(Received for publication, December 5, 1996, and in revised form, February 19, 1997)
From the Department of Environmental Health,
University of Washington, Seattle, Washington 98195 and Bristol-Myers
Squibb Pharmaceutical Research Institute,
Seattle, Washington 98121
The role of intracellular oxidative stress in the
mechanism of action of phosphotyrosine phosphatase (PTP)
inhibitors was studied using three vanadium-based compounds.
Sodium orthovanadate (Na3VO4),
sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) (pV(phen), and bis(maltolato)-oxovanadium(IV) (BMOV) differentially induced oxidative stress in lymphocytes. Treatment with pV(phen), which caused
intracellular oxidation, induced strong protein tyrosine phosphorylation compared with Na3VO4 and BMOV.
Syk family kinases and the mitogen-activated protein kinase erk2 were
rapidly activated by pV(phen) but not by BMOV or
Na3VO4. In contrast, both BMOV and pV(phen)
strongly activated NF-B. The antioxidant pyrrolidine dithiocarbamate
(PDTC) greatly diminished the intracellular oxidation and protein
phosphotyrosine accumulation induced by pV(phen). Pretreatment of cells
with PDTC reduced and delayed the activation of Syk kinases and erk2.
However, NF-
B activation by pV(phen) was markedly enhanced in
lymphocytes pretreated with PDTC, and another antioxidant,
N-acetylcysteine, did not prevent the activation of NF-
B
by BMOV. These results indicate a role for oxidative stress in the
biological effects of some PTP inhibitors, whereas NF-
B activation
by PTP inhibitors is mediated by mechanisms independent of
intracellular redox status.
Lymphocyte signal transduction requires the activation of protein tyrosine kinases (PTKs),1 with subsequent assembly of signaling complexes, generation of second messengers, activation of transcription factors, and gene expression (1, 2). The balance of protein tyrosine phosphorylation within the cell is controlled by the relative activities of the PTKs and PTPs in the signaling network (3). Besides dephosphorylating a variety of PTK substrates, PTPs have been shown to directly modulate the activities of PTKs (4, 5). Thus PTPs serve a crucial function in lymphocytes by controlling both the initiation and termination of receptor-based signals.
The inhibition of PTPs reveals PTK substrates on which phosphotyrosine accumulates in the absence of receptor engagement (6). Some of these substrates are key phosphoproteins in lymphocyte signal transduction pathways, suggesting that PTKs involved in transmission of receptor signals are activated by the absence of PTP regulation (6-9). However, many of the PTP inhibitors used thus far to explore lymphocyte signal transduction pathways are redox-active compounds. For example, phenylarsine oxide, a thiol-reactive compound, and H2O2, which generates hydroxyl radicals, both act as potent PTP inhibitors (6, 10). The role of intracellular oxidation in the mechanism of action of PTP inhibitors is unknown, a question this study addresses.
Vanadium-based PTP inhibitors, which have been extensively studied as
insulin mimetic agents, stimulate glucose uptake and fatty acid
synthesis in adipocytes and mimic receptor-based signals in lymphocytes
(11-15). The widely used PTP inhibitor pervanadate is a peroxovanadium
compound generated by reaction of H2O2 with orthovanadate. Pervanadate is much more potent than
H2O2 or Na3VO4, causing
strong and rapid accumulation of intracellular protein phosphotyrosine
and activating Src family kinases in T-lymphocytes (7, 8). Cytokine
signaling via the transcription factor Stat1 is also mimicked by
pervanadate but not orthovanadate or vanadyl sulfate (16). Since
pervanadate is highly unstable and must be used within minutes of
generation (17, 18), the molecular mechanism of its action has not been
extensively studied but rather has been attributed solely to effects on
PTP activity.
The development of stabilized peroxovanadium compounds (19) such as
pV(phen) (Fig. 1A) provides the opportunity to more fully
characterize the action of peroxovanadium within the cell. The action
of vanadium can also be modified by organic ligands that facilitate
cellular targeting and biochemical specificity (20, 21); for example,
we found that BMOV (Fig. 1B) induced apoptosis in B cell lineages but enhanced the activation of T cell
lineages (15). In this study, we compare the action of pV(phen), BMOV,
and Na3VO4 on lymphocyte signal transduction
pathways and show that redox-dependent effects such as
early kinase activation occur in concert with the redox-independent
activation of NF-B.
Treatment of a B cell line (Ramos) or a T cell line (Jurkat) with
pV(phen) caused strong increases in intracellular oxidation, while
equimolar doses of Na3VO4 or BMOV produced only
small changes in cellular redox status. pV(phen) caused strong and
rapid induction of protein tyrosine phosphorylation in both cell lines.
