From the
Most of the inflammatory and proviral effects of tumor necrosis
factor (TNF) are mediated through the activation of the nuclear
transcription factor NF-
PTPase
inhibitors also blocked NF-
Tumor necrosis factor (TNF)
The
activation of NF-
The role of PPases in cytokine
receptor signaling has been investigated using various chemical
inhibitors such as phenylarsine oxide (PAO), diamide, and pervanadate,
which inhibit tyrosine PPase, and okadaic acid and calyculin, which
inhibit serine-threonine PPase
(10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) .
The growth modulatory effects of TNF have been shown to be abolished by
vanadate
(25) . In contrast, pervanadate and okadaic acid have
been shown to mimic TNF for activation of NF-
We recently demonstrated that
erbstatin, an inhibitor of protein-tyrosine kinase, inhibits
TNF-dependent NF-
Electrophoretic mobility shift assays (EMSA) were performed by
incubating 4-5 µg of nuclear extract with 16 fmol of
The EMSA
for AP-1 and Sp1 were performed as described for NF-
The activation of NF-
Visualization of
radioactive bands were carried out by a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA) using Imagequant software.
In this report, we used inhibitors of PPases to examine the
role of protein phosphorylation in the TNF-dependent activation of
NF-
Whether PTPase inhibitors specifically block the activation of
NF-
It has
been shown in rat hepatocytes that PAO can lower intracellular ATP
levels
(13) . Rotenone and antimycin A are also known to lower
intracellular ATP levels by inhibiting the mitochondrial electron
transport chain. To distinguish the ATP-lowering effects of PAO from
its ability to inhibit PTPase, we examined the effect of rotenone and
antimycin A on the TNF-dependent NF-
The results shown here clearly indicate that inhibitors of
PTPase completely block the TNF-dependent activation of NF-
TNF is one of the most potent
activators of NF-
What role protein PTPase plays in the TNF-dependent activation of
NF-
How PTPase inhibitors block the activation of NF-
In insulin and T cell receptor systems, PAO has been
shown to inhibit the tyrosine phosphatase activity of CD45
(IC
Besides inhibiting PTPase, PAO affects the signal
transduction pathway via several other mechanisms. In T cells, PAO was
found to induce tyrosine phosphorylation and calcium mobilization
independent of its effects on the protein-tyrosine phosphatase CD45
(18) . This drug was also found to inhibit insulin-dependent
activation of p21 in fibroblast
(10) , inhibit cellular uptake
of ligands (epidermal growth factor, insulin, and asialofetuin), and
decrease intracellular ATP levels
(12, 13, 51, 52) . Some of these
effects, however, required higher concentrations (30 µM)
than the 1.6 µM used in our studies for maximum effect.
Our results indicate that PAO's inhibitory action is not due to a
decrease in intracellular ATP since rotenone and antimycin A, agents
also known to lower cellular ATP by inhibiting electron transport
chain, had no effect. We also found that in our system PAO had no
effect either on TNF receptors or on the receptor-mediated uptake of
the ligand (data not shown).
We found that, like PAO, diamide and
pervanadate, which are likewise inhibitors of tyrosine phosphatases
(20) , could also suppress TNF-mediated NF-
Recently it was reported that
serine protease inhibitors, L-tosylamido-2-phenylethyl
chloromethyl ketone and
N
The role of phosphatases in the
action of TNF is much less understood. We showed that the growth
modulatory effects of TNF are completely inhibited by phosphatase
inhibitors
(25) . In these studies, both vanadate and okadaic
acid inhibited both the growth-stimulatory and growth-inhibitory
effects of TNF. Since okadaic acid had no effect on NF-
In summary, the suppressive
effect of PTPase inhibitors on NF-
-We thank Dr. Rinee Mukherjee for assistance
provided at the early stage of this project. We also thank Drs. Werner
Green and Tapas Mukhopadhyay for supplying antibodies against p50 and
p65 proteins and Dr. Bryant Darnay for help on Western blotting and for
critically reading the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B. How TNF activates NF-
B, however,
is not well understood. We examined the role of protein phosphatases in
the TNF-dependent activation of NF-
B. Treatment of human myeloid
ML-1a cells with TNF rapidly activated (within 30 min) NF-
B; this
effect was abolished by treating cells with inhibitors of
protein-tyrosine phosphatase (PTPase), including phenylarsine oxide
(PAO), pervanadate, and diamide. The inhibition was dependent on the
dose and occurred whether added before or at the same time as TNF. PAO
also inhibited the activation even when added 15 min after the TNF
treatment of cells. In contrast to inhibitors of PTPase, okadaic acid
and calyculin A, which block serine-threonine phosphatase, had no
effect. The effect of PTPase inhibitors was not due to the modulation
of TNF receptors. Since both dithiothreitol and dimercaptopropanol
reversed the inhibitory effect of PAO, critical sulfhydryl groups in
the PTPase must be involved in NF-
B activation by TNF.
