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Originally published online as doi:10.1189/jlb.0505252 on December 14, 2006

Published online before print December 14, 2006
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(Journal of Leukocyte Biology. 2007;81:835-844.)
© 2007 by Society for Leukocyte Biology

Role of protein tyrosine phosphatases in the regulation of interferon-{gamma}-induced macrophage nitric oxide generation: implication of ERK pathway and AP-1 activation

Julie Blanchette*, Philippe Pouliot{dagger} and Martin Olivier*,{dagger},1

* Centre de Recherche en Infectiologie and Département de Biologie Médicale, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, Université Laval, Ste-Foy, Québec, Canada; and
{dagger} Centre for the Study of Host Resistance, The Research Institute of the McGill University Health Centre, Departments of Experimental Medicine, Microbiology and Immunology, Faculty of Medicine, McGill University, Montréal, Québec, Canada

1 Correspondence: McGill University, Department of Microbiology and Immunology, Duff Medical Building, Room 511, 3775 University Street, Montréal, Québec, Canada H3A 2B4. E-mail: martin.olivier{at}mcgill.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NO is a potent molecule involved in the cytotoxic events mediated by macrophages (MØ) against microorganisms. We reported previously that inhibition of MØ protein tyrosine phosphatases (PTPs) mediates a protective effect against Leishmania infection, which was NO-dependent. Herein, we show that the PTP inhibitors of the peroxovanadium (pV) class, bpV(phen) and bpV(pic), can similarly increase murine MØ IFN-{gamma}-induced NO generation. Using various second messenger (JAK2, MEK, Erk1/Erk2, and p38) antagonists, we found that the Erk1/Erk2 pathway was the principal pathway submitted to regulation by PTPs in the context of IFN-{gamma}-driven MØ activation and increase in NO production. We observed that bpV(phen) increases inducible NO synthase (iNOS) expression, resulting in enhanced NO production, whereas the bpV(pic) increase of NO production does not seem to result from a modulation of iNOS expression. Transcription factors STAT-1{alpha} and NF-{kappa}B, recognized for their importance in NO generation, were not affected by the pV treatment. However, AP-1 was strongly activated by bpV(phen) but not by bpV(pic). Collectively, our results suggest that increased IFN-{gamma}-induced NO production, observed after bpV(phen) treatment, involves the activation of the transcription factor AP-1 by Erk1/Erk2- and stress-activated protein kinase/JNK-dependent transduction mechanisms.

Key Words: protein tyrosine kinases • transcription factors • peroxovanadiums • second messengers


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Known as an important messenger, NO [1 ] is the product of catalytic transformation of L-arginine to L-citrulline by NO synthase (NOS). It is an important, gaseous-free radical molecule involved in various biological systems (reviewed in refs. [1 , 2 ]). Its physiological and cellular effects include vasorelaxation [3 ], inhibition of platelet aggregation [4 ], neurotransmission [5 ], as well as antimicrobial and cytotoxic functions of rodent macrophages (MØ) against intracellular pathogens [6 7 8 ], viruses [9 , 10 ], and tumor cells [11 ]. Three isoforms of NOS have been identified: the constitutively expressed neuronal NOS (nNOS) [12 ], the endothelial NOS (eNOS) [13 ], and the inducible NOS (iNOS; also known as NOS2) [14 15 16 ]. nNOS and eNOS are Ca2+/calmodulin-dependent enzymes [17 , 18 ], whereas iNOS activity is not modulated by Ca2+. Consequently, the transcriptional control of this gene is of prime importance for the regulation of its enzymatic activity [19 20 21 ].

NO implication in the control of Leishmania infection is well documented [22 23 24 ]. It is interesting that it has been reported that this parasite can inhibit MØ NO generation to survive and propagate within its mammalian host [25 ]. In previous studies, we and others showed that IFN-{gamma}-triggered, JAK2-dependent signaling was altered in Leishmania-infected MØ, a result that could explain the inhibition observed previously in IFN-{gamma}-induced NO generation in the same MØ [26 , 27 ]. To understand better how the inhibition of NO production was affected by this abnormal JAK2 signaling pathway, our team investigated the mechanism previously, whereby Leishmania regulated this signal pathway negatively. We have already showed that JAK2 inhibition was accompanied by rapid activation of the protein tyrosine phosphatase (PTP) Src homology 2 domain PTP 1 (SHP-1) and their molecular association [26 ]. This clearly suggested that host PTPs and more precisely, SHP-1 play a pivotal role in the establishment and propagation of Leishmania. In an in vivo context, we and others [23 , 24 , 28 ] have shown, using the PTP inhibitors peroxovanadium (pV) and SHP-1-deficient, motheaten mice, that Leishmania infections cannot progress normally in vitro and in vivo in the absence of host PTPs. It is interesting that in our model, the iNOS antagonists restored the ability of this pathogen to propagate within a pV-treated host [23 , 24 ]. These results clearly show the relevance of our in vitro observations about JAK-2 inhibition (in MØ) to an in vivo mouse model and may explain the incapacity of the parasite to infect mice with inhibited PTP activity [24 ] (or with deficient SHP-1 activity [23 ]), where JAK-2 inhibition by PTPs cannot be achieved.

