DNAX-activating protein 10 (DAP10) membrane adaptor associates with receptor for advanced glycation end products (RAGE) and modulates the RAGE-triggered signaling pathway in human keratinocytes.

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

Design and synthesis of a series of α-benzyl phenylpropanoic acid-type peroxisome proliferator-activated receptor (PPAR) gamma partial agonists with improved aqueous solubility.

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.

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.

Preclinical safety and efficacy of in situ REIC/Dkk-3 gene therapy for prostate cancer.

The preclinical safety and therapeutic efficacy of adenoviral vectors that express the REIC/Dkk-3 tumor suppressor gene (Ad-REIC) was examined for use in prostate cancer gene therapy. The Ad-human (h) and mouse (m) REIC were previously demonstrated to induce strong anti-cancer effects in vitro and in vivo, and we herein report the results of two in vivo studies. First, intra-tumor Ad-hREIC administration was examined for toxicity and therapeutic effects in a subcutaneous tumor model using the PC3 prostate cancer cell line. Second, intra-prostatic Ad-mREIC administration was tested for toxicity in normal mice. The whole-body and spleen weights, hematological and serum chemistry parameters, and histological evaluation of tissues from throughout the body were analyzed. Both experiments indicated that there was no significant difference in the examined parameters between the Ad-REIC-treated group and the control (PBS- or Ad-LacZ-treated) group. In the in vitro analysis using PC3 cells, a significant apoptotic effect was observed after Ad-hREIC treatment. Confirming this observation, the robust anti-tumor efficacy of Ad-hREIC was demonstrated in the in vivo subcutaneous prostate cancer model. Based on the results of these preclinical experiments, we consider the adenovirus-mediated REIC/Dkk-3 in situ gene therapy to be safe and useful for the clinical treatment of prostate cancer.

Tumor suppressor REIC/Dkk-3 interacts with the dynein light chain, Tctex-1.

REIC/Dkk-3 is a member of the Dickkopf family proteins known as Wnt-antagonists, and REIC/Dkk-3 expression is downregulated in a broad range of cancer types. REIC/Dkk-3 acts as a tumor suppressor in multiple cancer cell lines by inducing apoptosis through endoplasmic reticulum (ER) stress signaling. However, the intracellular interaction partners of REIC/Dkk-3 have not been fully elucidated. By employing yeast two-hybrid screening, we identified the human dynein light chain, Tctex-1, as a novel interaction partner of REIC/Dkk-3. We further disclosed that the interaction involves the 136-157 amino acid region of REIC/Dkk-3 by using the mammalian two-hybrid system. Interestingly, this binding region of REIC/Dkk-3 with Tctex-1 contains an amino acid sequence motif [-E-X-G-R-R-X-H-] which was previously reported as the Tctex-1 binding domain of dynein intermediate chain (DIC). Immunocytochemistry demonstrated that both REIC/Dkk-3 and Tctex-1 were localized around the ER of human fibroblasts, and the similar distribution pattern of the proteins suggests that their interaction occurs around the ER. This is the first study showing the interaction of a Dickkopf family protein with a dynein motor complex protein. The link between REIC/Dkk-3 and Tctex-1 may be of significance for understanding the molecular functions of the proteins in ER stress signaling and intracellular dynein motor dynamics, respectively.

Expression of REIC/Dkk-3 in normal and hyperproliferative epidermis.

Dickkopf (Dkk) family members are known as Wnt modulators involved in the development, cell growth/differentiation and cancer. REIC/Dkk-3, which does not interfere with Wnt signalling, has been proposed to be a tumor suppressor gene, but its physiological function has remained unclear. In this study, we analysed the expression of REIC/Dkk-3 in normal interfollicular epidermis (IFE) and hyperproliferative epidermis. REIC/Dkk-3 was expressed in human and mouse IFE, being localized at the interface of upper spinous layer and granular layer. Skin cancer cell lines lost REIC/Dkk-3 expression as reported previously. When we analysed patient samples, REIC/Dkk-3 expression was down-regulated in the hyperproliferative epidermis including skin cancers and non-cancerous proliferative diseases. REIC/Dkk-3 expression was also suppressed in the regenerative and inflammative epidermis of model mice. These findings will certainly contribute to the extension of studies on REIC/Dkk-3.