B activation induced by phorbol ester,
ceramide, and interleukin-1 but not that activated by okadaic acid. The
degradation of I
B protein, a critical step in NF-
B
activation, was also abolished by the PTPase inhibitors as revealed by
immunoblotting. Thus, overall, we demonstrate that PTPase is involved
either directly or indirectly in the pathway leading to the activation
of NF-
B.
(
)
is a
pleiotropic cytokine involved in modulation of growth and
differentiation, inflammation, viral replication, and septic shock (for
references see Refs. 1 and 2). How TNF signals for these wide variety
of effects is not yet understood, but the role of protein kinases,
protein phosphatases (PPase), phospholipase C and D, sphingomyelinase,
and superoxide radicals in the action of TNF has been demonstrated (for
references, see Refs. 1 and 2). The ability of TNF to induce
replication of human immunodeficiency virus-1, septic shock, and
inflammation appears to be mediated through the activation of a nuclear
transcription factor, NF-
B (for references, see Ref. 3).
B, in general, has been shown to require
dissociation of a cytoplasmic heterodimer consisting of p50 and p65
polypeptides from an inhibitory subunit termed I
B. Following
dissociation, the heterodimer translocates to the nucleus, and I
B
is rapidly degraded (for references, see Ref. 3). The mechanisms that
induce the dissociation of the heterodimeric complex and the
degradation of I
B are poorly understood, but they may involve
changes in the phosphorylation state of I
B
(4, 5, 6) . It has been demonstrated that
phosphorylation of I
B precedes its degradation
(5, 6) , but neither the kinase responsible for I
B
phosphorylation nor the protease involved in I
B degradation have
been isolated. The role of several serine-threonine protein kinases
including protein kinase C, Raf-1 protein kinase, and double-stranded
RNA activated protein kinase have been implicated
(7, 8, 9) . The I
B phosphorylation sites
also have not been identified.
B
(26, 27, 28) .
B activation, thus suggesting a role for
tyrosine-phosphorylated substrates in this activation
(29) .
Herbimycin A, another inhibitor of protein-tyrosine kinase, has also
been shown to inhibit interleukin-1-mediated NF-
B activation
(30) . By using purified I
B, it has been shown that I
B
loses its inhibiting activity upon phosphatase treatment, thus leading
to the suggestion that NF-
B activation in intact cells may depend
not only on phosphate transfer but also on phosphate removal from
I
B
(31) . What role protein phosphatase plays in
ligand-mediated activation of NF-
B in intact cells, however, is
not understood. In the present report, we have used the tyrosine PPase
inhibitors PAO, diamide, and pervanadate and the serine-threonine PPase
inhibitors okadaic acid and calyculin A to study their role in the TNF
signaling pathway leading to NF-
B activation. The results indicate
that tyrosine PPase are involved in the regulation of NF-
B
activation by TNF.
Materials
Penicillin, streptomycin, RPMI 1640
medium, and fetal calf serum were obtained from Life Technologies, Inc.
Carrier-free NaI was purchased from Amersham Corp.
Glycine, NaCl, bovine serum albumin, sodium orthovanadate, rotenone,
antimycin A, and gelatin were obtained from Sigma; calyculin and
okadaic acid were obtained from LC Laboratories (Woburn, MA), and
phenylarsine oxide was from Aldrich. Bacteria-derived recombinant human
TNF, purified to homogeneity with a specific activity of 5
10
units/mg, was kindly provided by Genentech, Inc. (South
San Francisco, CA). A rabbit polyclonal antibody against I
B
was a kind gift from Dr. Werner Greene of the University of California,
San Francisco, CA. Pervanadate was prepared from orthovanadate as
described previously
(56) . Antibodies against NF-
B
subunits p50 and p65 and double-stranded oligonucleotides having AP-1
and Sp1 consensus sequences were obtained from Santa Cruz
Biotechnology, Santa Cruz, CA.
