From the Department of Molecular Biology, Unit for Molecular Signal Transduction in Inflammation, Flanders Interuniversity Institute for Biotechnology and Ghent University, 9000 Ghent, Belgium
Received for publication, March 13, 2001
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ABSTRACT |
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Cathepsin B has previously been shown to
proteolytically activate the proinflammatory caspase-11 in
vitro. Here we show that cathepsin B is not involved in
activation of caspase-11 induced by lipopolysaccharide (LPS) and
subsequent maturation of interleukin (IL)-1 Chronic inflammatory diseases are caused by prolonged production
of several proinflammatory cytokines such as interleukin (IL1)-1, IL-2, IL-6, IL-8,
and tumor necrosis factor (TNF), as well as genes encoding nitric oxide
synthase, cell adhesion molecules, and immunoreceptors (1). The
expression of many of these genes is regulated by the nuclear factor
In addition to transcriptional regulation, translational or
post-translational regulation is also involved in the production of a
number of inflammatory mediators. For example, the maturation and
secretion of IL-1 Cells and Reagents--
The murine macrophage cell lines Mf4/4
(12) and pU5.18 (ATCC, Manassas, VA) were maintained in RPMI
1640 medium supplemented with 10% fetal calf serum and 1 mM sodium pyruvate. z-FA.fmk and SB203580 were obtained
from Calbiochem-Novabiochem. E64 was purchased from Roche
Molecular Biochemicals. LPS originated from Sigma, and anti-IL-1 Measurement of Cathepsin B Activity--
To determine cathepsin
B activity in total cell extracts, cells were lysed in 10 mM Tris/HCl, pH 7, 1% Nonidet P-40, 200 mM NaCl, 5 mM EDTA, and 1 mM phenylmethylsulfonyl
fluoride. Cleared cell extracts (25 µg of cellular protein) were
incubated for 1 h at 30 °C in cell-free system buffer (10 mM Hepes/NaOH, pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM
KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM
dithiothreitol) with 50 µM of the fluorogenic cathepsin B
substrate benzyloxycarbonyl-Ala-Arg-Arg-aminotrifluoromethylcoumarin (z-ARR.afc) (Enzyme Systems Products). The release of free
7-amino-4-trifluoromethylcoumarin (afc) was monitored for 60 min in a
fluorometer (CytoFluor; PerSeptive Biosystems, Cambridge, MA) at an
excitation wavelength of 409 nm and an emission wavelength of 505 nm.
Data are expressed as increase in fluorescence as a function of time
( IL-1 Bioassay--
Macrophages were seeded in 6-well plates at
5 × 105 cells/well. After 24 h,
cells were treated for 1 h with cathepsin B inhibitors before LPS
stimulation (1 µg/ml). Supernatant was harvested after 48 h and
assayed for IL-1 in a CTLL cell proliferation assay (13).
Western Blot Analysis--
Cells were lysed in 10 mM
Tris/HCl, pH 7, 1% Nonidet P-40, 200 mM NaCl, 5 mM EDTA, and 1 mM phenylmethylsulfonyl
fluoride. Lysates were cleared by centrifugation; cell extracts (100 µg of total protein) were subjected to SDS-polyacrylamide gel
electrophoresis and transferred to Hybond nitrocellulose membranes
(Amersham Pharmacia Biotech). These were blocked with 5% dry milk in
phosphate-buffered saline containing 0.1% Tween 20 and were probed
with primary and secondary antibodies according to the manufacturer's
instructions. Immunoreactivity was revealed with the enhanced
chemiluminescence method (ECL; Amersham Pharmacia Biotech).
Transient Transfection and Reporter Gene Assays--
Mf4/4 cells
were seeded at a density of 4 × 105 cells/6-well
plate. They were transfected with the LipofectAMINE transfection method
(Life Technologies, Inc.) using p( NF- Measurement of Cytokine mRNA Expression--
Total RNA was
extracted from 2 × 106 Mf4/4 cells with RNAzol
(Biotecx Laboratories, Houston, TX) and then analyzed for specific cytokine mRNA expression with a RiboQuant custom mouse template set
(PharMingen, San Diego, CA) as described in the manufacturer's protocol.