S100A11, a dual growth regulator of epidermal keratinocytes.

S100A11, a member of the family of S100 proteins, is a dimmer, each monomer of which has two EF-hands. Expression of S100A11 is ubiquitous in various tissues at different levels, with a high expression level in the skin. We have analyzed functions of S100A11 mainly in normal human keratinocytes (NHK) as a model cell system of human epithelial cells. High Ca(2+) and transforming growth factor-ß (TGF-ß), two representative growth suppressors for NHK, need a common S100A11-mediated pathway in addition to unique pathways (NFAT1-mediated pathway for high Ca(2+) and Smad-mediated pathway for TGF-ß) for exhibiting a growth inhibitory effect. S100A11 has another action point for growth suppression in NHK. Annexin A1 (ANXA1) complexed with S100A11 efficiently binds to and inhibits cytosolic phospholipase A2 (cPLA2), the activity of which is needed for the growth of NHK. On exposure of NHK to epidermal growth factor (EGF), ANXA1 is cleaved at 12Trp, and this truncated ANXA1 loses binding capacity to S100A11, resulting in maintenance of an active state of cPLA2. On the other hand, we found that S100A11 is actively secreted by NHK. Extracellular S100A11 acts on NHK to enhance the production of EGF family proteins, resulting in growth stimulation. These findings indicate that S100A11 plays a dual role in growth regulation, being suppressive in cells and being promotive from outside of cells.

A novel tumor suppressor, REIC/Dkk-3 gene identified by our in vitro transformation model of normal human fibroblasts works as a potent therapeutic anti-tumor agent.

Reduced Expression in Immortalized Cell (REIC) was cloned by subtractive hybridization method as a gene whose expression is reduced in many human immortalized and neoplastic tumor cells. The REIC, when over-expressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on a wide variety of human cancers through a mechanism triggered by ER-stress-mediated JNK activation. In addition to this direct effect on cancer cells, Ad-REIC exerted another cytotoxicity on human cancers, an indirect host-mediated effect due to overproduction of IL-7 by mis-targeted normal cells. This "one-bullet two-arms" finding may lead to a powerful new therapeutic approach to the treatment of human cancers.

Down-regulation of BiP/GRP78 sensitizes resistant prostate cancer cells to gene-therapeutic overexpression of REIC/Dkk-3.

We have recently shown that an adenovirus carrying REIC/Dkk-3 (Ad-REIC) exhibits a potent tumor-specific cell-killing function for various human cancers. It has also become evident that some human cancers are resistant to Ad-REIC-induced apoptosis. The aim of the present study was to determine the molecular mechanisms of resistance to Ad-REIC. First, we isolated resistant clones from a human prostate cancer cell line, PC3, after repeated exposure to Ad-REIC. Infection efficiency of the adenovirus vector and expression level of REIC/Dkk-3 in the resistant clones were similar to those in the parental PC3 cells. By screening for alteration in levels and functional status of proteins involved in Ad-REIC-induced apoptosis, we found that BiP/GRP78, an ER-residing chaperone protein, was expressed at higher levels consistently among resistant cells. Expression levels of BiP and rates of apoptosis induced by Ad-REIC were inversely correlated. Down-regulation of BiP with siRNA sensitized the resistant cells to Ad-REIC in vivo as well as in culture. These results indicate that BiP is a major determinant of resistance to Ad-REIC-induced apoptosis. Thus BiP is useful for diagnosis of inherent and acquired resistance of cancers and also as a target molecule to overcome resistance to the gene therapeutic Ad-REIC.

Transcriptional regulation of a brown adipocyte-specific gene, UCP1, by KLF11 and KLF15.

