Proteasome-mediated Degradation of STAT1α following Infection of Macrophages with Leishmania donovani*

Activation of the Janus-activated kinase 2 (JAK2)/STAT1α signaling pathway is repressed in Leishmania-infected macrophages. This represents an important mechanism by which this parasite subverts the microbicidal functions of the cell to promote its own survival and propagation. We recently provided evidence that the protein tyrosine phosphatase (PTP) SHP-1 was responsible for JAK2 inactivation. However, STAT1 translocation to the nucleus was not restored in the absence of SHP-1. In the present study, we have used B10R macrophages to study the mechanism by which this Leishmania-induced STAT1 inactivation occurs. STAT1α nuclear localization was shown to be rapidly reduced by the infection. Western blot analysis revealed that cellular STAT1α, but not STAT3, was degraded. Using PTP inhibitors and an immortalized bone marrow-derived macrophage cell line from SHP-1-deficient mice, we showed that STAT1 inactivation was independent of PTP activity. However, inhibition of macrophage proteasome activity significantly rescued Leishmania-induced STAT1α degradation. We further demonstrated that degradation was receptor-mediated and involved protein kinase Cα. All Leishmania species tested (L. major, L. donovani, L. mexicana, L. braziliensis), but not the related parasite Trypanosoma cruzi, caused STAT1α degradation. Collectively, results from this study revealed a new mechanism for STAT1 regulation by a microbial pathogen, which favors its establishment and propagation within the host.

Intracellular protozoan parasites of the Leishmania genus are the etiological agents of leishmaniasis, a condition that causes considerable worldwide morbidity and mortality, with pathologies ranging from disfiguring cutaneous to lethal visceral afflictions (1). Survival of these pathogens within the phagocytic cells of the host requires rapid alteration of macrophage signal transduction, resulting in abnormal immune functions (reviewed in Refs. 2 and 3). For instance, macrophage dysfunctions, such as failure to respond to interferon ␥ (IFN␥), 1 have been linked to altered signaling cascades, including Ca 2ϩ -, PKC (protein kinase C)-, MAPK (mitogen-activated protein kinase)-, and JAK2 (Janus-activated kinase 2)-dependent pathways (4 -11). Manipulation of these signaling pathways distorts the activities of transcription factors, such as STAT1␣, which is important for the expression of IFN␥-induced genes, such as inducible nitric-oxide synthase and major histocompatibility complex class II (12).
We have previously shown that the unresponsiveness of the JAK2 signaling pathway in Leishmania donovani-infected macrophages upon IFN␥ stimulation was due to the ability of the parasite to activate a particular protein tyrosine phosphatase (PTP), SHP-1 (4). SHP-1 is a known inhibitor of several tyrosine kinase-dependent pathways, including the JAK2/ STAT1␣ pathway, where it dephosphorylates JAK2 (4,13,14). However, our observation that IFN␥-stimulated STAT1␣ activity is also reduced in SHP-1-deficient macrophages following L. donovani infection indicates that Leishmania employs further mechanisms to inhibit STAT1 activity. 2 Aside from SHP-1, numerous physiological and pathogeninduced proteins have been shown to regulate JAK/STAT pathways (15). Indeed, alternative splicing of the STAT1 mRNA itself produces a dominant negative variant, STAT1␤, which binds the same promoter elements as STAT1␣ but lacks a crucial transcription activation domain (16,17). The intracellular bacterium Mycobacterium avium appears to induce STAT1␤ to inhibit macrophage STAT1␣ activity (18). Members of the suppressor of cytokine signaling (SOCS) family of proteins are induced by stimulation with various cytokines and provide negative feedback to the response of the cell to cytokine treatment (19,20). SOCS proteins are capable of acting as ubiquitin ligases, and inhibition of signaling by proteasomemediated receptor degradation has been proposed to contribute to inactivation of various STATs (21)(22)(23)(24)(25)(26)(27). Although IFN␥-activated STAT1 has been shown to be ubiquitinated and protein levels have been stabilized by inhibitors of the proteasome, details of this mechanism and whether the SOCS family is involved have not been determined (28). SOCS family members have also been reported to prevent STAT phosphorylation by 1 The abbreviations used are: IFN␥, interferon ␥; CR3, complement receptor 3; EMSA, electrophoretic mobility shift assay; GAS/ISRE, ␥-activated sequence/interferon stimulation response element; JAK, Janusactivated kinase; PKC, protein kinase C; PTP, protein tyrosine phosphatase; PIAS-1, protein inhibitor of activated STAT1; SOCS-1, suppressor of cytokine signalling-1; STAT, signal transducer and activator of transcription; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bpV(phen), potassium bispexoxol(1, 10phenanthroline) oxovanadate. 2 G. Forget, D. J. Gregory, and M. Olivier, manuscript submitted.
