Involvement of peptidyl-prolyl isomerase Pin1 in the inhibitory effect of fluvastatin on endothelin-1-induced cardiomyocyte hypertrophy

Aims

Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions.

Main methods

We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [³H]-leucine incorporation.

Key findings

Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [³H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin.

Significance

This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.     

The prolyl isomerase Pin1 modulates development of CD8+ cDC in mice

Background

Pin1 has previously been described to regulate cells that participate in both innate and adaptive immunity. Thus far, however, no role for Pin1 has been described in modulating conventional dendritic cells, innate antigen presenting cells that potently activate naïve T cells, thereby bridging innate and adaptive immune responses.

Methodology/Principal Findings

When challenged with LPS, Pin1-null mice failed to accumulate spleen conventional dendritic cells (cDC). Analysis of steady-state spleen DC populations revealed that Pin1-null mice had fewer CD8+ cDC. This defect was recapitulated by culturing Pin1-null bone marrow with the DC-instructive cytokine Flt3 Ligand. Additionally, injection of Flt3 Ligand for 9 days failed to induce robust expansion of CD8+ cDC in Pin1-null mice. Upon infection with Listeria monocytogenes, Pin1-null mice were defective in stimulating proliferation of adoptively transferred WT CD8+ T cells, suggesting that decreases in Pin1 null CD8+ cDC may affect T cell responses to infection in vivo. Finally, upon analyzing expression of proteins involved in DC development, elevated expression of PU.1 was detected in Pin1-null cells, which resulted from an increase in PU.1 protein half-life.

Conclusions/Significance

We have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity.     

Pin1 positively affects tumorigenesis of esophageal squamous cell carcinoma and correlates with poor survival of patients

Background

Pin1 promotes oncogenesis by regulating multiple oncogenic signaling. In this study, we investigated the involvement of Pin1 in tumor progression and in the prognosis of human esophageal squamous cell carcinoma (ESCC).

Results

We observed that proliferation, clonogenicity and tumorigenesis of CE81T cells were inhibited by Pin1 knockdown. We next analyzed Pin1 expression in clinical ESCC specimens. When compared to the corresponding non-tumor part, Pin1 protein and mRNA levels in tumor part were higher in 84% and 62% patients, respectively. By immunohistochemistry, we identified that high Pin1 expression was associated with higher primary tumor stage (p = 0.035), higher overall cancer stage (p = 0.047) and poor overall survival (p < 0.001). Furthermore, the association between expression of Pin1 and levels of β-catenin and cyclin D in cell line and clinical specimens was evaluated. β-catenin and cyclin D1 were decreased in CE81T cells with Pin1 knockdown. Cyclin D1 level correlated with Pin1 expression in clinical ESCC specimens.

Conclusions

Pin1 upregulation was associated with advanced stage and poor prognosis of ESCC. Pin1 knockdown inhibited aggressiveness of ESCC cells. β-catenin and cyclin D1 were positively regulated by Pin1. These results indicated that targeting Pin1 pathway could represent a potential modality for treating ESCC.     

The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1.

The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man. Here we demonstrate that depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B. We show biochemically that this phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1. Although Pin1 could interact with Plx1 during interphase and mitosis, only the phosphorylated, mitotically active form of Cdc25 was able to bind Pin1, an event we have recapitulated using in vitro phosphorylated Cdc25. Taken together, these data suggest that Pin1 may modulate cell cycle control through interaction with Cdc25 and its activator, Plx1.     