z-FA.fmk Inhibits IL-1 z-FA.fmk Inhibits the LPS-induced Production of IL-1 z-FA.fmk Inhibits NF-
We subsequently analyzed the effect of z-FA.fmk on LPS-induced NF-
An alternative explanation for the z-FA.fmk-induced mobility shift
could be that z-FA.fmk inhibits the potential proteolytic processing of
one of the components of the NF- z-FA.fmk Has No Effect on the LPS-induced Activation of p38
MAP Kinase--
We and others previously demonstrated that p38 MAP
kinase plays an essential role in the transactivation of NF- Cathepsin B is a cysteine protease implicated in a number of
inflammatory diseases and pathological conditions, such as bronchitis, rheumatoid arthritis, acute pancreatitis, and cancer progression (5,
6). The main function of cathepsin B is the degradation of proteins
that have entered the lysosomal system from outside the cell (via
endocytosis or phagocytosis) or from other compartments within the cell
(autophagy). In addition, activation of a number of proteins such as
procollagenase, proplasminogen activator, and prorenin has been
described (20-22). Recently, we demonstrated that purified cathepsin B
is also able to proteolytically activate the proinflammatory caspase-11
in vitro (4). The latter results prompted us to investigate
the role of cathepsin B in the activation of caspase-11 and the
subsequent production of IL-1 in LPS-stimulated macrophages. For these
experiments we made use of z-FA.fmk and E64, which are frequently used
inhibitors of cathepsin B (23, 24). However, none of these inhibitors
was able to prevent the LPS-induced processing of caspase-11 in
macrophages. To our surprise, z-FA.fmk completely inhibited the
LPS-induced production of IL-1 Our observations illustrate that results obtained with the so-called
specific cathepsin B inhibitor z-FA.fmk should be interpreted with care
and verified by other inhibitors before any conclusion on the role of
cathepsin B can be drawn. For example, cathepsin B has been implicated
in rheumatoid arthritis, and z-FA.fmk has been used successfully to
treat that disease (11). However, the role of NF- Our studies also revealed that the band pattern obtained in EMSA for
NF- In conclusion, we have demonstrated that z-FA.fmk can behave as a
powerful inhibitor of NF- in macrophages.
Nevertheless, we found that the cathepsin B inhibitor
benzyloxycarbonyl-Phe-Ala-fluoromethylketone (z-FA.fmk) prevents
LPS-induced production of IL-1
, IL-1
, and tumor necrosis factor
at the transcriptional level. The latter was not because of cathepsin B
inhibition, but was mediated by inhibition of the transactivation
potential of the nuclear factor
B (NF-
B). z-FA.fmk did not
prevent LPS-induced activation of p38 mitogen-activated protein kinase,
which was shown to be involved in NF-
B transactivation in response
to LPS. These results suggest that the previously described therapeutic
effect of z-FA.fmk in the treatment of rheumatoid arthritis might not
only result from inhibition of cathepsin B but also implicates an
important contribution from the inhibition of
NF-
B-dependent gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B (NF-
B). In almost all cell types, NF-
B exists as a dimer of
a p50 and p65 subunit that is retained in an inactive cytoplasmic
complex by binding to a third inhibitory subunit (I
B). NF-
B can
be activated by many stimuli including lipopolysaccharide (LPS), TNF,
and IL-1, which induce phosphorylation and proteolytic degradation of
the I
B subunit. This allows NF-
B to translocate to the nucleus,
where it can bind to specific recognition sequences in the promoter
region of various genes and stimulate their transcription.
is controlled by caspase-1 (2), which itself is
activated by caspase-11 (3). The critical role of caspase-1 and
caspase-11 in inflammation is well illustrated by the finding that
caspase-1- or caspase-11-deficient mice are resistant to LPS-induced
septic shock (3). Recently, we showed that caspase-11 could be
activated by cathepsin B in vitro (4). Cathepsin B is
implicated in a number of inflammatory diseases and pathological conditions, such as bronchitis, rheumatoid arthritis, acute
pancreatitis, and cancer progression (5, 6). In the case of rheumatoid arthritis, it is well documented that cytokines induce the secretion of
lysosomal proteases of the cathepsin family by the main cellular population of rheumatoid synovial membranes and fluids (7-9). Inhibitors of these proteases abrogate the articular degradation and
maintenance of rheumatoid lesions and have already proven their
therapeutic potential in diseases of excessive bone resorption, such as
rheumatoid arthritis or osteoporosis (10).
