Hiroshi Murai

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK‐3β and β‐catenin and promotes GSK‐3β‐dependent phosphorylation of β‐catenin

Glycogen synthase kinase‐3 (GSK‐3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. We have isolated here a GSK‐3β‐interacting protein from a rat brain cDNA library using a yeast two‐hybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and dishevelled (Dsh) homologous domains in its N‐ and C‐terminal regions, respectively. The predicted amino acid sequence of this GSK‐3β‐interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, we termed this protein rAxin (rat Axin). rAxin interacted directly with, and was phosphorylated by, GSK‐3β. rAxin also interacted directly with the armadillo repeats of β‐catenin. The binding site of rAxin for GSK‐3β was distinct from the β‐catenin‐binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted GSK‐3β‐dependent phosphorylation of β‐catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK‐3β and β‐catenin and mediating the signal from GSK‐3β to β‐catenin.

Suppression of plasminogen activator inhibitor-1 by RNA interference attenuates pulmonary fibrosis

Background and aim There is a growing body of evidence demonstrating that plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. In fact, PAI-1 knockout mice are protected from bleomycin-induced pulmonary fibrosis. This study was conducted to determine whether the intrapulmonary administration of small interfering RNA (siRNA) targeting PAI-1 (PAI-1-siRNA) limits the development of bleomycin-induced pulmonary fibrosis. Methods Lung biopsies from patients with idiopathic pulmonary fibrosis (IPF) were stained for PAI-1. The distribution of siRNA in the lung, the PAI-1 level in bronchoalveolar (BAL) fluid and the extent of fibrotic changes in the lung were evaluated following the intranasal administration of PAI-1-siRNA in a mouse model of bleomycin-induced pulmonary fibrosis. The effect of PAI-1-siRNA on the epithelial to mesenchymal transition (EMT) was also evaluated using a mouse lung epithelial cell line, LA-4. Results PAI-1 was overexpressed in the hyperplastic type 2 pneumocytes lining the honeycomb lesions of patients with IPF. The single intranasal instillation of PAI-1-siRNA resulted in the diffuse uptake of siRNA into the epithelial cells lining the dense fibrotic lesions. The repeated administration of PAI-1-siRNA initiated during either the inflammatory or the fibrotic phase into bleomycin-injured mice reduced the PAI-1 level in BAL fluid and limited the accumulation of collagen in the lungs. EMT induced by transforming growth factor β (TGFβ) in LA-4 cells was inhibited by transfection with PAI-1-siRNA. Conclusions The direct suppression of PAI-1 in the lung by the intrapulmonary administration of PAI-1-siRNA attenuated the development and progression of pulmonary fibrosis. The inhibition of EMT may be, at least in part, involved in this effect.

Tyrosine dephosphorylation of glycogen synthase kinase‐3 is involved in its extracellular signal‐dependent inactivation

We examined whether extracellular signals regulate glycogen synthase kinase‐3 (GSK‐3) activity through tyrosine dephosphorylation of GSK‐3. In resting Chinese hamster ovary cells overexpressing the human insulin receptor (CHO‐IR cells), GSK‐3 was tyrosine‐phosphorylated and active. Insulin and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) induced inactivation and tyrosine dephosphorylation of GSK‐3. It is known that Ser‐9 of GSK‐3β is phosphorylated in response to insulin and that the phosphorylation of this amino acid residue causes inactivation of GSK‐3β. However, the ectopically expressed GSK‐3βΔ9, in which the N‐terminal nine amino acids of GSK‐3β were deleted, was still inactivated and tyrosine‐dephosphorylated in response to insulin. Protein phosphatase 2A treatment partially reversed insulin‐induced GSK‐3β inactivation, but did not change GSK‐βΔ9 inactivation. In CHO‐IR cells where protein kinase C was down‐regulated, TPA neither inactivated nor tyrosine‐dephosphorylated GSK‐3. However, insulin inactivated and tyrosine‐dephosphorylated GSK‐3, although to a lesser degree than in the control cells. These results suggest that in addition to serine phosphorylation, tyrosine dephosphorylation of GSK‐3 is also important for the regulation of GSK‐3 activity in response to extracellular signals and that insulin regulates GSK‐3 activity through both protein kinase C‐dependent as well as protein kinase C‐independent pathways.

KL-6 concentration in pulmonary epithelial lining fluid is a useful prognostic indicator in patients with acute respiratory distress syndrome

BackgroundKL-6 is a mucin-like glycoprotein expressed on the surface of alveolar type II cells. Elevated concentrations of KL-6 in serum and epithelial lining fluid (ELF) in patients with acute respiratory distress syndrome (ARDS) have been previously reported; however, kinetics and prognostic significance of KL-6 have not been extensively studied. This study was conducted to clarify these points in ARDS patients.MethodsThirty-two patients with ARDS who received mechanical ventilation under intubation were studied for 28 days. ELF and blood were obtained from each patient at multiple time points after the diagnosis of ARDS. ELF was collected using a bronchoscopic microsampling procedure, and ELF and serum KL-6 concentrations were measured.ResultsKL-6 levels in ELF on days 0 to 3 after ARDS diagnosis were significantly higher in nonsurvivors than in survivors, and thereafter, there was no difference in concentrations between the two groups. Serum KL-6 levels did not show statistically significant differences between nonsurvivors and survivors at any time point. When the highest KL-6 levels in ELF and serum sample from each patient were examined, KL-6 levels in both ELF and serum were significantly higher in nonsurvivors than in survivors. The optimal cut-off values were set at 3453 U/mL for ELF and 530 U/mL for serum by receiver operating characteristic (ROC) curve analyses. Patients with KL-6 concentrations in ELF higher than 3453 U/mL or serum concentrations higher than 530 U/mL had significantly lower survival rates up to 90 days after ARDS diagnosis.ConclusionsELF and serum KL-6 concentrations were found to be good indicators of clinical outcome in ARDS patients. Particularly, KL-6 levels in ELF measured during the early period after the diagnosis were useful for predicting prognosis in ARDS patients.