Regulation of PRDX1 peroxidase activity by Pin1

Pin1 isomerizes the phosphorylated Ser/Thr-Pro peptide bonds and regulates the functions of its binding proteins by inducing conformational changes. Involvement of Pin1 in the aging process has been suggested based on the phenotype of Pin1-knockout mice and its interaction with lifespan regulator protein, p66^Shc. In this study, we utilize a proteomic approach and identify peroxiredoxin 1 (PRDX1), another regulator of aging, as a novel Pin1 binding protein. Pin1 binds to PRDX1 through interacting with the phospho-Thr⁹⁰-Pro⁹¹ motif of PRDX1, and this interaction is abolished when the Thr⁹⁰ of PRDX1 is mutated. The Pin1 binding motif, Thr-Pro, is conserved in the 2-Cys PRDXs, PRDX1–4 and the interactions between Pin1 and PRDX2–4 are also demonstrated. An increase in hydrogen peroxide buildup and a decrease in the peroxidase activity of 2-Cys PRDXs were observed in Pin1^?/? mouse embryonic fibroblasts (MEFs), with the activity of PRDXs restored when Pin1 was re-introduced into the cells. Phosphorylation of PRDX1 at Thr⁹⁰ has been shown to inhibit its peroxidase activity; however, how exactly the activity of PRDX1 is regulated by phosphorylation still remains unknown. Here, we demonstrate that Pin1 facilitates the protein phosphatase 2A-mediated dephosphorylation of PRDX1, which helps to explain the accumulation of the inactive phosphorylated form of PRDX1 in Pin1^?/? MEFs. Collectively, we identify Pin1 as a novel PRDX1 binding protein and propose a mechanism for Pin1 in regulating the metabolism of reactive oxygen species in cells.     

Role of Pin1 in neointima formation: Down-regulation of Nrf2-dependent heme oxygenase-1 expression by Pin1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr–Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMC proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guidewire injury. Exposure of VSMCS to platelet-derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMCs, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. In contrast, the activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblasts (MEFs), compared to Pin1-wild-type MEFs. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3 weeks (1 mg/kg, two times a week) significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMC proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.     

Proteomic screen in the simple metazoan <Emphasis Type="Italic">Hydra</Emphasis> identifies 14-3-3 binding proteins implicated in cellular metabolism,cytoskeletal organisation and Ca<Superscript>2+</Superscript> signalling

Background  

14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca²⁺ signalling.     

Phosphorylated guanine nucleotide exchange factor C3G,induced by pervanadate and Src family kinases localizes to the Golgi and subcortical actin cytoskeleton

Background  

The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context.     

Molecular basis for an ancient partnership between prolyl isomerase Pin1 and phosphatase inhibitor-2

Pin1 is a prolyl isomerase that recognizes phosphorylated Ser/Thr-Pro sites, and phosphatase inhibitor-2 (I-2) is phosphorylated during mitosis at a PSpTP site that is expected to be a Pin1 substrate. However, we previously discovered I-2, but not phospho-I-2, bound to Pin1 as an allosteric modifier of Pin1 substrate specificity [Li, M., et al. (2008) Biochemistry 47, 292]. Here, we use binding assays and NMR spectroscopy to map the interactions on Pin1 and I-2 to elucidate the organization of this complex. Despite having sequences that are ～50% identical, human, Xenopus, and Drosophila I-2 proteins all exhibited identical, saturable binding to GST-Pin1 with K(0.5) values of 0.3 μM. The (1)H-(15)N heteronuclear single-quantum coherence spectra for both the WW domain and isomerase domain of Pin1 showed distinctive shifts upon addition of I-2. Conversely, as shown by NMR spectroscopy, specific regions of I-2 were affected by addition of Pin1. A single-residue I68A substitution in I-2 weakened binding to Pin1 by half and essentially eliminated binding to the isolated WW domain. On the other hand, truncation of I-2 to residue 152 had a minimal effect on binding to the WW domain but eliminated binding to the isomerase domain. Size exclusion chromatography revealed that wild-type I-2 and Pin1 formed a large (>300 kDa) complex and I-2(I68A) formed a complex of half the size that we propose are a heterotetramer and a heterodimer, respectively. Pin1 and I-2 are conserved among eukaryotes from yeast to humans, and we propose they make up an ancient partnership that provides a means for regulating Pin1 specificity and function.     