Benzyloxycarbonyl-Phe-Ala-fluoromethylketone (z-FA.fmk) is a peptide
inhibitor that irreversibly alkylates the active cysteine residue in
cathepsin B, thereby inhibiting its proteolytic activity. In
vivo, z-FA.fmk has been shown to inhibit the severity of
inflammation, as well as the extent of cartilage and bone damage in
adjuvant-induced arthritis (11). In the present study we demonstrate
that z-FA.fmk is also a potent inhibitor of
NF-
B-dependent gene expression in macrophages, which might contribute to its described anti-inflammatory activity.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
antibody was from R & D Systems (Minneapolis, MN). Polyclonal
anti-caspase-11 antibodies were raised in rabbits against purified
murine caspase-11. Antibodies against p50, p65, and I
B were from
Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal phosphospecific
and polyclonal p38 mitogen-activated protein (MAP) kinase antibodies
were purchased from New England Biolabs (Beverly, MA).
F/min).
B)3LUC, which contains the luciferase gene after three copies of the NF-
B recognition sequence, and a minimal promoter (14). 48 h after transfection, cells were trypsinized and seeded at 1.2 × 105
cells/24-well plate. 66 h after transfection, cells were treated for 1 h with 50 µM z-FA.fmk, E64, or solvent
Me2SO, after which they were either noninduced or
induced for 6 h with 1 µg/ml LPS. Inducible promoter activity
was determined by measuring the luciferase activity present in cell
extracts as described previously (14).
B Gel Retardation Assay--
Mf4/4 cells (106
cells/6-well plate) were pretreated with or without 50 µM
z-FA.fmk for 1 h and stimulated with LPS (1 µg/ml for 1 h).
Nuclear extract preparations and electrophoretic mobility shift assays
(EMSA) were performed essentially as described previously (15).
Extraction buffers contained several protease inhibitors including 2 mM Pefabloc (Pentapharm, Basel, Switzerland), 0.15 IU/ml
aprotinin, and 10 µg/ml leupeptin. NF-
B DNA binding was analyzed
by incubating 8 µg of nuclear proteins for 30 min with an
NF-
B-specific 32P-labeled oligonucleotide probe
(5'-agctATGTGGGATTTTCCCATGAGCagct-3'), containing the NF-
B
recognition sequence of the immunoglobulin
-chain promoter.
Protein·DNA complexes were separated on a 4% native
polyacrylamide gel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Production in LPS-stimulated Macrophages
Independently of Cathepsin B--
In line with our previous findings
that cathepsin B can activate caspase-11 in vitro (4), we
studied the effect of two cathepsin B inhibitors, viz. E64
and z-FA.fmk, on the LPS-induced activation of procaspase-11 and
production of IL-1
by macrophages, which is known to require
caspase-11 (3). Mf4/4 cells were preincubated for 1 h with 50 µM E64 or z-FA.fmk and subsequently stimulated with 1 µg/ml LPS for 48 h. As expected, pretreatment of Mf4/4 cells
with z-FA.fmk or E64 completely inhibited cathepsin B activity over a
period of 1 to 72 h as measured with the fluorogenic substrate
z-ARR.afc (see Fig. 1A and
data not shown). However, although the LPS-induced production of
IL-1
could be completely inhibited by z-FA.fmk, E64 treatment had no
effect at all. Similarly, pretreatment of the cells with the cathepsin
B-specific inhibitor CA-074Me had no effect (data not shown). Identical
results were obtained when IL-1
was measured in the supernatant in
an IL-1-dependent CTLL proliferation assay (Fig.
