© 1997 American Heart Association, Inc.
Articles |
N-
-Tosyl-L-Lysine Chloromethylketone Prevents Expression of iNOS in Vascular Smooth Muscle by Blocking Activation of NF-
B
From the Center of Physiology, University Clinic of Frankfurt, Frankfurt, Germany.
Correspondence to V.B. Schini-Kerth, PhD, Zentrum der Physiologie, Klinikum der JWG–Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany.
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Abstract |
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Abstract Certain cytokines and lipopolysaccharide stimulate expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle, an event that is regulated at the transcriptional level and appears to involve several transcription factors, including nuclear factor
B (NF-B). Since
proteases play an essential role in NF-B activation, experiments
were designed to clarify, in both cultured rat aortic smooth muscle
cells (SMCs) and isolated rat aortas, whether protease
inhibitors affect the interleukin-1ß (IL-1ß)–elicited
expression of iNOS. The formation of NO was assessed by nitrite release
in cultured SMCs and the attenuation of
phenylephrine-induced contraction in aortic rings, the
expression of iNOS by Western blot analysis and reverse
transcription–polymerase chain reaction, and NF-B activity in
nuclear extracts by gel electrophoretic mobility shift assay. Exposure
of cultured SMCs to IL-1ß increased NF-B binding activity within
30 minutes and was associated with nitrite accumulation and the
appearance of iNOS protein 24 hours later. These responses were
abolished in cells that had been exposed to the cytokine in the
presence of the protease inhibitor
N--tosyl-L-lysine
chloromethylketone. Aprotinin and
p-toluenesulfonyl-L-arginine methyl ester, two
other protease inhibitors, also reduced the
cytokine-stimulated release of nitrite and the level of iNOS
protein. Exposure of rat aortic segments without
endothelium to IL-1ß activated NF-B within
30 minutes and was associated with the appearance of iNOS mRNA and an
attenuation of phenylephrine-induced contraction 6
hours later. These responses were blunted when the segments were
incubated with the cytokine and
N--tosyl-L-lysine chloromethyl ketone. The
present observations indicate that protease inhibitors
prevent iNOS expression in both cultured and native vascular SMCs by
blocking the activation of NF-B.
Key Words: inducible nitric oxide synthase • interleukin-1ß • nuclear factor-B • vascular reactivity • vascular smooth muscle
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Nitric oxide is an important physiological regulator of mammalian vascular function in health and disease. In normal blood vessels, NO is generated by a constitutively expressed, calcium/calmodulin–dependent NOS that is localized predominantly in the endothelium.1 Endothelium-derived NO plays a vital role in the local regulation of vascular tone, prevents platelet activation, and helps to maintain VSMCs in a quiescent state.2 3 4
NO can also be generated after transcriptional upregulation of iNOS by
certain cytokines, such as IL-1ß, TNF-, and
endotoxin.5 This sequence of events probably accounts for
the release of NO from denuded blood vessels that have been exposed in
vitro to inflammatory mediators or injured in vivo by either a balloon
catheter or endotoxin.6 7 8 9 Although iNOS expression has
been demonstrated in endothelial cells, fibroblasts,
and macrophages,10 11 12 VSMCs are likely to be the
major cellular source of NO in endothelium-deficient or
injured arteries. Unlike endothelial NOS, iNOS is not
activated by calcium but generates NO at a maximal rate over
long periods of time.6 Similar to the protective roles of
endothelium-derived NO, it is conceivable that
continuous formation of discrete amounts of NO by VSMCs may be
important for maintaining vascular tone, patency, and
nonthrombogenicity at sites of endothelial damage.