Given these previous results, we were interested to know the mode of action of pV compounds in their modulation of IFN-induced NO generation. Knowing the importance of the interaction between the MØ and the Leishmania parasite [29 ], we were interested to identify how the pV compounds are able to modulate NO generation in the context of the MØ signaling. pV compounds are the most potent PTP inhibitors developed to date and are chemically defined as pervanadate derivatives, each containing an oxo ligand, one or two peroxo anions in the inner coordination sphere of vanadium, and an ancillary ligand [30 ]. In a different context, they are known to activate the insulin receptor kinase and mimic insulin biological action in vivo [31 ]. Moreover, they have the capacity to inhibit the proliferation of neuronal cell lines in vitro [32 ] and to activate immune cell responses [33 ], such as IFN-{gamma}-induced MØ NO generation [26 ]. We used two different pV compounds previously to successfully prevent Leishmania infection [24 ]. However, although both compounds resulted in the same reduction of infection, we observed a different profile of tyrosil phosphorylation in liver proteins, suggesting that both compounds act differently on host cells [24 ]. Therefore, we were interested in comparing the effects that the PTP inhibitors of the pV class, bpV(phen) and bpV(pic), could mediate in the IFN-induced NO generation.

The use of pV compounds [bpV(phen) and bpV(pic)] has permitted us to obtain clear evidence of the role played by PTPs in the regulation of NO generation. Enhanced, IFN-{gamma}-induced NO generation in bpV(phen)-treated MØ could be attributed to a selective modulation of Erk1/Erk2 and stress-activated protein kinases (SAPKs)/JNK, leading to AP-1 activation, correlated by increased iNOS expression. By contrast, JAK2, MEK, and p38 kinases, as well as the transcription factors STAT-1{alpha} and NF-{kappa}B, were not modulated significantly by bpV(phen)-mediated inhibition of host PTPs. Of interest, our study suggests further that bpV(pic)-mediated NO generation enhancement involves an event independent of iNOS expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Isotopes were obtained from Valeant Pharmaceuticals International (Montréal, PQ, Canada). Recombinant murine IFN-{gamma} (2x105 units/ml) was purchased from Gibco-BRL (Burlington, ON, Canada). The anti-Tyr(P) antibody (Clone 4G10) was purchased from Upstate Biotechnology Inc. (Walthem, MA, USA), and anti-iNOS antibody was purchased from Cedarlane Laboratories Ltd. (Hornby, ON, Canada). The pV compounds K[VO(O2)2(1,10-phenanthroline)] · 3H2O [bpV(phen)] and K2[VO(O2)2(pyridine-2-carboxylate)] · 2H2O [bpV(pic)] used in this study were synthesized as described previously [28 ] and kindly provided by Dr. Barry I. Posner (McGill University, Montréal, Canada). The Erk1/Erk2 MAPK inhibitor apigenin, MEK inhibitor PD98059, the MAPK p38 inhibitor SB203580, and NF-{kappa}B inhibitor BAY 11-7082 were purchased from Calbiochem (Boston, MA, USA). The JAK2 inhibitor AG490 was purchased from BioMol International (Plymouth Meeting, PA, USA), and the NF-{kappa}B inhibitor sodium salicylate and rapamycin were purchased from Sigma-Aldrich (Oakville, ON, Canada). The oligonucleotides specific for AP-1 and NF-{kappa}B binding were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the iNOS IFN-{gamma}-activated site (GAS)-containing oligonucleotide was synthesized in our laboratory based on Gao et al. [34 ].

Cell culture
The murine MØ cell line J774 was maintained in DMEM (Invitrogen Life Technologies, Burlington, ON, Canada) supplemented with 10% FBS (Hyclone, Technology Inc., Logan, UT, USA) plus streptomycin (100 µg/ml) and 2 mM L-glutamine at 37°C and 5% CO2. MØ cell line J774 was obtained from the American Type Culture Collection (Manassas, VA, USA; J774A-1, ATCC #TIB-67).

NO production
Briefly, J774 MØ were seeded in 24-well dishes (5x105 cells/well) and cultured in the presence or absence of bpV(phen) or bpV(pic) (0–10 µM) for 1 h prior to IFN-{gamma} stimulation (100 units/ml, 24 h). In some experiments, cells were treated with various second messenger inhibitors 1 h prior to stimulation as described above. The NO generation was evaluated by measuring the nitrite accumulation in the culture medium by the Griess reaction as reported previously [28 ].