1B) or in cell extracts by Western blotting with
IL-1
-specific antibody (Fig. 1C, upper panel). The slightly lower IL-1 levels, detected with the bioassay in the
supernatant of E64-treated cells, are because of a limited toxic effect
of E64 on the CTLL cells used for the bioassay. In line with the above
results, proteolytic activation of procaspase-11 with the generation of
the p36 subunit still occurs in LPS-stimulated macrophages in the
presence of z-FA.fmk or E64 (Fig. 1C, lower panel). Similar observations were made in pU5.18 macrophages. Measurement of a dose response in this cell line demonstrated that
z-FA.fmk inhibits LPS-induced IL-1
production at a dose of 25-50
µM (Fig. 2), which is over
100 times higher than that required to inhibit cathepsin B (data not
shown). In conclusion, these results exclude the involvement of
cathepsin B in the LPS-induced signaling pathway leading to IL-1
production by macrophages, which is in contrast to the previously
described effect of cathepsin B in vitro (4). Moreover, our
observations also demonstrate that z-FA.fmk is able to inhibit
LPS-induced IL-1 production by a novel mechanism.
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Fig. 1.
Effect of z-FA.fmk and E64 on the production
of IL-1 and the activation of caspase-11 in
LPS-stimulated Mf4/4 cells. A, cathepsin B activity.
Cells were treated for 48 h with 50 µM of the
indicated inhibitors, and cathepsin B activity was measured in cell
extracts (25 µg of cellular protein) with the fluorogenic substrate
z-ARR.afc as described under "Materials and Methods." Data are
expressed as increase in fluorescence as a function of time
(
F/min). Me2SO (DMSO),
used as a solvent for the inhibitors, served as a negative control.
Results are representative of three independent experiments (S.D.
<10%). B, IL-1
production in cell supernatant. Cells
were incubated with 50 µM of the indicated inhibitors
1 h before stimulation with 1 µg/ml LPS for 48 h.
Me2SO, used as a solvent for the inhibitors, served as a
negative control. Supernatants were harvested and assayed in an
IL-1-dependent bioassay. Because the latter does not allow
to distinguish between IL-1
and IL-1
, IL-1
was
specifically detected by measuring the activity neutralized with
specific IL-1
-neutralizing antibodies. Results are representative of
three independent experiments (S.D. <10%). C,
up-regulation of IL-1
(upper panel) and
activation/up-regulation of caspase-11 (lower panel).
Lysates were prepared from cells treated as in B. 100 µg
of protein was subjected to SDS-polyacrylamide gel electrophoresis and
immunoblotting with anti-IL-1
and anti-caspase-11. The faster
migrating band detected with anti-caspase-11 corresponds to the p36
subunit generated by proteolytic removal of the C-terminal p10
subunit.
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Fig. 2.
Dose response of LPS-stimulated pU5.18
macrophages to z-FA.fmk. pU5.18 macrophages were incubated for
1 h with the indicated concentrations of z-FA.fmk prior to
stimulation with 1 µg/ml LPS for 48 h. Supernatants were
harvested and assayed in an IL-1-dependent bioassay.
Results are representative of three independent experiments (S.D.
<10%).
and Other
Cytokines at the Transcriptional Level--
It has previously been
demonstrated that the LPS-induced expression of several cytokines can
be regulated at the transcriptional, translational, and
post-translational levels (16). In the case of IL-1
, we demonstrated
by Western blotting that z-FA.fmk inhibits synthesis of the inactive
IL-1
precursor (Fig. 1C). To investigate whether z-FA.fmk
inhibits IL-1
synthesis at the transcriptional level, mRNA was
isolated from Mf4/4 cells that were stimulated with LPS for 3 h in
the absence or presence of 50 µM z-FA.fmk and analyzed
for the expression of several cytokine mRNAs in a RiboQuant assay.
This revealed that z-FA.fmk inhibits the LPS-induced synthesis of
IL-1
at the mRNA level (Fig. 3).
Also, the expression of IL-1
and TNF was strongly inhibited by
z-FA.fmk, whereas IL-6 synthesis, as well as transcription of the
housekeeping genes L32 and glyceraldehyde-3-phosphate
dehydrogenase, were not affected (Fig. 3).
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Fig. 3.
Effect of z-FA.fmk on mRNA levels of
several cytokines in LPS-stimulated macrophages. Mf4/4 cells were
incubated for 1 h with 50 µM z-FA.fmk prior to
stimulation with 1 µg/ml LPS for 3 h. Me2SO
(DMSO) served as a negative control. mRNA expression of
several cytokines was analyzed using a RiboQuant custom mouse template
set. IFN, interferon.
