 |
INTRODUCTION |
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-
(TNF-
).1
Following binding its receptor on the plasma membrane, TNF-
initiates de novo transcription of genetic networks, in
part, through activating nuclear translocation of the cytoplasmic
transcription factor nuclear factor-
B (NF-
B) (4, 5). NF-
B, a
multiprotein complex inactivated in the cytoplasm by association with
its I
B inhibitor, translocates into the nucleus following
dissociation of the NF-
B·I
B complex. TNF-
modifies
NF-
B·I
B association through a process initiated by
inducible I
B
serine phosphorylation on its amino-terminal
regulatory domain, a modification coupled to I
B polyubiquitination
(Ubn) on adjacent lysine residues (6). NF-
B·I
B
dissociation requires I
B proteolysis because phosphorylated and
ubiquitinated I
B still inactivates NF-
B (Ref. 6 and references therein).
Presently, the ubiquitin-proteasome system has been the only pathway
identified in mediating cytokine-inducible I
B proteolysis. Pretreatment with cell-permeant proteasome inhibitors blocks
TNF-
-inducible I
B proteolysis concomitantly with the accumulation
of Ubn- and phosphorylated I
B intermediates (6, 8).
Independently, inducible I
B
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 I
B
proteolysis. First, in pre-B lymphocytes, c-Rel:NF-
B1 is
constitutively nuclear as the consequence of a
calcium-dependent proteolytic activity that preferentially
affects I
B
(rather than I
B
(10)). Second, we have observed
a non-proteasome-dependent pathway mediating inducible
I
B
proteolysis (and NF-
B activation) following respiratory syncytial virus infection of human airway epithelial cells (11). However, whether additional nonproteasome-dependent
pathways participate in cytokine-inducible NF-
B activation have not
been explored.
These studies prompted us to examine whether
nonproteasome-dependent pathways participate in
cytokine-inducible I
B
degradation. Here we investigate the
proteolytic mechanism involved in a well characterized model of NF-
B
activation in TNF-
-stimulated HepG2 hepatocytes, where NF-
B
activation mediates the expression of acute phase reactants (12, 13).
By using calpain and proteasome-selective inhibitors, we demonstrate
that inducible I
B
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-
rapidly activates
cytosolic calpain proteolytic activity. In subcellular fractionations
of TNF-
-stimulated cells, the catalytic m-calpain subunit
translocates from the particulate into the cytosolic fraction (the
latter containing I
B
) coincidentally with I
B
proteolysis. Moreover, TNF-
-inducible I
B
proteolysis occurs in cells
conditionally deficient in the ubiquitin-proteasome pathway, and in
cells expressing I
B
mutations deficient in
proteasome-dependent processing. Together, these data
implicate calpains are a parallel pathway in mediating I
B
proteolysis and NF-
B activation.
 |
EXPERIMENTAL PROCEDURES |
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-
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-
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-
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-
stimulation.
Antibodies and Western Immunoblots--
Antibodies used were to
I
B
(sc-203, recognizing amino acids 6-20, and sc-371,
recognizing amino acids 297-317), I
B
(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
and
subunits, corresponding to the human
proteasome subunit
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-
-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
. Both RING 10 and
,
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-
(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-
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 I
B
Proteolytic Assay in
Cytosolic S100 Extract--
Two hundred µg of protein
100,000 × g supernatant (S100) was
incubated with 150 ng of recombinant human I
B
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. I
B
degradation was
quantitated by Western immunoblot.
I
B Proteolysis and NF-
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). I
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-
B-binding site from
the angiotensinogen promoter as described (12).
 |
RESULTS |
Non-proteasomal Component for TNF-
-inducible I
B
Proteolysis Is Sensitive to Calpain Inhibitors--
We have previously
shown that administration of TNF-
(30 ng/ml) to HepG2 cells induces
rapid I
B (
and
) proteolysis and NF-
B translocation,
maximally detectable 15 min following addition of hormone (12). To
examine initially if proteasome-independent pathways for I
B
proteolysis exist, I
B abundance was assayed by Western immunoblots
in protease inhibitor pretreated cells harvested 15 min after TNF-
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 I
B isoform. In the absence of
protease inhibitors, TNF-
produced rapid proteolysis of both 37-kDa
I
B
and 49-kDa I
B
(compare lanes 1 and
2). Pretreatment with lactacystin, Z-LLF, or Z-LLL blocked
I
B
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
I
B
proteolysis (6).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Calpain inhibitors affect inducible I B
proteolysis. a, cytoplasmic extracts from control
(lane 1) or TNF- -treated cells (15 min, lanes
2-8) were analyzed by Western immunoblot using a mixture of I B and I B antibodies (indicated at
left). Cells were stimulated following preincubation with
indicated protease inhibitors (doses under "Experimental
Procedures"). Compared with TNF- treatment alone, I B
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- induced I B degradation in Hep G2 cells.
Apparent I B half-life is 1-3 min following TNF- treatment,
preceded by I B phosphorylation (I B P).
