The Role of the Peptidyl-Prolyl Isomerase Pin1 in Immune Cell Function

Critical Reviews™ in Immunology, 28(1):45–60 (2008)

Pinning Down Signaling in the Immune System: The Role of the Peptidyl-Prolyl Isomerase Pin1 in Immune Cell Function Stephane Esnault,1 Zong-Jian Shen,1 & James S. Malter* Waisman Center for Developmental Disabilities, Department of Pathology and Laboratory Medicine and UW–Madison School of Medicine and Public Health; 1both authors contributed equally to this work * Address all correspondence to James S. Malter, MD, 509T, Waisman Center, 1500 Highland Ave., Madison, WI 53705; Tel.: 608-262-8888; Fax: 608263-3301; [email protected] Referee: Peter E. Shaw, Professor of Biochemistry, School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, UK

ABSTRACT: The peptidyl prolyl isomerase (PPIase) Pin1 has been recently implicated in cell cycle control and neuropathologies. There is now growing evidence that Pin1 plays an important role in the immune system and does so differentially from the related PPIases, cyclophilinA and FKBP. This review describes how Pin1 modulates cytokine expression by activated T cells and eosinophils and participates in T-cell and eosinophil apoptotic decisions both in vitro and in vivo. We highlight several possible immunologic diseases, including asthma, as well as organ transplant rejection, where anti-Pin1 therapeutics may be of value. KEY WORDS: Pin1, mRNA decay, eosinophil, lymphocyte, asthma, transplantation

I. INTRODUCTION An effective response to foreign antigens requires widespread activation of the immune system. Under most conditions, T lymphocytes become stimulated in a major histocompatibility complex (MHC)–restricted manner through engagement of CD3, CD28, CD40, and other cell surface receptors to initiate a complex web of intracellular signaling, which rapidly culminates in the production of a variety of soluble cytokine mediators and, eventually, T-cell division. The goals of immunosuppressive therapies essential for the survival of mismatched organ grafts or the prevention of autoimmunity include interference with cell proliferation or cytokine production.1–3 Recently, we and others have identified a new player in the cascade from cell-surface receptors to cell proliferation/survival and cytokine expression.4,5 In this review, we focus on the role of the peptidyl-prolyl Received: 6/26/07; Accepted: 10/23/07 1040-8401/08/$35.00 © 2008 28 by Begell House, Volume Number 1 Inc.

isomerase (PPIase) Pin1 in mediating intracellular signaling by activated immune cells. Recent advances have suggested that along with reversible phosphorylation, protein isomerization can play a critical role in signal transduction as well as effector protein function. One of the first events after T-cell receptor and co-receptor engagement is the triggering of a variety of tyrosine-, threonine-, and serine-directed protein kinases. These include those directly associated with cell surface receptors such as PI3K, Zap-70, and LCK6,7; those slightly downstream, such as Akt, Raf, and PKC8; or those even further downstream, such as the IKK, MEK, or MAPK families.9 When signal transduction has successfully reached the nucleus and initiated proliferative responses, cyclin-dependent kinases (CDKs) are also activated, triggering the reversible phosphorylation of a variety of nuclear as well as cytoplasmic substrates.10

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Despite these increasingly well-characterized serine, threonine, or tyrosine-directed kinase events, the mechanisms that underlie how phosphorylation changes protein function remain murky. When assayed in vitro, phosphorylated proteins often display modest changes in biologic activity compared to their nonphosphorylated precursors. These data suggest that additional post-translational modifications that are dependent on phosphorylation may participate in the alteration of protein function. A little over 10 years ago, a new family of proline-directed peptidyl-isomerases were identified,11 which participate in signal transduction by isomerizing a subgroup of proteins at selective Ser-Pro or Thr-Pro peptide bonds. The goal of this review is to discuss the role of one such isomerase, Pin1, as a mediator of immune cell function and cytokine expression. II. IDENTIFICATION AND CHARACTERIZATION OF PIN1 A novel PPIase was first characterized from E. coli in 1994,12 with a mass of approximately 10 kDa. The Km was comparable to other cyclophilins from E. coli, but the substrate specificity was more similar to FKBP. Notably, enzymatic activity was not inhibited by cyclosporin A nor FK506, suggesting a unique isomerase. Later that year, a complete protein sequence was obtained and named Parvulin.13 The eukaryotic version (Pin1) was identified 2 years later as an interacting protein with never in mitosis gene A (NIMA),11 a mitotic kinase expressed by Aspergillus. Pin1 and prokaryotic Parvulin reversibly catalyze the isomerization of peptide bonds immediately N-terminal to proline. The conversion between cis and trans alters target protein conformation, function, and stability.14,15 Pin1 preferentially binds to Ser-Pro or Thr-Pro (S/T-P) motifs through an N-terminal 40 amino acid WW (Trp-Trp) domain. 16 The peptidyl-prolyl isomerase (PPIase) is C-terminal (118 amino acids) and connected to the WW domain via a short (5 amino acid) flexible linker. Although Pin1 shows some activity towards S/T-P, phosphorylation by a variety of proline-directed kinases (including Cdk, PKC, and MAPK) increases the rate of isomerization by as much as 1000-fold.17 Pin1 is ubiquitously expressed in

