Hirotada Akiyama

Pin1 Down-regulates Transforming Growth Factor-β (TGF-β) Signaling by Inducing Degradation of Smad Proteins

Transforming growth factor-β (TGF-β) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-β signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-β and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-β-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-β signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

Ablation of a peptidyl prolyl isomerase Pin1 from p53-null mice accelerated thymic hyperplasia by increasing the level of the intracellular form of Notch1.

Tumor suppressor p53 is essential for checkpoint control in response to a variety of genotoxic stresses. DNA damage leads to phosphorylation on the Ser/Thr-Pro motifs of p53, which facilitates interaction with Pin1, a pSer/pThr-Pro-specific peptidyl prolyl isomerase. Pin1 is required for the timely activation of p53, resulting in apoptosis or cell cycle arrest. To investigate the physiological relationship between Pin1 and p53, we created Pin1−/−p53−/− mice. These p53-deficient mice spontaneously developed lymphomas, mainly of thymic origin, as well as generalized lymphoma infiltration into other organs, including the liver, kidneys and lungs. Ablation of Pin1, in addition to p53, accelerated the thymic hyperplasia, but the thymocytes in these Pin1−/−p53−/− mice did not infiltrate other organs. The thymocytes in 12-week-old Pin1−/−p53−/− mice were CD4−CD8− (double negative) and had significantly higher levels of the intracellular form of Notch1 (NIC) than the thymocytes of p53−/− or wild-type mice. Presenilin-1, a cleavage enzyme for NIC generation from full-length Notch1 was increased in the thymocytes of Pin1−/−p53−/− mice. Pin1 depletion also inhibited the degradation of NIC by proteasomes. These results suggest that both Pin1 and p53 control the normal proliferation and differentiation of thymocytes by regulating the NIC level.

Involvement of the mouse Prp19 gene in neuronal/astroglial cell fate decisions.

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19β, a splice variant of mouse PRP19α corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The β-variant possesses an additional 19 amino acid residues inframe in the N-terminal region of the α-variant. The forced expression of the α-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the β-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the β-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the β-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the α-variant gene plays a pivotal role in neural differentiation and that the β-variant participates in neuronal/astroglial cell fate decisions.

Comparative analysis of enzyme activities and mRNA levels of peptidyl prolyl cis/trans isomerases in various organs of wild type and Pin1−/− mice

We investigated the enzyme activity of peptidyl prolyl cis/trans isomerases (PPIases) in brain, testis, lung, liver, and mouse embryonic fibroblasts (MEF) of Pin1 +/+ and Pin1 −/− mice. The aim of this study is to determine if other PPIases can substitute for the loss of Pin1 activity in Pin1 −/− mice and what influence Pin1 depletion has on the activities of other PPIases members. The results show that high PPIase activities of Pin1 are found in organs that have the tendency to develop Pin1 knockout phenotypes and, therefore, provide for the first time an enzymological basis for these observations. Furthermore we determined the specific activity (k cat/K M) of endogenous Pin1 and found that it is strongly reduced as compared with the recombinant protein in all investigated organs. These results suggest that posttranslational modifications may influence the PPIase activity in vivo. The activities originating from cyclophilin and FKBP are not influenced by the Pin1 knockout, but a basal enzymatic activity towards phosphorylated substrates could be found in Pin1 −/− lysates. Real time PCR experiments of all PPIases in different mouse organs and MEF of Pin1 +/+ and Pin1 −/− mice support the finding and reveal the specific expression profiles of PPIases in mice.

A Novel Role for hGas7b in Microtubular Maintenance POSSIBLE IMPLICATION IN TAU-ASSOCIATED PATHOLOGY IN ALZHEIMER DISEASE

Here, we report a novel role for hGas7b (human growth arrest specific protein 7b) in the regulation of microtubules. Using a bioinformatic approach, we studied the actin-binding protein hGas7b with a structural similarity to the WW domain of a peptidyl prolyl cis/trans isomerase, Pin1, that facilitates microtubule assembly. Thus, we have demonstrated that hGas7b binds Tau at the WW motif and that the hGas7b/Tau protein complex interacts with the microtubules, promoting tubulin polymerization. Tau, in turn, contributes to protein stability of hGas7b. Furthermore, we observed decreased levels of hGas7b in the brains from patients with Alzheimer disease. These results suggest an important role for hGas7b in microtubular maintenance and possible implication in Alzheimer disease.