Bottom panel, I B half-life in lactacystin-pretreated
cells (10 µM for 1 h). TNF- induces I B
degradation in the time-dependent manner even in the
absence of detectable 26 S proteasome activity. ~30-kDa I B
intermediate produced is indicated with arrow
(left) detected on longer exposure. c, combined
effect of proteasome and calpain inhibition completely blocks inducible
I B 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- -stimulated (15 min). Rel B staining is used as an internal
control for protein recovery. TNF- -induced I B degradation is
totally blocked by the combined treatment of Z-LLY and lactacystin.
d, calpastatin blocks NF- 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- -stimulated cells is shown. e,
capastatin expression blocks I B proteolysis. HepG2 cells
transfected with IL-2 receptor expression plasmid ± pcDNA
I-capastatin expression plasmid were TNF- -stimulated and
transfectants affinity purified. Shown is a Western blot of I B
(top) and control -actin (bottom). Lane
1, control-treated; lanes 2-4, TNF- -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 I B signal to -actin in lane
2 is 30%; lane 3 is 35%, and lane 4 is
50%.
|
|
In contrast, I
B
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 I
B
proteolysis. Surprisingly, moreover, the calpain inhibitors Z-LnL and
E64 also partially blocked I
B
proteolysis, even under conditions
where I
B
proteolysis was unaffected (compare lane 2 with 6 and 7). These data suggest a parallel
contribution of calpain-like proteases in TNF-
-inducible I
B
hydrolysis.
To define the kinetics of proteasome-independent pathways mediating
I
B
proteolysis, I
B
half-life in TNF-
-treated cells was
compared in cells containing with that in cells lacking proteasome activity (Fig. 1b). In cells not treated with protease
inhibitors, I
B
proteolysis is rapid (t1/2 of 1-3 min), occurring coincidentally with the generation of
phosphorylated I
B
intermediates (Fig. 1b,
I
B
P). In cells pretreated with the potent
irreversible proteasome inhibitor lactacystin, I
B
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 I
B
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 I
B
proteolysis. In the presence of both
inhibitor types, I
B
proteolysis was completely blocked with
accumulation of non- and phosphorylated I
B
intermediates (Fig.
1c). We note consistently that I
B
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-
B activation, the effect of transiently expressed calpastatin was determined on NF-
B-dependent
reporter activity in transient cotransfection assay (18, 22). We have previously shown that the human IL-8 promoter is TNF-
-inducible in a
manner solely dependent on a high affinity NF-
B site (23). Cotransfection of calpastatin expression plasmid (pcDNA
I-calpastatin) did not affect basal IL-8/CAT activity but significantly
blocked TNF-
-inducible CAT activity (Fig. 1d). As a
control, the effect of calpastatin on I
B
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-
.
Following isolation of transfected cells, a Western immunoblot was done
to determine changes in I
B
in cytosolic lysates (Fig.
1e). In the presence of 2.5 µg of pcDNA I-calpastatin,
I
B
proteolysis was inhibited by ~50%. Combined, these data
strongly suggest a parallel contribution of the calpain system in
TNF-
-inducible proteolysis of I
B
and NF-
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-
-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-
-stimulated cells.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 2.
Specificity of inhibitors on proteasome and
calpain activities in vitro. a, proteasome
activity in cell lysates. Control and TNF- -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-
-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)).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of TNF- 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- 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- (30 ng/ml), TNF- + Z-LnL (100 µM), or TNF- + lactacystin (10 µM, 30 min). The basal activity in each
cell was measured for use as its own control (open bars)
prior to TNF- stimulation (solid bars). Increased
proteolytic rate occurred only for TNF- (p < 0.001)
and TNF- with lactacystin (p < 0.05). c,
kinetics of change in intracellular fluorescence intensity in
TNF- -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-
increased calpain activity ~2.2-fold
over the basal rate within 1 min of exposure (Fig. 3a). The TNF-
-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-
-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-
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-
Induces Changes in m-Calpain Abundance--
For m-calpain
proteolytic activity to be relevant for I
B
proteolysis, we sought
to determine the subcellular distribution of m-calpain in control and
TNF-
-stimulated cells. For this, particulate (S100
pellet) and cytosolic (S100 supernatant) fractions were
prepared at various times following TNF-
treatment by
ultracentrifugation at 100,000 × g and analyzed for
both 80-kDa m-calpain catalytic subunit and I
B
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-
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 I
B
and that changes in m-calpain subunit occurred concomitantly with I
B
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).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 4.
Calpain particulate-cytosol translocation and
hydrolysis of recombinant human I B (rhI B ). a,
Western immunoblot of m-calpain 80-kDa catalytic subunit and I B
in particulate (100,000 × g pellet, Ref. 18) and
cytosolic (100,000 × g supernatant) fractions of
TNF- -treated cells. Relative to control, in TNF- -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 I B by
TNF- -treated S100 cytosolic extract. Top
panel, time course. Bottom panel, effect of calpain
inhibitors. S100-induced rhI B 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-
-treated
cells containing translocated m-calpain catalytic subunit (determined by Western immunoblot, Fig. 4) also contain I
B
proteolytic
activity, an in vitro protease assay was established (Fig.
