alpha at 37 °C. Identically treated wild-type E1-corrected H38-5 cells were taken as control. For protease inhibitor experiments, 6-h temperature-restricted ts20b cells were pretreated for 1 h at 37 °C without or with 50 µM Z-LnL, 100 µM E64, 100 µM Z-LLY, 10 µM Lacta, and 100 µM PMSF prior to TNF-alpha stimulation.

Antibodies and Western Immunoblots-- Antibodies used were to Ikappa Balpha (sc-203, recognizing amino acids 6-20, and sc-371, recognizing amino acids 297-317), Ikappa Bbeta (sc-945, recognizing amino acids 339-358), Rel A (sc-109), Rel B (sc-226) from Santa Cruz Biotech and m-calpain (Research Diagnostics, Inc.). Subunit-specific rabbit polyclonal antibodies to recombinant Methanosarcina thermophilia alpha  and beta  subunits, corresponding to the human proteasome subunit zeta  and RING 10, respectively, were obtained from Calbiochem. 200 µg of cytosolic or nuclear extracts were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene membranes (12). Following incubation with primary antibody, antigens were detected in the enhanced chemiluminescent assay (Amersham Pharmacia Biotech) following the manufacturer's recommendations.

In Vitro Protease Assays-- For proteasome activity, 200 µg of HepG2 lysates from control or TNF-alpha -treated (30 ng/ml, 15 min) were incubated with 60 µM in 1 ml of ATP-containing reaction buffer (15) in the presence or absence of indicated protease inhibitor (30 min, 30 °C). AMC released was quantitated by measuring fluorescence emission intensity at 440 nm (Iex, 365 nm) normalizing to standards. Results are mean ± S.D. of three experiments. To confirm Suc-LLVY-AMC hydrolyzing activity was dependent on proteasome activity, HepG2 lysate was proteasome-depleted by ultracentrifugation (150,000 × g, 26 h, 4 °C). Western blots before or after ultracentrifugation were done to detect the core proteasome subunits, RING 10 and zeta . Both RING 10 and zeta , present in the crude supernatant, were lost in the S150 supernatant and recovered in the 150,000 × g pellet. By contrast, the 80-kDa m-calpain catalytic subunit remained in the S150 supernatant. Ninety four percent of the AMC generation was lost in the S150 supernatant, indicating that proteasome activity was selectively being measured by this assay. For measurement of m-calpain caseinolytic activity, 0.025 units/ml purified human erythrocyte m-calpain was incubated with phosphorylated bovine casein (2 mg/ml) in the presence or absence of indicated inhibitors under standard conditions (4 mM CaCl2, 10 mM dithiothreitol, at 30 °C, 1 h). Hydrolysis was quantitated by Coomassie Brilliant Blue colorimetric assay (16). Similar results were obtained with purified rabbit skeletal muscle m-calpain (not shown).

Calpain Assay in Intact Cells-- For HepG2 and ts20b cells, calpain activity was measured by the rate of generation of the fluorescent product, AMC, from intracellular thiol-conjugated Boc-Leu-Met-CMAC (17). Cells were dispersed, grown on glass coverslips, continuously superfused with physiologic saline solution at 37 °C, and sequentially imaged with a quantitative fluorescence imaging system (18). At t = 0, Boc-Leu-Met-CMAC (10 µM, Molecular Probes) was introduced into the superfusion solution, and mean fluorescence intensity (excitation 350 nm, emission 470 nm) of individual cells was measured at 60-s intervals. At 10 min, TNF-alpha (30 ng/ml) was added to the superfusion solution with 10 µM Boc-Leu-Met-CMAC. The slope of the fluorescence change with respect to time represents the intracellular calpain activity (17). Hydrolysis of the thiol-conjugated substrate was rate-limiting for the generation of fluorescent product as shown by comparing the initial rate of cell fluorescence increase after exposure to Boc-Leu-Met-CMAC with that produced by CMAC. CMAC requires only the thiol conjugation step, not hydrolysis, for fluorescence. There was a 35.8-fold increase in the AMC fluorescence rate compared with Boc-Leu-Met-CMAC, demonstrating that hydrolysis and not conjugation was rate-limiting. For calpain assays in whole cell populations, suspension cultures of HepG2 cells were loaded with 10 µM Boc-Leu-Met-CMAC, and changes in intracellular fluorescence was measured prior to and after TNF-alpha addition at 37 °C using a FACS Vantage system. Cellular fluorescence of AMC was measured using a 360-nm excitation filter and a 405-nm long-pass emission filter.

