Hitoshi Murata

TIRAP, an Adaptor Protein for TLR2/4, Transduces a Signal from RAGE Phosphorylated upon Ligand Binding

The receptor for advanced glycation end products (RAGE) is thought to be involved in the pathogenesis of a broad range of inflammatory, degenerative and hyperproliferative diseases. It binds to diverse ligands and activates multiple intracellular signaling pathways. Despite these pivotal functions, molecular events just downstream of ligand-activated RAGE have been surprisingly unknown. Here we show that the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCζ upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules. Blocking of the function of TIRAP and MyD88 largely abrogated intracellular signaling from ligand-activated RAGE. Our findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions.

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.

Overexpression of REIC/Dkk-3 in Normal Fibroblasts Suppresses Tumor Growth via Induction of Interleukin-7

We previously showed that the tumor suppressor gene REIC/Dkk-3, when overexpressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on human cancers through a mechanism triggered by endoplasmic reticulum stress. Adenovirus vectors show no target cell specificity and thus may elicit unfavorable side effects through infection of normal cells even upon intra-tumoral injection. In this study, we examined possible effects of Ad-REIC on normal cells. We found that infection of normal human fibroblasts (NHF) did not cause apoptosis but induced production of interleukin (IL)-7. The induction was triggered by endoplasmic reticulum stress and mediated through IRE1α, ASK1, p38, and IRF-1. When Ad-REIC-infected NHF were transplanted in a mixture with untreated human prostate cancer cells, the growth of the cancer cells was significantly suppressed. Injection of an IL-7 antibody partially abrogated the suppressive effect of Ad-REIC-infected NHF. These results indicate that Ad-REIC has another arm against human cancer, an indirect host-mediated effect because of overproduction of IL-7 by mis-targeted NHF, in addition to its direct effect on cancer cells.

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 A2 (cPLA2), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca2+-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA2, resulting in inhibition of cPLA2 activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp12, 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.

Exploiting protein cationization techniques in future drug development

The development of a method for the efficient intracellular delivery of inherently non-permeable proteins is needed for manipulation of cellular phenotypes or the discovery of protein-based drugs. It has been demonstrated that proteins artificially cationized by chemical conjugation show efficient intracellular delivery via adsorptive-mediated endocytosis and then can exert their biological activity in cells. Studies have also revealed that cationic peptides known as cell-penetrating peptides (CPPs) provide a means to deliver molecules into mammalian cells. Although the internalization mechanisms remain controversial, it is now becoming clear that the main port of entry into cells by CPPs also involves adsorptive-mediated endocytosis rather than the direct penetration of the plasma membrane. As the mammalian cell membrane possesses an abundance of negatively charged glycoproteins and glycosphingolipids, cationization of proteins is a reasonable choice to endow them with the ability for intracellular delivery. Cationization of proteins is usually accompanied by drastic changes in protein properties, structure and biological activities. Recently developed sophisticated protein chemistry can minimize these side effects. Therefore, protein cationization techniques will hopefully prove to be powerful tools for innovative research and drug discovery. In this review, techniques for cationization of proteins and their intracellular delivery, as well as some of their potential therapeutic applications, are discussed.

SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria

Dysfunction of PINK1 causes familial Parkinsons disease. Recent work suggested that accumulation of PINK1 on damaged mitochondria is a critical step for mitophagy. It was not clear, however, how PINK1 is stabilized. PINK1 forms a complex with SARM1 and TRAF6, which is important for stabilization of PINK1 and induction of mitophagy.

NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions

Mutations of the PTEN-induced putative kinase 1 (PINK1) gene are a cause of autosomal recessive forms of Parkinson’s disease. Recent studies have revealed that PINK1 is an essential factor for controlling mitochondrial quality, and that it protects cells from oxidative stresses. Although there has been considerable progress in the elucidation of various aspects of PINK1 protein regulation such as activation, stability and degradation, the transcriptional regulation of PINK1 mRNA under stress conditions remains unclear. In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions. Damaged mitochondria arising from stress conditions induced NRF2-dependent transcription of the PINK1 gene through production of reactive oxygen species (ROS). Either an ROS scavenger or forced expression of KEAP1, a potent inhibitory partner to NRF2, restricted PINK1 expression induced by activated NRF2. Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death. The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates.

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

Intraperitoneal administration of an adenovirus vector carrying REIC/Dkk-3 suppresses peritoneal dissemination of scirrhous gastric carcinoma

Expression levels of the novel tumor suppressor gene REIC/Dkk-3 are reduced in many human cancers. We have previously showed that an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) induced apoptosis of cancer cells selectively and exerted bystander antitumor effects via ER stress. We examined possible effects of Ad-REIC in a peritoneal dissemination model of scirrhous gastric carcinoma (SGC). Among various types of gastric cancer, SGC continues to be associated with the worst prognosis due to a high incidence of metastases in the peritoneal cavity. We found that a single intraperitoneal injection of Ad-REIC suppressed tumor dissemination and disease progression. Immunomodulation by Ad-REIC led to recruitment of natural killer cells inside tumor nodules. We conclude that Ad-REIC gene therapy may be a potential tool in combinatorial approaches to achieve curative effects in SGC.