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mammals, with the highest protein and activity levels seen in the brain and gonads.18 Despite genetic ablation, the existence of residual isomerase activity towards Pin1-specific target substrates suggests the presence of additional Pin1 family members.19 Therefore, Pin1 is a unique S/T-P-directed isomerase that has a narrow and highly selective target population capable of providing an additional level of regulatory control over prolinedirected phosphorylation/dephosphorylation events. Given these data, it is not surprising that initial studies focused on the role of Pin1 in cell cycle control. Deletion of Ess1, the saccharomyces ortholog of Pin1, induced mitotic arrest,20 whereas cell cycle transit varied by the amount of Pin1 present in Xenopus egg extracts.21,22 In mammalian systems, Pin1 is often overexpressed in tumors,23 has been positively correlated with metastatic potential,15 and interacts with many important cell cycle regulators and master control proteins, including p5324 and NF-κB.25 However, by promoting the degradation of key oncogenic proteins, including myc26 and cyclinE,27 Pin1 may suppress tumorigenesis as well. Sequence analysis revealed that Pin1 is highly conserved, with the human homologue capable of complementing either Drosophila or yeast knockout (KO) mutants.28 Pin1 KO is lethal in some lower organisms (S. cerevisiae, C. albicans, and A. nidulans) but causes much less obvious phenotypic changes in higher organisms. The Pin1 homologue in D. melanogaster29 is called dodo. Dodo-deficient flies show developmental defects in egg chamber patterning30 secondary to accelerated, proteasomal decay of the key transcriptional repressor CF2. To better understand these phenotypes, Pin1 KO mice have been produced by several labs.31 The original mice were in a mixed background and showed similarities to cyclinD-deficient animals,32 including accelerated aging, retinal and testicular atrophy, and decreased body weight. Central nervous system phenotypes included early degeneration of hippocampal and cortical neurons possibly secondary to abnormal regulation/processing of amyloid precursor protein (APP) and tau,33 both of which interact with Pin1. More recent lines in a pure C57/6J background demonstrate infertility and germ cell atrophy.34 Neuronal phenotypes have not been explored in this mouse

Critical Reviews™ in Immunology

model. Therefore, Pin1 plays important roles in mammalian germ cell development, the maintenance of neuronal integrity, and tumorigenesis by participating in the timing of protein transactivation and turnover. III. PIN1 FUNCTION IN LYMPHOCYTES Relatively little is known about the role and function of Pin1 in the mammalian immune system. Given the importance of rapid, orderly, and yet controlled cell cycle entry and progression for the normal expansion and function of lymphocytes, it would be surprising if Pin1 was not involved. A. Pin1 Regulates Transcription Interleukin-2 (IL-2) is a critical signaling cytokine that controls the mammalian immune response. IL-2 expression regulates the survival, clonal proliferation, and differentiation of T cells after stimulation by a foreign antigen. The production of IL-2 in response to T-cell stimulation is modulated by at least four families of transcriptional activators: NF-AT, NF-κB, Oct, and AP1 (c-Jun and c-Fos),35 many of which interact with and are regulated by Pin1. For example, Pin1 binds to and regulates the transcriptional activity of c-Jun after Ser63-Pro64 and Ser73-Pro74 motifs are phosphorylated by activated JNK or oncogenic Ras.36 c-Jun then shows enhanced transcriptional activity toward the cyclin D1 promoter.36 In a similar fashion, Pin1 modulates Erk-phosphorylated c-Fos transcriptional activity in NIH 3T3 mouse fibroblasts.37 NF-AT is hyperphosphorylated in the cytoplasm of resting T cells. Activation leads to NF-AT dephosphorylation by the calmodulindependent protein phosphatase calcineurin, translocation into the nucleus, and binding to the promoters of numerous cytokine genes, culminating in their transcription. Overexpression of Pin1 in Jurkat cells leads to the inhibition of NF-AT-dependent genes.38 Pin1 binds to and likely isomerizes phosphorylated NF-AT, blocking its dephosphorylation by calcineurin and reducing its transcriptional activity.38