However, when NOS induction becomes exaggerated, the release of copious
amounts of NO will cause loss of vascular tone and result in severe
hypotension, such as occurs in septic shock.8
NF-B appears to mediate the cytokine- and
lipopolysaccharide-induced gene transcription that is involved
in the inflammatory response.13 The NF-B family of
transcription factors, including p65 (RelA), c-Rel, RelB, p50, and p52,
share a structurally homologous N-terminal Rel domain that
encodes DNA binding and dimerization functions.14 15 In
addition, p65, c-Rel, and RelB contain potent
transactivator domains.16 The various NF-B
subunits interact to form homodimers and heterodimers, the
best-characterized form of NF-B being a heterodimer composed of a
p50 and a p65 subunit.17 NF-B is constitutively
expressed in the cytosol of many cells albeit bound to
inhibitory protein(s) IB.18 Although the
signal-transduction cascade linking cytokine- and
lipopolysaccharide-receptor stimulation to activation and
nuclear translocation of NF-B is not entirely clear, a final step
involves phosphorylation and proteolytic degradation of
IB.19 20 21 22 Recent studies have suggested that activation
of NF-B also plays a pivotal role in the induction of iNOS. Cognate
sequences for NF-B have been identified in the promoters of both the
human and the mouse gene encoding iNOS.23 24 25 In addition,
the NF-B binding site upstream from the TATA box in the iNOS
promoter of the murine gene has been shown to be necessary but not
sufficient for maximal transcriptional activation of this gene by
lipopolysaccharide.23 25 Recently, protease
inhibitors (eg, TLCK) and antioxidants (eg, pyrrolidine
dithiocarbamate) prevented lipopolysaccharide from stimulating the
expression of iNOS in macrophages presumably by inhibiting the
activation of NF-B.26 27 28 Therefore, we designed
experiments in cultured and native VSMCs to test the hypothesis that
protease inhibitors prevent induction of NOS by interfering
with NF-B activation.
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Methods |
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Cell Culture
SMCs were isolated by elastase and collagenase digestion of thoracic aortas from male Wistar rats and characterized by immunocytochemical techniques using a monoclonal antibody against smooth muscle
-actin.29 Cells were cultured in minimum
essential medium containing Earle's salts, 2 mmol/L glutamine,
5 mmol/L Tris-NaOH, 5 mmol/L HEPES-NaOH, (both at pH 7.3),
100 U/mL penicillin, 100 µg/mL streptomycin, and 10% (vol/vol) fetal
bovine serum. Confluent cultures of SMCs were serially passaged with
0.05% trypsin/0.02% EDTA. All experiments were performed with
confluent cultures of cells at passage 4 or higher. SMCs were seeded
into either 24-well multiwell plates for measurement of nitrite
production or Petri dishes (60-mm diameter) for detection of
iNOS protein by Western blot analysis, iNOS mRNA by RT-PCR, and
NF-B binding activity by EMSA. After the cells had reached
confluence, the medium was replaced with serum-free culture medium
containing 0.1% (wt/vol) fatty acid–free BSA. After 24 hours the
incubation medium was replaced again with serum-free culture medium,
and the SMCs were then exposed to IL-1ß for 24 hours or as
indicated.
Preparation of Isolated Blood Vessels
Male Wistar rats were killed and their thoracic aortas removed
and placed in a modified Krebs-Ringer bicarbonate solution containing
(in mmol/L) NaCl 119, KCl 4.7, MgSO4 1.2,
KH2PO4 1.2, CaCl2 1.6,
NaHCO3 25, calcium EDTA 0.026, and glucose 11.1, with
sodium diclofenac (10-6 mol/L) added to prevent
production of vasoactive prostanoids. The aortas were cleaned
of loose connective tissue and cut into either rings (3 to 4 mm
long) for the organ chamber study or segments (5 to 6 mm long) for
both the EMSA and determination of iNOS mRNA by RT-PCR. The
endothelium was removed mechanically from both types of
aortic preparation. Rings were incubated in serum-free culture medium
containing BSA and polymyxin B (1 µg/mL) with or without TLCK,
IL-1ß, or a combination of both for 6 hours at 37°C before the
organ chamber experiment. For both EMSA and RT-PCR, each treatment was
applied to a batch of four segments that had been obtained from four
different rat aortas. Each group of aortic segments was incubated in
serum-free culture medium containing BSA at 37°C. After a 30-minute
rest, the medium was replaced again with fresh, serum-free medium and
the aortic segments were incubated with or without TLCK, IL-1ß, or
both for 30 minutes or as indicated.