Intra-airway administration of small interfering RNA targeting plasminogen activator inhibitor-1 attenuates allergic asthma in mice.

Recent studies suggest that plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of the fibrinolytic system, may promote the development of asthma. To further investigate the significance of PAI-1 in the pathogenesis of asthma and determine the possibility that PAI-1 could be a therapeutic target for asthma, this study was conducted. First, PAI-1 levels in induced sputum (IS) from asthmatic subjects and healthy controls were measured. In asthmatic subjects, IS PAI-1 levels were elevated, compared with that of healthy controls, and were significantly higher in patients with long-duration asthma compared with short-duration asthma. PAI-1 levels were also found to correlate with IS transforming growth factor-β levels. Then, acute and chronic asthma models induced by ovalbumin were established in PAI-1-deficient mice and wild-type mice that received intra-airway administrations of small interfering RNA against PAI-1 (PAI-1-siRNA). We could demonstrate that eosinophilic airway inflammation and airway hyperresponsiveness were reduced in an acute asthma model, and airway remodeling was suppressed in a chronic asthma model in both PAI-1-deficient mice and wild-type mice that received intra-airway administration of PAI-1-siRNA. These results indicate that PAI-1 is strongly involved in the pathogenesis of asthma, and intra-airway administration of PAI-1-siRNA may be able to become a new therapeutic approach for asthma.

SK-216, an Inhibitor of Plasminogen Activator Inhibitor-1, Limits Tumor Progression and Angiogenesis

Plasminogen activator inhibitor-1 (PAI-1), which can be produced by host and tumor cells in the tumor microenvironment, is intimately involved in tumor progression. In the present study, to pursue the possibility that PAI-1 could be a therapeutic target in the management of malignancy, SK-216, a specific PAI-1 inhibitor, was orally administered to wild-type mice that were subcutaneously implanted or intravenously injected with either PAI-1–secreting Lewis lung carcinoma (LLC) or PAI-1–nonsecreting B16 melanoma cells. The systemic administration of SK-216 was found to reduce the size of subcutaneous tumors and the extent of metastases, regardless of PAI-1 secretion levels from the tumor cells. SK-216 also reduced the extent of angiogenesis in the tumors and inhibited VEGF-induced migration and tube formation by human umbilical vein endothelial cells in vitro. Then, to determine whether host or tumor PAI-1 was more crucial in tumor progression and angiogenesis, PAI-1–deficient or wild-type mice were subcutaneously implanted or intravenously injected with LLC or PAI-1 knockdown LLC cells. Tumor progression was shown to be controlled by the presence of host PAI-1 and not affected by the PAI-1 levels in the tumors. Similarly, host PAI-1 played a more crucial role in tumor angiogenesis than did tumor PAI-1. These observations suggest that regardless of the PAI-1 levels in the tumor, the systemic administration of SK-216 exerts an antitumor effect through its interaction with host PAI-1. This antitumor effect might be mediated by the antiangiogenic properties of SK-216. Mol Cancer Ther; 12(11); 2378–88. ©2013 AACR.

Effect of increasing respiratory rate on airway resistance and reactance in COPD patients

Airway resistance and reactance measured by forced oscillometry have been used to measure the severity of airway obstruction in chronic obstructive pulmonary disease (COPD) patients. The aims of this study were to assess the effects of tachypnoea on airway resistance and reactance and to correlate these with the severity of dyspnoea. We also evaluated the effects of short‐acting β2‐agonist (SABA) on these measurements.

Whole-blood incubation method to study neutrophil cytoskeletal dynamics.

To reduce artifactual effects in the study of filamentous (F)-actin dynamics in neutrophils, we have developed a whole-blood incubation method. Neutrophils in whole blood contained significantly less basal F-actin than did separated neutrophils. Although the peak relative F-actin content of neutrophils in whole blood after formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation was significantly higher than that of separated neutrophils at 10(-9) to 10(-6) M fMLP concentrations (p < 0.05), there was no significant difference in increase in mean fluorescence intensity and the EC50 (concentration of stimulant giving a half-maximum response). On the other hand, the EC50 of platelet-activating factor (PAF) between separated neutrophils and whole-blood-incubated neutrophils differed significantly (1.6 +/- 1.1 x 10(-9) M in separated neutrophils and 2.0 +/- 0.7 x 10(-8) M in whole-blood-incubated neutrophils, p < 0.05). The whole-blood incubation method described presently reduces the sample volume, cost and time needed to separate neutrophils, prevents neutrophil activation during separation, and reserves all blood components that may affect neutrophil function. For these reasons, the conditions adopted in the present method are thought to simulate well neutrophils circulating in vivo and the method would be preferable to other neutrophil function tests performed to study actin dynamics.