Calpain-dependent Ikappa Balpha Proteolytic Assay in Cytosolic S100 Extract-- Two hundred µg of protein 100,000 × g supernatant (S100) was incubated with 150 ng of recombinant human Ikappa Balpha for indicated times in Reaction Buffer (RB, 25 mM HEPES, pH 7.2, 65 mM KCl, 2 mM MgCl2, 1.5 mM CaCl2, 2 mM dithiothreitol) at 32 °C in a final volume of 60 µl. Ikappa Balpha degradation was quantitated by Western immunoblot.

Ikappa B Proteolysis and NF-kappa B Activation Assay in Broken Cells-- Indicated amounts of purified rabbit skeletal muscle m-calpain was added to a mixture of 200 µg of HepG2 cytosol and 1 × 106 sucrose cushion-purified nuclei (12) in 10% glycerol-containing RB (100 µl, 32 °C). Ikappa B proteolysis in cytoplasmic extract and nuclear Rel A was analyzed by Western immunoblot following sucrose-cushion purification of nuclei (12). Gel mobility shift assay was performed using the NF-kappa B-binding site from the angiotensinogen promoter as described (12).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Non-proteasomal Component for TNF-alpha -inducible Ikappa Balpha Proteolysis Is Sensitive to Calpain Inhibitors-- We have previously shown that administration of TNF-alpha (30 ng/ml) to HepG2 cells induces rapid Ikappa B (alpha  and beta ) proteolysis and NF-kappa B translocation, maximally detectable 15 min following addition of hormone (12). To examine initially if proteasome-independent pathways for Ikappa B proteolysis exist, Ikappa B abundance was assayed by Western immunoblots in protease inhibitor pretreated cells harvested 15 min after TNF-alpha stimulation. A battery of previously characterized proteasome (lactacystin, Z-LLF, Z-LLL (8, 15, 19)), calpain (Z-LnL (20), E64), and nonspecific serine protease (PMSF) inhibitors were used (Fig. 1a). In data not shown, pretreatment with these agents had no effect on constitutive (nonstimulated) levels of either Ikappa B isoform. In the absence of protease inhibitors, TNF-alpha produced rapid proteolysis of both 37-kDa Ikappa Balpha and 49-kDa Ikappa Bbeta (compare lanes 1 and 2). Pretreatment with lactacystin, Z-LLF, or Z-LLL blocked Ikappa Bbeta processing (compare lane 2 with 3-5), whereas Z-LnL, E64, and PMSF had no effect. These observations are consistent with a predominant role of the proteasome pathway mediating Ikappa Bbeta proteolysis (6).


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Fig. 1.   Calpain inhibitors affect inducible Ikappa Balpha proteolysis. a, cytoplasmic extracts from control (lane 1) or TNF-alpha -treated cells (15 min, lanes 2-8) were analyzed by Western immunoblot using a mixture of Ikappa Balpha and Ikappa Bbeta antibodies (indicated at left). Cells were stimulated following preincubation with indicated protease inhibitors (doses under "Experimental Procedures"). Compared with TNF-alpha treatment alone, Ikappa Balpha proteolysis is blocked by Lacta (43%), Z-LLF (46%), Z-LLL (100%), Z-LnL (56%), E64 (35%), and PMSF (11%), respectively. b, top panel, TNF-alpha induced Ikappa Balpha degradation in Hep G2 cells. Apparent Ikappa Balpha half-life is 1-3 min following TNF-alpha treatment, preceded by Ikappa Balpha phosphorylation (Ikappa Balpha P). Bottom panel, Ikappa Balpha half-life in lactacystin-pretreated cells (10 µM for 1 h). TNF-alpha induces Ikappa Balpha degradation in the time-dependent manner even in the absence of detectable 26 S proteasome activity. ~30-kDa Ikappa Balpha intermediate produced is indicated with arrow (left) detected on longer exposure. c, combined effect of proteasome and calpain inhibition completely blocks inducible Ikappa Balpha proteolysis. HepG2 cells pretreated for 1 h with the cell-permeant irreversible calpain inhibitor Z-LLY (21) (50 µM) and/or 10 µM lactacystin were TNF-alpha -stimulated (15 min). Rel B staining is used as an internal control for protein recovery. TNF-alpha -induced Ikappa Balpha degradation is totally blocked by the combined treatment of Z-LLY and lactacystin. d, calpastatin blocks NF-kappa B dependent reporter activity. HepG2 cells were cotransfected with -162/+44 human IL-8/CAT reporter, internal control SV40-driven alkaline phosphatase reporter, and either empty pcDNA I- or pcDNA I calpastatin expression vector. Normalized CAT/alkaline phosphatase activity (x ± S.D.) in control and TNF-alpha -stimulated cells is shown. e, capastatin expression blocks Ikappa Balpha proteolysis. HepG2 cells transfected with IL-2 receptor expression plasmid ± pcDNA I-capastatin expression plasmid were TNF-alpha -stimulated and transfectants affinity purified. Shown is a Western blot of Ikappa Balpha (top) and control beta -actin (bottom). Lane 1, control-treated; lanes 2-4, TNF-alpha -treated. Lanes 1 and 2, 0 µg of pcDNA I-calpastatin; lane 3, 0.5 µg; lane 4, 2.5 µg. Relative to control cells, normalized Ikappa Balpha signal to beta -actin in lane 2 is 30%; lane 3 is 35%, and lane 4 is 50%.

