The peptidyl-prolyl isomerase Pin1 regulates the stability of ... - Nature

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The peptidyl-prolyl isomerase Pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils Zhong-Jian Shen1,2, Stephane Esnault1,2 & James S Malter1 The infiltration, accumulation and degranulation of eosinophils in the lung represents a hallmark of active asthma. In vivo or in vitro eosinophil activation triggers the secretion of the antiapoptotic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We now identify Pin1, a cis-trans isomerase, as an essential component of the ribonucleoprotein complex responsible for GM-CSF mRNA stabilization, cytokine secretion and the survival of activated eosinophils. Pin1 regulated the association of the AU-rich element–binding proteins AUF1 and hnRNP C with GM-CSF mRNA, accelerating or slowing decay, respectively. These data indicate Pin1 is a key mediator of GM-CSF production.

Considerable eosinophilic infiltration and accumulation in the lung are an invariable feature of allergic inflammation in asthma1. Along with neutrophils and macrophages, eosinophils constitute the dominant cell population in the brochoalveolar lavage (BAL) fluid of active asthmatics or in rodent models of asthma. Eosinophils typically increase by 20-fold within a few days of allergen challenge2 and secrete proinflammatory mediators, enzymes, vasoactive substances and cytokines into the airway and parenchyma, which probably contributes to additional inflammatory cell infiltration, airway remodeling and, ultimately, fibrosis1,2. Despite those morphological, physiological and molecular findings, the function of eosinophils in the pathogenesis of asthma has remained controversial. Patients who receive antibody to interleukin 5 (anti-IL-5) have no change in lung function despite a substantial decrease in airway eosinophilia3. However, eosinophils in the lung parenchyma remain essentially unchanged3. Notably, systemic eosinophil ablation prevents both acute and chronic peribronchiolar fibrosis and scarring in mouse asthma models4,5. Thus, the influx and persistence of eosinophils in asthma probably drives short and long-term fibrotic pathology. Unlike peripheral blood eosinophils, which have a brief (approximately 3-day) lifespan, airway eosinophils have prolonged survival and activation. Those phenotypic changes are maintained by cytokines released in the lung after allergen challenge6. Of those molecules, granulocyte-macrophage colony-stimulating factor (GM-CSF) is critical, functioning as an ‘eosinophilopoietic’ factor but also promoting eosinophil survival, maturation and function7,8. Once in the lung, or after in vitro activation, eosinophils produce and release GM-CSF9. A variety of extracellular matrix proteoglycans, including hyaluronic acid and fibronectin, as well as cytokines such as tumor necrosis

factor, which are increased considerably in the airways of asthmatic lung10–12, cause release of GM-CSF by eosinophils13,14. Mechanistically, hyaluronic acid or tumor necrosis factor plus fibronectin induces GM-CSF mRNA stabilization and accumulation in peripheral blood eosinophils9,13. Eosinophils from the airways of active asthmatics have a similar phenotype9, suggesting that both in vitro and in vivo activation events culminate in secretion of GM-CSF via this pathway. In a variety of inflammatory cells, expression of GM-CSF is regulated at the post-transcriptional level through 3¢ untranslated region AU-rich elements (AREs)14,15 found in many unstable cytokine and proto-oncogene mRNAs. Various ARE-binding proteins have been identified that promote (tristetraprolin)16 or prevent (HuR and YB-1)17,18 decay of GM-CSF mRNA. AUF1, an ARE-binding protein with four isoforms of 37, 40, 42 and 44 kilodaltons (p37, p40, p42 and p44, respectively) has been suggested to be both a stabilizer and destabilizer of ARE-containing mRNA19–21. Whereas mitogenactivated protein kinase–dependent signaling pathways have been linked to alterations in the decay of ARE mRNA14, it remains unclear how these functionally opposing ARE-binding proteins are regulated, ultimately culminating in the accumulation of GM-CSF mRNA, cytokine secretion and eosinophil survival. Cyclophilin A and FK506BP, two members of the peptidyl-prolyl isomerase (PPIase) family, may be involved in the regulation of cytokine production in T cells22,23. These ubiquitously expressed PPIases, which are targets of the commonly used immunosuppressants cyclosporin A and FK506, respectively24,25, are highly conserved, multifunctional proteins that specifically bind to target proteins containing X-Pro motifs (where X is any amino acid) and catalyze the cis-trans isomerization of that peptide bond26. Isomerization

1The

Waisman Center for Developmental Disabilities, the Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine, Madison, Wisconsin 53705, USA. 2These authors contributed equally to this work. Correspondence should be addressed to J.S.M. ([email protected]).

Received 30 June; accepted 30 August; published online 6 November 2005; doi:10.1038/ni1266