Release of Nitrite
Nitrite accumulation was determined by formation of diazo
compounds and the resulting change in absorbance at 570 nm. Aliquots
(150 µL) of conditioned medium from confluent cells were collected
and mixed with an equal volume of Griess reagent [1% sulfanilamide
and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride
in 2% phosphoric acid]. The mixture was incubated at 20°C for 10
minutes. Concentrations were determined relative to a standard curve
constructed by analyzing aqueous solutions of various concentrations of
sodium nitrite, and background nitrite values corresponding to
nonconditioned medium were subtracted from experimental values.
EMSA
Cells were washed twice with cold HEPES-Tyrode's solution,
harvested by scraping, and incubated in 200 µL of buffer A (HEPES
10 mmol/L, pH 7.9; KCl 10 mmol/L; EDTA 0.1 mmol/L; EGTA
0.1 mmol/L; DL-DTT 1 mmol/L; leupeptin 2 µg/mL;
pepstatin A 2 µg/mL; trypsin inhibitor 10 µg/mL; and
PMSF 88 µg/mL) for 15 minutes at 4°C. The aortic segments were
rapidly frozen and homogenized in liquid N2
with a porcelain mortar and pestle. The frozen tissue powder was
dissolved in 200 µL of ice-cold buffer A for 5 minutes at 4°C.
Thereafter, Nonidet P-40 (0.6%) was added to both types of
preparation. The crude nuclei released by lysis were collected by
microcentrifugation at 15 000g for 30
seconds. The nuclear pellets were resuspended in 50 µL of buffer B
(HEPES 20 mmol/L, pH 7.9; KCl 400 mmol/L; EDTA 1 mmol/L;
EGTA 1 mmol/L; glycerol 10% [wt/vol]; DTT 1 mmol/L; PMSF
88 µg/mL; and 20 µg/mL each of leupeptin, pepstatin A, trypsin
inhibitor, antipain, chymostatin, and aprotinin). Nuclei
were shaken for 15 minutes at 4°C and clarified by
microcentrifugation at 15 000g for 5
minutes. The resulting supernatants contained 1 to 5 mg/mL protein as
assessed by Bradford assay (Bio-Rad Laboratories GmbH) with BSA as the
standard. Nuclear extracts were frozen in liquid N2 and
stored at -70°C. A double-stranded 22-mer
oligonucleotide containing the most common NF-B
consensus sequence ([underlined]
5'-AGTTGAGGGGACTTTCCCAGGC-3', Promega30 ) was
end-labeled with [
-32P]ATP (3000 Ci/mmol, Hartmann
Analytik), using T4 polynucleotide kinase. Binding
reactions were set up with 10 000 counts per minute
32P-labeled DNA, HEPES-NaOH 5 mmol/L (pH 7.5), NaCl
100 mmol/L, DTT 1 mmol/L, 5% glycerol, EDTA 1 mmol/L, 1
µg poly(dI/dC), and 10 to 15 µg of nuclear extract. The binding
reactions were performed at 20°C for 30 minutes and separated by
electrophoresis on a 6% nondenaturating polyacrylamide gel at
135 V for 2 hours in TBE buffer (Tris-borate 89 mmol/L and EDTA
1 mmol/L). On the vacuum-dried gels, the protein-DNA complexes
were visualized by autoradiography and analyzed
by scanning densitometry (ImageMaster, Pharmacia). Competition studies
were performed by adding a twofold excess of unlabeled double-stranded
oligonucleotides to the binding reaction 10 minutes
before the labeled oligonucleotide was added. Samples
were subjected to electrophoresis as described above.