binding upstream receptors and preventing autophosphorylation or by competing for STAT binding (29 -31). Viruses have developed a wide range of mechanisms for inhibiting IFN␥ signaling, including production of decoy receptors and dominant negative intermediates (32). Of particular interest, paramyxovirus-encoded proteins have been shown to cause specific degradation of STAT1 by the proteasome by recruiting a cellular E3-ubiquitin-protein isopeptide ligase (33)(34)(35). However, other groups have reported that this degradation is celltype-specific and suggested that inhibition of STAT1 phosphorylation is a more important mechanism (32,36). To our knowledge, no microbial pathogen has been reported to repress STAT1␣ by proteasome-mediated degradation.
In the present study, we investigated the mechanisms whereby Leishmania infection causes macrophage STAT1␣ inactivation. Our results show that abnormal STAT1␣ nuclear translocation in Leishmania-infected macrophages could be the consequence of rapid and sustained STAT1␣ protein diminution and that this may contribute to the lack of response to IFN␥. The depletion was shown to be specific to STAT1␣, as STAT3 levels were unchanged, and was triggered by the early interaction between the pathogen and host cell. Our study further implicates protein kinase C␣ (PKC␣)-dependent signaling and proteasomes (but not PTPs) in this process. Together, these results argue for a direct role of the proteasome pathway in the specific proteolysis of STAT1␣ in macrophages infected by the protozoan parasite Leishmania, representing a new mechanism whereby microbial pathogens can subvert microbicidal activity.
Immunofluorescence Staining-Experiments were performed in chamber slides. Cells (1 ϫ 10 5 ) were gently washed three times with cold phosphate-buffered saline and fixed with 200 l of methanol at Ϫ20°C for 10 min. The slides were blocked with 200 l of phosphatebuffered saline ϩ 0.5% bovine serum albumin and then treated with 100 l of anti-STAT1␣ in phosphate-buffered saline ϩ 0.5% bovine serum albumin for 1 h at room temperature in a humid, dark chamber, washed three times, and then treated with fluorescein isothiocyanateconjugated secondary antibody for 1 h. After the final washes, coverslips were mounted on the chamber slides using 90% glycerol, 10% phosphate-buffered saline, and 2.5% DABCO (1,4-diazobicyclo-(2,2,2)octane). Pictures were taken using an immunofluorescence microscope at 400ϫ magnification.
Two-dimensional Electrophoresis-After infection, 5 ϫ 10 6 cells were washed and lysed in 1 ml of buffer (50 mM Tris, pH 8, 137 mM NaCl, 1% Triton X-100, and Complete TM protease inhibitor mixture (Roche Applied Science)). STAT1␣-containing complexes were precipitated with 1 g of ␣-STAT1␣ overnight at 4°C and AG plus-agarose beads (Santa Cruz Biotechnology) for 1 h, all at 4°C on a rotating platform. Immunoprecipitates were then washed four times with immunoprecipitation lysis buffer, and the remaining bead complexes were eluted in 45 l of 2D rehydration buffer (40 mM Tris, 8 M urea, 4% (w/v) CHAPS) for 30 min at 37°C. Isoelectric focusing was performed using the IPGPhor isoelectric focusing system 18-cm Immobiline immobilized pH gradient strips, pH 3-10 (Amersham Biosciences), according to manufacturer's instructions. Proteins were resolved by 12% SDS-PAGE and visualized by Vorum silver staining.