By comparison, BMOV or Na3VO4 did not
appreciably affect global tyrosine phosphorylation in the doses and
times assayed. Syk tyrosine kinase was activated in cells by pV(phen)
treatment via an indirect mechanism, since the isolated kinase was not
activated by treatment in vitro. The MAPK erk2 was activated
by pV(phen) but not by BMOV or Na3VO4 in both
cell lines. Both BMOV and pV(phen) strongly activated the transcription
factor NF-B. Pretreatment with antioxidants prevented development of
intracellular oxidation and greatly diminished both the
pV(phen)-induced accumulation of protein tyrosine phosphorylation and
the activation of Syk, ZAP-70, and erk2 following pV(phen) treatment.
Surprisingly, antioxidant treatment did not prevent the activation of
NF-
B by BMOV or pV(phen). These results indicate that for many of
the effects of pV(phen), the generation of oxidative stress is
necessary. Here, we bring together results from vanadium compounds that
differ in their ability to induce intracellular oxidation, along with
effects of antioxidants on PTP inhibitor action, to provide novel
evidence for the redox-independent activation of NF-
B triggered by
PTP inhibition.
The human B cell lymphoma line Ramos and the human T cell leukemia line Jurkat were cultured in RPMI 1640 media (Life Technologies, Inc.) supplemented with 10% fetal bovine serum.
Sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) (pV(phen)) and
bis(maltolato)oxovanadium(IV) (BMOV) were synthesized as described
previously (15, 19), and the products were characterized by infrared
and mass spectroscopy. Pyrrolidine dithiocarbamate (PDTC) and sodium
orthovanadate were purchased from Aldrich. N-Acetylcysteine (NAC), MES, trypan blue, and electrophoresis reagents were from Sigma.
Dichlorofluorescin diacetate (DCF-DA) was purchased from Molecular
Probes (Eugene, OR). Goat anti-mouse IgG-horseradish peroxidase
conjugate, goat anti-rabbit IgG-horseradish peroxidase conjugate,
p-nitrophenyl phosphate (pNPP), and dithiothreitol (DTT)
were from Boehringer Mannheim. Mouse monoclonal anti-phosphotyrosine (4G10), rabbit anti-porcine Syk, rabbit anti-human Zap-70, mouse anti-human MAPK (erk2), and GST-PTP1B fusion protein were from Upstate
Biotechnology (Lake Placid, NY). Rabbit anti-IgM F(ab)2 was from Jackson Immunoresearch Labs (Westgrove, PA). Rabbit
anti-MAD3/I
B
was from Santa Cruz Biotechnology (Santa Cruz, CA).
The rabbit anti-human Syk serum used for kinase assays was a kind gift
of Dr. Joseph Bolen of Bristol-Myers Squibb. The anti-human CD3
monoclonal G19-4, and the anti-human CD45 monoclonals 1TA6 and 9.4 have
been previously described (22, 23).
Jurkat or Ramos cells were treated with PTP inhibitors in the presence of 4 µM DCF-DA (24) in serum-free RPMI 1640 (no phenol red) supplemented with 10 mM HEPES buffer (Life Technologies, Inc.). Cells were incubated at 37 °C for 2 h and then washed with phosphate-buffered saline. The development of DCF fluorescence was measured on a Cytofluor 2300 fluorescence plate reader (Molecular Dynamics, Sunnydale, CA; excitation = 485/20 nm, emission = 530/20 nm). Alternatively, DCF fluorescence was measured in cells using flow cytometry (FACSCAN, Becton Dickenson, Mountain View, CA). For PDTC-treated cells, a 60-min incubation with PDTC preceded the addition of pV(phen). Following each plate assay, cell viability was determined by trypan blue exclusion; in flow cytometric studies non-viable cells were gated out of the sample population on the basis of light scattering characteristics.
Analysis of Phosphatase ActivityActivity of a GST-phosphotyrosine phosphatase 1B fusion protein (PTP1B) was assessed against the phosphatase substrate pNPP (Boehringer Mannheim). The PTP1B was supplied bound to reduced glutathione beads. For IC50 determinations, the PTP1B was eluted from beads with three changes of elution buffer (5 mM reduced glutathione, 5 mM DTT, 1% bovine serum albumin, 150 mM NaCl, 25 mM imidazole, pH 7.2), and activity against pNPP was assayed in PTP buffer (60 mM MES, 5% glycerol, 3 mg/ml pNPP) in the absence or presence of varying concentrations of inhibitor. The rate of pNP formation was measured over 15 min following the addition of substrate, and the Vmax (A405-725/min) was recorded. In a second set of experiments, the enzyme-agarose was incubated on ice for 30 min in the presence of 5 µM inhibitor and then repeatedly washed with buffer containing 10 mM EDTA and no inhibitor. The PTP1B was then suspended in PTP buffer without inhibitors and activity against pNPP was assayed as above.