Expression of iNOS mRNA by RT-PCR
The aortic segments were rapidly frozen and
homogenized in liquid N2, and total RNA was
isolated by the method of Chomczynski and Sacchi.31 For
annealing 1 µg of pdN6 to 2 µg total RNA, the reaction mixture (20
µL) was heated to 95°C for 5 minutes and rapidly cooled on ice. RT
was performed with 200 U reverse transcriptase (Life Technology) in
1 mmol/L DTT, 500 µmol/L dNTP, and reaction buffer for 1
hour at 37°C in a final volume of 50 µL. In some reaction mixtures,
reverse transcriptase was omitted to control for contaminating genomic
DNA or cDNA. After denaturation at 95°C for 7 minutes, the
synthesized cDNA was subjected to a 30-cycle PCR. The antisense primer
corresponded to 5'-TCATTGTACTCTGAGGGCTGACACA-3' of the murine
macrophage iNOS cDNA32 and the sense primer to
5'-GCCTTCAACACCAAGGTTGTCTGCA-3'. For normalization of cDNA amounts,
GAPDH transcripts were amplified by PCR in the same PCR reaction. The
antisense primer corresponded to 5'-AGATCCACAACGGATACATT-3' of the rat
and human sequences33 and the sense primer to
5'-TATGACAACTCCCTCAAGAT-3'. The PCR reaction contained 0.4
µmol/L of each primer, 200 µmol/L of each dNTP, 1 U
Taq polymerase (Pharmacia), reaction buffer, and 5 µL cDNA
in a final volume of 50 µL. For Southern blot analysis, the
PCR products were fractionated by size by agarose gel
electrophoresis, transferred to nylon membranes, and hybridized with a
32P-labeled fragment obtained from a plasmid containing
mouse macrophage iNOS cDNA and a 32P-labeled GAPDH
fragment isolated from the PCR reaction. iNOS and GAPDH mRNAs were
localized by autoradiography and analyzed by
scanning densitometry.
Expression of iNOS Protein
Cells were washed twice with cold HEPES-Tyrode's solution,
harvested by scraping, and collected by
microcentrifugation at 3000g for 3 minutes
at 4°C. The cell pellets were resuspended in double-distilled water
and lysed by five freeze/thaw cycles (freezing in liquid N2
and thawing in a 37°C water bath). An equal volume of
homogenization buffer [Tris-HCl 100 mmol/L
(pH 7.4); KCl 2.3% (wt/vol); EDTA 2 mmol/L; DTT 0.2 mmol/L;
PMSF 8.8 µg/mL; and 2 µg/mL each of leupeptin, pepstatin A, trypsin
inhibitor, antipain, chymostatin, and aprotinin] was added
to the cell homogenates, and the cytosols were clarified by
centrifugation at 10 000g for 10 minutes at
4°C. The resulting supernatants contained 1 to 5 mg/mL protein. The
cytosolic fractions were subjected to SDS-PAGE (8% gradient gel). The
separated proteins were electrophoretically transferred to
nitrocellulose membranes (Bio-Rad Laboratories). Nitrocellulose blots
were first incubated with a primary polyclonal rabbit antibody against
iNOS from murine macrophage (dilution 1:2000, overnight at
4°C; kindly provided by Dr Pfeilschifter, JWG–University Clinic,
Frankfurt, Germany) and then with a secondary polyclonal donkey
anti-rabbit immunoglobulin antibody conjugated to horseradish
peroxidase (Amersham International plc) for 1 hour at 20°C. The
immunocomplex was developed using an enhanced horseradish
peroxidase/luminol chemiluminescence reaction (ECL Western blotting
detection reagents, Amersham International plc) and detected with x-ray
film. Prestained molecular-mass markers (Bio-Rad Laboratories) were
used as standards for SDS-PAGE immunoblot analysis.
The autoradiographs were analyzed by scanning densitometry.