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NF-κB subunits p65/Rel and p50 accumulate in the nucleus after degradation of IκBα, which binds and traps NF-κB in the cytoplasm. Pin1 binds to pThr254-Pro255 in p65 and prevents p65/IκBα interaction. This increases the accumulation and stability of p65 in the nucleus, with enhanced NF-κB dependent transcription.25 In addition, Pin1 may prevent NF-κB from SOCS-1mediated ubiquitination.25 Consistent with these findings, mouse embryonic fibroblasts (MEF) from Pin1–/– were resistant to NF-κB activation by cytokines. Therefore, Pin1 favors NF-κB transcriptional activity but suppresses the function of NF-AT. Typically, type 2 cytokine expression (IL-4) is upregulated by NF-AT, whereas both transcription factors (NF-AT and NF-κB) are involved in type 1 cytokine expression (IFN-γ).39 These data suggest that Pin1 blockade would preferentially inhibit type 1 immune responses. In addition to NF-AT, Pin1 attenuates the transcription factor interferon-regulatory factor 3 (IRF3). IRF3 is ubiquitously expressed and is responsible for IFN-β expression during the innate immune response after viral or bacterial infection.40 Pin1 suppresses IRF3 dimerization, which accelerates protein catabolism through ubiquitinylation.5 In MEF from KO or wild-type (WT) mice, the repression of IRF3 by Pin1 decreased IFN-β expression and reduced resistance to viral infection.5 Altogether, these data suggest a complex interplay of Pin1-dependent immune responses, which affect both type 1 and type 2 cytokines. B. Pin1 Regulates mRNA Stability in T Cells In resting T cells, despite basal granulocytemacrophage colony-stimulating factor (GM-CSF) mRNA transcription, cytoplasmic mRNA fails to accumulate. Upon mitogen treatment with phorbol ester or anti-CD3 and anti-CD28 antibodies, T cells stabilize GM-CSF mRNAs, accounting for much of their cytoplasmic accumulation and rapid translation.41 Both rapid decay and mitogen-driven stabilization requires the presence of 3′ untranslated region (3′ UTR) adenosine-uridine rich elements (AREs).42 Alteration or deletion of the AREs significantly enhanced the stability of mutant

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mRNAs.43–45 Chimeric globin mRNAs fused to the AREs from GM-CSF decayed at rates similar to wild-type GM-CSF,42 demonstrating that these elements were sufficient. The underlying molecular mechanisms for ARE mRNA regulation are slowly emerging. Multiple groups have shown the interaction of nuclear and cytoplasmic proteins with the AREs. These tend to be high-affinity events, which, like scaffolding proteins, recruit additional cofactors through protein–protein interactions. Occupancy of the ARE has been associated with both more or less rapid mRNA decay, implying the existence of both stabilizing and destabilizing protein activities.46–48 The Hu family appears to block ARE mRNA decay, whereas TTP, BRF1, or KSRP have the opposite effect. Interestingly, AUF-1/hnRNP D has shown both activities, depending on the circumstances and cell type.49,50 As many different ARE mRNAs can be simultaneously co-expressed and differentially stabilized, there must exist mechanisms to exert mRNA specific control on select mRNA cis elements. Although the mechanisms remain obscure, they likely involve the recruitment and participation of auxiliary or accessory proteins such as 14-3-3, which blocks KSRP interaction with the exoribonucleotic exosomal complex,51 as well as post-transcriptional modification (phosphorylation) of ARE-binding and adapter proteins.. We hypothesized that Pin1 could function as a phosphorylation sensor and modify either AREbinding and/or adapter proteins rapidly after activation. Indeed, we recently showed that Pin1 isomerase activity is required for the elaboration of GM-CSF by anti-CD3 plus anti-CD28-activated human primary CD4+ T lymphocytes.52 Mechanistically, Pin1 regulates GM-CSF mRNA decay by interacting with and modulating the mRNA binding activity of AUF1. Pin1 is weakly expressed by in vitro–activated macrophages and appears not to play a role in GM-CSF expression by these cells. In contrast, Pin1 is highly expressed by resting CD4+ T lymphocytes. Pin1 levels were unaffected by anti-CD3 plus anti-CD28-mediated activation, but PPIase activity was significantly increased. Unlike GM-CSF, IL-4 and TGF-β mRNAs, which lack AU-rich elements, were unaffected by Pin1.52 This suggests that in T cells, Pin1 regulates only the most labile AU-rich mRNAs. Indeed, in anti-CD3 plus anti-CD28-