Immune Complex Phosphatase AssaysFor CD45 studies, cells were pretreated with PDTC for 1 h, followed by incubation with pV(phen) for up to 3 h. Proteins were extracted with Nonidet P-40 lysis buffer, and the mAb 1TA6 was used to immunoprecipitate CD45. After extensive washing, the immune complex was resuspended in PTP buffer containing no inhibitors, and activity against pNPP was assayed as described above.
Analysis of Tyrosine PhosphorylationCells were lysed and blotted by standard methods. Polyvinylidene difluoride membranes were stained with affinity purified anti-phosphotyrosine antibodies, with antibody binding detected by 125I-protein A followed by autoradiography, as described previously (25). Alternatively, some blots were stained with the monoclonal antibody 4G10 (Upstate Biotechnology, Lake Placid, NY) followed by enhanced chemiluminescence (ECL, Amersham Corp.).
Analysis of Syk Kinase ActivitySyk was immunoprecipitated from Ramos cell lysates with rabbit anti-human Syk (N-terminal region), and the immune complex was incubated for 10 min at room temperature in the presence of [32P]ATP as described previously (25). Proteins were separated by gel electrophoresis, transferred to polyvinylidene difluoride membrane, then incubated for 1 h in 1 M KOH at 55 °C to hydrolyze phosphoserine and phosphothreonine. The blots were dried, and autoradiography was performed. Following this exposure, the blots were rewetted and stained with rabbit anti-porcine Syk, followed by ECL to quantify Syk protein levels.
Detection of NF-Jurkat cells were washed
once in phosphate-buffered saline followed by preparation of nuclear
extracts using a modification of the procedure of Dignam et
al. (26). Briefly, 1 × 107 cells were lysed in
buffer A (10 mM HEPES, 1.5 mM
MgCl2, 10 mM NaCl, 0.25% Nonidet P-40, pH 7.5)
for 5 min at 4 °C, followed by centrifugation at 4000 rpm for 2 min.
The supernatant (cytosol) was removed and the nuclei extracted with
buffer C (20 mM HEPES, 25% glycerol, 0.42 M
NaCl, 0.2 mM EDTA, 1.5 mM MgCl2,
0.25% Nonidet P-40, pH 7.5). The nuclei were vortexed vigorously
several times over 20 min, followed by centrifugation at 14,000 rpm for
5 min. The supernatant (nuclear extract) was removed and diluted 1:2 with buffer D (20 mM HEPES, 50 mM KCl, 0.2 mM EDTA, 20% glycerol, pH 7.5) and frozen at 80 °C
until use. Gel mobility shift assays were carried out on 5-15 µg of
nuclear extract. Samples were mixed with 1.5 µg of poly(dI-dC) and
2-3 × 104 cpm of double-stranded oligonucleotide
probe (5
-AGTTGAGGGGACTTTCCCAGGC-3
) for 30 min at room temperature
prior to loading on the gel. Samples were resolved on a 6%
non-denaturing Tris borate EDTA polyacrylamide gel (Novex, San Diego,
CA); the gels were dried and labeled protein was detected by
PhosphorImager analysis (Molecular Dynamics, Sunnydale, CA).
Treatment with micromolar concentrations of pV(phen) led to
dose-dependent increases in protein phosphotyrosine in
Jurkat (Fig. 2A) and Ramos cells (Fig.
2B). Treatment with BMOV resulted in slightly increased
phosphotyrosine levels in Ramos cells; however, the induction was
substantially less than previously observed with longer treatment times
(15). Treatment with Na3VO4 caused very little
change in either cell line.
pV(phen) differs from Na3VO4 and BMOV by the
presence of two peroxy groups, which alter its chemical reactivity and
may contribute to the inhibition of PTP activity. We considered that
generation of oxidative stress might be important, as the peroxy groups
might participate in intermolecular redox cycling. The reduced
fluorescein compound DCF-DA has been widely used to assess
intracellular oxidation (24); we employed this probe to compare the
effects of the three vanadium PTP inhibitors on cellular oxidation
state. Jurkat cells were treated with DCF-DA and exposed to
Na3VO4, BMOV, or pV(phen) for up to 2 h,
and DCF oxidation was measured by flow cytometry. The pV(phen)
treatment greatly induced intracellular DCF fluorescence compared with
controls and to cells treated with equimolar concentrations of
Na3VO4 or BMOV (Fig.