B-mediated Gene Expression in
LPS-stimulated Macrophages--
It has previously been demonstrated
that the transcription of the TNF and IL-1
genes is, at least
partly, dependent on the activation of the transcription factor NF-
B
(17). To investigate whether z-FA.fmk inhibits LPS-induced IL-1 and TNF
production by preventing LPS-induced NF-
B activation, we analyzed
its effect on NF-
B-dependent luciferase reporter gene
expression in Mf4/4 cells that were transiently transfected with
p(
B)3LUC and stimulated with LPS. Pretreatment of Mf4/4
cells with 50 µM z-FA.fmk for 1 h significantly
inhibited LPS-induced luciferase reporter gene expression (Fig.
4). As a control, expression of a
noninducible cytomegalovirus-regulated luciferase reporter was not
blocked by z-FA.fmk (data not shown). These results suggest that
inhibition of NF-
B activity by z-FA.fmk contributes to the
inhibitory effect of z-FA.fmk on the expression of IL-1
and several
other cytokines in LPS-stimulated macrophages.
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Fig. 4.
Effect of z-FA.fmk on the LPS-induced
activation of NF- B. A,
NF-
B-dependent reporter gene activity. Mf4/4 cells were
transiently transfected with 100 ng p(
B)3LUC and
preincubated for 1 h with 50 µM z-FA.fmk prior to
stimulation with 1 µg/ml LPS for 6 h. Luciferase (Luc) activity
in cell extracts was measured as described under "Materials and
Methods." B, EMSA for NF-
B. Mf4/4 cells were incubated
for 1 h with z-FA.fmk prior to stimulation with 1 µg/ml LPS for
30 min. Equal amounts of nuclear cell extracts were incubated with a
32P-labeled oligonucleotide containing the NF-
B-binding
site of the immunoglobulin
-chain gene and analyzed by EMSA.
Arrows indicate the activated NF-
B complex and the
constitutively expressed recombination-binding protein J
(RBP-J
). Nuclear extracts were preincubated for 20 min
with 2 µg of purified polyclonal antibodies against p50 and p65
(lanes 4-6 and 7-9, respectively). Partial
disappearance of the two NF-
B complexes indicates that they are both
composed of p50 and p65. p50/p65* indicates the NF-
B
complex formed by a truncated form of the p65 subunit.
B
activation by measuring the amount of nuclear NF-
B in EMSA. Mf4/4
cells stimulated with LPS translocate NF-
B into the nucleus,
resulting in a defined band in EMSA (Fig. 4B, lane
2). Pretreatment of Mf4/4 cells with z-FA.fmk induced a slower
migrating band in EMSA (Fig. 4B, lane 3), and the
faster migrating band disappeared. This might result from a change in
the composition of the NF-
B complex. However, pretreatment of the
nuclear extracts with polyclonal antibodies against p50 and p65 led to
a reduction in intensity of both bands, demonstrating that p50 and p65
are present in both inducible complexes (Fig. 4B,
lanes 4-9).
B complex during nuclear extract
preparation, even in the presence of general protease inhibitors such
as Pefabloc, aprotinin, and leupeptin. To check this possibility we
analyzed nuclear extracts of LPS-treated MF4/4 cells for the presence
of intact p50 and p65 by immunoblotting. As expected, LPS induced
nuclear translocation of p65 and p50, even in the presence of z-FA.fmk.
However, in the absence of z-FA.fmk, p65 was cleaved to products of 31 and 33 kDa (Fig. 5A). Cleavage products of p50 could not be detected (Fig. 5B), although
p50 levels were slightly increased in LPS-stimulated cells pretreated with z-FA.fmk. In contrast, p65 cleavage was not detected when cells
were directly lysed in Laemmli buffer (Fig. 5C). Analysis of
the same extracts for I
B showed LPS-induced I
B degradation, which
could not be prevented by z-FA.fmk pretreatment (Fig. 5D). In conclusion, these results further demonstrate that inhibition of
NF-
B-mediated gene expression by z-FA.fmk is not because of inhibition of the nuclear translocation of NF-
B in response to LPS.
Moreover, the specific degradation of p65 in nuclear extracts of
LPS-treated cells and its inhibition by z-FA.fmk pretreatment demonstrates that the slower migrating NF-
B complex, visible in EMSA
of LPS-treated cells pretreated with z-FA.fmk (Fig. 4B), results from the z-FA.fmk-mediated inhibition of p65 proteolysis during
nuclear extraction. These data also demonstrate the risk of
misinterpretation of mobility shifts liable to occur in EMSA.