Cell Lines
The cell lines employed in these
studies included ML-1a, a human myelomonoblastic leukemia cell line
kindly provided by Dr. Ken Takeda of Showa University, Japan; U-937, a
human histiocytic lymphoma cell line; and L-929, a murine fibrosarcoma,
both obtained from American Type Cell Culture Collection (Rockville,
MD). Human diploid fibroblasts were kindly provided by Dr. Olivia
Perriera-Smith (Baylor College of Medicine, Houston, TX). All cell
types were routinely grown in RPMI 1640 medium except L-929 cells,
which were grown in Eagle's minimum essential medium; both media
were supplemented with glutamine (2 mM), gentamicin (50
µg/ml), and fetal bovine serum (10%). The cells were seeded at a
density of 1 10
cells/ml in T25 flasks (Falcon
3013, Becton Dickinson Labware, Lincoln Park, NJ) containing 10 ml of
medium and grown at 37 °C in an atmosphere of 95% air and 5%
CO
. Cell cultures were split every 3 or 4 days.
Occasionally, cells were tested for Mycoplasma contamination using the
DNA-based assay kit purchased from Gen-Probe (San Diego, CA).
Electrophoretic Mobility Shift Assays
ML-1a cells
(2 10
cells/ml) were treated separately with
different concentrations of an activator at 37 °C. Nuclear extracts
were then prepared according to Schreiber et al.(32) .
Briefly, 2
10
cells were washed with cold
phosphate-buffered saline and suspended in 0.4 ml of lysis buffer (10
mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA,
0.1 mM EGTA, 1 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride, 2.0 µg/ml leupeptin, 2.0 µg/ml
aprotinin, and 0.5 mg/ml benzamidine). The cells were allowed to swell
on ice for 15 min, after which 12.5 µl of 10% Nonidet P-40 was
added. The tube was then vigorously vortexed for 10 s, and the
homogenate was centrifuged for 30 s in a Microfuge. The nuclear pellet
was resuspended in 25 µl of ice-cold nuclear extraction buffer (20
mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1
mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl
fluoride, 2.0 µg/ml leupeptin, 2.0 µg/ml aprotinin, and 0.5
mg/ml benzamidine), and the tube was incubated on ice for 30 min with
intermittent mixing. This nuclear extract (NE) was then centrifuged for
5 min in a Microfuge at 4 °C, and the supernatant was either used
immediately or stored at -70 °C for later use. The protein
content was measured by the method of Bradford
(33) .
P-end labeled 45-mer double-stranded NF-
B
oligonucleotide from the human immunodeficiency virus-1 long terminal
repeat (5`-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3`)
(34) in the presence of 1-2 µg of poly(dI-dC) in a
binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5
mM DTT, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl)
(35, 36) for 20 min at 37 °C. The DNA-protein
complex formed was separated from free oligonucleotide on 4.5 or 7.5%
native polyacrylamide gel using buffer containing 50 mM Tris,
200 mM glycine, pH 8.5, and 1 mM EDTA
(37) ,
and then the gel was dried. A mutated oligonucleotide was used to
examine the specificity of binding of NF-
B to the DNA. For
supershift assay, NE were incubated with the antibodies for 15 min at
room temperature before analyzing the NF-
B by EMSA.
B except that
2-3 µg of poly(dI-dC) was used in the reaction mixture.
B in vitro was performed by
treating cytoplasmic extracts with 0.8% deoxycholate for 5 min followed
by the addition of Nonidet P-40 to a final concentration of 1.6%. These
extracts were then analyzed for NF-
B by EMSA.
Western Blotting for I
After the
NF-B
B activation reaction described above, postnuclear extracts
were resolved on 9% SDS-polyacrylamide gels, electrotransferred to
nitrocellulose, probed with a rabbit polyclonal antibody against
I
B
and detected by chemiluminescence (ECL, Amersham Corp.)
(29) .