3A). Cells pretreated with the antioxidant pyrrolidine
dithiocarbamate (PDTC, 100 µM) had lower basal oxidation and did not develop high DCF fluorescence following 2-h treatments with
pV(phen), orthovanadate, or BMOV (Fig. 3B). In both Jurkat and Ramos cells, the increased DCF fluorescence in pV(phen) treated cells was time-dependent (see Fig.
4B) and dose-dependent (data not shown).
Pretreatment of the cells with PDTC in a range of concentrations from
25 to 300 µM effectively eliminated the intracellular oxidation (data not shown).
Intracellular Oxidation by pV(phen) Is Independent of Kinase Activation
As the mechanism for development of intracellular oxidation following pV(phen) treatment was not known, we considered the possibility that the redox changes could be triggered by a kinase-dependent event. Examples of this type of mechanism have been found in other cell-signaling systems (27, 28). We pretreated Jurkat and Ramos cells with the tyrosine kinase inhibitor herbimycin A, which blocks antigen-receptor induced tyrosine phosphorylation (2). Herbimycin A treatment completely prevented the induction of protein tyrosine phosphorylation by pV(phen) (Fig. 4A). The DCF-DA assay was performed on cells pretreated with herbimycin A and control cells treated with Na3VO4, BMOV, or pV(phen). The dose- and time-dependent pattern of intracellular oxidation was similar for herbimycin A-pretreated cells and control cells treated with each of the different PTP inhibitors, although the herbimycin A-treated cells developed slightly enhanced intracellular oxidation compared with cells with no pretreatment (Fig. 4B). This finding confirms that the accumulation of protein phosphotyrosine following pV(phen) treatment requires kinase activation; however, intracellular oxidation occurs by a mechanism that is independent of PTK activity.
pV(phen)-induced Phosphotyrosine Accumulation Is Dependent Upon Intracellular OxidationPretreatment of Ramos or Jurkat cells
with the antioxidant PDTC inhibited the induction of protein
phosphotyrosine by pV(phen). A time course was performed on Ramos
cells; treatment for 2-8 h with pV(phen) resulted in the
time-dependent accumulation of intracellular
phosphotyrosine. Pretreatment with PDTC both delayed and decreased the
response (Fig. 5A). The widely used
antioxidants NAC and DTT could not be used because they rapidly
degraded the pV(phen) in vitro. Analysis of the absorbance
spectra of pV(phen) indicated no degradation of the pV(phen) by PDTC at
concentrations 10-fold higher than those used in this study (data not
shown). None of the PDTC doses employed in this study interfered with protein tyrosine phosphorylation following antigen receptor stimulation in B cells (Fig. 5B) or T cells (data not shown).
Pretreatment with 300 µM PDTC prior to receptor
stimulation did not interfere with Syk, Zap-70 activation, or erk2
activation.2 The PDTC treatment induced the
tyrosine phosphorylation of a few protein substrates; however, compared
with the phosphotyrosine increases stimulated by pV(phen) or receptor
engagement this effect was small.
1,10-Phenanthroline Increases Intracellular Oxidation but Not Protein Tyrosine Phosphorylation
The ligand 1,10-phenanthroline (PA) has been shown to chelate a variety of redox-active metals including copper (29); we therefore considered the possibility that PA was dissociating from the pV(phen) complex and inducing the generation of reactive oxygen within the cell. PA treatment did enhance intracellular oxidation, albeit to lower levels than pV(phen) (data not shown). However, protein tyrosine phosphorylation within the cell was not increased by PA even in combination with equimolar Na3VO4 (data not shown). Our results suggest that 1) the presence of oxidative stress alone is not enough to account for the massive protein tyrosine phosphorylation seen in pV(phen)-treated cells; and 2) the action of pV(phen) is particular to its structure and cannot be duplicated by the combination of PA plus Na3VO4.
pV(phen) Irreversibly Inhibits PTP1B and CD45 Through an Oxidative MechanismPTPs contain an active site thiol which must be
maintained in reduced form for catalysis to occur (10, 18, 30). We
postulated that the peroxy groups on pV(phen) could target this
catalytic cysteine and inactivate the enzyme through thiol oxidation.