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Fig. 5.
Proteolysis of p65 by a z-FA.fmk-sensitive
protease. A and B, nuclear expression of p65
and p50. Nuclear extracts (100 µg) from Mf4/4 cells, stimulated with
1 µg/ml LPS for 30 min in the absence or presence of 50 µM z-FA.fmk, were analyzed for p65 (A) and p50
(B) expression by immunoblotting. p65 and truncated forms of
p65 are indicated as p65 and p65*, respectively.
C and D, immunoblot analysis of p65 and I B in
total cell extracts of Mf4/4 cells treated as in A and
B but immediately lysed in Laemmli buffer.
B in
response to TNF, whereas it is not involved in the pathway leading to
nuclear translocation of NF-
B (18, 19). Because z-FA.fmk also seems to interfere specifically with the transactivation of NF-
B in response to LPS, we analyzed whether p38 MAP kinase is also involved in
NF-
B-dependent gene expression in response to LPS and
whether z-FA.fmk affects the activation of p38 MAP kinase. Pretreatment of LPS-stimulated Mf4/4 cells with the p38 MAP kinase inhibitor SB203580 showed that p38 MAP kinase was also involved in the enhanced transcriptional activity of NF-
B in response to LPS (Fig.
6A). However, z-FA.fmk did not
affect the LPS-induced phosphorylation of p38 MAP kinase in Mf4/4
cells, excluding the fact that z-FA.fmk prevents NF-
B
transactivation by inhibiting the LPS-induced activation of p38 MAP
kinase.
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Fig. 6.
A, role of p38 MAP kinase in LPS-induced
NF- B-dependent gene expression. Mf4/4 cells were
transiently transfected with 100 ng p(
B)3LUC and
incubated for 1 h with 10 µM SB203580 or 50 µM z-FA.fmk prior to stimulation for 6 h with LPS.
Cell extracts were analyzed for luciferase (Luc) activity.
B, z-FA.fmk does not interfere with LPS-induced activation
of p38 MAP kinase. Mf4/4 cells were incubated for 1 h with 50 µM z-FA.fmk and treated for 15 min with 1 µg/ml LPS.
Total lysates were separated by SDS-polyacrylamide gel electrophoresis,
and activated p38 MAP kinase was detected by Western blotting with a
polyclonal antibody specifically recognizing phosphorylated p38 MAP
kinase. As a control for the amount of protein loaded, total expression
of p38 MAP kinase was revealed with a polyclonal anti-p38 MAP kinase
antibody and found to be equal in all samples (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. Also, IL-1
and TNF synthesis was
abolished by pretreatment with z-FA.fmk, whereas IL-6 and caspase-11
production was not affected. Because E64 and CA-074Me did not modulate
the LPS-induced production of these cytokines, we conclude that this
effect of z-FA.fmk is independent of cathepsin B. Moreover, we were
unable to detect an effect of LPS on cathepsin B activity in the cells
studied (data not shown). z-FA.fmk was found to interfere with the
production of IL-1 and TNF by preventing the transcription activation
potential of NF-
B. It is still unclear why the expression of IL-6 is
only minimally affected by z-FA.fmk, because IL-6 expression has
previously been shown to be NF-
B-dependent (25). This
suggests the involvement of gene-specific regulatory mechanisms of
NF-
B-dependent gene expression, as previously
demonstrated in the case of TNF (26). This also fits with our
observation that z-FA.fmk does not prevent the I
B kinase pathway
leading to degradation of the NF-
B inhibitor I
B and subsequent
nuclear translocation of NF-
B. In contrast, z-FA.fmk seems to
specifically interfere with a signaling pathway or molecule that leads
to the transactivation of NF-
B and that might indeed be gene
promoter-specific. The p38 MAP kinase pathway has previously been shown
to fulfill such an NF-
B transactivation function in response to TNF
(18). Here we could demonstrate a similar role for p38 MAP kinase in
activating NF-
B-dependent gene expression in response to
LPS. However, z-FA.fmk did not interfere with the LPS-induced
activation of p38 MAP kinase. Although we cannot exclude an effect of
z-FA.fmk on the activation of downstream substrates of p38 MAP kinase,
the above observations make it rather unlikely that z-FA.fmk modulates
NF-
B-dependent gene expression by interfering with the
p38 MAP kinase pathway. The fluoromethyl ketone group of z-FA.fmk is
known to be very reactive with activated nucleophiles (27), and the