Western blotting
Cells were collected (106–107) and disrupted in ice-cold lysis buffer [20 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 10% glycerol (v/v), 1% Nonidet P-40 (NP-40; v/v), 10 µM NaF, 1 mM sodium ortho-vanadate, 100 µg/ml PMSF, and protease inhibitors (25 µg/ml aprotinin and leupeptin)]. The lysates (30 µg/lane) were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) as described previously [28 ]. Specific proteins and their state of phosphorylation were detected with antibodies directed against iNOS (Cedarlane Laboratories Ltd.), phosphotyrosine-JAK2, phosphotyrosine-Erk1/Erk2 (p42/p44), phophotyrosine-MEK (p43), and phosphotyrosine-p38 (BioSource International, Camarillo, CA, USA) and p-Y-STAT-1 and p-Ser727-STAT-1 (kindly provided by Dr. David Frank, Dana Farber Cancer Institute, Boston, MA, USA). To monitor the amount of protein loaded in each lane, membranes were stripped and reprobed with anti-JAK2 antibody (C-20 rabbit polyclonal IgG) or anti-STAT-1 antibody (C-111 mouse monoclonal IgG), purchased from Santa Cruz Biotechnology, and anti-p42/p44, anti-MEK (p43), or anti-p38 antibodies, purchased from BioSource International. Proteins were detected with antimouse or antirabbit HRP-conjugated antibodies (Amersham Biosciences, Piscataway, NJ, USA), and visualization was done using the ECL Western blotting detection system (Amersham Biosciences).

Northern blot analysis
Expression of the iNOS gene in IFN-{gamma}-stimulated J774 MØ (100 units/ml, 0–8 h), treated or not with pV compounds and specific inhibitors, was evaluated by Northern blot of total mRNA as reported previously [26 ]. Briefly, after stimulation, cells were washed twice with PBS, and total RNA was extracted with TRIzol (Gibco-BRL). RNA (10 µg) was then subjected to electrophoresis on 1% agarose gels, transferred onto Hybond-N filter paper, and hybridized with random, primer-labeled cDNA probes. Equal RNA loading was confirmed by hybridization with a GAPDH cDNA probe kindly provided by Dr. Danuta Radzioch (McGill University). All washes were performed under stringent conditions, and transcripts were visualized by autoradiography.

EMSA
Cells grown to a density of 2 x 106 cells per flask were treated as indicated. Reactions were stopped by addition of ice-cold PBS. In brief, sedimented cells were resuspended in 400 µl cold buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2 mM PMSF). After 15 min on ice, the lysates were vortexed for 10 s, and 25 µl NP-40 (10%) was added to each sample before being centrifuged for 30 s at 12,000 g. The supernatants were discarded, and the cell pellets were resuspended in 50 µl cold buffer C[20 mM Hepes-KOH, pH 7.9, 25% glycerol (v/v), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF] and incubated on ice for 15 min. Cellular debris was removed by centrifugation at 12,000 g for 5 min at 4°C, and the supernatant containing nuclear proteins was stored at –70°C until further use. Then, 6 µg nuclear extract protein was subjected to electrophoresis on a 4% polyacrylamide gel. All the samples were labeled with [{gamma}-32P]ATP containing an oligonucleotide directed toward the nuclear factors of interest. After migration, the gel was dried and exposed to X-ray film (Kodak, Rochester, NY, USA). The sequences of oligonucleotides used were 5'-AGTTGAGGGGACTTTCCCAGGC-3' for NF-{kappa}B, 5'-AGCTCGCGTGACTCAGCTG-3' for the factor AP-1, and 5'-CTTTTCCCCTAACAC-3' for iNOS GAS {GAS/IFN-stimulated response element (ISRE)/iNOS [33 ]}.

Statistical analyses
Statistically significant differences were identified using the ANOVA and the Fisher least significant difference test module of SAS software (Version 6.07, SAS Institute, Cary, NC, USA). P values of <0.05 were deemed statistically significant. All data are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modulation of IFN-{gamma}-induced NO generation by PTP inhibitors bpV(phen) and bpV(pic)
To extend our previous findings about the modulation of MØ NO generation by the PTP inhibitor bpV(phen) [28 ], we analyzed the role of PTP in the modulation of signaling events that regulate NO using the pV compounds bpV(phen) and bpV(pic). As depicted in Figure 1 , both compounds similarly enhanced MØ NO generation in response to IFN-{gamma} in a dose-dependent manner, corroborating our previous report about bpV(phen) [28 ]. Of interest, whereas bpV(phen) per se, at doses of 5–10 µM, was able to induce a significant production of NO, bpV(pic) was a weak NO inducer when used alone. This observation suggests that different PTPs are inhibited by these two pV compounds and that this variability may lead to a different modulation of iNOS expression and subsequent NO generation. This suggested a differential capacity of bpV(phen) and bpV(pic) to target different PTPs is supported by our previous observation of distinct tyrosyl phosphorylation profiles of liver proteins in mice treated with these compounds [24 ].