Preliminary experiments showed that control PTP1B activity was slightly
decreased if the enzyme was assayed in the absence of antioxidants;
however, pV(phen) was a much more potent PTP inhibitor under these
conditions. Therefore we tested the ability of pV(phen) to irreversibly
inhibit the PTP1B enzyme-agarose complex under nonreducing conditions. PTP1B was completely inhibited by incubation with 5 µM of
pV(phen), BMOV, and Na3VO4. However, PTP1B
activity was recovered if the BMOV- or
Na3VO4-treated enzyme-agarose was washed
repeatedly in buffer containing 10 mM EDTA, whereas
pV(phen) inhibited PTP1B irreversibly (Fig.
6A). This inhibition appears to be due to thiol oxidation
because washing the pV(phen)-treated enzyme with 5 mM DTT
or 1% 2-mercaptoethanol in the wash buffer could partially restore
enzyme activity.2 To test the relevance of this finding in
lymphocytes, the PTP CD45 was immunoprecipitated from control Jurkat
and Ramos cells and cells treated with 25 µM pV(phen) for
3 h prior to lysis. After extensive washing, immune complex
phosphatase assays were performed. Jurkat cells had significantly
higher CD45 activity than did Ramos; however, CD45 from both cell types
was completely inactivated if the cells were treated with pV(phen)
(Fig. 6B). To test the ability of PDTC to protect CD45
activity in situ, Jurkat cells were pretreated with 100 µM PDTC prior to treatment with pV(phen). Cells were
lysed, and CD45 was immunoprecipitated and assayed as described above.
Pretreatment of the lymphocytes with PDTC prevented the complete
inactivation of CD45 in pV(phen)-treated cells, although substantial
inhibition was observed. PDTC did not alter the CD45 activity from
control cells (Fig. 6C). These results suggest that the
antioxidant treatment may inhibit the phosphotyrosine accumulation
within the cell by protecting PTPs against irreversible inhibition.
The antioxidant PDTC decreased both the induction of oxidative stress and the accumulation of phosphotyrosine in cells treated with pV(phen). To test if PDTC were interfering with the ability of pV(phen) to inhibit phosphatases, PTP1B activity against the substrate pNPP was assessed in the presence of PDTC and the three vanadium PTP inhibitors. PTP1B was inhibited by nanomolar concentrations of pV(phen), BMOV, and Na3VO4 in the presence of 300 µM PDTC (Fig. 6D). This result is consistent with assays using PTP1B against a phosphotyrosine peptide substrate (data not shown). Taken together, our results indicate that PDTC does not block the direct inhibition of PTPs by the vanadium compounds but rather prevents irreversible inhibition by protecting against the oxidation of sensitive thiols in the PTPs.
pV(phen) Activation of Protein Kinases Is Oxidation-dependentOxidative stress is known to
alter lymphocyte signal transduction pathways by activating protein
kinases (for review see Ref. 31), including activation of the PTKs Syk
and ZAP-70 (23, 25) as well as MAP kinases (32). The combination of
H2O2 and vanadate, but neither compound alone,
was shown to activate the Src family kinases p59fyn and
p56lck (9). We hypothesized that since pV(phen) increases
intracellular oxidation, it might be activating PTKs in a similar
manner to H2O2; this kinase activation combined
with phosphatase inhibition could be responsible for the massive
accumulation of phosphotyrosine seen in pV(phen)-treated cells. To
address this question, we first performed immune complex kinase assays
on Syk PTK immunoprecipitated from Ramos cell lysates. Cells were
treated with pV(phen) overnight prior to cell lysis, or, alternatively,
the pV(phen) was added to the immunoprecipitated Syk kinase just prior
to the kinase assay. Syk from pV(phen)-treated cells was highly
activated, much more than Syk from cells activated by receptor
engagement (Fig. 7A). The protein levels of
immunoprecipitated Syk did not change due to treatment (Fig.
7B). Interestingly, addition of pV(phen) directly to the
kinase assay did not activate Syk, indicating the activation of Syk by
pV(phen) is an indirect effect.
We also examined the the role of intracellular oxidation in the
activation of Syk and ZAP-70 by pV(phen). As Syk and ZAP-70 are
activated by tyrosine phosphorylation following antigen receptor engagement, the phosphotyrosine status of these proteins was used as a
marker for their activation as has been previously described (25, 33).
Treatment with pV(phen) resulted in the time-dependent activation of Syk (Fig. 8A, top panels) and
ZAP-70.2 Pretreatment with PDTC inhibited the activation of
these kinases (Fig. 8A), although some activation of Syk
occurred at later time points, consistent with the late induction of
tyrosine phosphorylation in whole cell lysates (Fig. 5A).