Vascular Reactivity Studies
Rings were suspended between two stainless steel stirrups in
organ chambers filled with 10 mL Krebs-Ringer's bicarbonate solution
(37°C, pH 7.4) bubbled with 95% O2 and 5%
CO2. One of the stirrups was anchored to the organ bath
(Schuler-Organbad, kindly made available by Hugo Sachs Elektronik), and
one was connected to a strain gauge (F30, Hugo Sachs Elektronik)
coupled to a recorder for measurement of isometric tension. The
aortic rings were stretched progressively to their optimal length for
maximal contraction (2 to 2.5 g) before phenylephrine
(10-6 mol/L) was added. Once the contraction "plateau"
elicited by phenylephrine was obtained, acetylcholine was
added to demonstrate the effective removal of the
endothelium. The organ chambers were rinsed three times
with control solution. After a 30-minute rest, a
concentration-contraction curve for phenylephrine or a
concentration-relaxation curve for SIN-1 in rings contracted with
phenylephrine (10-6 mol/L) was
constructed.
Statistical Analysis
Results are expressed as mean±SEM. Relaxations to SIN-1 are
expressed as a percent of contraction to phenylephrine. The
negative logarithm of the effective molar concentration of SIN-1
causing 50% relaxation (IC50) was calculated for each
concentration-contraction curve. Statistical evaluation of the data was
performed by Student's t test for paired observations; when
more than two treatments were compared, ANOVA followed by Fisher's
protected least significant difference test was used. A value of
P<.05 was considered statistically significant.
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Results |
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Experiments With Cultured Rat Aortic SMCs
Release of Nitrite
The release of nitrite caused by exposure of cultured rat aortic SMCs to IL-1ß for 24 hours was inhibited in a concentration-dependent manner by the addition of TLCK, aprotinin, or L-TAME (Fig 1
). The relative order of potency was
TLCK>aprotinin>L-TAME. Exposure of cells to a protease
inhibitor alone minimally affected the basal release of
nitrite during a 24-hour incubation (Fig 1). The inhibitory
effect of TLCK and aprotinin (10-4 mol/L) on the release
of nitrite evoked by IL-1ß required the presence of the protease
inhibitor during the induction of NOS (Fig 2
and data not shown).
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indicates a
significantly greater release of nitrite than that evoked by
simultaneous treatment with IL-1ß and TLCK (0 h).
Expression of iNOS Protein
Exposure of VSMCs to IL-1ß for 24 hours was associated with a
substantial level of iNOS protein (Fig 3). However, only
a little iNOS protein was detected in cells that had been exposed to a
combination of IL-1ß and TLCK. The level of iNOS protein was reduced
by 43±11% in cells that were exposed simultaneously to
IL-1ß and aprotinin (Fig 3). In untreated cells and in cells that had
been treated with either TLCK or aprotinin alone, no iNOS protein was
detected (Fig 3).
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NF-B Binding Activity
EMSAs indicated the presence of low-level NF-B binding activity
in nuclear extracts from unstimulated rat aortic SMCs (Figs 4 and 5). The level of NF-B–DNA
complex increased in a transient manner after exposure of the cells to
IL-1ß (Fig 4). A marked increase in the level of NF-B–DNA complex
was found after only 10 minutes of exposure to IL-1ß. Thereafter, the
signal continued to increase and reached a peak value by 30 minutes.
Afterward, NF-B complex levels decreased to baseline within 4 hours
(Fig 4). Both basal and IL-1ß–stimulated NF-B–DNA binding
activities appeared predominantly as two bands of slightly different
mobility and were competed by excess unlabeled
oligonucleotide, demonstrating the specificity of the
binding to DNA (Fig 4 and data not shown). NF-B binding activity was
reduced in cells that had been exposed to IL-1ß in combination with
increasing concentrations of TLCK for 30 minutes (Fig 5). Exposure of
cells to TLCK alone did not stimulate NF-B binding activity (Fig 5).