Prolyl Isomerase Pin1 Regulates Mouse Embryonic Fibroblast Differentiation into Adipose Cells

Background A peptidyl prolyl cis/trans isomerase, Pin1, regulates insulin signal transduction. Pin1 reduces responses to insulin stimulation by binding CRTC2 (CREB-regulated transcriptional co-activator 2) and PPARγ (peroxisome prolifereator- activated receptor γ), but conversely enhances insulin signaling by binding IRS-1 (insulin receptor substrate-1), Akt kinase, and Smad3. Therefore, it is still unclear whether Pin1 inhibits or enhances adipose cell differentiation. Methodology/Principal Findings Pin1−/− and wild-type mice were fed with high fat diets and adipose tissue weight was measured. Compared to wild-type mice, Pin1−/− mice had lower adipose tissue weight, while the weight of other tissues was similar. Mouse embryo fibroblasts (MEFs), prepared from both groups of mice, were induced to differentiate into adipose cells by stimulation with insulin. However, the rate of differentiation of MEFs from Pin1−/− mice was less than that of MEFs from wild-type mice. The rate of insulin-induced MEF cell differentiation in Pin1−/− mice was restored by increasing expression of Pin1. We found that Pin1 binds to phosphoThr172- and phosphoSer271-Pro sites in CREB suppress the activity in COS-7 cells. Conclusion and Significance Pin1 enhanced the uptake of triglycerides and the differentiation of MEF cells into adipose cells in response to insulin stimulation. Results of this study suggest that Pin1 down-regulation could be a potential approach in obesity-related dysfunctions, such as high blood pressure, diabetes, non-alcoholic steatohepatitis.

Prolyl Isomerase Pin1 Protects Mice from Endotoxin Shock

Background Prolyl isomerase Pin1 may be involved in innate immunity against microbial infection, but the mechanism how Pin1 controls the innate immunity is poorly understood. Methodology/Principal Findings Injection of lipopolysaccharide (LPS) into the mice induces inflammatory pulmonary disorder and sometimes the serious damages lead to death. Comparing to the wild-type (WT) mice, the Pin1−/− mice showed more serious damages in lung and the lower survival rate after the LPS injection. We compared the levels of typical inflammatory cytokines. Pin1−/− mice overreacted to the LPS injection to produce inflammatory cytokines, especially IL-6 more than WT mice. We showed that Pin1 binds phosphorylated PU.1 and they localize together in a nucleus. These results suggest that Pin1 controls the transcriptional activity of PU.1 and suppresses overreaction of macrophage that causes serious damages in lung. Conclusions/Significance Pin1 may protect the mice from serious inflammation by LPS injection by attenuating the increase of IL-6 transcription of the mouse macrophages.

Direct Optical Microscopic Observation of the Microtubule Polymerization Intermediate Sheet Structure in the Presence of Gas7

The process of microtubule elongation is thought to consist of two stages-formation of a tubulin sheet structure and its closure into a tube. However, real-time observation of this process has been difficult. Here, by utilizing phospho-tau binding protein Gas7 (growth-arrest-specific protein 7), we visualized the polymer transformation process by dark-field microscopy. Upon elongation, thin and flexible structures, often similar to a curved hook, appeared at the end of microtubules. Electron microscopic observations supported the idea that these flexible structures are tubulin sheets. They maintained their length until they gradually became thick and rigid beginning in the central portion, resulting in straight microtubules. In the absence of Gas7, the sheet-like structure was rarely observed; moreover, when observed, it was fragile and engaged in typical dynamic instability. With Gas7, no catastrophe was observed. These results suggest that Gas7 enhances microtubule polymerization by stabilizing sheet intermediates and is a useful tool for analyzing microtubule transformation.

Onco-suppressor p53 protein prevents an Alzheimer disease mouse model, Pin1 -null mouse from the increase of presenilin-1

The onco-suppressor p53 protein is essential for checkpoint control in response to a variety of genotoxic stresses. The DNA damage leads to phosphorylation on Ser/Thr-Pro motifs of p53, which facilitates the interaction with Pinl, a pSer/pThr-Pro-specific peptidyl prolyl isomerase. Pinl is required for the timely accumulation and activation of p53 resulting in apoptosis or cell cycle arrest. Recently it has been indicated that Pin1-null (Pin1-/-) mouse is useful as an Alzheimer disease mouse model. To investigate the pathological relationship between Pinl and p53, we created Pin1-/-p53-/- mice. The thymocytes in 12-week-old Pin1-/-p53-/- mice had significantly higher levels of the intracellular form of Notchl (NIC) than the thymocytes of p53-/- or wild-type mice. Presenilin-1, a cleavage enzyme for NIC generation from full length Notchl was increased in the thymocytes of Pin1-/-p53-/- mice. Presenilin-1 is counted as the enzyme for amyloid β peptides generation in the brain. In the brain of 12-week-old Pin1-/-p53-/- mice, the levels of presenilin-1 were also more than Pin1-/- mice, and disruption of p53 increased presenilin-1 levels of Pin1-/- mice. From these results, it was suggested that p53 might down-regulate presenilin-1 expression and suppress the Aβ generation from Aβ peptide precursor in Pinl-/- mice.