Effect of L. donovani Infection on STAT1␣ Nuclear Localiza-
tion-Previous studies have reported that JAK2/STAT1 signaling is altered in Leishmania-infected macrophages (4, 6). Although JAK2 tyrosyl phosphorylation was shown to be inhibited, the effect on STAT1␣ activity has not been reported. We therefore first established whether STAT1 activation by IFN␥ stimulation was affected in Leishmania-infected macrophages. As shown in Fig. 1, EMSA allowed us to observe increased nuclear STAT1 DNA binding activity in response to IFN␥. However, this induction was prevented in Leishmaniainfected macrophages. Interestingly, the STAT1 nuclear signal in non-stimulated macrophages was substantially diminished in infected cells in comparison to uninfected ones.
It was important to determine the time frame in which this STAT1 down-regulation occurred. As indicated in Fig. 2A, STAT1 nuclear signal faded rapidly in L. donovani-infected cells in the absence of IFN␥, and was almost completely undetected 6 h post-infection. This contrasted with IFN␥ treatment of uninfected cells, which resulted in a significant, time-dependent increase in STAT1 translocation.
Lower nuclear levels of STAT1 during infection could result from its retention in the cytoplasm, for example, due to a failure to phosphorylate tyrosine 701. It could also reflect a reduction in total cellular STAT1 protein. To address these two possibilities, we monitored STAT1␣ protein level as well as its phosphorylation on residue Tyr-701 in whole cell lysates. Consistent with the EMSA, we observed that Leishmania infection, unlike IFN␥ stimulation, did not lead to STAT1␣ Tyr-701 residue phosphorylation (Fig. 2B). Importantly, we noticed that STAT1␣ protein levels decreased rapidly in the presence of Leishmania and in some cases disappeared completely by the third hour of infection. This observation permitted us to conclude that Leishmania-mediated STAT1␣ degradation is responsible for the absence of STAT1 from the nucleus. Of utmost interest, it appeared that this phenomenon was specific to STAT1␣, because STAT3 protein level was unchanged by L. donovani infection.
Disappearance of STAT1␣ from the nucleus was further reinforced by microscopic observation using immunofluorescence staining. Cells were infected with Leishmania or treated with IFN␥ (100 units/ml; positive control for STAT1␣ activation) for 30 min. STAT1␣ protein seemed equally distributed in resting macrophages (Fig. 3, top left panel). As expected, IFN␥ stimulation led to clustering of STAT1␣ in the nucleus (Fig. 3, top right panel). Importantly, in Leishmania-infected macrophages (Fig. 3, bottom left panel), a different pattern was revealed in that nuclear STAT1␣ was less abundant with diffuse localization in the cytoplasm, suggesting that STAT1␣ degradation may start in the nucleus.
Protein Tyrosine Phosphatases and SHP-1 Are Not Involved in STAT1␣ Inactivation-Many studies have reported evidence for a role for PTPs in STAT1 regulation (21,22,25,44). As we have previously shown, Leishmania infection leads to PTP and SHP-1 activation (4,37,40). It was legitimate to test whether the STAT1␣ inactivation seen here was dependent upon these molecules. As shown in Fig. 4A, our results demonstrated that inhibition of PTPs by bpV(phen) did not prevent the rapid disappearance of STAT1 triggered by Leishmania infection. In addition, we further demonstrated that the PTP SHP-1 is not involved in STAT1␣ inactivation by comparing immortalized macrophages from motheaten (SHP-1 Ϫ/Ϫ ) mice with their wildtype equivalents (37). As seen in Fig. 4B, the pattern of STAT1 nuclear degradation was similar between wild-type and SHP-1-deficient cells, except for the basal translocation in resting cells. Indeed, the STAT1 signal was stronger in the absence of SHP-1. This is consistent with the fact that SHP-1 is known to regulate JAK2 phosphorylation (13). Thus, in its absence, higher JAK2 activity increases STAT1 nuclear translocation.