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alters protein folding and biological activity. 24 a 100 b 80 c Time (h) 4 Pin1, a member of the PPIase family, is the – – HA HA+Jug 80 60 only enzyme known to specifically isomerize 60 p32 40 Caspase 3 40 phosphorylated Ser-Pro or phosphorylated p17 20 20 Thr-Pro bonds and to do so with selectivity 0 0 1,000-fold more than that for unphosphoryβ-actin (µM) (µM) 27 lated substrates . That notable specificity HA+CsA GM-CSF+CsA HA+Jug GM-CSF+Jug results from an N-terminal Trp-Trp (WW) domain and a C-terminal PPIase Figure 1 Pin1 is required for eosinophil survival. (a,b) Viability of purified eosinophils left untreated (R) domain27. So far, Pin1 has been linked or treated for 4 d with 100 mg/ml of hyaluronic acid alone (HA); hyaluronic acid plus cyclosporin A to cell cycle control28, neurodegeneration29 (HA+CsA), 100 pg/ml of recombinant human GM-CSF alone (GM-CSF); recombinant human GM-CSF plus cyclosporin A (GM-CSF+CsA); hyaluronic acid plus juglone (HA+Jug); or recombinant human and tumorigenesis30. GM-CSF plus juglone (GM-CSF+Jug). Concentrations of cyclosporin A and juglone, horizontal axes. Here we have identified another function Cell viability is expressed as the percentage of the initial cells in culture. Data represent three for Pin1 as an essential component of the independent experiments with different donors. Error bars indicate + s.d. (c) Immunoblot of lysates ribonucleoprotein complex responsible for of cells left untreated (–) or treated for 24 h with hyaluronic acid alone (as in a,b above) or together stabilization of GM-CSF mRNA, cytokine with 1 mM juglone. Anti–caspase 3 detects the ‘pro-form’ (p32) and the cleaved, active form (p17). secretion and, ultimately, the survival of b-actin, internal loading control. activated eosinophils. Pin1 regulated the association of the ARE-binding proteins AUF1 and hnRNP C with concentrations of cyclosporin A and juglone to cultures and measured GM-CSF mRNA in response to external stimuli. In resting eosino- cell survival. Cyclosporin A induced eosinophil apoptosis despite phils, GM-CSF mRNA was mainly present in complex with AUF1- activation with hyaluronic acid or recombinant human GM-CSF. Pin1 ribonucleoproteins, but after cell activation was bound to hnRNP However, recombinant human GM-CSF was able to counteract the C. The p37 isoform of AUF1 also interacted with PM-Scl75 in resting proapoptotic effect of a low concentration (0.1 mM) of juglone but not activated cells. PM-Scl75 is a component of the mammalian (Fig. 1a,b). These data suggest that Pin1 inhibition prevented release ribonuclease (RNAse) machinery known as the exosome. Thus, the of GM-CSF by eosinophils after hyaluronic acid treatment. To activation-sensitive interaction between p37 and PM-Scl75 also deter- assess that possibility, we measured GM-CSF mRNA by RT-PCR mines if GM-CSF mRNA is targeted to and degraded by the exosome. followed by Southern blot to detect rare cDNA. Treatment with These data indicate Pin1 is a key mediator of GM-CSF production and hyaluronic acid consistently increased GM-CSF mRNA by threeto fivefold, but that was unaffected by cyclosporin A at a conceneosinophil survival. tration of 1.6 mM or 16 mM. However, juglone reduced the abundance of GM-CSF mRNA to that of untreated control RESULTS cultures in a dose-dependent way (Fig. 2a,b). These data suggest Pin1 is required for eosinophil survival The PPIase inhibitor cyclosporin A has been linked to the expression Pin1 is required for GM-CSF mRNA upregulation, cytokine of GM-CSF by airway cells31,32, but the effects of juglone, a specific secretion and enhanced eosinophil survival, whereas cyclosporin and irreversible Pin1 inhibitor33, are unknown. As secreted GM-CSF is A induces eosinophil apoptosis through a non–GM-CSF– essential for eosinophil survival in vitro, we evaluated the effect of the dependent mechanism. It remained possible that the effects of juglone were nonspecific. PPIase inhibitors FK506, cyclosporin A and juglone on cell survival after activation with hyaluronic acid. Hyaluronic acid causes stabiliza- Therefore, we transduced eosinophils with a construct consisting of tion of GM-CSF mRNA, culminating in cytokine secretion13. We the WW domain of Pin1 fused at the N terminus to a TAT penetratin incubated purified peripheral blood eosinophils with hyaluronic acid tag (TAT-ww-Pin1)34. The WW domain functions as a dominant alone or with hyaluronic acid plus various concentrations of cyclo- negative element by blocking endogenous Pin1 activity35. When added sporin A, juglone or FK506 and determined cell viability at day 4. along with hyaluronic acid, TAT-ww-Pin1 completely prevented Consistent with published results13, the survival of untreated, control upregulation of GM-CSF mRNA (Fig. 2c), whereas a fusion protein eosinophils was 5–20% (depending on the donor), which increased of TAT plus green fluorescent protein had no effect. Eosinophil by three- to fivefold after hyaluronic acid treatment (Fig. 1a,b). Pre- survival closely paralleled the abundance of GM-CSF mRNA; cultures treatment with anti-GM-CSF completely prevented the enhanced treated with TAT-ww-Pin1 were indistinguishable from untreated, eosinophil survival (data not shown), demonstrating hyaluronic resting control cultures (Fig. 2d). However, supplementation of acid–induced secretion of GM-CSF. Cyclosporin A induced eosino- TAT-ww-Pin1–treated cultures with recombinant human GM-CSF phil cell death (approximately 10% survival at a concentration of fully restored survival (Fig. 2d). Therefore, Pin1 participates in 16 mM), as did juglone (5% survival at a concentration of 0.1 or hyaluronic acid–induced GM-CSF mRNA transcription and/or stabi1.0 mM; Fig. 1a,b), whereas FK506 had no effect (data not shown). We lity. Pin1 inhibition, by preventing GM-CSF release, accelerates evaluated by immunoblot whether the eosinophil death was apoptotic eosinophil apoptosis. Eosinophil activation with hyaluronic acid or tumor necrosis factor and was triggered through a caspase-dependent process. We found activated caspase 3 in control, untreated cultures but not in cultures plus fibronectin reduces the rate of GM-CSF mRNA decay9,13. Given treated with hyaluronic acid (Fig. 1c). Blockade of Pin1 with juglone those data and the rapid kinetics of GM-CSF mRNA clearance after also resulted in caspase 3 cleavage. Therefore, hyaluronic acid, through treatment with juglone (Fig. 2b), we activated eosinophils with the induction of GM-CSF, prevented caspase 3–mediated apoptosis. hyaluronic acid or hyaluronic acid plus juglone before adding actinoThe decreased survival could have reflected inhibition of GM-CSF mycin D to inhibit further transcription. We determined the rate of secretion, blockade of GM-CSF receptor signaling or induction of GM-CSF mRNA decay by RT-PCR followed by Southern blot. In the apoptosis through a GM-CSF–independent mechanism. Thus, absence of juglone (that is, with hyaluronic acid alone), GM-CSF we added recombinant human GM-CSF (100 pg/ml) and various mRNA was extremely stable (a half-life of more than 80 min); this