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activated splenocytes from Pin1 KO mice, mRNA expression was inversely correlated to the number of functional 3′ UTR AREs.52,53 AUF1 contains a phosphorylated Ser83-Pro84, which regulates binding to AU-rich, destabilizing elements.54 All four isoforms of AUF1 co-precipitate with Pin1.4,52 HuR, another Ser-Pro containing ARE-binding protein, was also co-immunoprecipitated with Pin1, but the association was RNA dependent, suggesting an indirect interaction mediated by GM-CSF mRNA. In activated T lymphocytes, Pin1 inhibition increased the percentage of GM-CSF mRNA associated with AUF1 and reduced the amount with HuR. Under these conditions, GM-CSF mRNA associated with the exosome, likely accounting for its rapid clearance.52 Dysregulated GM-CSF expression is likely relevant for many pathologies. Macrophage accumulation with severe tissue damage and inflammatory mediator release,55 autoimmune gastritis,56 lethal myeloproliferative syndrome,57 or eosinophilia58 all occur in the context of GM-CSF overexpression. In addition, GM-CSF polymorphisms are highly associated with asthma,59 and patients treated with GM-CSF experience rheumatoid disease secondary to T-cell inflammation.60 C. Pin1 and T-Cell Apoptosis Regulation of T-cell apoptosis is critical for lymphocyte homeostasis and immune function. Activated T-cell autonomous death (ACAD) is determined by the ratio of anti- and proapoptotic Bcl2 family members acting on the mitochondria. To shut down antigen-activated T lymphocyte immune responses, the proapoptotic BH3-only Bcl2 homologues—Bim, Bax, and Bak—antagonize prosurvival Bcl2.61 How Bim is regulated in T cells remains unclear, especially as apoptosis is not associated with upregulation of Bim after an immune response to staphylococcal enterotoxin B (SEB).62 However, Pin1 associates in vitro with ERK-phosphorylated BimEL.63 Bcl2 can be phosphorylated on two serine-proline motifs and becomes associated with Pin1.64 On the mitochondria, PKCα phosphorylates Bcl2 on Ser70, suppressing apoptosis.65 Under these conditions, Pin1 may isomerize Bcl2, leading to Pin1 degradation by the proteasome, dephosphorylation of


Bcl2, and loss of prosurvival functionality.66 Through its modulation of NF-κB and p73, Pin1 activity may indirectly affect multiple apoptotic proteins. These include NF-κB-dependent antiapoptotic Bcl2 family members67 as well as p73 proapoptotic ones, including Bax.68,69 Our studies have shown that activated human peripheral blood mononuclear cells (PBMC) or rat splenocytes cultured in vitro showed levels of apoptosis similar to those of the untreated controls after Pin1 blockade with the naphthoquinone, juglone.53 This suggests that Pin1 activity does not affect the apoptosis of primary mononuclear immune cells. Thus, the role of Pin1 in the regulation of pro- and antiapoptotic Bcl2 family members remains ill-defined, particularly in primary T cells rather than in malignant cell lines. D. Pin1 Regulation of T-Cell Proliferation In addition to the promotion of T-cell proliferation by its effect on cytokine production, Pin1 is likely involved in IL-2 signaling that leads to cell division. The mitogenic effects of IL-2 require c-Myc, cyclinD2 and -3, cdk4 and -6, and other proteins.70–72 As noted above, in cell lines Pin1 binds phosphorylated c-Jun and regulates its ability to induce cyclin D1 expression. Pin1 and cyclin D1 protein levels were positively correlated in human breast cancer.36 Additionally, embryonic fibroblasts from Pin1 KO mice grow normally but are unable to re-enter the cell cycle after G0 arrest.31 In contrast, Pin1 promotes the elimination of c-Myc by allowing PP2A to dephosphorylate Ser62, causing degradation by the proteasome.26 In this situation, Pin1 acts as a repressor for proliferation. Therefore, Pin1 can play opposite roles depending on the situation, cell type, and proliferative status. Interestingly, in Pin1 KO mice we have observed no differences in the numbers of splenic or thymic CD3, CD4, CD8, or regulatory T-cell populations or activation marker expression after stimulation (Ref. 53 and Esnault and Malter, unpublished observation), suggesting that the immune systems of WT and KO mice develop similarly. Whether other Pin1-like activities expressed by T cells underlie these observations is unclear. Double-KO (Pin1–/– and p53–/–) mice