Receptor Binding Assay
TNF receptor binding and
ligand internalization studies were carried out as described previously
(38, 39) .
B. For most of the studies, human ML-1a cells were used because
their response to TNF for activation of NF-
B has been well
characterized in our laboratory
(40) . The time of incubation
and the concentration of the drugs used in our studies were found to
have no effect either on the cell viability or on the TNF receptors or
on the receptor-dependent ligand internalization (data not shown).
PAO Inhibits TNF-dependent NF-
PAO has been shown to be a specific inhibitor of PTPase.
To determine the effect of PAO on TNF-dependent NF-B Activation in ML-1a
Cells
B activation,
ML-1a cells were coincubated with different concentrations of PAO along
with TNF (0.1 nM) for 30 min at 37 °C and then examined
for NF-
B activation by electrophoretic mobility shift assay. The
results in Fig. 1 a indicate that 0.6 µM PAO
had minimum effect, but 1.2 µM PAO inhibited most of the
TNF response. This concentration of PAO by itself did not activate
NF-
B. To gain further insight into the effect of PAO, we first
incubated the cells with TNF and then added the inhibitor at 0 (same
time as TNF), 10, 15, and 20 min later. Cells were incubated with
either TNF or TNF and PAO together for a total of 30 min and then
analyzed for NF-
B activation. As shown in Fig. 1 b,
coincubation of cells with PAO and TNF together completely blocked
activation. The inhibition of the TNF response could be noted even when
PAO was added as late as 15 min after the addition of the cytokine.
These results thus indicate that PAO is a fast acting inhibitor and
could block the intermediate stages of TNF action.
Figure 1:
Dose response and kinetics of PAO and
diamide for the inhibition of TNF-dependent NF-B. a,
ML-1a cells (2
10
/ml) were coincubated at 37 °C
for 30 min with different concentrations (0.6-2.4
µM) of PAO and 0.1 nM TNF. b, ML-1a
cells (2
10
/ml) were incubated with 0.1 nM
TNF for 30 min at 37 °C, and during this incubation 1.8
µM PAO was added at different times. c, ML-1a
cells (2
10
/ml) were coincubated with various
concentrations of diamide ranging from 0.01 to 0.5 mM and 0.1
nM TNF for 30 min at 37 °C. After these treatments,
nuclear extracts were prepared and then NF-
B was assayed as
described under ``Experimental
Procedures.''
TNF-dependent NF-
In addition to PAO, we examined the effect of
pervanadate and diamide, which are also known to inhibit tyrosine
PPase. As shown in Fig. 1 c, cotreatment of ML-1a cells
for 30 min with diamide also blocked the TNF-dependent activation of
NF-B Activation Is Blocked by other
PTPase Inhibitors
B in a concentration-dependent manner. The effect of
pervanadate on the TNF-dependent NF-
B activation is shown in
Fig. 2
. These results show that pervanadate also blocked
TNF-mediated NF-
B activation. Because a recent report suggested
that in certain cells pervanadate itself could activate NF-
B
(26) , we investigated the effects of this agent on
TNF-dependent NF-
B activation in more detail. As shown in
Fig. 2a, up to 100 µM pervanadate by itself
did not activate NF-
B but 50 µM of this agent was
sufficient to completely inhibit the TNF-induced NF-
B activation.
Furthermore, preincubation with pervanadate for as little as 5 min was
sufficient to completely inhibit the TNF response
(Fig. 2 b). We also tested the effect of pervanadate when
added after the addition of TNF. The inhibition of TNF-dependent
NF-
B activation could be noted even when pervanadate was added 5
min after addition of TNF, but the maximum effect was observed only
when it was added prior to TNF (Fig. 2 c). These results
somewhat differ from those of PAO, perhaps because of the lower
cellular permeability of pervanadate
(21, 22, 23) .
Figure 2:
Effect of pervanadate at different
concentrations ( a) and at different times ( b and
c) on TNF-dependent NF-B. a, ML-1a cells (2
10
/ml) were preincubated at 37 °C for 30 min
with different concentrations (1-100 µM) of
pervanadate and then tested for NF-
B activation either with or
without 0.1 nM TNF. b, ML-1a cells (2
10
/ml) were preincubated at 37 °C with 100
µM pervanadate for different times and then tested for
NF-
B activation at 37 °C for 30 min either with or without 0.1
nM TNF. c, ML-1a cells (2
10
/ml)
were incubated at 37 °C for 30 min with TNF (0.1 nM), and
during this incubation 100 µM pervanadate was added at
different times and then tested for NF-
B activation by EMSA as
mentioned under ``Experimental Procedures.'' C indicates TNF-treated control.