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Figure 2 Pin1 inhibition accelerates the decay of GM-CSF mRNA. (a–c) RT-PCR and Southern blot analysis for GM-CSF mRNA (CSF2; top) and gel electrophoresis of ACTB RT-PCR products (bottom). NC, PCR without added cDNA. (a) Eosinophils treated for 4 h before lysis as described in Figure 1a with hyaluronic acid and either cyclosporin A or juglone (concentrations, above lanes). (b) Cells treated for 4 h with hyaluronic acid alone or with 1 mM juglone added at various times (above lanes) before collection. (c) Cells left untreated (–) or incubated for 4 h with hyaluronic acid alone or with 160 or 320 nM TAT–green fluorescent protein (GFP) or TAT-ww-Pin1 (WW). Data in a–c are representative of at least three independent experiments with different donors. (d) Cell viability. Control, untreated eosinophils (–) or cells treated with hyaluronic acid or recombinant human GM-CSF, with or without 160 nM TAT–green fluorescent protein or 160 nM TAT-ww-Pin1, were cultured for 4 d before viability assay as described in Figure 1. Error bars indicate + s.d. of three independent experiments with different donors. (e) RT-PCR followed by Southern blot for GM-CSF cDNA. Cells were treated for 4 h with hyaluronic acid with or without juglone added after 3 h of culture, followed by the addition of 5 mg/ml of actinomycin D. Cells were then collected (time, above lanes) for 30 (hyaluronic acid) or 33 (hyaluronic acid plus juglone) cycles of PCR. Middle, ethidium bromide–stained gel of ACTB cDNA (30 PCR cycles). Bottom, calculated half-life (t1/2) ± s.d. of GM-CSF mRNA from three independent experiments with different donors.

stability decreased by 75% (a half-life of about 21 min) after Pin1 blockade (Fig. 2e). Therefore, Pin1 is ‘downstream’ of hyaluronic acid–mediated activation and controls cytokine secretion and cell survival by regulating the decay of GM-CSF mRNA. Pin1 associates with AUF1 Emerging data has linked AU-rich mRNA-binding proteins (AREbinding proteins) to the control of cytokine mRNA decay. Both stabilizing and destabilizing ARE-binding proteins have been defined16–21. However, the means by which one protein is replaced by another after cell activation have remained obscure. Most AREbinding proteins are phosphorylated proteins; many of these contain potential Pin1-recognition sites (Ser-Pro or Thr-Pro; Supplementary Table 1 online). We speculated that Pin1 might regulate the decay of GM-CSF mRNA through physical interactions with different AREbinding proteins with subsequent modulation of binding activity or protein-protein interactions by phosphorylation-dependent PPIase activity. Therefore, we immunoprecipitated cytoplasmic extracts with anti-Pin1 followed by immunoblot with antibodies to various antiARE-binding proteins (Supplementary Table 1 online). All four isoforms of AUF1 consistently precipitated together with Pin1. Immunoprecipitation with anti-AUF1 also precipitated Pin1 (data not shown). Treatment of the immunoprecipitation pellets with RNAse had no effect on the protein-protein interactions (data not shown), suggesting that the binding of Pin1 to AUF1 was independent of mRNA. HuR, the ARE-binding protein linked to stabilization of ARE mRNA17, was also coimmunoprecipitated (data not shown), whereas we did not detect the remaining ARE-binding proteins in any circumstances (data not shown). Of the four coimmunoprecipitated isoforms of AUF1 (p45, p42, p40 and p37; Fig. 3a), only p45 and p40 have a Pin1 isomerization site (Ser83-Pro84)36. These data suggest that p42 and p37 AUF1 associate indirectly with Pin1, possibly through protein-protein interactions with p40 and p45. These observations indicate that Pin1 interacts with all AUF1 isoforms independently of GM-CSF mRNA. We next examined the effects of hyaluronic acid and juglone on the interactions between AUF1 and Pin1. Hyaluronic acid had no effect on