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lack thymic CD4+ or CD8+ cells.73 However, there was no mention of the immunologic phenotype of the single Pin1–/– mice. E. Pin1 in B Lymphocytes Although the function of Pin1 has not been explored in B lymphocytes, many proteins regulated by Pin1 in T cells are relevant to B cells as well. For example, NF-κB activity is tightly linked to B-cell survival through antiapoptotic protein expression (Bcl-XL).74 In contrast, Bruton’s tyrosine kinase (Btk) is expressed in all hematopoietic cells except T lymphocytes and is important for membrane translocation during B-cell receptor (BCR) signaling.75 Btk is a Tec family, nonreceptor tyrosine kinase member. Following BCR engagement, Btk translocates to the cell membrane and becomes phosphorylated on two tyrosine residues (Y223 and Y551). Mutations in the Btk gene cause human Xlinked agammaglobulinemia and murine X-linked immunodeficiency.76 Although these mutations disrupt Btk interaction with inositol phosphates and B-cell signaling,77 Pin1 associates with Btk at two phosphorylated serines and decreases Btk expression level through a proteasome and lysosomeindependent process.78 Very recently, Pin1 was shown to bind and to control Bcl-6 expression in B cells.79 Lymph nodes from Bcl-6 KO mice fail to form germinal centers and produce low-affinity antibodies.80 Conversely, Bcl-6 overexpression induces B-cell tumors, partially by inhibiting the p53dependent response to DNA damage.81 Therefore, Pin1 likely participates in B-cell function and immune responses. F. Pin1 in a Type 1 Immune Response The type 1 proinflammatory cytokines, IFN-γ and IL-2, are produced in large amounts after antigen presentation during infection82 or allograft rejection.1 These inflammatory cytokines structure the type and amplitude of the T-cell response. Ex vivo, splenocytes from Pin1 KO mice activated with anti-CD3 plus anti-CD28 showed significantly less IFN-γ, IL-2 mRNA, and protein compared to WT.53 IFN-γ and IL-2 mRNAs were less stable in anti-CD3/anti-CD28

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activated KO than WT splenocytes, whereas the stability of CXCL-10 mRNA, which lacks AREs, was unchanged. Therefore, Pin1 is necessary for ARE-mediated cytokine mRNA stabilization after T-cell co-stimulation. The relatively specific and irreversible inhibitor of Pin1, juglone suppressed cytokine mRNA expression by splenocytes after mitogenic antibodies or ionophore/PMA by as much as 500-fold (IFN-γ). However, juglone had no significant effect on the abundance of IL-2, IFN-γ, or the housekeeping RNAs in Pin1 KO splenocytes.53 Coincident with mRNA, cytokines were dramatically reduced in the culture supernatants as well. These data suggested that Pin1 blockade might be helpful to suppress alloimmunity against MHC/ HLA mismatched organ transplants. The expression of these cytokines has been highly correlated with rejection in both animal models and humans.83,84 In addition, because drugs targeting cyclophilins, including cyclosporin A and FK506, respectively, are the current mainstays of clinical immunosuppression after organ transplantation, we evaluated if Pin1 blockade had similar effects on organ rejection. We used the widely employed and strongly immunogenic rat orthotopic, single left lung transplantation model.85,86 The recipient (WKY) differs by a Class I MHC antigen from the donor (F344). Therefore, nonimmunosuppressed animals experience profound acute rejection within several days and chronic rejection with alveolar, pleural, and peribronchial collagen deposition and eventual organ loss within 1–2 weeks. At day 7 or 14, animals treated by intraperitoneal (IP) injection of 1 mg/kg juglone showed minimal gross signs of rejection and were typically indistinguishable from the contralateral control. Microscopically, the untreated transplanted lung showed severe rejection with acute inflammatory cell infiltration predominantly composed of neutrophils, lymphocytes, and macrophages. Alveolar architecture was totally effaced and the small airways packed with inflammatory cells. These changes were completely absent in juglone-treated animals (Fig. 1). No significant differences were found in the relative proportions of CD4, CD8, or γδ T cells in bronchoalveolar lavage fluids (BALF) from the native right lung or in the transplanted left lung. This is in contrast to collagen V–tolerized animals that show predominantly

