©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein-tyrosine Phosphatase Inhibitors Block Tumor Necrosis Factor-dependent Activation of the Nuclear Transcription Factor NF-B (*)

Sanjaya Singh , Bharat B. Aggarwal (§)

From the (1) Cytokine Research Laboratory, Department of Clinical Immunology and Biological Therapy, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Most of the inflammatory and proviral effects of tumor necrosis factor (TNF) are mediated through the activation of the nuclear transcription factor NF-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.

PTPase inhibitors also blocked NF-B activation induced by phorbol ester, ceramide, and interleukin-1 but not that activated by okadaic acid. The degradation of IB 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.


INTRODUCTION

Tumor necrosis factor (TNF)() 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).

The activation of NF-B, in general, has been shown to require dissociation of a cytoplasmic heterodimer consisting of p50 and p65 polypeptides from an inhibitory subunit termed IB. Following dissociation, the heterodimer translocates to the nucleus, and IB is rapidly degraded (for references, see Ref. 3). The mechanisms that induce the dissociation of the heterodimeric complex and the degradation of IB are poorly understood, but they may involve changes in the phosphorylation state of IB (4, 5, 6) . It has been demonstrated that phosphorylation of IB precedes its degradation (5, 6) , but neither the kinase responsible for IB phosphorylation nor the protease involved in IB 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 IB phosphorylation sites also have not been identified.

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-B (26, 27, 28) .

We recently demonstrated that erbstatin, an inhibitor of protein-tyrosine kinase, inhibits TNF-dependent NF-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 IB, it has been shown that IB 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 IB (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.


EXPERIMENTAL PROCEDURES

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 IB 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) .

Electrophoretic mobility shift assays (EMSA) were performed by incubating 4-5 µg of nuclear extract with 16 fmol of 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.

The EMSA for AP-1 and Sp1 were performed as described for NF-B except that 2-3 µg of poly(dI-dC) was used in the reaction mixture.

The activation of NF-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.

Visualization of radioactive bands were carried out by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using Imagequant software.

Western Blotting for IB

After the NF-B activation reaction described above, postnuclear extracts were resolved on 9% SDS-polyacrylamide gels, electrotransferred to nitrocellulose, probed with a rabbit polyclonal antibody against IB 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) .


RESULTS

In this report, we used inhibitors of PPases to examine the role of protein phosphorylation in the TNF-dependent activation of NF-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-B Activation in ML-1a Cells

PAO has been shown to be a specific inhibitor of PTPase. To determine the effect of PAO on TNF-dependent NF-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-B Activation Is Blocked by other PTPase Inhibitors

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 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-B Activation Is Not Blocked by Inhibitors of Serine-Threonine PPase

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. 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-B Activation Are Specific

Several experiments were carried out to demonstrate that the effect of various PTPase inhibitors on the TNF-mediated NF-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.

Whether PTPase inhibitors specifically block the activation of NF-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.

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-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-B

It has been shown that besides TNF, NF-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 IB

It has been shown that activation of NF-B requires the dissociation of an inhibitory protein, IB, which then undergoes proteolytic degradation (3) . We sought to determine whether the inhibitory action of PAO was due to inhibition of IB degradation. The cytoplasmic levels of IB protein were examined by Western blot analysis by using IB-specific antibodies. The results shown in Fig. 7indicate that TNF treatment of ML-1a cells caused IB 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 IB. 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 IB degradation (Fig. 7). As was the case for NF-B activation, DTT and DMP neutralized the effect of PAO on IB degradation. Thus the effect of various inhibitors of NF-B activation coincides with their effect on the degradation of IB, 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 IBa in cytosolic fractions by Western blot analysis as described under ``Experimental Procedures.'' The arrow indicates the position of IB.




DISCUSSION

The results shown here clearly indicate that inhibitors of PTPase completely block the TNF-dependent activation of NF-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 IB degradation.

TNF is one of the most potent activators of NF-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 IB 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) .

What role protein PTPase plays in the TNF-dependent activation of NF-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.

How PTPase inhibitors block the activation of NF-B induced by TNF is not clear. The degradation of IB, 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 IB has also been shown to be essential for its degradation, it is possible that the inhibitor suppresses the phosphorylation of IB. 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.

In insulin and T cell receptor systems, PAO has been shown to inhibit the tyrosine phosphatase activity of CD45 (IC, 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.

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-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) .

Recently it was reported that serine protease inhibitors, L-tosylamido-2-phenylethyl chloromethyl ketone and N- 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.

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-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, IB, essential for the activation of NF-B. By using purified IB, it has been shown that NF-B activation in intact cells may depend not only on phosphate transfer onto IB but also on phosphate removal from IB (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.

In summary, the suppressive effect of PTPase inhibitors on NF-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.


FOOTNOTES

*
This work was supported in part by The Clayton Foundation for Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 713-792-3503; Fax: 713-794-1613.

The abbreviations used are: TNF, tumor necrosis factor; p60 (also referred to as p55), TNF receptor I or TNF receptor type B; p80 (also referred to as p75), TNF receptor II or TNF receptor type A; PTPase, protein-tyrosine phosphatase; PPase, protein phosphatase; PAO, phenylarsine oxide; DTT, dithiothreitol; DMP, 2,3-dimercaptopropanol; NE, nuclear extract; EMSA, electrophoretic mobility shift assay; PMA, phorbol 12-myristate 13-acetate; IL, interleukin.


ACKNOWLEDGEMENTS

-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.


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