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the amount of cytoplasmic Pin1 or its binding to AUF1 (Fig. 3a). However, juglone rapidly reduced both Pin1 and AUF1 (Fig. 3a). By 120 min after treatment, p45, p42 and p40 as well as Pin1 had essentially disappeared from the cytoplasm (Fig. 3b). Transduction of resting or hyaluronic acid–activated eosinophils with TAT-ww-Pin1 had no effect on the abundance of Pin1 or AUF1 (data not shown), suggesting that irreversible enzymatic inhibition of Pin1 by juglone induced degradation of both proteins. AUF1 and Pin1 are degraded in the proteasome37,38. As this process is very rapid, it could account for the kinetics of the loss of Pin1 and AUF1 after treatment with juglone. To address that possibility, we exposed eosinophils to the proteasome inhibitor MG132 for 4 h, added juglone 10 min before collecting cells, and analyzed lysates by immunoblot. Juglone-induced degradation of p45, p42 and p40 AUF1 was completely blocked by MG132 (Fig. 3c). At the highest concentrations of MG132, AUF1 accumulation was above the amounts in untreated, control cells, suggesting normal catabolism of this protein occurred in the proteasome. In these conditions, MG132 only partially prevented Pin1 degradation, indicating clearance through another proteolytic system(s). The steady-state abundance of GM-CSF mRNA was reduced after treatment with juglone (Figs. 2 and 3d) but was maintained after treatment with MG132 (Fig. 3d). These data are consistent with prior observations that decay of ARE mRNA requires proteasome activity37. As transcription of many mRNAs is unaffected by prolonged exposure of cells to MG132 (ref. 39), the increased abundance of GM-CSF mRNA presumably reflected stabilization. Pin1 regulates AUF1–GM-CSF mRNA interactions AUF1 binds GM-CSF mRNA in many cell types40. Paradoxically, binding has been associated with both stabilization and destabilization of ARE mRNA19–21. We evaluated the ‘partitioning’ of GM-CSF mRNA with AUF1 in resting eosinophils after hyaluronic acid activation or treatment with hyaluronic acid plus Pin1 inhibitors (Fig. 4). RT-PCR followed by Southern blot of pellets immunoprecipitated by anti-AUF1 demonstrated the association of a substantial percentage of cellular GM-CSF mRNA with AUF1 in resting cells (Fig. 4a, right),

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ARTICLES Jug IP: Pin1 IgG PC which was mostly reduced after treatment a Jug – Input b Time (min) 10 30 60 120 270 – + – – + + – with hyaluronic acid. Brief exposure to HA – + + + + + + HA – + + – + + – – juglone (10 min; Fig. 4a) or TAT-ww-Pin1 p45 p45 p42 p42 (90 min; Fig. 4d) had little effect on the AUF1 AUF1 p40 p40 p37 p37 steady-state abundance of GM-CSF tranβ-actin Pin1 scripts (Fig. 4a, left) but caused reassociation of GM-CSF mRNA with AUF1 (Fig. 4a, β-actin Pin1 right). To confirm that those results reflected c MG132 (µM) – – – 20 100 d in vivo interactions, we did crosslinking Jug (1 µM) – – + + + immunoprecipitation analysis41. We treated MG132 (20 µM) – – – + + + HA (100 µg/ml) – + + + + PC intact eosinophils with hyaluronic acid for Jug (1 µM) – – + + – – p45 p42 HA (100 µg/ml) – + + + + – AUF1 4 h or with hyaluronic acid for 4 h plus p40 p37 CSF2 juglone for 10 min and irradiated the cells Pin1 with ultraviolet light to covalently crosslink β-actin ACTB physically adjacent mRNA-protein complexes. We treated purified cytoplasmic Figure 3 Pin1 associates with AUF1. (a) Immunoblot for AUF1 and Pin1. Eosinophils were left lysates with RNAse T1 to cleave unprotected untreated or were treated for 4 h with hyaluronic acid, with or without juglone; cell lysates were mRNA before immunoprecipitation with immunoprecipitated (IP) with anti-Pin1 followed by immunoblot (antibodies, right margin). Input, anti-AUF1 or anti–hnRNP C and denaturing 10% of total lysate before immunoprecipitation; IgG, nonimmune IgG (negative control); PC, HeLa extracts (positive control). (b) Immunoblot of cytoplasmic protein from eosinophils treated for 4 h with electrophoresis. We eluted AUF1 and hnRNP hyaluronic acid, with or without juglone (times, above lanes). (c) Immunoblots of lysates of cells left C from gels and assessed the presence of GM- untreated or treated for 4 h with hyaluronic acid alone or with juglone (final 10 min) or 20 or 100 mM CSF mRNA by RT-PCR followed by Southern MG132 (MG; 4 h) and juglone (final 10 min). PC, HeLa cell lysates (positive control). (d) RT-PCR blot (Fig. 4b,c). AUF1 associated in vivo with followed by Southern blot of eosinophils treated as described in c except that only 20 mM MG132 GM-CSF mRNA only in cells treated with was used; cells were lysed with TriReagent for total RNA isolation. Data are representative of at least hyaluronic acid plus juglone but not in those three independent experiments with different donors. treated with hyaluronic acid alone, whereas Given the propensity of cytoplasmic mRNA to be associated with the reverse was true for the association of hnRNP C with GM-CSF mRNA. Thus, these experiments confirm the data obtained with protein, we sought to determine if another protein replaced AUF1 immunoprecipitation and RT-PCR followed by Southern blot after hyaluronic acid treatment. One possibility was hnRNP C, which (Fig. 4a) and eliminate the possibility that the mRNA-protein inter- shows increased binding to GM-CSF mRNA after eosinophil activaactions occurred in vitro after cell lysis. These observations collectively tion13. Therefore, we used RT-PCR followed by Southern blot to assess suggest that AUF1–GM-CSF mRNA interactions triggered rapid decay GM-CSF mRNA in cytoplasmic extracts immunoprecipitated by anti– in resting cells. Hyaluronic acid–mediated cell activation prevented hnRNP C. Depending on the donor, GM-CSF mRNA showed variable this interaction but only when Pin1 was enzymatically functional. association with hnRNP C in untreated eosinophils. Generally, donors A reasonable conclusion is that hyaluronic acid induces Pin1-mediated with allergic symptoms had more GM-CSF mRNA associated with isomerization of AUF1, which is associated with loss of its mRNA- hnRNP C before in vitro activation (data not shown). Consistent with binding activity. published observations13, hyaluronic acid induced a rapid increase in

