In this study, we identified a heretofore unknown role for Pin1 as a mediator of GM-CSF and IL-5 signaling through the direct regulation of Bax. These results clarify how Bax function is regulated by cytokine signaling in terminally differentiated, growth factor-dependent eosinophils and suggest Pin1 could be a new therapeutic target for the treatment of asthma as well as other eosinophilic diseases.
Eosinophilic airway inflammation is a hallmark of asthma which often culminates in subepithelial fibrosis with variable airway obstruction. Drugs such as steroids that reduce airway and parenchymal eosinophilic accumulation are the mainstay of therapy for asthmatics. A variety of monoclonal antibody based therapies (anti-IL-5, anti-selectin and anti-chemokine receptor)37
can block eosinophil development, trafficking and survival. Multiple doses of anti-IL-5 substantially depleted peripheral blood eosinophils, but had modest effects on pulmonary eosinophils38
. This may reflect both the loss of IL-5 receptor β-subunit from pulmonary eosinophils39
and persistently high expression of other anti-apoptotic cytokines such as GM-CSF, which is induced by allergen exposure22
. Previously, we showed that Pin1 is essential for the expression of GM-CSF13,40
, which along with IL-5, contributes to eosinophil survival in vitro
. The data here suggest that Pin1 blockade in vivo
would not only reduce GM-CSF production13,40
but would also activate the intrinsic apoptosis machinery to accelerate eosinophil death.
Numerous pro- and anti-apoptotic molecules are expressed by and implicated in eosinophil death25
. However, it has been unclear how pro-survival cytokines attenuate the eosinophil apoptosis. GM-CSF and IL-5 induce expression of Bcl-2 and Bcl-xL10
, but neither protein are detectable in eosinophils by immunoblotting even after treatment with IL-5 or GM-CSF (data not shown). Therefore, it is unlikely that the transcriptional induction of these proteins is of a sufficient magnitude or rapidity to account for GM-CSF or IL-5 prolonged survival. On the other hand, freshly purified human eosinophils from every donor evaluated expressed high amounts of Pin1 and Bax. After cell activation, Pin1 reproducibly interacted with Bax, in a manner prevented by inhibition of Pin1 PPIase activity. As Bax inhibition prevented cell death after GM-CSF withdrawal or Pin1 blockade, Bax, rather than Bak and Bad, is the dominant proapoptotic multidomain Bcl-2 family protein responsible for orchestrating eosinophil death25
. In support of this conclusion, allergen challenged Bax-deficient mice accumulated nearly 2-fold more airway eosinophils after allergen challenge than wild-type mice41
. Similarly, Bax was reduced in bronchial mucosal biopsies from active asthmatics42
. These results suggest that the normal, rapid turnover of peripheral blood eosinophils likely reflects Bax activation in the absence of adequate pro-survival signaling or Pin1 activity.
Increasing evidence suggests that Pin1 plays an important role in neuronal and tumor cell apoptosis16
. Pin1 accelerated neuronal apoptosis by enhancing the expression and function of proapoptotic Bcl-2 family member BimEL19
. Moreover, Pin1 physically interacted with Bcl-2 in cancer cells arrested in M phase. Cell death was associated with increased amounts of hyper-phosphorylated Bcl-2 and p53, with the latter transactivating Bax gene expression17
. In contrast, in Alzheimer’s disease, neuronal apoptosis was associated with the absence of nuclear Pin118
. These and our data suggests that Pin1 plays a complex role in apoptosis of dividing as well as quiescent, terminally differentiated cells. Unlike neurons, tumor cell lines and neutrophils, which express abundant quantities of several Bcl-2 family proteins43-45
, eosinophils expressed high quantities of Bax but very low amounts of other pro- or anti-apoptotic Bcl-2 family proteins25
. Thus eosinophils employ a seemingly less complex apoptotic signaling pathway. Whether Pin1 regulates other pro-apoptotic Bcl-2 family members has not been investigated. Our preliminary results showing interactions with c-IAP2 and caspase 8 suggest Pin1 may play a broader role, perhaps in a cell type-specific manner, in the overall regulation of apoptotic decisions.