The structure of the leukemia drug imatinib bound to human quinone reductase 2 (NQO2)

Background  

Imatinib represents the first in a class of drugs targeted against chronic myelogenous leukemia to enter the clinic, showing excellent efficacy and specificity for Abl, Kit, and PDGFR kinases. Recent screens carried out to find off-target proteins that bind to imatinib identified the oxidoreductase NQO2, a flavoprotein that is phosphorylated in a chronic myelogenous leukemia cell line.     

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases

Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1^+/+), hemizygous (Pin1^+/?) or knock-out (Pin1^?/?) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.     

The intracellular threonine of amyloid precursor protein that is essential for docking of Pin1 is dispensable for developmental function

Background

Processing of Aβ-precursor protein (APP) plays an important role in Alzheimer''s Disease (AD) pathogenesis. Thr residue at amino acid 668 of the APP intracellular domain (AID) is highly conserved. When phosphorylated, this residue generates a binding site for Pin1. The interaction of APP with Pin1 has been involved in AD pathogenesis.

Methodology/Principal Findings

To dissect the functions of this sequence in vivo, we created an APP knock-in allele, in which Thr⁶⁶⁸ is replaced by an Ala (T⁶⁶⁸A). Doubly deficient APP/APP-like protein 2 (APLP2) mice present postnatal lethality and neuromuscular synapse defects. Previous work has shown that the APP intracellular domain is necessary for preventing early lethality and neuromuscular junctions (NMJ) defects. Crossing the T⁶⁶⁸A allele into the APLP2 knockout background showed that mutation of Thr⁶⁶⁸ does not cause a defective phenotype. Notably, the T⁶⁶⁸A mutant APP is able to bind Mint1.

Conclusions/Significance

Our results argue against an important role of the Thr⁶⁶⁸ residue in the essential function of APP in developmental regulation. Furthermore, they indicate that phosphorylation at this residue is not functionally involved in those APP-mediated functions that prevent (NMJ) defects and early lethality in APLP2 null mice.     