TNF-dependent NF-
It is known that okadaic
acid is a specific inhibitor of serine-threonine PPase, therefore, we
investigated the effect of this inhibitor on TNF-mediated NF-B Activation Is Not Blocked by
Inhibitors of Serine-Threonine PPase
B
activation. Results of this experiment show that okadaic acid has no
effect on the NF-
B activation by the cytokine (Fig. 3).
Similarly, calyculin A, which is also a specific inhibitor of
serine-threonine PPase, had no effect on the activation (data not
shown). Since there are reports that demonstrate that okadaic acid can
activate NF-
B in certain cells
(27, 28) , the
effect of this agent was examined more in detail. The results shown in
Fig. 3a indicate that when ML-1a cells are exposed to
500 nM okadaic acid for different times, significant
activation of NF-
B occurs only after a 60-min treatment. These
results are consistent with published reports
(27, 28) .
When cells were first treated with okadaic acid for different times and
then exposed to TNF for 30 min, the ability of TNF to activate
NF-
B at all time points remained unchanged
(Fig. 3 a). Similarly when examined for the dose
response, the effect of okadaic acid by itself was dose-dependent, but
it did not affect the TNF-dependent activation. As pervanadate
inhibited the TNF-dependent activation of NF-
B, we investigated
its effect on the okadaic acid-mediated activation of NF-
B
(Fig. 3 c). These results show that pervanadate has no
effect on the NF-
B activation induced by okadaic acid, thus
suggesting that the effects of pervanadate on the TNF response is
specific and that the pathway leading to NF-
B activation by TNF
differs from that of okadaic acid.
Figure 3:
Effect of okadaic acid at different times
( a) and at different concentrations ( b) by itself and
on TNF-mediated activation of NF- kB. a, ML-1a cells (2
10
/ml) were incubated for different times with 500
nM okadaic acid and then tested for NF-
B activation
either with or without 0.1 nM TNF. b, ML-1a cells (2
10
/ml) were preincubated at 37 °C for 30 min
with different concentrations of okadaic acid and then tested for
NF-
B activation after treatment for 30 min at 37 °C either
with or without 0.1 nM TNF by EMSA as mentioned under
``Experimental Procedures.'' c, effect of
pervanadate on the okadaic acid-induced activation of NF-
B. ML-1a
cells (2
10
/ml) were preincubated for 30 min with
or without 50 µM pervanadate and then treated with okadaic
acid (500 nM) for either 15 or 60 min and then assayed for
NF-
B as described.
Effects of PTPase Inhibitors on TNF-dependent NF-
Several experiments were carried out to
demonstrate that the effect of various PTPase inhibitors on the
TNF-mediated NF-B
Activation Are Specific
B activation was specific (Fig. 4). In order
to determine that the band observed by EMSA was indeed due to p50-p65
heterodimer, we incubated the nuclear extracts from TNF-activated cells
with antibody to either p50 (NF-
B1) or p65 (Rel A) subunits and
then carried out EMSA. This technique is also referred as supershift
assay. The results from this experiment (Fig. 4 a) show
that antibodies to either subunit of NF-
B can shift the band to
higher molecular weight, thus suggesting that the TNF-activated complex
consist of both p50 and p65 subunits.
Figure 4:
Specificity of the effect of PTPase
inhibitors on the NF-B activation. For panela,
NEs were prepared from untreated or TNF- (0.1 nM) treated
ML-1a cells (2
10
/ml), incubated for 15 min with
antibodies, and then assayed for NF-
B as described under
``Experimental Procedures.'' For panelb,
NEs were incubated with different inhibitors (pervanadate, 50
µM; PAO, 2.4 µM; and diamide, 0.3
mM) for 15 min and then analyzed for NF-
B by EMSA. For
panelc, cells were treated with different inhibitors
alone or in combination with TNF. The concentrations of the inhibitors
used was same as in panelb. In lanes3 and 4, cells were treated with TNF for 5 and 20 min,
respectively. Cytoplasmic extracts were treated with 0.8% deoxycholate
and then analyzed for NF-
B by EMSA as described. For paneld, cells were treated as described for panelc; NE were prepared and then used for EMSA of AP-1 and
Sp1 transcription factors as described. In lane8, NE
from untreated cells were incubated with 100-fold in excess of cold DNA
in order to determine the specificity.