The blots were stripped and stained with anti-Syk; recovery of Syk
protein was similar for all treatments (Fig. 8A, bottom
panel). These results, taken together with the finding that
pV(phen) does not directly activate Syk, indicate that there is a
redox-sensitive step in the signaling pathway which occurs upstream of
Syk family kinases.
To assess the downstream effects of pV(phen) treatment, we investigated the activation of erk2, a p42 MAPK isoform, which phosphorylates the transcription factor Elk-1 (34). The Ras pathway has been shown to be sensitive to oxidative stress in rat PC12 cells (35), and in NIH3T3 cells the antioxidant NAC inhibits the activation of MAPK by H2O2, x-irradiation, and phorbol esters (32). Furthermore, the MAPK pathway is negatively regulated by the dual-specificity phosphatase MKP-1 (36, 37). MAP kinase activation state was detected by a shift in electrophoretic mobility of the phosphorylated protein. Treatment with pV(phen) resulted in a dose-dependent shift to the activated form of erk2 in both Jurkat T cells and Ramos B cells (Fig. 8B). Pretreatment with PDTC caused a very slight activation of MAPK but prevented any further activation by pV(phen). Neither Na3VO4 nor BMOV induced the activation of MAPK (data not shown).
Accumulation of Phosphotyrosine Is Delayed Compared with CD45 InhibitionTo assess the relative effects of phosphatase
inhibition and kinase activation by pV(phen), we treated Jurkat cells
with 25 µM PTP inhibitor and assayed CD45 activity and
phosphotyrosine accumulation after 30, 60, 90, or 120 min treatment.
CD45 was completely inactivated in cells treated with pV(phen) for 30 min or longer, whereas cells treated with
Na3VO4 for 120 min had only partly inhibited
CD45 activity (Fig. 9A). Recovery of CD45
from Jurkat cells was similar for all treatments (Fig. 9B).
The accumulation of phosphotyrosine in pV(phen)-treated cells was
delayed compared with inactivation of CD45 (Fig. 9C),
suggesting some secondary mechanism was primarily responsible for the
strong activation of cellular phosphorylation pathways. The induction
of oxidative stress by pV(phen) is also time-dependent (see
Fig. 4B) and is required both for kinase activation and for
the accumulation of cellular phosphotyrosine. Furthermore, no
phosphotyrosine accumulation was seen in
Na3VO4-treated cells even though the CD45 from
these cells was partially inhibited following 120 min of treatment. These results, taken together with the studies above, suggest that
peroxovanadium compounds act through the generation of intracellular oxidative stress which induces cellular signaling pathways and that
these effects are mediated by the activation of kinases.
pV(phen) Induces Tyrosine Phosphorylation of I
The
transcription factor NF-B, which controls many of the functional
effects of lymphocyte activation, is bound to the inhibitory protein
I
B
in the cytosol of unactivated cells. I
B
prevents translocation of NF-
B to the nucleus and is degraded via the ubiquitin/proteosome pathway following a variety of treatments (38).
Recently it has been reported that hypoxia induces the activation of
NF-
B by a mechanism which involves I
B
phosphorylation on
tyrosine (39). Further studies indicated that treatment with pervanadate, but not orthovanadate or H2O2
alone, resulted in I
B
tyrosine phosphorylation and activation of
NF-
B without causing I
B
degradation (40). We found that in
Jurkat cells treated with pV(phen), I
B
was
tyrosine-phosphorylated and shifted to a lower mobility form, whereas
in cells treated with BMOV, no tyrosine phosphorylation of I
B
was
observed (Fig. 10). Treatment with 300 µM
H2O2, which activated NF-
B, did not cause
tyrosine phosphorylation of I
B
(data not shown). Pretreatment of
the cells with the antioxidant PDTC prevented the tyrosine
phosphorylation of I
B
(Fig. 10A), whereas this
pretreatment did not prevent the pV(phen)-induced activation of NF-
B
(see below). Interestingly, we observed significant reduction of
I
B
protein levels following pV(phen) or BMOV treatment (Fig.
10B). These results suggest that peroxovanadium compounds
cause the induction of I
B
tyrosine phosphorylation via a
mechanism which requires intracellular oxidation; however, I
B
tyrosine phosphorylation is not necessary for the activation of NF-
B
by PTP inhibitors.
BMOV and pV(phen) Activate NF-
Numerous reports have demonstrated that
NF-B is sensitive to intracellular redox changes, and the
antioxidants PDTC and NAC have been widely used to inhibit NF-
B
activation (41, 42). We have previously reported that the combination
of H2O2 and vanadate can activate NF-
B (9).