STAT1␣ Is Degraded via the Proteasome Pathway-Because STAT1␣ proteins apparently underwent proteolysis during Leishmania infection, we addressed the role of proteasomes in this process. Macrophages were treated with increasing doses of MG-132 and clasto-lactacystin ␤-lactone (c-lactacystin), a more specific proteasome inhibitor, before infection with Leishmania. As seen in EMSA experiments in Fig. 5A, STAT1 reappeared in the nucleus when MG-132 and c-lactacystin were used. Moreover, these inhibitors also restored STAT1␣ protein level in whole cell lysates during infection (Fig. 5B). These results allowed us to conclude that, in the case of Leishmania infection, STAT1␣ is degraded via the proteasome.

Implication of Different Leishmania Species and Macrophage Receptors in STAT1␣ Inactivation-
The present study has demonstrated so far that infection of macrophages with L. donovani causes STAT1␣ inactivation by proteasomal degradation. It was important to verify whether other Leishmania species or other protozoan pathogens had the same ability. As seen in Fig. 6, A and B, all tested Leishmania species (L. donovani, L. major, L. mexicana, and L. braziliensis) caused STAT1␣ degradation. However, T. cruzi, a protozoan of the same Trypanosomatidae family as Leishmania, had no effect on STAT1. Moreover, phagocytosis of the malaria pigment hemozoin by macrophages had no effect either. Thus, the interaction of Leishmania species with the macrophages specifically affects STAT1␣ degradation and cannot be generalized to other similar pathogen/macrophage interactions or other forms of phagocytosis.
The degradation that we observed started as early as 30 min after parasite contact, an insufficient time for parasite internalization. We therefore asked whether macrophage receptors previously implicated in Leishmania/macrophage interactions had a role to play in STAT1␣ inactivation. Proteasomal targeting of STAT1␣ could hence be triggered by receptor signaling. Several receptors, such as the complement receptor 3 (CR3), the receptor for the Fc portion of immunoglobulins ␥ (Fc␥R), and the mannose-fucose receptor (mannose R) are known to bind Leishmania (45)(46)(47). Using blocking antibodies against these three receptors, we inhibited their ligation to Leishmania and observed the effects on STAT1 nuclear translocation. Results suggested that all three had a potential role to play in STAT1 inactivation (Fig. 6C), because the translocation of STAT1 was restored when receptors were blocked before infection, although the effect of blocking the mannose-fucose receptor was partial.
The two most abundant parasite surface molecules that can bind to these receptors are the lipophosphoglycan and the surface protease gp63 (47). We investigated whether they could be implicated in STAT1␣ inactivation by using knock-out Leishmania parasites for each molecule. Surprisingly, neither molecule seemed to alter STAT1 nuclear translocation (data not shown). However, we cannot rule out the possibility of redundancy between lipophosphoglycan and gp63.
The PKC␣-dependent Pathway Is Involved in STAT1␣ Inhibition-Because results showed that triggering of macrophage receptors has a role to play in STAT1␣ degradation, we investigated whether downstream signaling was induced following receptor binding. One second messenger that is activated by both CR3 and Fc␥R binding is the protein kinase C␣ (PKC␣) (48). Additionally, a previous report linked PKC␣ to activation of proteasome-dependent pathways leading to STAT3 degradation (49). We therefore investigated whether Leishmania infection activated PKC␣ by measuring its phosphorylation. As seen in Fig. 7A, interaction between Leishmania and macrophages for as little as 10 min provoked phosphorylation of PKC␣, which was maintained for at least 1 h after infection. To assess whether PKC␣ activation in Leishmania-infected cells induced STAT1␣ degradation by the proteasome, we treated macrophages with increasing doses of a PKC inhibitor (Gö6976, IC 50 , 3 nM for PKC␣). As seen in Fig. 7B, inhibition of PKC␣ significantly restored STAT1 nuclear translocation in infected macrophages. Thus, PKC␣-dependent signaling seems to lead to STAT1␣ targeting to the proteasome in Leishmaniainfected macrophages.
Identification of Proteins That Target STAT1␣ to the Proteasome during Leishmania Infection-To identify which factors bind to STAT1␣ and target it to the proteasome pathway, we used a proteomic approach using two-dimensional electro-phoresis of STAT1␣ immunoprecipitates. Fig. 8 shows that there were different spot patterns between uninfected and infected cells (top left versus top right panels). Indeed, many proteins either appeared or seemed up-regulated during Leishmania infection, whereas some others showed changed phosphorylation or disappeared. Of interest, we observed that almost no proteins were detected at 3 h post-infection. This is consistent with the fact that STAT1␣ is almost entirely degraded by this time and, thus, immunoprecipitation could not take place.