Effect of Pin1 or Microtubule Binding on Dephosphorylation of FTDP-17 Mutant Tau

Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of tauopathies, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), is directly associated with mutations of the gene tau. However, it is unknown why mutant Tau is highly phosphorylated in the patient brain. In contrast to in vivo high phosphorylation, FTDP-17 Tau is phosphorylated less than wild-type Tau in vitro. Because phosphorylation is a balance between kinase and phosphatase activities, we investigated dephosphorylation of mutant Tau proteins, P301L and R406W. Tau phosphorylated by Cdk5-p25 was dephosphorylated by protein phosphatases in rat brain extracts. Compared with wild-type Tau, R406W was dephosphorylated faster and P301L slower. The two-dimensional phosphopeptide map analysis suggested that faster dephosphorylation of R406W was due to a lack of phosphorylation at Ser-404, which is relatively resistant to dephosphorylation. We studied the effect of the peptidyl-prolyl isomerase Pin1 or microtubule binding on dephosphorylation of wild-type Tau, P301L, and R406W in vitro. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins. Dephosphorylation of wild-type Tau was reduced in brain extracts of Pin1-knockout mice, and this reduction was not observed with P301L and R406W. On the other hand, binding to microtubules almost abolished dephosphorylation of wild-type and mutant Tau proteins. These results demonstrate that mutation of Tau and its association with microtubules may change the conformation of Tau, thereby suppressing dephosphorylation and potentially contributing to the etiology of tauopathies.One of hallmarks of Alzheimer disease (AD)³ pathology is neurofibrillary tangles, which are composed of paired helical filaments (PHFs), aggregates of the abnormally phosphorylated microtubule-associated protein Tau. Intracellular inclusions comprising Tau are also found in several other neurodegenerative diseases, including Pick disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), collectively called tauopathies (1–3). Identification of Tau as a causative gene of the inherited tauopathy FTDP-17 reveals that Tau mutation is sufficient to cause disease (4–6). However, the impact Tau mutations have on neurodegeneration remains unknown.Tau proteins in inclusions are hyperphosphorylated, and extensive studies have identified the phosphorylation sites; for example, more than 20 sites have been identified in PHF-Tau obtained from AD brains (7, 8). Tau can be phosphorylated by a variety of protein kinases, including glycogen synthase kinase 3β (GSK3β), cyclin-dependent kinase 5 (Cdk5), mitogen-activated protein kinase, cAMP-dependent protein kinase (PKA), microtubule affinity regulating kinase, and others (9–11). Tau is predominantly phosphorylated on the Ser or Thr residue in Ser/Thr-Pro sequences, suggesting the involvement of proline-directed protein kinases such as GSK3β and Cdk5 in hyperphosphorylation. A critical question is how mutations in Tau induce hyperphosphorylation in brain (12). Early phosphorylation experiments in vitro and in cultured cells have shown that mutant Tau is less phosphorylated than wild-type (WT) Tau (13–18). However, two later studies demonstrated higher phosphorylation of mutant Tau using brain extracts as a source of protein kinases in the presence of protein phosphatase inhibitor okadaic acid (19) or in immortalized cortical cells (20). However, it is not fully understood how mutant Tau becomes highly phosphorylated in vivo.Tau hyperphosphorylation could also be attributed to reduced dephosphorylation activity. Tau is dephosphorylated in vitro by any of the major four classes of protein phosphatases, PP1, PP2A, PP2B, and PP2C, but PP2A is thought to be the major protein phosphatase that regulates Tau phosphorylation state in brains (21–23). PP2A activity reportedly is decreased in AD brain (24–26), and highly phosphorylated Tau in PHF is relatively resistant to dephosphorylation by PP2A (27). Few studies have been done on dephosphorylation of mutant Tau, however, and thus the mechanism remains unclear. One putative factor involved in mutant Tau dephosphorylation is the peptidyl-prolyl isomerase Pin1. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins (28, 29). Pin1 is involved in AD pathogenesis as shown by the fact that it is found in neurofibrillary tangles and that Tau is hyperphosphorylated in Pin1-deficient mouse brains (30). Pin1 is indicated to facilitate Tau dephosphorylation via PP2A by binding to the phospho-Thr-231-Pro or phospho-Thr-212-Pro site (31–33). The effect of Pin1 on the stability of mutant Tau was recently reported (34), but a detailed analysis of Pin1 action on mutant Tau has not been reported. Another possible factor affecting dephosphorylation of mutant Tau is the binding to microtubules. We previously showed that phosphorylation of Tau is stimulated upon binding to microtubules (35). We thus hypothesized that binding to microtubules may also affect the extent of Tau dephosphorylation.Here, we examined the effects of Pin1 and binding to microtubules on dephosphorylation of WT and FTDP-17 mutant (P301L and R406W) Tau proteins that had been phosphorylated by Cdk5-p25 or Cdk5-p35. P301L and R406W are two distinct types of FTDP-17 mutants that have been studied well. We show for the first time how the regulation of Tau dephosphorylation can contribute to the observed Tau hyperphosphorylation in tauopathies.     

Pin1 mediates neural-specific activation of the mitochondrial apoptotic machinery

Apoptosis of neurons plays fundamental roles in brain development and disease. Although neurons share with other cell types components of the mitochondrial apoptotic machinery, how this machinery is specifically activated in neurons remains poorly understood. Remarkably, phosphorylation of the BH3-only protein BIMEL at Ser65 triggers apoptosis in neurons but suppresses cell death in non-neural cells. Here, we report that the prolyl isomerase Pin1 interacts with Ser65-phosphorylated BIMEL in neurons. Pin1 is enriched at the mitochondrial membrane in neurons, where it forms a physical complex with the neuron-specific JNK scaffold protein JIP3. Activation of JNK signaling induces the dissociation of Pin1 from JIP3 and concomitantly promotes Pin1 binding to phosphorylated BIMEL. The interaction of Pin1 with phosphorylated BIMEL stabilizes BIMEL and thereby activates neuronal apoptosis. These findings define a neural-specific mechanism of cell death whereby Pin1 couples phosphorylation of BH3-only proteins to activation of the mitochondrial apoptotic machinery.     

Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.