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Figure 4 GM-CSF mRNA ‘partitions’ between AUF1 and hnRNP C. (a) RT-PCR and Southern blot of ACTB and CSF2 cDNA and immunoblot of AUF1 and b-actin. Cells were left untreated or were treated for 4 h with hyaluronic acid, with or without juglone (final 10 min), and were lysed; 40% of the lysate was used for total RNA isolation and the remaining 60% was used for AUF1 immunoprecipitation. NC, PCR without added cDNA. (b,c) Crosslinking immunoprecipitation assay of eosinophils treated with hyaluronic acid, with or without juglone (final 10 min). Samples were immunoprecipitated with antiAUF1 (b) or anti–hnRNP C (c). The final PCR products were analyzed by Southern blot with radiolabeled GM-CSF cDNA probes. (d) RT-PCR and Southern blot (above) and immunoblot (below). Cells were left untreated or were treated for 4 h with hyaluronic acid, with or without 100 nM TAT-ww-Pin1 (WW; times, above lanes), before lysis and immunoprecipitation (antibodies, top); 40% of the lysate was used for RT-PCR and Southern blot (genes detected, right margin) and the remaining 60% was used for immunoblot (proteins detected, right margin). PC, PCR followed by Southern blot of positive control GM-CSF cDNA; RPS26, ribosomal protein S26. Data are representative of at least three independent experiments with different donors.

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Hyaluronic acid increases Pin1 activity As the association of Pin1 and AUF1 was unaffected by hyaluronic acid (Fig. 3a) despite the loss of GM-CSF mRNA from the protein complex (Fig. 4), we hypothesized that either AUF1 or Pin1 was modified by hyaluronic acid–mediated signaling. Phorbol esters trigger the dephosphorylation of Ser83 and Ser87 of p40 AUF1, which is concomitant with the stabilization of IL-1b and tumor necrosis factor mRNA42. Hyaluronic acid stabilizes GM-CSF mRNA through an extracellular signal–regulated kinase (Erk)–mediated pathway14. Therefore, we obtained cytoplasmic extracts from control or hyaluronic acid–treated eosinophils or hyaluronic acid–treated cells exposed to the Erk inhibitor PD98059 and analyzed the extracts by immunoblot with antibody to phosphorylated serine, anti-AUF1 or anti-Pin1. Hyaluronic acid treatment consistently increased AUF1 phosphorylation, which was partially counteracted by treatment with PD98059 (Fig. 5a). Treatment with hyaluronic acid reproducibly dephosphorylated Pin1, which increases its isomerase activity35. In addition, Pin1 has 1000-fold more isomerase activity with phosphorylated than with unphosphorylated Ser-Pro or Thr-Pro substrates27. To directly confirm that the Pin1 dephosphorylation altered enzymatic activity, we did isomerase assays33. We prepared lysates from resting eosinophils treated for 4 h with hyaluronic acid or with hyaluronic acid plus juglone and incubated the cells with a tetrapeptide target composed of Ala-Glu-Pro-Phe-para-nitroanilide. After isomerization from cis to trans, the terminal 4-nitroanilide group can be cleaved by chymotrypsin and can be detected by absorbance at 390 nm. Pin1 activity was consistently increased by hyaluronic acid and inhibited by juglone (Fig. 5b). These data suggest that the combination of post-translational modifications of Pin1 and AUF1 induced by hyaluronic acid resulted in AUF1 isomerization with loss of binding to GM-CSF mRNA. Pin1 mediates binding of p37 AUF1 to the exosome The function of the exosome and proteasome in ARE mRNA regulation remains controversial, with both being associated with decay37,43. AUF1 reportedly associates with the exosome, a multiprotein complex with ribonuclease activity that has been linked to the rapid decay of ARE-containing mRNA43. We evaluated that possibility by analyzing AUF1 immunoprecipitates for the presence of the exosome-associated protein PM-Scl75 (Fig. 6). As expected, Pin1 was precipitated in resting cells, as was PM-Scl75 (Fig. 6a). Immunoprecipitation with anti-PM-Scl75 confirmed RNA-independent association with p37 but

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the binding of GM-CSF mRNA to hnRNP C (Fig. 4d). Incubation of cells with TAT-wwPin1 or juglone rapidly reduced the amount of GM-CSF mRNA associated with hnRNP C and increased that associated with AUF1 (Fig. 4a,d). Notably, the association of Pin1 with AUF1 was unaffected by hyaluronic acid (Fig. 3a) or TAT-ww-Pin1 (data not shown). In addition, we were unable to precipitate hnRNP C using anti-Pin1 (data not shown). These data suggest that in the absence of Pin1 activity, either in resting cells or after treatment with TAT-ww-Pin1 or juglone, AUF1 bound to most cytoplasmic GM-CSF mRNA. After treatment with hyaluronic acid, this process was reversed, resulting in hnRNP C–GM-CSF mRNA complexes.