DISCUSSION
Leishmania is known for its ability to down-regulate numerous macrophage functions that are detrimental to its survival and persistence (3, 10, 50 -52). Some of these are generally induced by cytokines such as IFN␥ (e.g. NO, major histocompatibility complex, interleukin-12). Thus, the parasite has evolved ways to inhibit the different signaling pathways activated by such stimulation, namely the JAK2/STAT1␣ and mitogen-activated protein kinase extracellular signal-regulated kinase-1/2 pathways (4 -7). Previous studies (4, 7) have demonstrated that the inhibition of JAK2 and extracellular signalregulated kinase-1/2 during infection was mediated by the PTP SHP-1. 2 The scope of the present study was to identify mechanisms that were responsible for this inactivation.
We observed that both the IFN␥-dependent STAT1 nuclear translocation and basal STAT1␣ protein level were inhibited in a time-dependent manner in Leishmania-infected macrophages. This occurred following infection with both New and Old World species of L. donovani, major, mexicana, and braziliensis, arguing for a conserved evolutionary trait among the genus. The fact that we observed no disappearance of STAT1␣ following phagocytosis of the malarial pigment hemozoin or infection with a different trypanosomatid (T. cruzi) indicates that the phenomenon is specific to Leishmania infection and is not a result of phagocytosis or general cell stress. Some studies have attributed inhibition of STATs by various stimuli to the action of a PTP on JAK proteins (21,22,25) or a nuclear PTP dephosphorylating STAT1 directly (44). This last report is further reinforced by a recent study describing a nuclear localization site in the SHP-1 C terminus region (53). From the present study, using immortalized macrophages from PTP SHP-1-deficient mice and their wild-type littermates (37) and the PTP inhibitor bpV(phen) (54), we can conclude that neither SHP-1 nor other PTPs are responsible for the disappearance of STAT1 from the nuclei of Leishmania-infected cells.
As well as PTPs, the activation of STAT1␣ following cytokine stimulation is limited by the protein inhibitor of activated STAT1 (PIAS-1). This repressor binds STAT1␣ and inhibits its DNA-binding capacity (55), perhaps by sumoylation (56,57). We monitored its involvement by verifying whether Leishmania infection could promote its attachment to STAT1␣. Despite repeated experiments, we have been unable to co-immunoprecipitate PIAS-1 and STAT1␣ from infected macrophages (data not shown), which indicates that the repression we observed was independent of PIAS-1. Instead, we found that the inhibition is due to specific proteolysis, because the STAT1␣ protein level was decreased with time in whole cell extracts of infected macrophages and had completely vanished 3 h post-infection. STAT3 was unaffected. Using immunofluorescence, we were able to demonstrate that STAT1␣ destruction probably started in the nucleus.
Members of the SOCS family, especially SOCS1, have been shown to repress STAT1␣ by binding to, and causing dephosphorylation of, upstream molecules, such as JAK2, and/or by recruiting a ubiquitin ligase complex, leading to proteasomemediated degradation (15,19). One could therefore propose that Leishmania infection induces expression of SOCS1, which binds to STAT1␣, resulting in its ubiquitination and degradation. However, a number of observations conflict with this theory. First, no direct interaction between SOCS proteins and STAT1␣ has been reported. Repression appears to occur by targeting upstream molecules. Second, SOCS1 is known to induce degradation of JAK2 (19,20), but the JAK2 protein level remains unchanged following infection (4,6). Third, we have been unable to detect increased SOCS1 expression in infected cells by Northern blot (data not shown). Another family member, SOCS3, has been shown to be transiently induced by L. donovani in human monocyte-derived macrophages (58). However, data from SOCS3 knock-out mice suggest that SOCS3 regulates STAT1␣ by mechanisms other than its stability (59,60). Taken together, these studies suggest that Leishmania-induced degradation of STAT1␣ is unlikely to be mediated by SOCS induction.