Figure 1
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  		Figure 1. Modulation of IFN-{gamma}-induced MØ NO generation by pV compounds. J774 MØ (5x105 cells/well) were cultured in triplicate in flat-bottom, 24-well culture plates and treated with increasing doses (1–10 µM) of bpV(phen) and bpV(pic) 1 h prior IFN-{gamma} stimulation (100 U/ml, 24 h). Thereafter, nitrite accumulation in the supernatants was determined by the Griess reaction (see Materials and Methods). This result is representative of one out of three experiments performed independently. *, Significant increase (P<0.05) of nitrite production compared with positive control (IFN-{gamma} alone). NIL, untreated cells.

 
Role of Erk1/Erk2 MAPKs in enhanced, IFN-{gamma}-induced NO generation by pV-treated MØ
We and others [35 36 37 38 ] have generated data suggesting an essential role for JAK2, Erk1/Erk2, and MEK kinases in the signaling events leading to NO generation by different cell types subjected to IFN-{gamma} stimulation. Based on these observations, the potential role played by these kinases on NO production in pV-treated cells was evaluated. Involvement of these pathways in pV-modulated, functional events was evaluated using selective inhibitors against JAK2 (AG490, 75 µM), Erk1/Erk2 (apigenin, 50 µM), MEK (PD98059, 40 µM), and p38 (SB203580, 0.1 µM) at maximal and subcytotoxic concentrations. As shown in Figure 2A , bpV(phen) and bpV(pic) enhancement of IFN-{gamma}-induced NO generation in MØ treated with AG490 was virtually unaltered for bpV(phen) and lightly reduced for bpV(pic) compared with the total inhibition of NO release in cells treated solely with bpV/IFN-{gamma}. Similar observations were noted for MØ treated with MEK and p38 inhibitors (Fig. 2C and 2D) , suggesting that the modulation of enhanced, IFN-{gamma}-induced NO generation by pV compounds does not involve JAK2, MEK, or p38 pathways. The results obtained with the MEK inhibitor PD98059 are consistent with a previous study in which Erk1/Erk2 MAPKs were shown to be activated in a MEK-independent manner in bpV(phen)-treated hepatocytes [39 ]. Of interest, a total inhibition of NO generation was observed in cells treated with the Erk1/Erk2 MAPK inhibitor apigenin (Fig. 2B) . This suggests that enhanced, IFN-{gamma}-induced NO production in both pV compound-treated MØ could be attributed to Erk1/Erk2-selective activation in response to host PTP inhibition. This evidence is reinforced further by the observation that the capacity of bpV(phen) per se to induce NO generation is blocked by apigenin treatment. Our results also suggest that inhibition of MØ PTP by bpV(pic) differentially modulates other signaling events responsible for enhanced NO generation in response to IFN-{gamma} stimulation: JAK2, MEK, and p38 kinase-selective inhibitors reduced NO generation partially but significantly (Fig. 2A 2C and 2D) as opposed to the lack of effect observed with bpV(phen). Collectively, these data suggest that negative regulation of Erk1/Erk2 phosphorylation by PTP plays a pivotal role in the control of IFN-{gamma}-mediated NO generation, although other pathways can be used.


Figure 2
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  		Figure 2. Effect of JAK2-, Erk1/Erk2-, MEK-, and p38-specific inhibitors on modulation of NO generation by pV compounds. MØ incubated in the presence of 10 µM bpV(phen) (PHEN) or bpV(pic) (PIC) were incubated in the presence of inhibitors prior to their stimulation with IFN-{gamma} (100 U/ml, 24 h). (A) JAK2 (AG490, 75 µM); (B) Erk1/Erk2 [apigenin (API), 50 µM]; (C) MEK [PD98059 (PD), 40 µM]; and (D) p38 [SB203580 (SB), 0.1 µM]. NO generation was monitored as described in Figure 1 . Results are representative of three experiments performed independently. *, Significant antagonist-mediated (P<0.05) reduction of nitrite production compared with bpV(phen) or bpV(pic), alone or in combination with IFN-{gamma}.