specificity of drugs like z-FA.fmk is only controlled by the structure
of the peptidyl portion of the reagent. Therefore, side reactions with
other proteins susceptible to alkylation are likely to occur. In this
context, we have previously shown that fluoromethyl ketone peptide
inhibitors of caspases can nonspecifically inhibit the activity of
several cathepsins (28). Recently, it has been reported that inhibition
of NF-
B-dependent gene expression by several reagents,
including helenalin and arsenite, is because of the irreversible
alkylation of a cysteine residue in the p65 NF-
B subunit or the
I
B kinase
and
subunits, respectively (29-31). In all cases,
this resulted in decreased DNA binding of NF-
B. As z-FA.fmk did not
affect the nuclear translocation and DNA binding of NF-
B, it is
unlikely that z-FA.fmk acts in a similar way. Moreover, other fmk-based
peptide inhibitors, such as
benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-DL-Asp-(OMe)-fmk and
benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fmk, did not have an effect on
NF-
B-dependent gene expression in response to LPS (data not shown).
B and IL-1 in
rheumatoid arthritis is also well documented (32). Therefore, it is
very likely that the therapeutic effect of z-FA.fmk in this disease is
at least partially mediated by its inhibitory effect on
NF-
B-dependent gene expression. These results, as well
as the fact that z-FA.fmk can be used in vivo without severe
side effects (11, 33-35), suggest z-FA.fmk to be a promising new tool
for the treatment of a number of other inflammatory diseases.
B can be influenced by the in vitro cleavage of the p65 NF-
B subunit by a z-FA.fmk-sensitive protease present in nuclear
extracts. Cleavage of p65 by this protease results in a p50·p65
complex, which induces a decreased shift in EMSA. These findings might
have important implications for the interpretation of results based on
EMSA with NF-
B-specific oligonucleotides. In many cases, the
appearance of two bands is explained to correspond to a p50·p50 and a
p50·p65 complex, respectively (15, 36). However, we showed that the
lower band can also correspond to a complex of p50 and truncated p65.
It should be mentioned that these effects could still be observed when
extracts were prepared in the presence of a mixture of general protease
inhibitors such as aprotinin, leupeptin, E64, and Pefabloc. Although we
described this effect in macrophages, we also noticed p65 cleavage in
other cells, such as the L929 fibrosarcoma cell line (data not
shown). A similar truncation of p65 has recently been reported when
extracts were prepared from acute lymphoblastic leukemia cells (37). In
the latter case, p65 cleavage could be prevented by
1-antitrypsin.
B-dependent gene expression in response to LPS, implicating that it is an interesting tool for the
development of novel anti-inflammatory treatments. Moreover, we also
propose the use of this inhibitor when preparing nuclear extracts for
EMSA with NF-
B-specific probes.
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FOOTNOTES |
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* This work was supported in part by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, the Interuniversitaire Attractiepolen, the Bijzonder Onderzoeksfonds, and the Belgische Federatie tegen Kanker.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.
Research fellow with the Vlaams Instituut voor de Bevordering van
het Wetenschappelijk-technologisch Onderzoek in de Industrie.
§ Research associate with the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.
¶ To whom correspondence should be addressed: Dept. of Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Tel.: 32-9-264-51-31; Fax: 32-9-264-53-48; E-mail: rudi.beyaert@dmb.rug.ac.be.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M102239200
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ABBREVIATIONS |
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The abbreviations used are:
IL, interleukin;
TNF, tumor necrosis factor;
afc, 7-amino-4-trifluoromethylcoumarin;
EMSA, electrophoretic mobility shift
assay;
fmk, fluoromethylketone;
LPS, lipopolysaccharide;
MAP, mitogen-activated protein;
NF-B, nuclear factor
B;
z-ARR.afc, benzyloxycarbonyl-Ala-Arg-Arg-afc;
z-FA.fmk, benzyloxycarbonyl-Phe-Ala-fmk.
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