Two studies have described proteasome-mediated degradation of STATs following cytokine stimulation. One study showed that, following phosphorylation in response to IFN␥, STAT1␣ is ubiquitinated and degraded (28). A second study showed that STAT3 was degraded during ciliary neurotrophic factor or 12-O-tetradecanoylphorbol-13-acetate stimulation (49). In both cases, the STAT was protected by inhibition of the proteasome. We report here that the use of two different proteasome inhibitors, MG-132 and the more specific clasto-lactacystin ␤-lactone, rescued STAT1␣ nuclear translocation as well as restored its general protein level in Leishmania-infected macrophages. We conclude that Leishmania infection results in proteasome-mediated degradation of STAT1␣. This contrasts with the normal mechanisms of repression of STAT1␣ following cytokine stimulation, where activity is reduced but stability is not (15,(21)(22)(23)(24)(25). Instead, it is reminiscent of the action of various paramyxoviral proteins, which cause ubiquitination and degradation of STAT1 by the proteasome (33)(34)(35). However, these viral proteins are expressed within the cell where they are able to recruit a cellular ubiquitin ligase complex by physical interaction (35). The rapidity of Leishmania-activated degradation means that it is unlikely to be caused by a parasite protein gaining access to the cell. Instead, the Leishmania must stimulate macrophage surface receptors to modulate intracellular signaling cascades to target STAT1␣ for degradation. Macrophage receptors known to bind to parasite surface molecules include CR3, Fc␥R, and the mannose-fucose receptor (45)(46)(47). Results from receptor blocking experiments demonstrated that all three could contribute to the lack of STAT1␣ in the nucleus. Indeed, STAT1␣ nuclear translocation was restored in Leishmania-infected cells when each receptor was blocked separately, although restoration was only partial following mannose-fucose receptor blocking. An explanation for this nonspecific result could be that Leishmania needs to bind to more than one receptor at a time for adequate cross-linking and subsequent signaling. To support this hypothesis, it has been demonstrated that Leishmania must attach to two different receptors for internalization to occur (61). As for the ligands of these receptors, all three are known to bind Leishmania lipophosphoglycan and gp63 (reviewed in Ref. 62). For this reason, we monitored the importance of these surface molecules in STAT1␣ inactivation by using specific knock-out parasite strains. The absence of either molecule did not affect STAT1␣ nuclear translocation (data not shown). However, this result does not rule out their possible involvement in this process, because both lipophosphoglycan and gp63 can bind to all three receptors and so may be redundant. In the absence of double knock-out parasites, we could not verify this hypothesis.
Both CR3 and Fc␥R ligation lead to PKC activation (48). Thus, this kinase could be important in STAT1␣ inactivation. Results showed that PKC␣ was activated in macrophages from 10 to 60 min following infection. This is consistent with the initiation of STAT1␣ proteolysis at 30 min post-infection. Using a PKC inhibitor (Gö6976), we were able to demonstrate PKC␣ involvement in our specific STAT1␣ nuclear inactivation. This is in accordance with a previous report showing PKC␣-dependent STAT3 degradation by the proteasome (49).
Finally, analysis of STAT1␣ immunoprecipitates by two-dimensional electrophoresis allowed us to see differences between patterns of proteins associated with STAT1␣ in resting macrophages and macrophages infected with Leishmania for 30 min. This is a good indication that Leishmania infection alters protein complex formation in macrophages, and the pattern is particularly suggestive of changes in the phosphorylation state of a number of proteins. Further identification of these spots will enable us to better understand the mechanisms underlying STAT1␣ degradation by the proteasome.
Overall, this study has shed light on a novel mechanism utilized by Leishmania to alter macrophage signaling, i.e. STAT1␣ degradation by the proteasome in the early stages of infection. This phenomenon seems to start by the triggering of three macrophage receptors (CR3, Fc␥R, and the mannosefucose receptor) by the parasite and subsequent PKC␣ induction. By allowing the degradation of STAT1␣, in addition to the inhibition of JAK2 by SHP-1, the parasite is able to inhibit the response of the host cell to IFN␥. This favors its persistence within the cell and propagation of the disease.