not with other AUF1 isoforms. Notably, this association was maximal in eosinophils treated for 10 min with juglone and was minimal after treatment with hyaluronic acid (Fig. 6b). Therefore, hyaluronic acid signaling decreased the association of p37 AUF1 with the exosome, presumably as a ‘prelude’ to stabilization of GM-CSF mRNA. Pin1 is activated in vivo after allergen challenge To establish the in vivo relevance of Pin1 in human asthma, we obtained BAL fluid and peripheral blood eosinophils from allergic donors after ‘segmental allergen challenge’. This procedure is a well accepted asthma paradigm involving aerosolized allergen delivery directly into a bronchial segment, followed by BAL 48 h later2. Typically, more than 50% of the recovered cells are eosinophils. Purified BAL fluid or peripheral blood eosinophils obtained IP

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a

CSF2 ACTB

Figure 7 In vivo activation increases Pin1 activity and eosinophil survival and modulates GM-CSF mRNA–protein interactions. Purified eosinophils (eos) from BAL fluid and peripheral blood were obtained from the same subject after allergen challenge. Control peripheral blood eosinophils (R) were obtained from healthy donors. (a) Viability of eosinophils after 3 d of culture, with juglone added at the initiation of culture. Data represent + s.d. (b) Pin1 activity measured as described in Figure 5b immediately after preparation of eosinophil lysates. Data represent ± s.d. (c) RT-PCR and Southern blot of GM-CSF cDNA and ethidium bromide–stained agarose gel analysis of RT-PCR for ACTB cDNA. ‘Resting’ peripheral blood eosinophils (Normal), BAL fluid eosinophils (BAL) or BAL fluid eosinophils incubated with juglone (BAL+Jug; final 10 min) were lysed. Input, 10% of lysates before immunoprecipitation; the remainder was used for immunoprecipitation (antibodies, above blot) followed by analysis. Data are representative of three independent experiments with different donors.

from the same donor after allergen challenge showed considerably prolonged survival compared with the survival of cells from untreated normal controls. In vitro incubation of BAL fluid eosinophils after antigen challenge with juglone (Fig. 7a) or anti-GM-CSF (data not shown and refs. 6,9) blocked prolonged survival. To confirm these events were dependent on Pin1, we measured PPIase activity in these cells. Pin1 activity was increased considerably in cytoplasmic lysates from BAL fluid eosinophils activated in vivo compared with that in resting, peripheral blood, control eosinophils (Fig. 7b). We obtained similar results with eosinophils activated in vitro with hyaluronic acid (Fig. 5b). Based on those data, we analyzed BAL fluid eosinophils by immunoprecipitation followed by RT-PCR and Southern blot. As seen with eosinophils activated in vitro, total GM-CSF mRNA was substantially increased compared with that of control cells and was associated selectively with hnRNP C (Fig. 7c). In these conditions, GM-CSF mRNA did not decay9. Brief (10-minute) treatment with juglone in vitro reversed these mRNA-protein interactions, resulting in reconstitution of GM-CSF mRNA-AUF1 complexes. DISCUSSION Prolonged eosinophil survival is an essential step in the late phase of allergic inflammation in asthma. In the peripheral blood, terminally differentiated eosinophils undergo rapid death unless cells encounter IL-5 and/or GM-CSF3,9. Once in the lung, however, eosinophils persist and, through the autocrine release of cytokines, resist apoptosis31. Published findings support the idea of a predominant function for GM-CSF in the intrapulmonary process. For example, the normally long-lived BAL fluid eosinophils obtained after allergen challenge rapidly die in the presence of anti-GM-CSF but are