In order to demonstrate that
PTPase inhibitors by themselves do not directly modify NF-B, we
incubated nuclear extracts from TNF-activated cells with PTPase
inhibitors and then examined their ability to bind DNA. The result of
this experiment shows that all three PTPase inhibitors at the
concentrations used in our studies do not modify the ability of
NF-
B to bind DNA (Fig. 4 b). We also examined the
effect of PTPase inhibitors on the cytoplasmic pool of NF-
B. For
this, cytoplasmic extracts were prepared from the cells treated with
different inhibitors. The NF-
B was activated by treatment with
deoxycholate, and then the ability of NF-
B to bind the DNA was
examined by EMSA. These results show that none of the PTPase inhibitors
had any effect on the ability of NF-
B to bind to the DNA
(Fig. 4 c). As expected, the NF-
B was absent in the
cytoplasmic extracts prepared from TNF-treated (for 20 min) cells.
B or other transcription factors also, was investigated. The
results in Fig. 4 d clearly show that PTPase inhibitors
had no effect on AP-1 and Sp1 transcription factors, thus indicating
that the effect of these agents on NF-
B are specific.
B activation. The results
shown in Fig. 5 a clearly demonstrate that these agents
had no effect either by themselves or with TNF on NF-
B activation.
These results suggest that the effect of PAO is through its action on
PTPase.
Figure 5:a, effect of ATP lowering agents
(antimycin A and rotenone) on TNF- induced activation of NF-B. ML-1a
cells (2 10
/ml) were preincubated for 30 min at 37
°C with either antimycin A (2 ng/ml) or rotenone (2 nM)
and then tested for NF-
B activation after exposure to 0.1
nM TNF for 30 min at 37 °C. b, effect of DTT and
DMP on the PAO- and diamide-induced inhibition of NF-B activation.
ML-1a (2
10
/ml) were incubated for 30 min with DTT
(100 µM), DMP (100 µM), TNF (0.1
nM), PAO (2.4 µM), diamide (0.5 mM), or
indicated combinations and then assayed for NF-
B activation as
described under ``Experimental
Procedures.''
Reducing Agents Reverse the Effect of PAO and
Diamide
Previously it has been shown that the biological effects
of both diamide and PAO are reversed by either DTT or
2,3-dimercaptopropanol (DMP)
(10, 12, 18, 20, 44) . To
determine if DTT and DMP could reverse the effect of PAO and diamide in
our system, ML-1a cells were treated with either PAO or diamide in the
presence and absence of either DTT or DMP and then examined for the
activation of NF-B by TNF. As shown in Fig. 5b, DTT and DMP
by themselves had no effect on TNF-dependent activation of NF-
B,
but they completely reversed the inhibition induced by PAO. These
reducing agents also blocked the inhibition induced by diamide but to a
lesser extent. Thus these results demonstrate the role of sulfhydryl
groups of the PTPase in the TNF-dependent activation of NF-
B.
PAO and Diamide Block Phorbol Ester-, Ceramide-, and
Interleukin-1-mediated Activation of NF-
It has been shown
that besides TNF, NF-B
B activation is also induced by phorbol ester
(PMA), ceramide (C8), and interleukin (IL)-1. Therefore we examined the
effect of various PTPase inhibitors on activation of the transcription
factor by these various agents. The results shown in
Fig. 6
indicate that PAO completely blocked PMA-, ceramide-, and
IL-1-induced activation of NF-
B. Diamide also inhibited activation
by all three inducers, most extensively that induced by PMA. Thus
PTPase inhibitors are general suppressors of NF-
B activation.