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CD4+ T cells.87 Therefore, Pin1 blockade can dramatically attenuate acute transplant rejection and prevent collagen deposition and lung effacement comparable to that seen in chronic graft rejection.53 Analysis of BALF IFN-γ and IL-2 in the animals one week after transplantation by ELISA showed highly significant reductions in IFN-γ and nearly significant reductions in IL-2. Draining lymph node and spleen also showed >90% reduction in CXCL-10 mRNA. These data suggest that Pin1 regulates cytokine production by

FIGURE 1. Pin1 is required for acute and chronic rejection. I and IV: Gross appearance of lungs from control, untreated transplants (I) and juglone-treated (1 mg/kg) (IV). Lungs are oriented so that transplant is on the left. II and V: Hematoxylin and eosin (H&E) stained sections from control untreated (II) or juglone-treated (V) 1 week after transplant. III and VI: Trichrome-stained sections of control, untreated (III) and juglone-treated (VI) 2 weeks after transplant. These are representative sections from 8 control and 8 juglone-treated transplant recipients in each set. (Reprinted from Esnault S, Braun RK, Shen ZJ, Xiang Z, Heninger E, Love RB, Sandor M, Malter JS. Pin1 modulates the type 1 immune response. PLoS ONE. 2:e226, © 2007, with permission from the Public Library of Science [PLoS].53)


T cells both in vitro as well as in vivo. To demonstrate a causal role for these cytokines in rejection, IFN-γ and CXCL-10 expression vectors were insufflated into donor lungs immediately before religature in the recipient. The forced expression of IFN-γ and CXCL-10 in juglone-treated rats resulted in severe cellular infiltration irrespective of Pin1 blockade. These results support a central role of IFN-γ and CXCL-10 in the process of acute rejection and suggest that cytokine suppression after Pin1 inhibition is likely responsible for graft sparing.53 Immunosuppressive drugs, particularly CsA, greatly improves graft survival but have renal damage as a common side effect.85 To avoid toxicity and enhance graft acceptance, immunosuppressants such as corticosteroids, calcineurin inhibitors, antimetabolites, and rapamycin are given in combination.2,88–90 Cyclosporine A and tacrolimus indirectly inhibit nuclear factor of T cells (NFAT), preventing the transcriptional upregulation of cytokine genes by activated T

cells.3 In contrast, Pin1 also suppresses expression partially through the regulation of cytokine mRNA stability. Given this distinct mode of action, combined suboptimal treatment of the transplanted animals with CsA and juglone provided excellent graft protection without identifiable cellular infiltrates (Fig. 2, Ref. 53). These data show that the combined inhibition of Pin1 and calcineurin is additive or synergistic and suggest that CsA dosage could be signifcantly reduced if Pin1 inhibition is added to the therapeutic regimen. Therefore, Pin1 influences the type I immune response by modulating the production of proinflammatory, T-cell cytokines, including IFN-γ and IL-2. Pin1 likely functions through direct interactions with and regulation of key AUUUAspecific, mRNA-binding proteins such as AUF1, HnRNP C, and HuR.4,52 Interference with Pin1 through pharmacologic or genetic means reduced IFN-γ and IL-2 production and substantially attenuated graft rejection. Our data predict that drug combinations directed at the production (CsA,

FIGURE 2. Pin1 and calcineurin inhibition are synergistic. Seven days after orthotopic lung transplant, organs were harvested, fixed, and stained with H&E. (I) Treated with CsA at 1 mg/kg/d for 3 days. (II) Treated with juglone at 0.1 mg/kg/d for 7 days. (III) Treated with CsA at 1 mg/kg/d for 3 days plus juglone at 0.1 mg/kg/d for 7 days. For all, gross appearance of the transplant at harvest is shown along the left, 5× and 20× representative sections stained with H&E are shown in the middle and right panels. (Reprinted from Esnault S, Braun RK, Shen ZJ, Xiang Z, Heninger E, Love RB, Sandor M, Malter JS. Pin1 modulates the type 1 immune response. PLoS ONE. 2:e226, © 2007, with permission from the Public Library of Science [PLoS].53)