 
Selective modulation of iNOS-dependent and independent pathways by bpV(phen) and bpV(pic), respectively
To understand the signaling mechanism underlying this pV-enhanced NO generation further, the modulation of iNOS expression by both PTP inhibitors was monitored in the same experimental context as described above. As depicted in Figure 3A 3B 3C 3D , bpV(pic)-treated MØ did not express iNOS mRNA and protein compared with NIL, whereas bpV(phen), as we reported previously [28 ], was, per se, a potent iNOS inducer. In addition, iNOS expression was only enhanced in response to IFN-{gamma} in cells pretreated with bpV(phen), whereas bpV(pic)-treated MØ showed iNOS expression similar to control cells stimulated solely with IFN-{gamma}. These results suggest that exacerbated NO generation observed in response to IFN-{gamma} by bpV(phen) and bpV(pic) involved iNOS expression-dependent and iNOS expression-independent events, respectively. The use of JAK2, Erk1/Erk2, MEK, and p38 kinase inhibitors has allowed us to further support the role played by Erk1/Erk2 in the NO exacerbation observed in pV-treated cells. Erk1/Erk2 inhibitor apigenin was the only one able to block bpV(phen)-mediated, enhanced iNOS expression (Fig. 3B) . All inhibitors used, with the exception of p38 inhibitor SB203580 (Fig. 3D) , were shown (Fig. 3A and 3C) to inhibit iNOS gene and protein expression totally or partially in naïve, IFN-{gamma}-stimulated, and bpV(pic)-treated MØ. Taken together, these results indicate that enhanced NO generated by bpV(pic)-treated cells in response to IFN-{gamma} uses an independent pathway, whereas the selective modulation of the MAPK Erk1/Erk2 by bpV(phen) results in enhanced iNOS expression (Fig. 3) and NO generation (Fig. 2) .


Figure 3
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  		Figure 3. Effect of kinase inhibitors on modulation of iNOS expression by pV compounds in IFN-{gamma}-stimulated cells, which were incubated with bpV(phen) or bpV(pic), alone or in combination with IFN-{gamma}, with or without a coincubation with JAK2 inhibitor AG490 (75 µM, A); Erk1/Erk2 inhibitor apigenin (50 µM, B); MEK inhibitor PD98059 (40 µM, C); and p38 inhibitor SB203580 (0.1 µM, D). The iNOS gene (8 h poststimulation) and protein expression (24 h poststimulation) have been monitored, respectively, by Northern and Western analyses as described in Materials and Methods. These results are representative of three separate experiments.

 
Capacity of bpV(phen) to induce selective AP-1 nuclear translocation over NF-{kappa}B and STAT-1{alpha} (GAS/ISRE/iNOS) in IFN-{gamma}-stimulated and unstimulated cells
Regarding the data reported above, we were interested in establishing the role of transcription factors (i.e., STAT-1{alpha}, NF-{kappa}B, AP-1) recognized for their implication in iNOS expression by MØ subjected to stimulation with IFN-{gamma} alone [36 ] or in combination with LPS [34 , 40 41 42 ]. As shown in Figure 4A and B, the GAS/ISRE/iNOS (STAT-1{alpha} for the specific site of iNOS) and NF-{kappa}B transcription factors are not induced by pV compounds tested but are translocated to the nucleus by IFN-{gamma} stimulation, as observed in naive cells under similar conditions. Similar results were observed at 15 min for NF-{kappa}B (data not shown). Of interest, we observed that cells treated with bpV(phen), followed or not by an IFN-{gamma} stimulation for 4 h, show an important degree of AP-1 nuclear translocation, whereas IFN-{gamma} per se or in combination with bpV(pic) was a weak AP-1 activator. This last piece of data suggests that selective activation of AP-1 by bpV(phen) could be responsible for the enhanced iNOS expression reported above and further reinforces our concept that bpV(pic) uses an iNOS-independent pathway to conduct its exacerbation effect on NO generation in response to IFN-{gamma}.


Figure 4
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  		Figure 4. Modulation of IFN-{gamma}-mediated nuclear translocation of STAT-1{alpha} (GAS/iNOS), NF-{kappa}B, and AP-1 transcription factors by pV compounds. Cells were treated with pV compounds and IFN-{gamma} as described in previous figures. Activation of STAT-1{alpha} was monitored for 15 min poststimulation using an oligonucleotide for the specific site of iNOS (GAS/iNOS; A) or 4 h poststimulation for NF-{kappa}B (B) and AP-1 (C) transcription factors. The last lane in each panel represents nuclear extracts of IFN-{gamma}-stimulated samples subjected to competition with its respective cold oligonucleotide (CO) to confirm signal specificity. Results are representative of three independent experiments.