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Experiments With Isolated Rat Aortas
Vascular Reactivity Studies
Exposure of aortic rings without endothelium to
IL-1ß for 6 hours caused a marked rightward shift of the
concentration-contraction curve and significantly reduced the maximal
contraction to phenylephrine (Fig 6A). No
such shift and reduction in maximal contraction were obtained with
rings that had been exposed to IL-1ß in combination with TLCK or TLCK
alone for 6 hours (Fig 6A). The possibility that the protective effect
of TLCK on vascular tone was due to inhibition of the cGMP effector
cascade is unlikely, because the NO donor SIN-1 evoked similar
concentration-relaxation curves in control rings and those that had
been incubated with TLCK for 6 hours before the organ chamber
experiments (IC50's were 9.5±2.9x10-7 mol/L
and 5.7±1.7x10-7 mol/L, respectively, without and with
TLCK; Fig 6B).
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Expression of iNOS mRNA
Southern hybridization of PCR products with an iNOS-specific
probe revealed that exposure of rat aortic segments without
endothelium to IL-1ß for 6 hours was associated with
the appearance of substantial levels of iNOS mRNA (Fig 7). However, only a little iNOS mRNA was detected in
aortic segments that had been exposed to IL-1ß in combination with
TLCK (10-4 mol/L, Fig 7). In untreated aortic segments and
those that had been treated with TLCK alone, no iNOS mRNA was detected
(Fig 7). In RT-PCR reactions without reverse transcriptase, neither
iNOS nor GAPDH PCR products were detectable (data not shown).
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NF-B Binding Activity
Similar to the findings obtained with cultured rat aortic SMCs,
low-level NF-B binding activity was found in nuclear extracts from
untreated rat aortic rings without endothelium (Fig 8). Exposure of rings to IL-1ß caused a time-dependent
increase in levels of the NF-B–DNA complex, which reached a peak by
30 minutes and persisted thereafter for the next 3.5 hours (Fig 8).
Both basal and IL-1ß–stimulated NF-B binding activities appeared
predominantly as two bands that were competed by excess unlabeled
oligonucleotide, which verified the specificity of the
binding to DNA (Fig 8). NF-B binding activity was reduced in aortic
rings that had been exposed to IL-1ß in combination with TLCK for 30
minutes (Fig 9). Exposure of aortic rings to TLCK alone
did not increase NF-B binding activity (Fig 9).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Certain cytokines such as IL-1ß induce expression of iNOS in VSMCs that results in substantial NO production and a depressed contractile response to vasoconstrictor compounds. In the present study, we found that various protease inhibitors inhibited iNOS expression and nitrite accumulation in cultured VSMCs. TLCK, which inhibits neutral proteases by its ability to react covalently with a histidine residue in the active center of these enzymes,34 completely blocked the cytokine-induced formation of NO. Aprotinin, a competitive inhibitor that forms a tight complex with various serine proteases,35 had similar effects though to a lesser extent. L-TAME, a competitive substrate for serine proteases,36 also prevented the induced release of nitrite but only at high concentrations >0.3 mmol/L. In addition, TLCK abolished IL-1ß–induced expression of iNOS and hyporeactivity to phenylephrine in rat aortic rings without endothelium. Altogether, these findings confirm and extend the observations that protease inhibitors of the chloromethylketone group block induction of NO release from a variety of cells other than VSMCs.37 38 39 40 41
The transcriptional activation of both murine and human genes encoding
iNOS is controlled by several common transcription factors that bind to
the upstream promoter region of the iNOS gene.23 24 Among
these transcription factors, a critical activator role has
been attributed to NF-B.23 25 26 27 28 Consistent
with this concept, IL-1ß induced NF-B activity in cultured and
native VSMCs, as indicated by the marked increase in the levels of
NF-B–DNA complexes in nuclear extracts (Reference 4242 and the
present findings). Basal and IL-1ß–stimulated NF-B–DNA
complexes appeared predominantly as two bands of slightly different
mobility (References 42 and 4342 43 and the present findings). Addition
of specific antibodies (directed against either the p65 or the p50
subunit) to nuclear extracts shifted the upper band, whereas the lower
band was shifted by p50 antibodies only, suggesting that the upper band
contained predominantly p65/p50 heterodimers and the lower band p50
homodimers (Reference 4343 and M. Hecker, unpublished data, 1995).