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insensitive to anti-IL-5 (ref. 9). This may reflect a loss of IL-5 receptor a-subunits44. Thus, understanding the mechanisms that underlie GM-CSF production by activated eosinophils is important for understanding asthma pathobiology. Here we have explored the function of PPIases and ARE-binding proteins in the regulation of GM-CSF production by eosinophils. Cyclophilin A and FK506-binding protein have been linked to the regulation of cytokine expression by activated T cells22,23. Cyclosporin A and FK506 bind to these PPIases and form a complex that inhibits calcineurin25,45. Unexpectedly, these reagents were relatively inactive against eosinophils, and neither affected GM-CSF mRNA accumulation induced by hyaluronic acid. Instead, our data have indicated that the third and least-studied member of the PPIase family, Pin1, is a critical and previously unknown mediator of GM-CSF mRNA stability and accumulation. Pin1 was originally linked to cell cycle control through interactions with many of the anti–mitotic protein monoclonal-2 antigens and mitotic phase proteins, including Cdc25, Wee1, Myt1, Nek2, Cdc27 and topoisomerase IIa27,28. Additional and more diverse functions, including the regulation of gene transcription through interactions with RNA polymerases I, II and III, the transcription factors NFAT , c-Jun and NF-kB, and the regulation of cell differentiation and signaling through the b-catenin–Wnt pathway27,28,46,47, have been reported. Finally, Pin1 has been linked to the isomerization of hyperphosphorylated tau protein characteristic of Alzheimer disease pathology29,48. Unlike cyclosporin A and FK506, which regulate NFAT indirectly through calcineurin25, the calciumand/or calmodulin-dependent phosphatase Pin1 binds directly to NFAT46. Those and other data have demonstrated the important but very restricted substrate specificity of Pin1 compared with that of cyclophilin A and FK506-binding protein, which probably reflects the inclusion of the WW domain35. Database searches have shown that many ARE-binding proteins linked to cytokine mRNA stability contain potential Pin1 isomerization sites, including the AUF1 isoforms, HuR17,49, KSRP50, tristetraprolin16,51, TIA-1 (ref. 52) and TIAR52. AUF1 undergoes reversible phosphorylation at Ser83, which has been correlated with alterations in IL-1 mRNA decay as well as the transactivation potential of the p40 isoform36,42. In addition, hyaluronic acid–mediated Erk signaling13, which culminates in the phosphorylation of target proteins at Ser-Pro and/or Thr-Pro moieties53, is obligatory for the stabilization of GM-CSF mRNA in eosinophils13,14. Those data suggest that AUF1 might be a Pin1 target. Immunoprecipitation of eosinophil cytoplasmic lysates demonstrated an interaction between Pin1 and all four AUF1 isoforms. Notably, only p45 and p40 AUF1 contain canonical WW domain interaction sites35, suggesting protein-protein interactions between isoforms or other components also participate in the formation of the ribonucleoprotein complex. There was no quantitative change in the Pin1-AUF1 complex after hyaluronic acid activation, despite a complete loss of associated GM-CSF mRNA. In those conditions, Pin1 was dephosphorylated, whereas p45 and p40 AUF1 were hyperphosphorylated. As AUF1 would be a better substrate after Ser83 phosphorylation and Pin1 activity increases after in vitro (hyaluronic acid) or in vivo activation, we propose that p45 and p40 are isomerized, preventing binding to GM-CSF mRNA. After activation, AUF1 was rapidly replaced by hnRNP C. In those conditions, GM-CSF mRNA was at least fourfold more stable than in unactivated control cells. AUF1 isoforms are involved in the decay of many ARE mRNAs, including GM-CSF, c-Myc and c-Fos19–21. However, the presence of four alternatively spliced isoforms and the absence of isoform-specific antibodies has hampered understanding of the function and regulation of AUF1. It is clear that the isoforms

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ARTICLES have distinct and opposing biological activity. Overexpression of p37 alone destabilizes ARE mRNA in a variety of mammalian cell lines20, but ARE mRNA is stabilized in NIH 3T3 cells when all four AUF1 isoforms are overexpressed19. Our data suggest that the conformation of p45 and p40 isoforms as well as their stoichiometry with p37 dictate the rate of GM-CSF mRNA decay. As p37 lacks Pin1 sites, we infer that it is regulated by the other three isoforms through protein-protein interactions. Such a mechanism would explain how overexpression of p37 in other systems or the loss of p45, p42 and p40 after juglone treatment noted here could destabilize ARE mRNA. Based on the results obtained here, we propose that moderately serine-phosphorylated p40 and p45 AUF1, in physical association with serine-phosphorylated Pin1 and unphosphorylated p42 and p37 AUF1, form a ribonucleoprotein complex with GM-CSF mRNA in resting eosinophils. Depending on the donor (and the activation status of their cells), some GM-CSF mRNA may be in complex with hnRNP C protein. When the ribonucleoprotein contains AUF1, rapid GMCSF mRNA decay ensues by targeting of p37–PM-Scl 75 to the exosome. In vitro or in vivo activation considerably reduces the p37–PM-Scl 75 interaction and triggers Pin1 dephosphorylation and activation with isomerization of hyperphosphorylated p45 and p40 AUF1 isoforms. The last events cause a reduction in the binding affinity of AUF1 and its replacement by hnRNP C on GM-CSF mRNA. We propose that increased association with hnRNP C causes GM-CSF mRNA stability to increase. Juglone-modified Pin1, along with p45, p42 and p40, are rapidly degraded by the proteasome, leaving p37 able to interact with GM-CSF mRNA. In these conditions, GM-CSF mRNA is also delivered to the exosome for decay through interactions with PM-Scl 75. In conclusion, our data indicate Pin1 is a critical regulator of cytokine mRNA turnover, which in turn controls the survival of activated eosinophils in the lungs of asthmatics. In addition, because AUF1 controls the decay of many other ARE mRNAs, Pin1 may be important in cytokine production by other activated cells as well. METHODS Reagents. Hyaluronic acid from human umbilical cords was purchased from ICN Pharmaceuticals. Cyclosporin A and juglone were from Sigma. FK506 was a gift from W. Burlingham (University of Wisconsin Medical School, Madison, Wisconsin). Recombinant human GM-CSF was from R&D Systems. Polyclonal anti-Pin1, anti-tristetraprolin, anti-TIA-1, anti-TIAR, anti-nucleolin and anti– hnRNP C were purchased from Santa Cruz Biotechnology; polyclonal anti-AUF1 was from Upstate Biotechnology; and polyclonal anti-GM-CSF was from R&D Systems. Monoclonal anti–phospho-serine was from Sigma (clone PSR-45); monoclonal anti-His6 (six histidine tags; 34698) was from Qiagen; monoclonal anti-HuR was from Molecular Probes (clone 19F12); and monoclonal anti-PM-Scl 75 was from J. Wilusz (Robert Wood Johnson Medical School, Piscataway, New Jersey). Proteasome inhibitor MG132 and protease inhibitor Cocktail Set-III were from Calbiochem. Horseradish peroxidase– conjugated anti-rabbit (secondary antibody) and the ECL immunoblot detection system (Amersham-Pharmacia) were used to detect primary antibodies. Erk inhibitor (PD98059) was supplied by New England Biolabs. Subjects and eosinophil preparation. Peripheral blood was obtained by venipuncture from normal or mildly atopic donors. Asthmatic subjects were used for allergen challenge. All participants have a clinical record at the University of Wisconsin Hospital and informed consent was obtained according to an approved University of Wisconsin Hospital Institutional Review Board protocol. Peripheral blood or BAL fluid eosinophils were purified with a negative immunomagnetic procedure as described54. Cells were used only when they were more than 99% pure. If there were few contaminating mononuclear cells, the cell population underwent a second separation by the addition of magnetic beads conjugated to monoclonal anti-CD14 (Miltenyi Biotech). After