In contrast, Ikappa Balpha proteolysis was partially blocked by the proteasome inhibitors lactacystin and Z-LLF (Fig. 1a, lanes 3 and 4). Of the presumed proteasome-selective inhibitors, only Z-LLL was a completely effective inhibitor of Ikappa Balpha proteolysis. Surprisingly, moreover, the calpain inhibitors Z-LnL and E64 also partially blocked Ikappa Balpha proteolysis, even under conditions where Ikappa Bbeta proteolysis was unaffected (compare lane 2 with 6 and 7). These data suggest a parallel contribution of calpain-like proteases in TNF-alpha -inducible Ikappa Balpha hydrolysis.

To define the kinetics of proteasome-independent pathways mediating Ikappa Balpha proteolysis, Ikappa Balpha half-life in TNF-alpha -treated cells was compared in cells containing with that in cells lacking proteasome activity (Fig. 1b). In cells not treated with protease inhibitors, Ikappa Balpha proteolysis is rapid (t1/2 of 1-3 min), occurring coincidentally with the generation of phosphorylated Ikappa Balpha intermediates (Fig. 1b, Ikappa Balpha P). In cells pretreated with the potent irreversible proteasome inhibitor lactacystin, Ikappa Balpha proteolysis occurs with a detectably slower half-life (t1/2 of 7-15 min) and is incomplete, with the appearance of a <30-kDa intermediate (Fig. 1b, bottom). To determine whether any pathway other than the combined calpain/proteasome account for Ikappa Balpha proteolysis, the additive effects of the specific irreversible calpain inhibitor Z-LLY (21) and lactacystin were studied (Fig. 1c). At saturating doses, neither Z-LLY nor lactacystin alone could completely inhibit Ikappa Balpha proteolysis. In the presence of both inhibitor types, Ikappa Balpha proteolysis was completely blocked with accumulation of non- and phosphorylated Ikappa Balpha intermediates (Fig. 1c). We note consistently that Ikappa Balpha P intermediate was detectable at the 15-min time point in the presence of proteasome inhibitors but not in calpain inhibitors (see "Discussion").

Enzymatic activity of calpains are influenced by the effect of endogenous calpain inhibitor, calpastatin. As additional evidence for the role of calpains in NF-kappa B activation, the effect of transiently expressed calpastatin was determined on NF-kappa B-dependent reporter activity in transient cotransfection assay (18, 22). We have previously shown that the human IL-8 promoter is TNF-alpha -inducible in a manner solely dependent on a high affinity NF-kappa B site (23). Cotransfection of calpastatin expression plasmid (pcDNA I-calpastatin) did not affect basal IL-8/CAT activity but significantly blocked TNF-alpha -inducible CAT activity (Fig. 1d). As a control, the effect of calpastatin on Ikappa Balpha steady state levels was measured in transient transfectants. HepG2 cells cotransfected with IL-2 receptor expression plasmid in the absence or presence of various concentrations of pcDNA I-calpastatin were stimulated with TNF-alpha . Following isolation of transfected cells, a Western immunoblot was done to determine changes in Ikappa Balpha in cytosolic lysates (Fig. 1e). In the presence of 2.5 µg of pcDNA I-calpastatin, Ikappa Balpha proteolysis was inhibited by ~50%. Combined, these data strongly suggest a parallel contribution of the calpain system in TNF-alpha -inducible proteolysis of Ikappa Balpha and NF-kappa B activation.