 
BpV(phen)-mediated phosphorylation of second messengers
As suggested by our results, bpV(phen)-mediated iNOS expression is Erk1/Erk2- and AP-1-dependent. To further decipher and support the implication of specific second messengers in the activation of AP-1 nuclear translocation by bpV(phen), the phosphorylation state of JAK2, STAT-1{alpha}, Erk1/Erk2 (p44/p42), MEK, p38, and SAPK/JNK (p54/p46) second messengers has been monitored. As shown in Figure 5 , bpV(phen) did not induce the phosphorylation of JAK2 or STAT-1{alpha} on its tyrosyl and seryl residues. These results correlate with our previous observations (Fig. 4) showing the incapacity of bpV(phen) to induce GAS/ISRE/iNOS nuclear translocation. Conversely, we observed that bpV(phen) can induce the phosphorylation of Erk1/Erk2, MEK, p38, and SAPK/JNK significantly. These results further reinforce our previous observations, suggesting that Erk1/Erk2 MAPKs are key players in bpV(phen)-mediated events leading to NO production synergism. Whereas bpV(phen) has shown a capacity to induce MEK and p38 phosphorylation, our previous results using specific inhibitors for these proteins did not affect the capacity of bpV(phen) to enhance, per se or in combination with IFN-{gamma}, the expression of iNOS and its subsequent NO generation. JNK has been shown to be involved in AP-1 activation (reviewed in ref. [43 ]). Based on these observations and on results obtained in the present study, we thought it was of importance to monitor whether bpV(phen) was capable or not to induce SAPK/JNK phosphorylation. As depicted at the bottom of Figure 5 , our results suggest that this kinase is modulated by bpV(phen) treatment. Collectively, these data demonstrate that bpV(phen) has the capacity to induce Erk1/Erk2 and SAPK/JNK phosphorylation, two second messengers that are potentially involved in bpV(phen)-mediated AP-1 activation and iNOS expression.


Figure 5
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  		Figure 5. Time-dependent activation of JAK2, STAT-1{alpha}, Erk1/Erk2, MEK, p38, and SAPK/JNK by IFN-{gamma} and bpV(phen) in J774 MØ. Cells were treated with IFN-{gamma} (100 U/ml) or bpV(phen) (10 µM) for various time periods (0.5–3 h). Cell lysates were subjected to Western blotting using antibodies specific for the phosphorylated forms of JAK2 ({alpha}-p-JAK2), STAT-1{alpha} tyrosyl 701 ({alpha}-p-STAT1-Tyr), and STAT-1{alpha} seryl 727 ({alpha}-p-STAT1-Ser) residues, Erk1/Erk2 MAPKs ({alpha}-p-p44 and {alpha}-p-p42), MEK ({alpha}-p-MEK), p38 ({alpha}-p-p38), and SAPK/JNK MAPKs ({alpha}-p-p54 and {alpha}-p-p46). Equal loading of proteins was monitored following stripping with antibodies detecting the nonphosphorylated form of the various kinases analyzed. Results depicted here are representative of three separate experiments performed similarly.

 
Role of Erk1/Erk2 in AP-1 nuclear translocation by bpV(phen) PTP inhibitor
To confirm that bpV(phen)-mediated Erk1/Erk2 activation is an important event being involved in AP-1 nuclear translocation, we monitored the effect of Erk1/Erk2 inhibitor apigenin on the induction of AP-1 nuclear translocation by bpV(phen) alone or in combination with IFN-{gamma} stimulation. As depicted in Figure 6 , we observed that treatment of MØ with apigenin completely inhibits the induction of AP-1 by bpV(phen), combined or not with IFN-{gamma} stimulation. This result strongly supports the fact that Erk1/Erk2 MAPKs play a pivotal role in the bpV(phen)-mediated iNOS expression using the transcription factor AP-1.


Figure 6
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  		Figure 6. Effect of Erk1/Erk2 inhibitor on AP-1 induction by bpV(phen) and IFN-{gamma} stimulations. The effect of Erk1/Erk2 inhibitor apigenin (50 µM) on AP-1 activation was evaluated in cells incubated with IFN-{gamma} (100 U/ml) and/or bpV(phen) (10 µM) for a 4-h period. AP-1 activation was monitored as described in Materials and Methods. This result is representative of three separate experiments performed similarly.

 
Inhibition of IFN-{gamma}-induced NO generation by rapamycin in naïve and pV-treated MØ
In an attempt to identify the iNOS-independent pathways modulated by bpV(pic), we used rapamycin, which has been reported to inhibit LPS-mediated NO generation without decreasing iNOS expression [44 ]. As shown in Figure 7 , enhanced NO generation was inhibited in a dose-dependent manner by rapamycin in all experimental groups. First, this suggests that IFN-{gamma} can also trigger signaling events involving the mammalian target of rapamycin (mTOR) and p70 S6 kinase, leading to iNOS phosphorylation and NO generation. Second, it suggests that both pV compounds can modulate this pathway. However, at the maximal, subcytotoxic dose used, the bpV(pic) effect on IFN-{gamma}-induced NO generation was still significantly detectable. Together, these data reinforce our hypothesis that this pV compound could modulate the activity of various cofactors necessary for iNOS activation and its consequent NO generation by affecting other PTPs rather than the one inhibited by bpV(phen).