Several growth factors and transcription factors have been reported to play important roles in brown adipocyte differentiation and modulation of thermogenic gene expression, especially the expression of UCP1. In this study, we focused on KLF11 and KLF15, which were expressed highly in brown adipose tissue. Our data demonstrated that KLF11 and KLF15 interacted directly with the UCP1 promoter using GC-box and GT-boxes, respectively. Co-transfection of KLF11 and KLF15 in the mesenchymal stem cell line muBM3.1 during brown adipocyte differentiation enhanced the expression level of UCP1. KLF11, but not KLF15, was essential for UCP1 expression during brown adipocyte differentiation of muBM3.1.

Immunological aspects of REIC/Dkk-3 in monocyte differentiation and tumor regression.

The REIC/Dkk-3 gene has been reported to be a tumor suppressor and the expression is significantly down-regulated in a broad range of cancer cell types. The protein is secretory, but the physiological function remains unclear. This study demonstrated that recombinant REIC/Dkk-3 protein induced the differentiation of human CD14+ monocytes into a novel cell type (REIC/Dkk-3Mo). REIC/Dkk-3Mo resembles immature dendritic cells generated with IL-4 and GM-CSF. Both these cell populations exhibit similar proportions of CD11c+, CD40+, CD86+ and HLA-DR+ cells and endocytic capacity, but REIC/Dkk-3Mo is negative for CD1a antigen. An analysis of the signal transducers and activators of transcription (STAT) pathways revealed that REIC/Dkk-3 induces phosphorylation of STAT 1 and STAT 3. Furthermore, intratumoral administration of REIC/Dkk-3 protein significantly suppressed tumor growth with CD11c+ and CD8+ (dendritic and killer T cell marker, respectively) cell accumulation and enhanced anti-cancer cytolytic activity of splenocytes. These data indicated a cytokine-like role of REIC/Dkk-3 protein in monocyte differentiation that might be exploited therapeutically.

S100C/A11 is a key mediator of Ca(2+)-induced growth inhibition of human epidermal keratinocytes.

An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at 10Thr and 94Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21CIP1/WAF1, a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21CIP1/WAF1. In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca(2+)-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

PKCalpha mediates TGFbeta-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11.

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825-835). This paper addresses a question whether transforming growth factor beta (TGFbeta) shares the pathway with high Ca2+. On exposure of the cells to TGFbeta1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C alpha (PKCalpha) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFbeta1-induced growth inhibition was almost completely mitigated when PKCalpha activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCalpha-S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFbeta1.

Suppression of carbon tetrachloride-induced liver fibrosis by transplantation of a clonal mesenchymal stem cell line derived from rat bone marrow.

Transplantation of hepatocytes or bone marrow-derived cells has been shown to ameliorate liver fibrosis in animal models, but no direct comparison of relative efficiency has been made. The aim of this study was to compare the efficiency of a bone marrow-derived clonal mesenchymal stem cell line established by us (rBM25/S3) with that of its adipogenic or hepatogenic differentiation derivative for suppression of rat liver fibrosis. After induction of differentiation of rBM25/S3 cells into adipogenic or hepatogenic cells in culture, we intrasplenically transplanted the three types of cells into rats (3 x 10(7) cells/rat) before and 4 weeks after initiation of carbon tetrachloride treatment (1 ml/kg body weight twice a week for 8 weeks) to induce liver fibrosis. Undifferentiated rBM25/S3 cells were the most effective for suppression of liver fibrosis, followed by the adipogenic cells and hepatogenic cells. Expression levels of MMP-2 and MMP-9 were also highest in undifferentiated rBM25/S3 cells. These results indicate that bone marrow-derived clonal mesenchymal stem cell lines are useful for further mechanistic studies on cell-mediated suppression of liver fibrosis and that such cell lines will provide information on an appropriate cell source for transplantation therapy for cirrhosis.

S100A8/A9, a key mediator for positive feedback growth stimulation of normal human keratinocytes.

