(Pro)renin receptor accelerates development of sarcopenia via activation of Wnt/YAP signaling axis

To extend life expectancy and ensure healthy aging, it is crucial to prevent and minimize age‐induced skeletal muscle atrophy, also known as sarcopenia. However, the disease's molecular mechanism remains unclear. The age‐related Wnt/β‐catenin signaling pathway has been recently shown to be activated by the (pro)renin receptor ((P)RR). We report here that (P)RR expression was increased in the atrophied skeletal muscles of aged mice and humans. Therefore, we developed a gain‐of‐function model of age‐related sarcopenia via transgenic expression of (P)RR under control of the CAG promoter. Consistent with our hypothesis, (P)RR‐Tg mice died early and exhibited muscle atrophy with histological features of sarcopenia. Moreover, Wnt/β‐catenin signaling was activated and the regenerative capacity of muscle progenitor cells after cardiotoxin injury was impaired due to cell fusion failure in (P)RR‐Tg mice. In vitro forced expression of (P)RR protein in C2C12 myoblast cells suppressed myotube formation by activating Wnt/β‐catenin signaling. Administration of Dickkopf‐related protein 1, an inhibitor of Wnt/β‐catenin signaling, and anti‐(P)RR neutralizing antibody, which inhibits binding of (P)RR to the Wnt receptor, significantly improved sarcopenia in (P)RR‐Tg mice. Furthermore, the use of anti‐(P)RR neutralizing antibodies significantly improved the regenerative ability of skeletal muscle in aged mice. Finally, we show that Yes‐associated protein (YAP) signaling, which is coordinately regulated by Wnt/β‐catenin, contributed to the development of (P)RR‐induced sarcopenia. The present study demonstrates the use of (P)RR‐Tg mice as a novel sarcopenia model, and shows that (P)RR‐Wnt‐YAP signaling plays a pivotal role in the pathogenesis of this disease.     

Truncation of annexin A1 is a regulatory lever for linking epidermal growth factor signaling with cytosolic phospholipase A2 in normal and malignant squamous epithelial cells

Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A(2) (cPLA(2)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca(2+)-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA(2), resulting in inhibition of cPLA(2) activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp(12), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.     

Quantitative proteomic analysis of rat liver for carcinogenicity prediction in a 28-day repeated dose study

The potential of quantitative proteomic analysis to predict carcinogenicity of chemical compounds was investigated. Using 2D-DIGE, we analyzed the effects of 63 chemical compounds on protein expression in the rat liver after 28 daily doses. Types of carcinogens were categorized depending on the species and organ specificity. The carcinogen characteristic proteins for each classification were identified by Welch's t value. For evaluation of the predictive concordance we used support vector machines. The rat hepatic carcinogen-specific classification gave higher concordance than the other classification. The generalization performance was measured by leave-one-out cross-validation. For genotoxic and non-genotoxic compounds, a concordance of 79.3 and 76.5%, respectively, was obtained by the top 30 ranked proteins with Welch's t value. Furthermore, we found that the increase of the expression level of the stress response proteins as the common feature of poorly predicted chemical compounds in the leave-20%-out cross-validation. Quantitative proteomics could be promising technique for developing biomarker panels that can be used for carcinogenicity prediction. The list of proteins identified in this study and the zoomed gel images of the top ranked proteins in statistic analysis are provided in Supplementary Data.     

Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2–PTEN–AKT Pathway and Placental Cell Proliferation

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2–PTEN–AKT pathway and placental cell proliferation.     

Cell swelling activates stress-activated protein kinases, p38 MAP kinase and JNK, in renal epithelial A6 cells

Osmotic shock is well recognized as one of the factors activating stress-activated protein kinases (SAPKs), p38 MAP kinase and c-Jun N-terminal kinases (JNKs). In renal epithelial A6 cells, hypo-osmotic shock transiently activated SAPKs with maximal activation at 5 min. A6 cells showed a regulatory volume decrease (RVD) after swelling when the cells were exposed to a hypo-osmotic solution. In contrast, activation of SAPKs was maintained over 90 min after hypo-osmotic shock in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl(-) channel blocker), which completely blocked the RVD and kept the cells continuously swelling. Exposure of the cells to a high K(+) iso-osmotic solution containing nystatin, which induces continuous cell swelling, also continuously activated SAPKs. Furthermore, membrane deformation induced by chlorpromazine activated SAPKs. These results suggest that changes in membrane tension by cell swelling or chlorpromazine, but not osmolality, are important steps for activation of SAPKs in A6 cells.     

Brap2 functions as a cytoplasmic retention protein for p21 during monocyte differentiation

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.     

Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells

The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.     

Application of short-form oral health impact profile on elderly Japanese

Objectives: The purpose of this study was to use the oral health impact profile (OHIP‐14) to evaluate the impact of oral disease on the quality of life of a group of independently‐living elderly persons in an urban area of Japan. Subjects: A total of 1244 participants of the Senior Citizen's College, who attended the lectures once a week. They were community‐dwelling, independently‐living people over 60 years of age. Measurements: Japanese version of the short‐form OHIP‐14. Results: Internal reliability for the 14 items overall was very high (Cronbach's α = 0.95). Report of ‘painful aching’ and ‘uncomfortable to eat’ were the two most highly scored items using the mean sum OHIP‐14 score. A multiple logistic regression analysis indicated that the sum OHIP‐14 score had significant associations with self‐assessment of general health, dental status, and a perceived need for dental treatment. However, age, gender, dissatisfaction with financial status or education level was not significantly associated with the sum OHIP‐14. Compared with that of other countries, the items were ranked similarly, whereas the perceived magnitudes of the problems were quite different from other population. Conclusions: The OHIP‐14 in Japanese had a high internal reliability, was significantly associated with dental status and comparable ranking for items when compared with studies from other countries.