Figure 7
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  		Figure 7. Dose-dependent inhibition of NO generation by rapamycin. Enhanced NO generation by IFN-{gamma}-stimulated cells treated with PTP inhibitors bpV(phen) (10 µM) and bpV(pic) (10 µM) was abolished in the presence of increasing doses of rapamycin (1, 10, and 25 µg/ml). Results are representative of three separate experiments performed similarly.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NO is an important cytotoxic molecule in the host immune response (reviewed in ref. [45 ]). We showed previously that activation of SHP-1 was responsible for down-regulation of IFN-{gamma}-induced MØ NO generation in Leishmania-infected cells [23 , 26 ]. We also demonstrated in mice that modulation of PTP activity with bpV(phen) and bpV(pic) leads to NO generation and confers protection against visceral and cutaneous leishmaniasis [23 , 24 , 28 ]. The present study investigated which step of JAK2/STAT-1{alpha}-dependent pathways and which MAPKs were regulated by phosphatases targeting protein tyrosyl residues. Results from our study suggest that enhanced, IFN-{gamma}-induced NO generation by bpV(phen)-treated MØ involves a selective activation of Erk1/Erk2 and SAPK/JNK MAPKs leading to AP-1 activation. Exacerbation of IFN-{gamma}-induced NO generation by bpV(pic) was found to be partially dependent on the signaling pathways involving JAK2, MEK, Erk1/Erk2, and p38 MAPKs and does not require an increased iNOS expression to conduct its exacerbating action toward NO generation. Taken together, these results suggest a mode of action for our models of leismnaniasis studied previously.

Previous studies using vanadate or pervanadate (a mixture of vanadate and H2O2) were suggestive for the implication of PTPs in the down-regulation of iNOS expression in rat hepatocytes [46 ], rat vascular smooth muscle tissue [47 ], and MØ [48 , 49 ]. To firmly establish the role played by PTPs in the regulation of signaling events leading to NO production, we have used the PTP inhibitors bpV(phen) and bpV(pic), which represent chemically defined pervanadate derivatives and are the most potent PTP inhibitors known to date [30 ]. These compounds were shown to exacerbate IFN-{gamma}-induced NO generation in a dose-dependent manner (see Fig. 1 ). Although bpV(phen) was able to increase NO production when used alone, the effect of bpV(pic) was only observed in the presence of IFN-{gamma}. These data are consistent with our previous findings in which we have shown the capacity of bpV(phen) and bpV(pic) in vivo [24 ] and bpV(phen) in vitro [28 ] to modulate NO generation.

Studies using various experimental systems have shown that PTPs are important for the regulation of several kinases, such as Erk1/Erk2 MAPKs [50 51 52 53 54 ], p38 MAPK [54 ], and JNK [55 ], all second messengers known for their implication in IFN-{gamma}- and/or LPS-induced NO generation [32 33 34 , 36 , 56 , 57 ]. In the present study, the use of selective antagonists against JAK2 and MAPK family members revealed that Erk1/Erk2 are the main players in the exacerbating effect modulated by pV compounds on MØ NO generation induced by IFN-{gamma}. Inhibition of JAK2, MEK, and p38 did not affect NO production by bpV(phen), whereas bpV(pic)-treated cells were affected partially but significantly in their capacity to respond to IFN-{gamma}. The effect of each inhibitor on iNOS expression supports our suggestion that Erk1/Erk2 are the main kinases responsible for bpV(phen)-induced NO generation in IFN-{gamma}-stimulated or unstimulated cells. bpV(pic) had an exacerbating effect on NO generation in MØ stimulated with IFN-{gamma}, but no increased iNOS expression was observed compared with cells stimulated with IFN-{gamma} alone. In addition, the level of iNOS expression observed in the presence of JAK2, MEK, and Erk1/Erk2 inhibitors in bpV(pic)-treated cells subjected to IFN-{gamma} stimulation was consistent with the inhibitory effect observed in cells stimulated with IFN-{gamma} alone. This set of experiments suggests that bpV(phen)-modulated NO generation involves increased iNOS expression as opposed to the iNOS expression-independent pathway proposed for bpV(pic)-treated cells in response to IFN-{gamma}.