Figure 6:
Effect of PAO and diamide on different
activators (TNF, PMA, ceramide, and IL-1) of NF-B. ML-1a cells (2
10
/ml) were incubated for 30 min at 37 °C with
PAO (1.8 µM), diamide (0.5 mM), TNF (0.1
nM), PMA (25 ng/ml), C8 ceramide (10 µM), IL-1
(100 ng/ml), or indicated combinations and then tested for NF-
B
activation as described under ``Experimental
Procedures.''
Myeloid cells express both the p60 and p80 forms of the TNF
receptor, whereas epithelial cells express primarily the p60 receptor
(41, 42) . To determine whether ML-1a results could be
extended to other cell lines, we examined the effect of PAO on
TNF-dependent NF-B activation in another myeloid cell line (U-937)
and two epithelial cell lines (L-929 and human diploid fibroblasts). We
found that treatment of cells with PAO completely abolished the
TNF-mediated activation of the transcription factor in all three lines
(data not shown), thus suggesting that the effect of PAO is not cell
type dependent.
PAO Inhibits TNF-dependent Degradation of
I
It has been shown that activation of NF-B
B requires
the dissociation of an inhibitory protein, I
B, which then
undergoes proteolytic degradation
(3) . We sought to determine
whether the inhibitory action of PAO was due to inhibition of I
B
degradation. The cytoplasmic levels of I
B protein were examined by
Western blot analysis by using I
B-specific antibodies. The results
shown in Fig. 7indicate that TNF treatment of ML-1a cells caused
I
B to disappear within 5 min; it returned to the control level by
45 min. The treatment of cells with TNF together with PAO abolished the
degradation of I
B. Since we found that the reducing agents DTT and
DMP reversed PAO's inhibitory effects on TNF-dependent activation
of NF-
B, we examined the effects of these agents on the inhibition
of I
B degradation (Fig. 7). As was the case for NF-
B
activation, DTT and DMP neutralized the effect of PAO on I
B
degradation. Thus the effect of various inhibitors of NF-
B
activation coincides with their effect on the degradation of I
B,
the inhibitory polypeptide chain.
Figure 7:
Effect of PAO, DTT, and DMP on TNF-induced
degradation of IB
. ML- 1a (2
10
/ml) were
incubated for different times with DTT (100 µM), DMP (100
µM), TNF (0.1 nM), PAO (2.4 µM), and
diamide (0.5 mM) in an indicated combinations and then assayed
for I
Ba in cytosolic fractions by Western blot analysis as
described under ``Experimental Procedures.'' The arrow indicates the position of I
B
.
B in a
time- and dose-dependent manner in both epithelial and myeloid cells.
NF-
B activation induced by various other agents such as phorbol
ester, ceramide, and IL-1 was also inhibited by these agents. Reducing
agents reversed the inhibition, thus suggesting the role of a critical
sulfhydryl group. Interestingly, serine-threonine PPase inhibitors had
no effect. The inhibition of NF-
B activation was accompanied by
the inhibition of I
B degradation.
B, but the mechanism by which the activation
occurs is not understood. Roles for ceramide, superoxide radicals,
proteases, and protein kinases in this process have been suggested
(3) . The phosphorylation of I
B has been shown to be
essential but not sufficient for the TNF-dependent activation of
NF-
B
(4, 6, 57) . The type of kinase
involved in the TNF-dependent activation of the transcription factor,
is not known. There have been some reports pointing to the
serine-threonine protein kinases in activation of NF-
B
(4, 5, 6, 7, 8, 9) .
Recent results from our laboratory, however, indicate that
TNF-dependent activation of NF-
B is dependent on
erbstatin-sensitive tyrosine kinase
(29) . Tyrosine kinases have
also been implicated in NF-
B activation by ultraviolet light,
lipopolysaccharide, hypoxia and v- src(45, 46, 47, 48, 49) .
B is not known, but our results clearly show that PAO-,
pervanadate-, and diamide-sensitive tyrosine PTPase may be important.
Interestingly neither okadaic acid nor calyculin A, inhibitors of
serine-threonine PPase, had any effect on TNF-mediated NF-
B
activation in ML-1a cells. Previously, it has been shown that okadaic
acid by itself can activate NF-
B in transformed human fibroblast
and Jurkat T cells
(27, 28) . In our ML-1a cell system,
we likewise found that okadaic acid by itself could activate NF-
B
but that this activation could not be blocked by pervanadate,
suggesting a difference in the pathway leading to NF-
B activation
by TNF and okadaic acid. Like okadaic acid, pervanadate has been shown
to activate NF-
B in Jurkat cells
(26) . The effect of
pervanadate on NF-
B activation noted in previous studies, may be
due to either the cell type or due to residual hydrogen peroxide (a
potent activator of NF-
B) remaining in pervanadate preparation.