Chloromethylketone derivatives have been shown to
prevent activation of NF-B in murine pre–B lymphocytes, 70Z/3
cells, and Jurkat cells.44 Therefore, it is likely that
inhibition of iNOS expression by protease inhibitors
results from the blocking of the activation of NF-B. In support of
this idea are the findings that the inhibitory effect of
TLCK on IL-1ß–stimulated formation of NO was associated with reduced
NF-B activity in both cultured and native VSMCs. Similar
inhibitory effects of
chloromethylketones have been found in the
IL-1ß–stimulated, insulin-producing rat cell line
RINm5F45 and murine peritoneal macrophages
stimulated by a combination of interferon gamma and
lipopolysaccharide.28 In addition,
chloromethylketones may also affect activation of
other transcription factors necessary for iNOS expression. The
mechanism whereby protease inhibitors block activation of
NF-B and induction of iNOS remains to be established. One
possibility is that these inhibitors prevent the
proteolytic degradation of IB.22 44 As a consequence,
the level of IB remains constant in activated cells, and
IB will continue to "mask" the nuclear localization sequences of
p50 and p65, thereby retaining the transcription factor in the
cytosol.44 Nevertheless, this possibility still needs to
be proved, since we were unable to visualize IB from rat aortic
smooth muscle preparations on immunoblots using three
different polyclonal antibodies raised against human IB (data not
shown). Although the proteases that catalyze the degradation of IB
have not yet been identified, specific proteasome
inhibitors were able to block the inducible degradation of
IB, suggesting involvement of the proteasome
complex.21 46 However, there is evidence that
chloromethylketones have little or no effect on
proteasome activity.47 Thus, the effect of TLCK on NF-B
activation may lie upstream from the proteolysis of IB. Both in
vitro and in vivo investigations have shown that degradation of IB
is preceded by its
phosphorylation.20 22 48 Although this
covalent modification of IB per se is insufficient to release the
inhibitor from NF-B, this step appears to be necessary
for NF-B activation, possibly by modifying IB into an appropriate
substrate for the proteasome complex.46
Chloromethylketones have been shown to prevent
phosphorylation of IB, suggesting that these
compounds may interfere with NF-B activation by inhibiting the
kinase(s) that phosphorylates IB.49 Many
kinases have been reported to phosphorylate IB and
activate NF-B in vitro, including protein kinase C and
cAMP-dependent protein kinase.19 50 Therefore, the
suppressive effect of chloromethylketones, which
are potent alkylating agents, on IB phosphorylation
could be due to their ability to inactivate
adenylate cyclase,51 cAMP-dependent protein
kinase,52 or protein kinase C.53
Consistent with this idea are the findings that
inhibitors of either protein kinase C or cAMP-dependent
protein kinases inhibited iNOS expression in IL-1ß–stimulated
VSMCs.54 55
In conclusion, various types of protease inhibitor were
found to prevent NF-B activation and iNOS expression in cultured and
native VSMCs. These effects may result from direct inhibition of IB
proteolysis or—since IB phosphorylation is an
essential prerequisite for IB degradation—potentially by an
indirect mechanism associated with reduced levels of IB
phosphorylation. Besides the iNOS gene, NF-B also
regulates a variety of other genes that are involved in immune and
inflammatory responses, including those encoding adhesion molecules
(eg, E-selectin, ICAM, and VCAM-1),13 cytokines
and their receptors (eg, IL-1, IL-2, IL-1 receptor, and
IL-6),14 and tissue factor (a protein that triggers the
coagulation protease cascades).56 Thus, protease
inhibitors, by blocking NF-B activation, could be of
therapeutic value for the treatment of the hyperdynamic circulatory
states in sepsis, thrombotic disorders, and inflammatory responses.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Deutsche Forschungsgemeinschaft Bu 436/4-3 (to R.B.), Schi 389/1-1 to 3 (to V.B.S.-K.), and a Training Grant from the Land Hessen (to M.B.). The authors thank Dr Agnieszka Bara and Martina Bruckmann for excellent technical assistance.
Received December 12, 1995; accepted June 24, 1996.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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