Rapamycin has been reported to inhibit LPS-induced NO production totally by RAW 264.7 MØ without decreasing iNOS protein level [44 ]. The fact that mTOR inhibition by rapamycin antagonizes the effect of LPS-mediated p70 S6 kinase activity suggests that it could be involved in NO generation, probably by increasing the iNOS protein phosphorylation state [44 , 58 ]. From these observations, we hypothesized that the mechanism whereby bpV(pic) exacerbates IFN-{gamma}-mediated NO generation, without further modulating iNOS expression, could be attributed to the inactivation of PTPs acting on kinases responsible for iNOS tyrosine phosphorylation [49 ] or on the modulation of cofactors implicated in NO generation such as NADPH, flavin mononucleotide, flavin adenine dinucleotide, and tetrahydrobiopterin, as well as calmodulin [59 ]. However, our study revealed that rapamycin blocked IFN-{gamma}-induced NO generation in a dose-dependent manner in cells treated or not with both pV compounds. It suggests that the status of iNOS tyrosine phosphorylation is not correlating with enhanced NO generation modulated by the PTP inhibitor used and further reinforces the importance to explore the impact of bpV(pic) on cofactor modulation.

STAT-1{alpha} and NF-{kappa}B have been recognized as the main transcription factors involved in signaling events leading to iNOS expression and NO generation in MØ stimulated with IFN-{gamma} alone or in combination with LPS [34 , 40 , 41 ]. Of interest, our results revealed that none of the pV compounds induced per se or in combination with IFN-{gamma} enhanced STAT-1{alpha} (GAS/ISRE/iNOS) or NF-{kappa}B nuclear translocation. However, knowing that the iNOS promoter contains two recognition sites for AP-1 [60 ] and that Erk1/Erk2 MAPKs have been shown to phosphorylate JunD and FosB [61 ] leading to AP-1 induction [61 , 62 ], the effect of pV compounds on AP-1 nuclear translocation was tested. Our data revealed that bpV(phen) was a powerful inducer of AP-1 activation per se or in combination with IFN-{gamma}, whereas bpV(pic)-mediated AP-1 activation was absent and consequently consistent with our previous observations, suggesting that the bpV(pic) exacerbating capacity for NO generation is mainly independent of iNOS expression. The importance of AP-1 in iNOS expression was also known in other MØ (RAW 264.7 [63 ]) and models such as neurons (PC12 cells [64 ]), and it is interesting that our data support AP-1 importance in the expression of iNOS. Collectively, this set of data suggests that AP-1 is the main transcription factor responsible for enhanced, bpV(phen)-modulated iNOS expression and NO generation.

Consistent with these results, we showed that MEK, Erk1/Erk2, p38, and SAPK/JNK phosphorylation states were enhanced markedly by bpV(phen) treatment, whereas JAK2 and STAT-1{alpha} phosphorylation states were not modified, conferring a biochemical cascade picture leading to AP-1 activation. Conversely, IFN-{gamma} was shown to trigger phosphorylation of all second messengers monitored except the SAPK/JNK kinase. Such results are consistent with the fact that IFN-{gamma} has been shown to be a weak inducer of AP-1. The fact that inhibition of MEK and p38 kinases did not subsequently affect the capacity of bpV(phen) to enhance IFN-{gamma}-mediated iNOS expression and resulting NO generation suggests that inhibition of MØ PTP by bpV(phen) allows for bypassing these kinases and to targeting Erk1/Erk2 and SAPK/JNK kinases selectively, resulting in AP-1-dependent NO generation. Our findings are supported by previous studies showing that Erk1/Erk2 MAPKs were involved in AP-1 induction in okadaic acid-treated cells [61 ] and that SAPK/JNK has been demonstrated to be a major player in AP-1 nuclear translocation [43 ]. Further supporting our findings, we showed that bpV(phen)-modulated AP-1 nuclear translocation was inhibited by the Erk1/Erk2 inhibitor apigenin, demonstrating a direct role of these kinases in AP-1 induction and subsequent NO generation.

Collectively, our findings suggest that bpV(phen) and bpV(pic) can up-regulate IFN-{gamma}-induced NO generation by targeting different PTPs involved in the regulation of various pathways leading to the generation of NO. Whereas bpV(phen) seems to target PTPs controlling MAPK signaling pathways, leading to enhanced iNOS expression, bpV(pic) was shown to modulate signaling events through a pathway independent of iNOS expression, suggesting that these inhibitors can selectively antagonize cellular PTPs regulating different signaling events. Finally, a better understanding of the role played by the PTPs in the regulation of MØ microbicidal functions, such as NO generation, could lead to the development of more selective molecules, with immunomodulatory capacity favoring control over infections and other types of diseases.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Canadian Institutes in Health Research (CIHR) to M.O., who is a member of a CIHR group in host-pathogen interaction. M.O. is a Fonds de Recherche en Santé du Québec (FRSQ) Senior Investigator and a Burroughs Wellcome Fund Fellow. J. B. and P. P. are recipients of a CIHR Ph.D. studentship. We thank Dr. David Frank (Dana Farber Cancer Institute) for kindly providing antibodies and Marc Bergeron for his help in editing the manuscript.

Received May 9, 2005; revised November 14, 2006; accepted November 17, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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