Specificity of Protease Inhibitors-- The specificity and effect of protease inhibitors for proteasome and calpain activities were directly measured in vitro (Fig. 2). Hydrolysis of the fluorogenic substrate Suc-LLVY-AMC was used to measure proteasome activity in whole cell lysates (15, 19). As described under "Experimental Procedures," following proteasome depletion, 94% of the Suc-LLVY-AMC hydrolyzing activity was depleted, indicating that the assay is measuring bona fide proteasome activity. As shown in Fig. 2a, in both control and TNF-alpha -treated cells, Suc LLVY-AMC hydrolytic activity was indistinguishable (12.5 and 13 nmol/min/mg, respectively). Also, greater than 90% inhibition of proteasome activity was seen following lactacystin, Z-LLF, and Z-LLL treatment, indicating these inhibitors potently inhibited (whereas Z-LnL, E64, Z-LLY and PMSF had no effect) cellular proteasome activity. This effect was consistent for either control or TNF-alpha -stimulated cells.


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Fig. 2.   Specificity of inhibitors on proteasome and calpain activities in vitro. a, proteasome activity in cell lysates. Control and TNF-alpha -stimulated proteasome activities are measured in the presence of the indicated inhibitors at concentrations described. b, m-calpain activity. Specific caseinolytic activity of purified rabbit skeletal muscle m-calpain in the presence or absence of indicated inhibitors. Lactacystin and PMSF have negligible effects on casein hydrolysis.

Caseinolytic activity of purified m-calpain was measured in the presence of the same inhibitors (Fig. 2b). Surprisingly, the presumed "selective" proteasome inhibitors Z-LLF and Z-LLL, as well as the calpain inhibitors Z-LnL, E64, and Z-LLY, were potent inhibitors of m-calpain. Only lactacystin, therefore, was able to differentiate calpain from proteasome activity, and Z-LnL, E64, and Z-LLY, conversely, were specific for caseinolytic activity of calpain, without effects on the proteasome.

TNF-alpha -inducible Changes in Intracellular Calpain Proteolytic Activity-- Direct measurement of dynamic changes in intracellular calpain activity in broken cells has been hampered due to the presence of endogenous calpastatin inhibitor that rapidly associates with active calpains following cell disruption. However, the recent development of a specific fluorescent assay using a cell-permeant calpain substrate to measure changes in calpain proteolytic activity has obviated the need for broken cell assays (17). After diffusion of the substrate, Boc-Leu-Met-CMAC, into cells, it is conjugated with glutathione (GSH) to form a membrane-impermeant, nonfluorescent calpain substrate. Following its hydrolysis, the unquenched fluorescent product (AMC-GSH) accumulates, where its rate of accumulation is a measure of intracellular calpain activity (17). Specificity of this assay for calpain has been previously demonstrated by its inhibition by the specific calpain inhibitor, Z-LLY, and independence from lysosomal, serine, or cathepsin proteases (17). A basal rate of generation of AMC-GSH was observed in individual cells (Fig. 3a, 1-10 min). We further examined the assay specificity by measuring the effects of inhibitors on basal proteolytic activity. Basal generation of the fluorescent proteolysis product is calcium-dependent due to the inhibitory effects of the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (76% inhibition, 100 µM (n = 30)) and is quantitatively inhibited by the calpain inhibitors, Z-LnL (59% inhibition at 100 mM (n = 40) and 93% at 200 µM (n = 30)) and Z-LLY (62% inhibition at 100 µM (n = 70)), and not inhibited by the proteasome inhibitor, lactacystin (0% inhibition at 10 µM (n = 20)).


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Fig. 3.   Effect of TNF-alpha on calpain activity in intact HepG2 cells. a, fluorescence intensity changes in a single cell. Cells were loaded with calpain substrate at time = 0. Accumulation of calpain hydrolytic product measured before and after TNF-alpha addition (arrow, at 10 min). b, relative stimulation of single cell calpain activity in the presence of protease inhibitors. Relative calpain activity was measured after no addition (control), TNF-alpha (30 ng/ml), TNF-alpha  + Z-LnL (100 µM), or TNF-alpha  + lactacystin (10 µM, 30 min). The basal activity in each cell was measured for use as its own control (open bars) prior to TNF-alpha stimulation (solid bars). Increased proteolytic rate occurred only for TNF-alpha (p < 0.001) and TNF-alpha with lactacystin (p < 0.05). c, kinetics of change in intracellular fluorescence intensity in TNF-alpha -exposed cell populations. FACS analysis. A representative experiment is shown (n = 6). Fluorescence intensity increased 2.5 ± 0.3 fold in >95% cell population.