B induced by
TNF is not clear. The degradation of I
B, essential for activation
of NF-
B, is also inhibited, suggesting that the drugs may inhibit
the protease involved in degradation. Alternatively, since the
phosphorylation of I
B has also been shown to be essential for its
degradation, it is possible that the inhibitor suppresses the
phosphorylation of I
B. This seems paradoxical, however, in view of
the reports that PAO, pervanadate, and diamide are all inhibitors of
dephosphorylation and thus should increase the levels of the
phosphorylated substrate. The paradoxical effects are also apparent
from reports that show that okadaic acid and pervanadate mimic TNF in
activating NF-
B
(26, 27, 28) . There are
reports, however, that show that PAO can stimulate the activity of
cytosolic protein-tyrosine kinases in NIH 3T3 cells
(11, 50) . If this was true in our system, one would
expect synergistic rather than antagonistic effects of TNF and PAO in
combination.
, 5-10 µM) and to increase the
levels of tyrosine-phosphorylated substrates without affecting the
activity of tyrosine kinases (Lck and Fyn)
(17, 43) .
Interestingly, in T cells, PAO increased the phosphorylation of the T
cell receptor at low concentration (1-3 µM), had no
effect at 6 µM, and completely inhibited its
phosphorylation at higher concentrations (above 12 µM)
(17) . The basis for this complex effect of PAO is not
understood. In adipocytes, PAO has been shown to induce the
phosphorylation of several proteins at serine residues, and this
correlated with inhibition of insulin-dependent glucose uptake by the
cells
(12) . Similarly in fibroblasts, pretreatment with PAO
leads to multiple insulin-induced tyrosine phosphorylation not observed
with insulin alone
(11) . This has led to the suggestion that
there are a number of tyrosine kinase substrates whose phosphorylation
is predominately regulated by phosphatases. Therefore, it is possible
that the PTPase inhibitors promote the phosphorylation of a kinase that
leads to its inactivation, which in turn prevents the activation of
NF-
B.
B activation.
All of these agents have been shown to block PTPase by interacting with
vicinal sulfhydryl groups of the enzyme. To reverse their inhibitory
effects, we used the reducing agents DTT and DMP, which in fact did
reverse the effects of PAO and diamide. These results are consistent
with a previous report that the signal transduction in natural killer
and LAK cells is inhibited by PAO, and this effect is competitively
fully blocked by DTT
(53) . The reversal of inhibitory effects
has also been reported with 2,3-dimercaptopropanol
(10, 12, 18) .
- p-tosyl-L-lysine
chloromethyl ketone, could modify cytoplasmic NF-
B in a manner
that it loses its ability to bind DNA
(57) . Our results,
however, show that PTPase inhibitors used here did not affect the
ability of NF-
B to bind DNA.
B
activation by TNF, it suggests that the NF-
B activation and growth
modulation by TNF are most likely independent responses, which is
consistent with previous reports
(54, 55) . Whether the
inhibitory effect of PTPase inhibitors is due to inactivation of PTPase
or activation of protein-tyrosine kinase or both is not certain. Since
besides inhibiting PTPase, PAO can also stimulate cytosolic
protein-tyrosine kinase
(11, 50) , it is possible that
both processes together lead to reduced phosphorylation of the key
inhibitory protein, I
B, essential for the activation of NF-
B.
By using purified I
B, it has been shown that NF-
B activation
in intact cells may depend not only on phosphate transfer onto I
B
but also on phosphate removal from I
B
(31) . Which type of
protein-tyrosine kinase or PK is essential for the activation or
inactivation of ligand-dependent activation of the nuclear
transcription factor is not known.
B activation implies that they
will also block the gene expression and inflammatory effects of TNF
that are dependent on NF-
B. In fact, the expression of TNF itself
has been shown to be dependent on NF-
B, which our studies suggest
should be inhibited by these drugs.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.