S100A8 and S100A9 are known to be up-regulated in hyperproliferative and psoriatic epidermis, but their function in epidermal keratinocytes remains largely unknown. Here we show that (1) S100A8 and S100A9 are secreted by cultured normal human keratinocytes (NHK) in a cytokine-dependent manner, (2) when applied to NHK, recombinant S100A8/A9 (a 1:1 mixture of S100A8 and S100A9) induced expression of a number of cytokine genes such as IL-8/CXCL8, CXCL1, CXCL2, CXCL3, CCL20, IL-6, and TNFalpha that are known to be up-regulated in psoriatic epidermis, (3) the S100A8/A9-induced cytokines in turn enhanced production and secretion of S100A8 and S100A9 by NHK, and (4) S100A8 and S100A8/A9 stimulated the growth of NHK at a concentration as low as 1 ng/ml. These results indicate the presence of a positive feedback loop for growth stimulation involving S100A8/A9 and cytokines in human epidermal keratinocytes, implicating the relevance of the positive feedback loop to the etiology of hyperproliferative skin diseases, including psoriasis.

An N-terminal 78 amino acid truncation of REIC/Dkk-3 effectively induces apoptosis.

Overexpression of REIC/Dkk-3 (a tumor suppressor gene) induces cancer cell apoptosis through endoplasmic reticulum (ER) stress. Therefore, the identification of the portion of REIC/Dkk-3 that causes ER stress may be essential for the development of cancer treatment based on REIC/Dkk-3. Here, we made several truncated forms of REIC/Dkk-3 and investigated their therapeutic potentials against prostate cancer. Among three truncated forms, a variant comprising the N-terminal 78 amino acid region of REIC/Dkk-3 ((1-78)REIC/Dkk-3) most strongly induced ER stress and apoptosis in human prostate cancer cells (PC3). For in vivo gene expression, we coupled a biodegradable polymer with naked DNA, which attained robust trans-gene expression in PC3-derived subcutaneous tumor. In therapeutic experiments, we demonstrated that multiple direct injections of polymer-conjugated (1-78)REIC/Dkk-3 plasmid provoke ER stress and significantly reduced the subcutaneous tumor volume compared with the control group. We suggest this non-viral strategy may be an effective alternative to viral gene therapy.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure.

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

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.

Reduction of cerebral infarction in rats by biliverdin associated with amelioration of oxidative stress.

Biliverdin (BV), one of the byproducts of heme catalysis through heme oxygenase (HO) system, is a scavenger of reactive oxygen species (ROS). We hypothesized that BV treatment could protect rat brain cells from oxidative injuries via its anti-oxidant efficacies. Cerebral infarction was induced by transient middle cerebral artery occlusion (tMCAO) for 90 min, followed by reperfusion. BV or vehicle was administered intraperitoneally immediately after reperfusion. The size of the cerebral infarction 2 days after tMCAO was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) stain. Superoxide generation 4 h after tMCAO was determined by detection of oxidized hydroethidine. In addition, the oxidative impairment of neurons were immunohistochemically assessed by stain for lipid peroxidation with 4-hydroxy-2-nonenal (4-HNE) and damaged DNA with 8-hydroxy-2'-deoxyguanosine (8-OHdG). BV treatment significantly reduced infarct volume of the cerebral cortices associated with less superoxide production and decreased oxidative injuries of brain cells. The present study demonstrated that treatment with BV ameliorated the oxidative injuries on neurons and decreased brain infarct size in rat tMCAO model.

Derivation of hepato-pancreatic intermediate progenitor cells from a clonal mesenchymal stem cell line of rat bone marrow origin.

We have recently established a clonal mesenchymal stem cell line (rBM25/S3) from adult rat bone marrow. The cells have practically unlimited proliferation capacity (over 300 PDL), maintaining multipotency for differentiation. In the present study, we examined the potential for rBM25/S3 cells to differentiate into insulin-secreting cells. When cultured in the presence of HGF and FGF-4 on Matrigel, rBM25/S3 cells expressed genes specific to pancreatic beta-cells as well as those specific to hepatocytes. They still maintained proliferation capacity with a doubling time of approximately 30 h. These hepato-pancreatic intermediate progenitor cells, but not the original undifferentiated rBM25/S3 cells, were induced by the overexpression of PDX-1 to produce significant amounts of insulin in a manner responding to glucose concentration in medium. The present culture system indicates a direction for further studies aimed at the realization of cell transplantation therapy for type I diabetes mellitus.