However, a recent study of PTP inhibitors showed that peroxovanadate
and even the sulfhydryl reactive compounds diamide and phenyl arsine
oxide were inhibitors of TNF-induced NF-
B activation (43). Therefore
it was of great interest to examine NF-
B responses to pV(phen) and
BMOV. We performed gel shift assays on nuclear extracts from Jurkat
cells treated with PTP inhibitors, H2O2, and
PDTC. Treatment with 25 µM pV(phen) activated NF-
B
more strongly than did 300 µM
H2O2 (Fig. 11A). In
cells pretreated with 300 µM PDTC, as expected, the
H2O2 activation of NF-
B was prevented.
However NF-
B activation by pV(phen) was actually enhanced in cells
pretreated with PDTC. We repeated the assay with the antioxidant NAC,
which has been shown to block NF-
B activation following a variety of
stimuli. BMOV can be used in combination with NAC, whereas pV(phen) is
not stable in the presence of NAC, preventing its use for this assay.
NF-
B was strongly activated in Jurkat cells treated with 200 µM BMOV, even though this PTP inhibitor does not induce
strong intracellular oxidation. Although NAC completely prevented the
activation of NF-
B in H2O2-treated cells, it
only partially reduced the NF-
B activation following BMOV treatment
(Fig. 11B). These results demonstrate the activation of
NF-
B by vanadium PTP inhibitors by a mechanism that is independent
of intracellular oxidation.
We have characterized oxidation-sensitive and
oxidation-independent effects of three structurally distinct vanadium
PTP inhibitors, pV(phen), BMOV, and Na3VO4.
Each of these compounds was shown to inactivate PTP1B at nanomolar
concentrations in vitro. In lymphocytes treated with the
peroxovanadium compound pV(phen), large increases in intracellular
oxidation were correlated with the strong induction of cellular
tyrosine phosphorylation and activation of kinases. Treatment with BMOV
or Na3VO4, which do not contain peroxy groups, did not cause intracellular oxidation and resulted in very little tyrosine phosphorylation. However, BMOV activated NF-B without inducing strong phosphotyrosine accumulation or oxidative stress. Both
the accumulation of phosphotyrosine and the activation of specific
kinases were diminished when PDTC was employed to prevent intracellular
oxidation. Whereas the H2O2-induced activation
of NF-
B was inhibited by the antioxidants PDTC and NAC, these
antioxidants failed to prevent NF-
B activation in cells treated with
PTP inhibitors. This suggests a dual mechanism of action for
redox-active PTP inhibitor compounds, in which the early activation of
kinases requires the development of intracellular oxidation, while
other important responses such as NF-
B activation occur through
redox-insensitive pathways.
Oxidative stress is known to alter lymphocyte signal transduction pathways by activating PTKs (23, 25). p21ras and the MAPK pathway are also sensitive to changes in cellular redox status (32, 35). We found that the activation of kinases by pV(phen) was dependent upon its ability to induce oxidative stress. The effect required the intracellular millieu, however, as pV(phen) added directly to Syk kinase did not alter its activity in vitro. Similarly, the antioxidant PDTC did not prevent the inhibition of PTP1B by pV(phen) in vitro, but it partially protected CD45 from inactivation by pV(phen) in cells. This indicates the presence of a redox-active mediator that is triggered by pV(phen) but not BMOV or Na3VO4. The redox-sensitive steps in lymphocyte signaling cascades are not known but could involve changes in the associations between key signaling proteins, alterations in protein conformation, or the generation of a second messenger such as a reactive oxygen species.
The transcription factor NF-B, which is essential for T cell
activation and B cell differentiation, is known to be activated by
oxidative stress. NF-
B activation following a wide variety of
stimuli has been prevented by the antioxidants NAC and PDTC (41, 44).
Since a variety of signals leading to NF-
B activation may be blocked
by antioxidants, a reactive oxygen species has been the postulated
second messenger in NF-
B activation pathways (45, 46). However, some
studies indicate that more complex regulation of NF-
B exists,
involving both oxidation-sensitive and redox- independent pathways. The
role of oxidation in the activation of NF-
B has been shown to vary
by cell type (47). For example, oxidative stress may play a role in
NF-
B activation by the serine-threonine phosphatase inhibitor
okadaic acid, but experiments using Jurkat cells and HeLa cells yielded
contradictory results (48, 49).
