A new cytosolic pathway from a Parkinson disease-associated kinase, BRPK/PINK1: activation of AKT via mTORC2.

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

Activity diary method for predicting energy expenditure as evaluated by a whole-body indirect human calorimeter.

In comparison with the energy expenditure determined by a whole-body indirect human calorimeter, which provides 24-h energy expenditure (TEE) with high precision and accuracy, the accuracy of predicting energy expenditure (EE) using an activity diary (AD) method was evaluated. Observed and predicted basal metabolic rate (BMR) as well as literature values for typical physical activities were used for TEE prediction. The effect of the number of recorded items in the activity diary on the accuracy of TEE was also examined. Additionally, predicted EE was divided into sleeping, exercise, and sedentary EE to evaluate the estimation errors in the AD method. Subjects were 20- to 69-y-old Japanese women (n = 20) and men (n= 21). Predicted TEE based on the AD was derived by applying the observed or predicted BMR to literature values for physical activities; i.e., relative metabolic rate (R.M.R.), physical activity ratio (PAR), and metabolic equivalent (MET). The BMR value observed for each subject was obtained by indirect calorimetry using a Douglas bag. The BMR for the subject was also estimated from the predictive equations in the 6th revision of the Recommended Dietary Allowances for the Japanese (1999). The correlations between observed and predicted TEE appeared stronger when using observed BMR than those using predicted BMR. Although the difference of mean values between the predicted and observed TEE was small, the limits of agreement between the predicted and observed TEE were around +/- 400 kcal. Predicted EE, excluding the time periods for exercise and rest laying down when determining BMR, showed similar results to those of TEE. Furthermore, the number of recorded items in the AD was not significantly correlated to the accuracy of the predicted TEE (r = -0.03). These findings indicate that the predicted TEE of the AD using observed or predicted BMR and literature values is favorably comparable to observed TEE using a whole-body human calorimeter on a group basis; however, its use as a proxy measure of TEE or EE on an individual basis may be limited.

Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization.

The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid ß. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling.

S100A11, an dual mediator for growth regulation of human keratinocytes.

We previously revealed a novel signal pathway involving S100A11 for inhibition of the growth of normal human keratinocytes (NHK) caused by high Ca(++) or transforming growth factor beta. Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p21(WAF1). In contrast, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11, and we provide herein evidence that 1) S100A11 is actively secreted by NHK; 2) extracellular S100A11 acts on NHK to enhance the production of epidermal growth factor family proteins, resulting in growth stimulation; 3) receptor for advanced glycation end products, nuclear factor-kappaB, Akt, and cAMP response element-binding protein are involved in the S100A11-triggered signal transduction; and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays a dual role in growth regulation of epithelial cells.

Evaluation of low-intensity physical activity by triaxial accelerometry.

OBJECTIVE: To develop regression-based equations that estimate physical activity ratios [energy expenditure (EE) per minute/sleeping metabolic rate] for low-to-moderate intensity activities using total acceleration obtained by triaxial accelerometry. RESEARCH METHODS AND PROCEDURES: Twenty-one Japanese adults were fitted with a triaxial accelerometer while also in a whole-body human calorimeter for 22.5 hours. The protocol time was composed of sleep (8 hours), four structured activity periods totaling 4 hours (sitting, standing, housework, and walking on a treadmill at speeds of 71 and 95 m/min, 2 x 30 minutes for each activity), and residual time (10.5 hours). Acceleration data (milligausse) from the different periods and their relationship to physical activity ratio obtained from the human calorimeter allowed for the development of EE equations for each activity. The EE equations were validated on the residual times, and the percentage difference for the prediction errors was calculated as (predicted value - measured value)/measured value x 100. RESULTS: Using data from triaxial accelerations and the ratio of horizontal to vertical accelerations, there was relatively high accuracy in identifying the four different periods of activity. The predicted EE (882 +/- 150 kcal/10.5 hours) was strongly correlated with the actual EE measured by human calorimetry (846 +/- 146 kcal/10.5 hours, r = 0.94 p < 0.01), although the predicted EE was slightly higher than the measured EE. DISCUSSION: Triaxial accelerometry, when total, vertical, and horizontal accelerations are utilized, can effectively evaluate different types of activities and estimate EE for low-intensity physical activities associated with modern lifestyles.

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.