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isolation, eosinophils were cultured at 37 1C in a humidified atmosphere of 5% CO2 and 95% air at a density of 1  106 cells/ml in RPMI 1640 medium, 10% FBS and 50 mg/ml of gentamycin (all from Life Technologies). Eosinophil viability. Eosinophils (1  106 cells/ml) were cultured in 96-well tissue culture plates (BD Biosciences). Cell viability was assessed by trypan blue exclusion on a hemocytometer. Where used, low-molecular-mass hyaluronic acid (100 mg/ml), hyaluronic acid plus Pin1 inhibitor (juglone or TATww-Pin1) or neutralizing anti-GM-CSF (5 mg/ml; ref. 13) was added at the initiation of culture unless indicated otherwise. RT-PCR and Southern blot. RT-PCR for GM-CSF (encoded by CSF2) was done as described13. PCR primers for b-actin mRNA (ACTB) were complementary to nucleotides 227–246 and 429–410, whereas those for GM-CSF mRNA corresponded to nucleotides 241–260 and 438–421; 30 cycles of PCR were done. Because the signals obtained by ethidium bromide gel electrophoresis were generally weak for the GM-CSF PCR products, Southern blots used a radioactively labeled GM-CSF cDNA probe . Immunoprecipitation and immunoblot. After activation, eosinophils were ‘snap-frozen’ at –801C and cytoplasmic lysates were prepared as described55. For immunoprecipitation, 2–5 mg of antibody was added to each sample, followed by incubation with rocking for 2–4 h at 4 1C. For experiments requiring ribonuclease treatment, RNAse A (10 mg/ml) and RNAse T1 (100 U/ml; both from Calbiochem) were added to the lysates immediately after preparation. Protein G–agarose beads (Sigma-Aldrich) were added and the incubation was continued overnight. Pellets were washed five times with lysis buffer, and at the last wash, the beads were split: 20% were dissolved in TriReagent (Molecular Research Center) for RNA extraction and 80% were dissolved in SDS-PAGE loading buffer for immunoblot. Crosslinking immunoprecipitation assay. After hyaluronic acid activation or treatment with hyaluronic acid with juglone added for the final 10 min, eosinophils were immediately crosslinked with ultraviolet irradiation, lysed and processed as described41, except that anti-AUF1 or anti–hnRNP C were used for immunoprecipitation and that the final PCR products were analyzed by Southern blot with radiolabeled GM-CSF cDNA rather than sequencing. Recombinant TAT proteins. The cDNA encoding enhanced green fluorescent protein or the WW domain of Pin1 (provided by K.P. Lu, Harvard University) was cloned in-frame into pHisTAT18,34 (provided by S. Dowdy, Washington University, St. Louis, Missouri). Proteins were expressed in Escherichia coli and were purified on a Ni2+ chelate column (Qiagen) as described by the manufacturer. Both TAT-linked proteins were more than 90% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels. Pin1 activity assay. Activity was measured as described33 with the following modifications. Purified eosinophils lysates were prepared by five cycles of freezing and thawing in a buffer containing 50 mM HEPES and 100 mM NaCl, pH 7.0. Total protein (10 mg in 10 ml) was mixed with 70 ml of HEPES–NaCl buffer supplemented with 2 mM dithiothreitol and 0.04 mg/ml of BSA. Then, 5 ml of a-chymotrypsin (60 mg/ml in 0.001 N HCl) was added, followed by thorough mixing. Finally, 5 ml of the tetrapeptide substrate Suc-Ala-Glu-ProPhe-pNa (Peptides International), dissolved in dimethylsulfoxide and preincubated at a concentration of 100 mg/ml in 480 mM LiCl and trifluoroethanol, was added. Absorption at 390 nM was measured over 30 min with a Beckman Coulter DU 800 spectrophotometer. Accession codes. BIND (http://bind.ca): 330941-44 and 330948-51. Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS We thank J. Sedgwick for eosinophils; K.P. Lu (Harvard University, Boston, Massachusetts) for the Pin1 WW domain cDNA; N. Jarjour for bronchoscopy samples; P. Bertics for critical reading of the manuscript; and members of the lab and the UW-Asthma SCOR group for suggestions. Supported by the National Institutes of Health (P50HL56396 to J.S.M.).

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ARTICLES COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.

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Published online at http://www.nature.com/natureimmunology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/

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