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FK506, steroids) and decay (juglone) of cytokine mRNAs would more effectively suppress alloimmunity and do so with reduced toxicity. More generally, the broad range of type 1 immune responses such as infectious diseases (intra- and extracellular pathogens) or autoimmunity (diabetes, multiple sclerosis, and lupus) that involve or require IFN-γ suggest a role for Pin1 in these processes as well. IV. PIN1 FUNCTION IN EOSINOPHILS The role of eosinophils (Eos) as effector inflammatory cells in asthma has been widely debated. In support, asthmatic lungs show large accumulations of Eos in the BAL and airway submucosa. Eos clearly contribute to submucosal matrix deposition and hyperplasia of the airway smooth muscle through the release of soluble mediators such as TGF-β1.91 Along with lymphocytes, activated Eos exacerbate the immune response through the production of Th2 cytokines and other potent proinflammatory mediators in the lung.91 Cytokines released from those cells, particularly IL-3, IL-5, and GM-CSF, regulate eosinophil priming, activation, and survival.91 Recently, we demonstrated that Pin1 mediates Eos GM-CSF expression and survival.4 However, reductions in circulating and pulmonary Eos by anti-IL-5 therapy did not reduce airway hyper-responsiveness or acute disease severity.92,93 Whether the therapeutic failure reflected inadequate Eos ablation or a lack of involvement of Eos in the pathophysiology, the results rekindled the controversy over the role of these cells in asthma. A. Role of Pin1 in Eos Survival There is an increasing consensus that Pin1 plays a significant role in cell cycle progression, apoptosis, and cell death in neurons and tumor cells.94 Primarily, Pin1 protects neurons by restoring normal Tau function,95 as well as enhancing the transcriptional activity of p53.96 However, Pin1 can also accelerate apoptosis by enhancing the expression and function of proapoptotic proteins, including BimEL,97 indicating multiple, diverse roles for Pin1 in apoptotic cell death.

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Eos, like neurons, are terminally differentiated, nondividing cells. In the absence of prosurvival growth factors such as GM-CSF, IL-3, or IL-5, Eos rapidly die.98 We observed a similar phenotype after Pin1 blockade, irrespective of the presence of growth factors.4 In addition, Pin1 was required for the production of GM-CSF, which is an essential prosurvival cytokine. Immunosuppressants FK506 and cyclosporine A (CsA), which act on related PPIases FKBP and cyclophilin A, respectively, reduced Eos survival but failed to attenuate GM-CSF production.4 Asthmatics treated with CsA showed reduced Eos numbers in bronchial biopsies, which was associated with the lack of T-cell-derived GM-CSF production.99,100 However, it remains unclear whether CsA directly inhibits GM-CSF production by T cells or has direct proapoptotic effects on Eos. The above data suggested that Pin1 was necessary for GM-CSF signaling. We recently found that juglone, an irreversible Pin1 inhibitor, could antagonize the antiapoptotic signaling of GMCSF and rapidly induce caspase-3 activation in Eos. Similarly, Pin1 null fibroblasts were sensitive to TNF-α-induced caspase-3 activation, although the mechanism was not defined. GM-CSF induces the transcription of Bcl-Xl,101 which can inhibit the function of BH3-only Bcl-2 family members (e.g., Bad, Bid, and Bim) and prevent downstream cytochrome c release from mitochondria.102 Potential Pin1 sites exist in Bcl-xL, and Pin1 binds Bcl-2 in cancer cells arrested in M phase,64 where Bcl-2 is hyperphosphorylated. However, in Eos, alternative mechanisms likely exist because these cells express very low levels of Bcl-xL and no Bcl-2. In Alzheimer’s disease, neuronal apoptosis is induced by depletion of nuclear Pin195 or associated with the upregulation of phospho-Bcl-264 and p53,103 whose activation induces Bax transcription.104 Alternatively, Pin1 may interact with and modulate the permeability of the mitochondrial membrane97 and/or the function of other Bcl-2 family members, including Mcl and Bax, which are highly expressed by Eos.105 Proteins of the Bcl family couple apoptotic signals from distinct cellular compartments and signaling pathways to the mitochondrial cell-death machinery.102 Conversely, IAPs (e.g., survivin and cIAP2) were induced by GM-CSF.106 These act downstream of mitochondria and suppress the processing of caspase 9 and 3.