In individual cells, TNF-alpha increased calpain activity ~2.2-fold over the basal rate within 1 min of exposure (Fig. 3a). The TNF-alpha -induced stimulation of calpain activity is blocked by the calpain inhibitor Z-LnL but not lactacystin (Fig. 3b), indicating an exact parallel of inhibitor specificity for intracellular calpain activity as for purified m-calpain in vitro (cf. Figs. 3b and 2c). This assay was also applied by FACS analysis to determine the portion of TNF-alpha -responsive HepG2 cells. As shown in Fig. 3c, in HepG2 populations, mean cellular fluorescence intensity, as an indicator of calpain activation, increased an average of 2.5 ± 0.3-fold in greater than 95% of cells following TNF-alpha administration for 60-800 s. Specificity of changes in mean fluorescence intensity also follows the same inhibitor profile as shown in the single cell assay (not shown).

TNF-alpha Induces Changes in m-Calpain Abundance-- For m-calpain proteolytic activity to be relevant for Ikappa Balpha proteolysis, we sought to determine the subcellular distribution of m-calpain in control and TNF-alpha -stimulated cells. For this, particulate (S100 pellet) and cytosolic (S100 supernatant) fractions were prepared at various times following TNF-alpha treatment by ultracentrifugation at 100,000 × g and analyzed for both 80-kDa m-calpain catalytic subunit and Ikappa Balpha by Western immunoblot (Fig. 4a). Although m-calpain could be detected in both cytosolic and particulate fractions, normalizing each fraction per microgram of protein, the highest specific activity of m-calpain was found in the particulate fraction. In the cytosolic fraction, m-calpain abundance increased 2-fold within 2 min following TNF-alpha stimulation in the cytosolic fraction. In both fractions, however, m-calpain abundance fell (compare 15-min time points with control, Fig. 4a). Importantly, we note that the cytosolic fraction contained Ikappa Balpha and that changes in m-calpain subunit occurred concomitantly with Ikappa Balpha proteolysis (Fig. 4a) and synchronously with calpain proteolytic activity (Fig. 3). Internal control immunostaining of inert Rel B in the same membrane was used to document equivalent protein loading (Fig. 4a, bottom).


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Fig. 4.   Calpain particulate-cytosol translocation and hydrolysis of recombinant human Ikappa Balpha (rhIkappa Balpha ). a, Western immunoblot of m-calpain 80-kDa catalytic subunit and Ikappa Balpha in particulate (100,000 × g pellet, Ref. 18) and cytosolic (100,000 × g supernatant) fractions of TNF-alpha -treated cells. Relative to control, in TNF-alpha -treated cytosol, m-calpain abundance rapidly increases 2.08-fold (2 min), 1.67-fold (5 min), and 1.37-fold (10 min) and thereafter decreases to 0.6-fold (15 min). In membrane extracts, m-calpain gradually decreases to 88% (2 min), 86% (5 min), 61% (10 min), and 62% (15 min). Control Rel B staining is shown (bottom). b, calpain-dependent degradation of recombinant Ikappa Balpha by TNF-alpha -treated S100 cytosolic extract. Top panel, time course. Bottom panel, effect of calpain inhibitors. S100-induced rhIkappa Balpha proteolysis is blocked by 5 mM EGTA (40%), 50 µM E64 (50%), 50 µM Z-LnL (81%) but not 50 µM lactacystin (Lact) or 100 µM PMSF.

To determine whether cytosolic fractions from TNF-alpha -treated cells containing translocated m-calpain catalytic subunit (determined by Western immunoblot, Fig. 4) also contain Ikappa Balpha proteolytic activity, an in vitro protease assay was established (Fig. 4b). In this assay, purified recombinant human Ikappa Balpha (rhIkappa Balpha ) was added to TNF-alpha -stimulated cytosolic lysates (S100 supernatant), and the effect on rhIkappa Balpha proteolysis was determined by Western immunoblot. We observed a time-dependent proteolysis of rhIkappa Balpha , a proteolysis that was blocked either by calcium chelation or the addition of calpain inhibitors, E64 or Z-LnL but not lactacystin or PMSF (Fig. 4b, bottom).

m-Calpain Is Sufficient for Ligand-independent Ikappa Balpha Proteolysis and NF-kappa B Translocation-- To determine whether m-calpain proteolyzed native Ikappa B within the NF-kappa B·Ikappa B complex and could produce ligand-independent NF-kappa B activation, activated m-calpain was added to broken cell lysates (containing nuclei, Fig. 5). m-Calpain produced a time-dependent degradation of endogenously expressed Ikappa Balpha (Fig. 5, top). Ikappa Balpha was proteolyzed into transiently stabilized carboxyl-terminal intermediates of ~30 kDa (arrow, Fig. 5), an intermediate also seen in TNF-alpha -stimulated cells lacking proteasome activity (see Fig. 1b). The effect of m-calpain was dose-dependent and required m-calpain proteolytic activity (Fig. 5, bottom). Inducible phosphorylation is apparently not required for calpain-induced Ikappa Balpha proteolysis because the phosphorylation-defective Ikappa Balpha mutant, S32/36A, is inducibly degraded, and nonphosphorylated recombinant Ikappa Balpha (Ikappa Balpha expressed in Escherichia coli) is efficiently degraded in vitro (not shown).