Tyrosine kinase activity also appears to be necessary for the induction
of NF-B (9, 23, 50). However, the role of tyrosine phosphorylation
in NF-
B activation is much less well defined than the classical
serine phosphorylation pathway leading to ubiquitination and
degradation of I
B
. Both the tyrosine phosphorylation of I
B
and activation of NF-
B following hypoxia have been reported to be
prevented by PTK inhibitors (39). Pervanadate was reported to activate
NF-
B through a mechanism that induced I
B
tyrosine phosphorylation and did not require the proteosomal degradation of
I
B
(40). We found that pV(phen) likewise induced the tyrosine phosphorylation of I
B
; however, cytosolic levels of I
B
were substantially decreased by pV(phen) treatment (Fig. 10B).
The degree of I
B
degradation in pV(phen)-treated cells was
similar to that seen following BMOV treatment, which did not induce the
tyrosine-phosphorylated form of I
B
. Antioxidant treatment reduced
the apparent degradation of I
B
and inhibited its tyrosine
phosphorylation without preventing the activation of NF-
B.
In the present study the activation of NF-B by
H2O2 was prevented by PDTC pretreatment,
whereas NF-
B activation by pV(phen), which exerts many of its
effects through an oxidative mechanism, was enhanced rather than
prevented by the antioxidant. By comparison, BMOV induced much smaller
amounts of intracellular oxidation, and pretreatment with the
antioxidant NAC did not block NF-
B activation, although it was
reduced. These results suggest that multiple pathways, including
redox-sensitive and redox-insensitive mechanisms, can lead to the
activation of NF-
B. It is also possible that pV(phen) and BMOV
affect NF-
B responses differently following nuclear translocation,
since DNA binding of NF-
B has been shown to be inhibited under
oxidizing conditions (51, 52). The strong intracellular oxidation seen
with pV(phen) may result in diminished DNA binding independent of
nuclear translocation of NF-
B, leading to a less than maximal
response. PDTC may potentially improve the NF-
B-DNA interaction by
blocking oxidative alteration of the DNA-binding site. However, even in
the absence of PDTC, treatment with 25 µM pV(phen)
induced greater NF-
B-DNA binding than treatment with 300 µM H2O2. The pV(phen)-induced
binding was enhanced in the presence of PDTC, whereas
H2O2 induced binding was totally abrogated.
Taken together, these results suggest the existence of an efficient,
redox-insensitive pathway for NF-
B activation which can be
stimulated by the inhibition of PTPs.
PDTC has been widely used as an antioxidant, and its ability to prevent oxidative stress may be determined by the combination of its functions as a metal chelator and a thiol-reducing agent (42). A recent report demonstrated that low dose treatments with PDTC could actually increase oxidative stress within thymocytes, due to increased cellular import of copper, a highly redox-active metal (53). We did not observe this effect within the PDTC dose and time regimes we tested; the PDTC acted only as an antioxidant in our hands. However, PDTC at high doses was toxic to cells despite its ability to prevent intracellular oxidation. The mechanism for this toxicity was not determined. In the short term, low dose experiments we performed, the PDTC treatments did not result in cell death.
Vanadium PTP inhibitors have been extensively used as tools for
elucidating the regulation of cellular signal transduction by
phosphatases. These compounds have been shown to normalize glucose
levels in animal models of diabetes mellitus and have achieved
favorable responses in human clinical trials (54). Likewise, they could
prove important in other applications for which the control of cellular
tyrosine phosphorylation is desired. In the present study, we found
that differences in the effects of three structurally distinct vanadium
PTP inhibitors could be partially explained by their ability to induce
intracellular oxidation. Our finding that antioxidant treatment can
protect cellular CD45 from total inactivation by pV(phen) suggests a
direct role for oxidative stress in the greatly increased potency of
peroxo derivatives of vanadate. A recent publication (18) comparing
pervanadate to Na3VO4 showed that in PTP1B the
active site cysteine was oxidized to cysteic acid by pervanadate,
whereas Na3VO4 did not permanently alter the
active site thiol. We have shown that while the development of
oxidative stress causes both the activation of kinases and the
irreversible inactivation PTPs, the contribution of kinase activation
is more important than PTP inhibition for the accumulation of
phosphotyrosine within the cell. However, the finding that oxidative
stress and phosphotyrosine accumulation are not required for NF-B
activation by vanadium PTP inhibitors suggests that PTP inhibition
activates signaling cascades which are more difficult to observe,
albeit quite important to understand. Our results demonstrate a role
for oxidative stress in many of the effects of PTP inhibition and
suggest the presence of a PTP-regulated, redox-independent pathway for
NF-
B activation.
We thank Peter Kiener for helpful discussions
of PTP action, Tony Hunter for advice and discussion about IB
,
and Derek Hewgill for expert technical assistance in the flow cytometry
studies.