These prosurvival proteins also contain Pin1 recognition sites (Shen and Malter, unpublished observation). Therefore, it is possible that Pin1 regulates intrinsic apoptotic signaling both upstream and downstream of mitochondrial events. B. Pin1 Controls GM-CSF Expression by Activated Eos Eosinophils display immune-effector functions in the airways of asthmatic individuals by elaborating a myriad of cytokines (up to 28, including IL-3, -4, -5, -9, -13, and GM-CSF), chemokines (eotaxin, MCP4, and RANTES), and profibrotic factors (TGF-β1, IL-11, and IL-17).107,108 These soluble mediators modulate the immune response by attracting and activating lymphocytes to the sites of inflammation.109 In addition to these Th2-type functions, Eos, like T cells, may express Th1-type cytokines as well,110 suggesting their potential significance and complexity in the context of allergic inflammation. The regulated expression of proinflammatory proteins is achieved by activation-inducible mRNA stabilization. In most cases, these mRNAs contain a 3′ UTR AUrich element (ARE), which destabilizes them in resting cells. For this reason, a major effort in the field has been to identify the molecular mechanisms by which different ARE mRNAs and their protein-binding partners (ARE-BPs) cooperatively regulate mRNA stability and translation. Recently, we showed that Pin1 is an essential component that, by isomerizing AUF1, controls the production of GM-CSF in Eos.4 Because this cytokine is an important determinant of Eos differentiation, survival, and function,111,112 Pin1 may contribute to the onset, development, and maintenance of allergic asthma. A variety of extracellular matrix proteoglycans (e.g., hyaluronic acid, HA), cytokines (TNF-α), and other mediators (fibronectin), which are increased in the airways of asthmatic lung,113–115 cause GM-CSF mRNA stabilization and cytokine release by eosinophils.4,116 The 3′ UTR ARE clearly functions as the dominant cis element in this process and can be occupied by multiple AREBPs, causing stabilization (HuR and YB-1)46,117 or destabilization (TPP)48 of the mRNA. Only AUF1, hnRNP C, YB-1, and HuR have been

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shown to bind GM-CSF mRNA in Eos.4,116 Whether the others are expressed and functional is unknown. There are high levels of AUF1 isoforms (p37, p40, p43, and p45) in Eos, with p37 the least abundant.4 AUF1 can destabilize AREcontaining mRNAs in vitro, including GM-CSF, IL, and TNF-α.118,119 Destabilization likely reflects recruitment of the exosome (a large complex of about ten 3′–5′ exonocleases and associated helicases) to the mRNA.120 p37 directly interacts with the exosome,4 and its overexpression has been associated with ARE mRNA destabilization. In resting Eos, GM-CSF mRNA and AUF1 interact, leading to rapid decay. Erk signaling rapidly activates Pin1.4 We reproducibly see Pin1 dephosphorylation and when measured in vitro, increased isomerase activity. These events are fairly rapid (75%, whereas none of the other inflammatory cell numbers were affected.130 Therefore, both BAL fluid as well as parenchyma showed significant and selective reductions in eosinophils. The attenuation in pulmonary eosinophils after Pin1 blockade may be due to reductions in chemoattractants or increased cell death. To better understand the observed changes, we measured BAL expression of critical cytokines, including GM-CSF, IL-3, 4, -5, or eotaxin, by qPCR or ELISA. GM-CSF and IL-5 mRNAs were significantly decreased by Pin1 inhibition, whereas

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IL-4 and eotaxin were unchanged.130 Moreover, GM-CSF and IL-5 mRNA expression were highly correlated with eosinophils in BAL fluid, whereas no correlation between any other cytokines and any other cell types was observed.130 We also observed significant increases in apoptotic BAL eosinophils after Pin1 inhibition.130 These suggest that reductions in GM-CSF and/or IL-5 led to increased apoptosis, culminating in fewer airway and parenchymal eosinophils. Interestingly, bone marrow differential counts showed a slight increase in eosinophil numbers after Pin1 blockade (3% in challenged but untreated controls versus 8% in juglone-treated animals), suggesting that eosinophil production remained intact or that eosinophils are confined to the bone marrow and are not recruited to the lung.130

IV. CONCLUSIONS There is growing recognition that proline-directed isomerases such as Pin1 function as key intermediates to amplify or suppress the action of a host of critical kinases and phosphatases. This functionality provides signaling cascades with the rapid and substantial capacity to selectively and specifically modulate protein function and longevity. As Pin1 targets function in multiple areas of immune cell metabolism, including apoptosis, transcription, and mRNA decay (among others), pharmacolgic interference may provide a novel approach to immunosuppression with potential value for the treatment of alloimmunity such organ rejection or asthma.

ACKNOWLEDGMENTS The authors wish to thank other members of the laboratory, our multiple co-authors, and the UW– Madison Immunology community for thoughtful discussions and suggestions. We also thank NIH (P50SCOR-Asthma) for support.

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