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Fig. 5.   Endogenous Ikappa Balpha proteolysis by purified m-calpain in broken cell assay. Top panel, time course. Western blot of Ikappa Balpha abundance following introduction of m-calpain in broken cell assay for indicated times. Arrow, 30-kDa Ikappa Balpha intermediate (cf. Fig. 1c). Bottom panel, dose response and sensitivity to calpain inhibitors. Calpain proteolytic activity is required for Ikappa Balpha proteolysis.

To determine whether calpains could result in NF-kappa B activation, nuclei were purified on sucrose cushions and nuclear proteins extracted. Gel mobility shift assays showed that m-calpain induced a time- and dose-dependent increase in Rel A:NF-kappa B1 DNA binding activity (Fig. 6a, indicated as complex 2, a species previously characterized by supershift assay (12)). To additionally demonstrate NF-kappa B translocation, changes in 65-kDa Rel A nuclear abundance was measured by Western immunoblot (Fig. 6b), where a 2.3-fold increase in Rel A in the m-calpain treated nuclei was seen. We therefore conclude that ligand-independent Ikappa Balpha proteolysis and NF-kappa B activation can be effected by m-calpain.


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Fig. 6.   Ligand-independent activation of Rel A:NF-kappa B1 by purified m-calpain catalytic subunit in broken cell assay. a, gel mobility shift assay for nuclear DNA binding activity. Purified m-calpain was added to broken cell assay at the indicated concentrations and times. Following incubation, nuclei were prepared by sucrose cushion centrifugation. Assays were performed using 15 µg of nuclear extracts binding to radiolabeled angiotensinogen NF-kappa B-binding site (12). Migration of RelA·NF-kappa B1 heterodimer (complex C2) demonstrated previously by supershift assay is shown (12). Left panel, time course. RelA:NF-kappa B1 DNA-binding activity increases 1.16- (5 min) and 2.07-fold (15 min). Right panel, dose response. b, Rel A nuclear translocation by m-calpain. Following calpain treatment of broken cells (0.1 units of m-calpain as in a), Rel A was extracted from sucrose cushion-purified nuclei and detected by Western blot. Rel A increases 2.3-fold (arrow). Nonspecific band (NS) as control for protein loading is indicated.

Degradation of Ikappa Balpha in Ubiquitin-Proteasome-defective Cell Lines-- As additional evidence for calpain-mediated, proteasome-independent pathway for Ikappa B proteolysis and NF-kappa B activation, we analyzed the effect of TNF-alpha in Balb/c 3T3 cells conditionally defective in the ubiquitin-proteasome pathway. ts20b cells express a temperature-sensitive E1 responsible for initial ATP-dependent step in the Ubn reaction (14, 18), whereas control H38-5 cells are corrected ts20b stably transfected with the wild-type E1 (14). Relative stimulation of calpain activity was observed in individual cells measuring hydrolysis of the cell-permeant calpain substrate Boc-Leu-Met-CMAC incubated with TNF-alpha (30 ng/ml). In ts20b cells, calpain activity increased from 1.70 ± 0.15 arbitrary units/min (n = 29) to 2.49 ± 0.15 arbitrary units/min (n = 30, 60 min, 32 °C) after TNF-alpha incubation (p = 0.0005, two-tailed t test). After E1 inactivation by culture in ts20b cells at the restrictive temperature (39 °C), TNF-alpha -induced Ikappa Balpha proteolysis was still detectable at 15 min and continued until 60 min (Fig. 7a, top). By contrast, identically treated H38-5 cells showed a more rapid Ikappa Balpha proteolysis with a nadir at 15 min, followed by its resynthesis over 60-120 min. This observation excludes nonspecific temperature effects on the TNF-alpha signaling pathway. In both cell types, DNA binding activity of the Rel A:NF-kappa B1 heterodimer was induced in parallel to Ikappa Balpha proteolysis (Fig. 7a, bottom). Finally, Ikappa Balpha proteolysis in restricted ts20b cells is blocked by calpain (Z-LnL, E64, and Z-LLY), and not by proteasome inhibitors (Fig. 7b). We note the slower kinetics of Ikappa Balpha proteolysis in the temperature-restricted ts20b cells are remarkably similar to those of lactacystin-treated HepG2 cells (Fig. 1b). Together, these data indicate that calpain-induced Ikappa Balpha turnover is slower than when both proteolytic systems are intact.