Despite the important role of Bax in apoptosis, the mechanisms controlling Bax conformational modifications, activation and mitochondrial translocation are not completely understood. Ku70, which interacts with the N-terminus of Bax26
, or humanin, which binds to the C-terminus of Bax46
, can both antagonize Bax activation. However, deficiency of Ku70 or humanin failed to induce Bax translocation to mitochondria or apoptosis in the absence of appropriate stimuli. Mitochondria-associated Bax interacts with clusterin, which prevents Bax oligomerization and cytochrome c release47
. In contrast, Pin1 selectively interacted with Thr-phosphorylated Bax in cytokine-stimulated cells. In the absence of Pin1 interactions or PPIase activity, irrespective of cytokine signaling, the 6A7 epitope became exposed and Bax translocated to the mitochondria. These data strongly suggest that at least in cytokine-dependent primary human eosinophils, Bax is suppressed by Pin1, likely through isomerization after Erk-mediated threonine phosphorylation.
Bax can be phosphorylated at three sites (Ser163-Tyr164, Thr167-Pro168 and Ser184-Lys185) which confer distinct phenotypes. Whereas phosphorylation of Ser163 induced cell death in neurons by promoting Bax translocation to mitochondria, phosphorylation of Ser184 reversed this phenotype32
. Ser184-phosphorylated Bax can hetero-dimerize with Mcl-1, Bcl-xL and A1, and thereby increase neutrophil survival32
. JNK- and p38-mediated Thr167 phosphorylation accelerated apoptosis in human tumor cells29
, whereas Erk1/2 protected melanoma cells from apoptosis by inhibiting Smac/DIABLO release from mitochondria33
. Thus, depending on the cell type, different MAP Kinases can modify Bax on Thr167 with disparate apoptotic endpoints.
Despite GM-CSF signaling, Bax was hypo-phosphorylated at Thr167 if Pin1 was inhibited. These results suggest that Pin1-mediated isomerization is required to stabilize phospho-Ser/Thr-Pro sites, possibly by preventing phosphatase access to these sites. Consistent with this hypothesis, Pin1 interacts with and is itself regulated by PP2A2
In primary human eosinophils, mutagenesis of Bax Thr167 to alanine increased Bax activity and reduced its interaction with Pin1 and sensitivity to pro-survival signaling. Mutagenesis of Bax Pro168 prevented Bax translocation to the mitochondria31
. The Thr167-Pro168 site is located in a hinge immediately adjacent to the Bax C-terminal transmembrane domain. Our results suggest Pin1 isomerizes phosphorylated Bax, and that this isomerization constrains the C- and N-termini within Bax. This notion is consistent with our inability to immunoprecipitate Bax with the active conformation-specific 6A7 antibody during pro-survival signaling. Upon cessation of cytokine signaling or blockade of Pin1 PPIase activity, the N-terminal domain of Bax is spontaneously exposed and Bax is activated.
Terminally differentiated eosinophils express high amounts of p23 Bax which is cleaved to p18 Bax during apoptosis or after Pin1 blockade. This process was completely blocked by GM-CSF or IL-5, or by a calpain inhibitor. p18 Bax is a more potent inducer of apoptosis than p23 Bax37
. p23 cleavage occurred subsequent to 6A7 epitope exposure, approximately 12 h after cytokine withdrawal. Similar events occurred in tumor cell lines treated with chemotherapeutic agents36
. The toxicity of p18 Bax may reflect exposure of the hydrophobic BH3 domain (amino acids 59-73), which facilitates oligomerization and death pore formation in the mitochondrial outer membrane48
. Consistent with these results, p18 Bax is detected exclusively in the mitochondrial fraction and is associated with increased calpain activation. An attractive hypothesis is that Pin1 maintains Bax in a calpain-resistant conformation, thereby reducing p18 generation.