4b). In this assay, purified recombinant human I
B
(rhI
B
) was added to TNF-
-stimulated cytosolic lysates
(S100 supernatant), and the effect on rhI
B
proteolysis was determined by Western immunoblot. We observed a
time-dependent proteolysis of rhI
B
, 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 I
B
Proteolysis
and NF-
B Translocation--
To determine whether m-calpain
proteolyzed native I
B within the NF-
B·I
B complex and could
produce ligand-independent NF-
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
I
B
(Fig. 5, top). I
B
was proteolyzed into
transiently stabilized carboxyl-terminal intermediates of ~30 kDa
(arrow, Fig. 5), an intermediate also seen in
TNF-
-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 I
B
proteolysis because the
phosphorylation-defective I
B
mutant, S32/36A, is inducibly
degraded, and nonphosphorylated recombinant I
B
(I
B
expressed in Escherichia coli) is efficiently degraded
in vitro (not shown).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 5.
Endogenous I B proteolysis by purified
m-calpain in broken cell assay. Top panel, time course.
Western blot of I B abundance following introduction of m-calpain
in broken cell assay for indicated times. Arrow, 30-kDa
I B intermediate (cf. Fig. 1c). Bottom
panel, dose response and sensitivity to calpain inhibitors.
Calpain proteolytic activity is required for I B
proteolysis.
|
|
To determine whether calpains could result in NF-
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-
B1 DNA
binding activity (Fig. 6a,
indicated as complex 2, a species previously characterized by
supershift assay (12)). To additionally demonstrate NF-
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 I
B
proteolysis and NF-
B
activation can be effected by m-calpain.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 6.
Ligand-independent activation of Rel
A:NF- 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- B-binding site (12). Migration of RelA·NF- B1 heterodimer
(complex C2) demonstrated previously by supershift assay is
shown (12). Left panel, time course. RelA:NF- 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 I
B
in Ubiquitin-Proteasome-defective Cell
Lines--
As additional evidence for calpain-mediated,
proteasome-independent pathway for I
B proteolysis and NF-
B
activation, we analyzed the effect of TNF-
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-
(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-
incubation (p = 0.0005, two-tailed t test). After E1 inactivation by culture in
ts20b cells at the restrictive temperature (39 °C), TNF-
-induced I
B
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 I
B
proteolysis with a nadir at 15 min, followed by its
resynthesis over 60-120 min. This observation excludes nonspecific
temperature effects on the TNF-
signaling pathway. In both cell
types, DNA binding activity of the Rel A:NF-
B1 heterodimer was
induced in parallel to I
B
proteolysis (Fig. 7a,
bottom). Finally, I
B
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 I
B
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 I
B
turnover is slower than
when both proteolytic systems are intact.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 7.
TNF- -induced I B degradation in
ubiquitin pathway-defective cells is calpain-dependent.
a, top, Western immunoblots for I B in E1-deficient
ts20b and wild-type H38-5 cells. Upon TNF- treatment,
relative to control values, I B 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- B1 DNA binding activity (C2) increased in
parallel with cytoplasmic I B proteolysis. b, effect of
protease inhibitors on inducible I B degradation in
ts20b cells. Western immunoblots are shown using I B
and Rel B (internal control) antibodies. Inducible I B degradation
is blocked by Z-LnL, E64, and Z-LLY, but not by lactacystin
(Lact) and PMSF, indicating I B proteolysis occurs via
a calpain-sensitive pathway.
|
|
 |
DISCUSSION |
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-
-inducible I
B
proteolysis and NF-
B activation. TNF-
, therefore, activates
NF-
B through the participation of two distinctly regulated
cytoplasmic (nonlysosomal) protease systems as follows: (i) the
constitutive proteasome pathway, where I
B
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-
.
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-
B activation is based
on the convergence of the following observations. 1) Inducible I
B
proteolysis is only be partially blocked by either calpain-selective or
proteasome-selective inhibitors and completely blocked by both. 2) In
TNF-
-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) I
B
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-
B
activation. 6) Calpain-dependent I
B
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-
-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 I
B
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 I
B
, following
TNF-
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 I
B
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-
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-
-induced calpain
activity for several reasons. First, TNF-
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-
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 I
B
to the emerging list of key regulatory proteins
acted upon by a parallel calpain-proteasome pathway. Of relevance to
I
B
, erythrocyte ankyrin itself is a substrate for calpain
proteolysis (18, 38).
The TNF-
-inducible calpain pathway mediating I
B
proteolysis
described here is probably distinct from the two previously reported
nonproteasome-dependent I
B
proteolytic pathways (10, 11). In the first report, constitutive I
B
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 I
B
proteolysis in our system (Fig. 1
and see Ref. 12). Second, potent calpain inhibitors MG132 and Z-LLF do
not have significant effects on I
B
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 I
B
proteolysis by the cytokine TNF-
. These data indicate that calpains contribute to rapid I
B
proteolysis through a mechanism involving changes in total cytosolic calpain proteolytic activity.