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Fig. 7.   TNF-alpha -induced Ikappa Balpha degradation in ubiquitin pathway-defective cells is calpain-dependent. a, top, Western immunoblots for Ikappa Balpha in E1-deficient ts20b and wild-type H38-5 cells. Upon TNF-alpha treatment, relative to control values, Ikappa Balpha was 83% (15 min), 11% (1 h), and 43% (2 h) in ts20b cells, and 43% (15 min), 63% (1 h), and 68% (2 h) of controls in H38-5 cells. Bottom, gel shift assay. Rel A:NF-kappa B1 DNA binding activity (C2) increased in parallel with cytoplasmic Ikappa Balpha proteolysis. b, effect of protease inhibitors on inducible Ikappa Balpha degradation in ts20b cells. Western immunoblots are shown using Ikappa Balpha and Rel B (internal control) antibodies. Inducible Ikappa Balpha degradation is blocked by Z-LnL, E64, and Z-LLY, but not by lactacystin (Lact) and PMSF, indicating Ikappa Balpha proteolysis occurs via a calpain-sensitive pathway.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Calpains are intracellular calcium-dependent cysteine proteases whose ubiquitously expressed subunits include milli (m)-calpain and micro (µ)-calpain. Although these heterodimeric isoforms have indistinguishable substrate affinities, m- and µ-calpain are found in distinct subcellular localizations and therefore may subserve distinct physiological roles (24, 25). Here we show for the first time that the calpain-calpastatin system is a parallel pathway partly responsible for TNF-alpha -inducible Ikappa B proteolysis and NF-kappa B activation. TNF-alpha , therefore, activates NF-kappa B through the participation of two distinctly regulated cytoplasmic (nonlysosomal) protease systems as follows: (i) the constitutive proteasome pathway, where Ikappa Balpha proteolysis is governed by its rate-limiting post-translational modification (coupled phosphorylation/ubiquitination), and (ii) the inducible calpain-calpastatin system, where protease activity is directly modified by TNF-alpha .

In the past, distinguishing between the effects of calpains and the proteasome in intracellular regulatory processes has been difficult because few selective inhibitors of the two cytoplasmic protease systems were identified. Data presented herein indicate that pathway-selective probes exist that can be used to dissect the parallel function of these protease systems in cytokine signaling. A role for the calpain-calpastatin pathway mediating NF-kappa B activation is based on the convergence of the following observations. 1) Inducible Ikappa Balpha proteolysis is only be partially blocked by either calpain-selective or proteasome-selective inhibitors and completely blocked by both. 2) In TNF-alpha -stimulated cells, a rapid (within 1 min), 2.2-fold increase in cytosolic calpain proteolytic activity in intact cells is measured. 3) Calpain proteolytic activity occurs indistinguishably with the particulate to cytosol redistribution of the catalytic m-calpain subunit. 4) Ikappa Balpha proteolysis occurs coincidentally with increases in m-calpain abundance in the cytosol. 5) Introduction of catalytically active m-calpain is sufficient to produce ligand-independent NF-kappa B activation. 6) Calpain-dependent Ikappa Balpha proteolysis is demonstrated in cells lacking proteasome activity (ts20b cells).

The mechanism for activation of calpains in intact cells is unknown. In vitro, calpains exposed to nonphysiological concentration of calcium acquire enzymatic activity through auto-proteolysis of its constituent subunits (1). In intact cells, evidence for autolytic activation or activation following changes in intracellular calcium concentration is weak. In other studies, calpains are known to be long-lived proteins with half-lives of >5 days; this observation would not be consistent with an autolytic protease (27). Our data indicates that TNF-alpha -stimulated calpain activity occurs in the absence of detectable autolysis because autolytic products are not detected at times when changes in protease activity can be measured. Moreover, calpain activation in the absence of detectable changes in intracellular calcium concentrations has been described in hepatocytes (26). In data not shown, we have not observed any changes in total intracytoplasmic calcium concentrations in HepG2 cells. Nevertheless, intracellular calcium is apparently required for calpain activity in intact cells, because intracellular calcium chelators block calpain activity and Ikappa Balpha proteolysis (Table I, data not shown).

One other mechanism for calpain activation could include changes in subcellular localization. Calpains are not randomly distributed throughout the cell. In cultured cell lines, m-calpain is distributed in a fine reticular network in the cytosol, implicating an association with cytoskeletal elements (28), and in the central nervous system, m-calpain content is membrane-associated (29). In cultured HepG2 cells, we observe consistently that m-calpain redistributes into the soluble cytoplasmic fraction, a fraction containing Ikappa Balpha , following TNF-alpha treatment. Whether redistribution is the mechanism for m-calpain activation will require additional investigation. Although m-calpain activity in the membrane fraction was previously thought to be important for proteolysis of protein kinase C (30), cytosolic calpain activity appears to be important in turnover of the p53 oncoprotein (18). Based on our subcellular fractionation experiments, the intracellular site of proteolysis of Ikappa Balpha probably also occurs in the cytosol.

Calpain activity is inducible following activation of other hormone receptors, including the hepatic purinergic receptor (17), and the pituitary thyrotropin-releasing hormone receptor (31), perhaps indicating a role for second messenger involvement. Others have shown that phospholipid mediators, including second messengers implicated in TNF-alpha signaling, can activate calpain catalytic activity through a mechanism that may involve their direct binding to the 30-kDa regulatory subunit (29, 32, 33). These lipids apparently lower calcium requirements to a range normally found intracellularly (29). Lipid mediators may be important intermediates for TNF-alpha -induced calpain activity for several reasons. First, TNF-alpha is known to increase ceramide production through its effects on acid sphingomyelinase activity in endosomal compartments (34); this second messenger has been linked to NF-kappa B activation (35). Second, ceramide directly stimulates intracellular calpain activity in permeabilized cells (32).

Calpains are increasingly recognized to be important regulators of intracellular signaling processes. Initially described in turnover of activated protein kinase C, erythrocyte ankyrin, and calmodulin-binding proteins (Ref. 1 and references therein), calpains have recently been implicated in mediating turnover of the c-Fos transcription factor (36, 37) and the tumor suppressor gene product p53 (18). Interestingly, both c-Fos and p53 were initially described to be proteasome substrates. Our data adds Ikappa Balpha to the emerging list of key regulatory proteins acted upon by a parallel calpain-proteasome pathway. Of relevance to Ikappa Balpha , erythrocyte ankyrin itself is a substrate for calpain proteolysis (18, 38).

The TNF-alpha -inducible calpain pathway mediating Ikappa Balpha proteolysis described here is probably distinct from the two previously reported nonproteasome-dependent Ikappa Balpha proteolytic pathways (10, 11). In the first report, constitutive Ikappa Balpha turnover in an undifferentiated pre-B lymphocytic cell line was not inhibited by the potent calpain inhibitors, calpain inhibitors I and II, or Z-LLF, agents that interfere with Ikappa Balpha proteolysis in our system (Fig. 1 and see Ref. 12). Second, potent calpain inhibitors MG132 and Z-LLF do not have significant effects on Ikappa Balpha proteolysis in respiratory syncytial virus-infected epithelial cells (11). The relationship of these pathways to calpain-calpastatin pathway, therefore, seems unlikely.

In summary, we implicate the calpain-calpastatin and proteasome pathways are parallel mechanisms mediating inducible Ikappa Balpha proteolysis by the cytokine TNF-alpha . These data indicate that calpains contribute to rapid Ikappa Balpha proteolysis through a mechanism involving changes in total cytosolic calpain proteolytic activity.

    ACKNOWLEDGEMENTS

We thank Drs. E. J. Corey for lactacystin; Mark Suto for Z-LLF; Harvey Ozer for ts20b and H38 cells; Lorie Schowalter for technical assistance; J. Griffin for FACS analysis; and E. A. Thompson, Jr., and L. Reuss for critical comments on the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Established Investigator of the American Heart Association. Supported by NHLBI Grant 55630, Council for Tobacco Research Grant 4017, and NIEHS Grant P30 ES06676 (to R. S. Lloyd, University of Texas Medical Branch). To whom correspondence should be addressed: MRB 8.138, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1060. Tel.: 409-772-2824; Fax: 409-772-8709; E-mail: arbrasie{at}utmb.edu.

The abbreviations used are: TNF-alpha , tumor necrosis factor-alpha ; AMC, 7-amino-4-methylcoumarin; E64, trans-epoxysuccinyl-L-leucylamido-(4-guanido)butane; Ikappa Balpha , inhibitor of NF-kappa B; NF-kappa B, nuclear factor-kappa B; 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.
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Abstract
Introduction
Procedures
Results
Discussion
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