Satoshi Waguri

Phosphorylation of p62 Activates the Keap1-Nrf2 Pathway during Selective Autophagy

The Keap1-Nrf2 system and autophagy are both involved in the oxidative-stress response, metabolic pathways, and innate immunity, and dysregulation of these processes is associated with pathogenic processes. However, the interplay between these two pathways remains largely unknown. Here, we show that phosphorylation of the autophagy-adaptor protein p62 markedly increases p62s binding affinity for Keap1, an adaptor of the Cul3-ubiquitin E3 ligasexa0complex responsible for degrading Nrf2. Thus, p62 phosphorylation induces expression of cytoprotective Nrf2 targets. p62 is assembled on selective autophagic cargos such as ubiquitinated organelles and subsequently phosphorylated in an mTORC1-dependent manner, implying coupling of the Keap1-Nrf2 system to autophagy. Furthermore, persistent activation of Nrf2 through accumulation of phosphorylated p62 contributes to the growth of human hepatocellular carcinomas (HCCs). These results demonstrate that selective autophagy and the Keap1-Nrf2 pathway are interdependent, and that inhibitors of the interaction between phosphorylated p62 and Keap1 have potential as therapeutic agents against human HCC.

Primary sensory neuronal expression of SLURP-1, an endogenous nicotinic acetylcholine receptor ligand.

Secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently identified, endogenous ligand of the alpha7 subunit of nicotinic acetylcholine receptors. SLURP-1 is also the causative gene for an autosomal recessive palmoplantar keratoderma, Mal de Meleda. Although the function of SLURP-1 in keratinocyte development and differentiation has been extensively studied, little is known about its role in the nervous system. In the present study, we analyzed SLURP-1 expression in the spinal cord of rats, as a number of studies suggest spinal nicotinic acetylcholine receptors are important modulators of pain transmission. We detected intense SLURP-1 immunoreactivity in the dorsal horn of the spinal cord, especially in lamina I and outer II. In dorsal root ganglia, SLURP-1 immunoreactivity was detected in small- to medium-sized neurons, where in situ hybridization also revealed the presence of SLURP-1 mRNA. Fluorescent labeling of SLURP-1 partially overlapped that of calcitonin-gene related peptide (CGRP) or substance P (SP) in both the spinal cord dorsal horn and glabrous skin, and electron microscopic analysis revealed colocalization of SLURP-1 with SP or CGRP, in large synaptic vesicles in terminals within the superficial layer of the spinal cord. Finally, sciatic nerve axotomy reduced levels of SLURP-1 immunoreactivity in parallel with that of SP and CGRP in the ipsilateral superficial dorsal horn. These findings suggest that SLURP-1 is expressed in a subset of primary peptidergic sensory neurons.

Involvement of Na+/Ca2+ exchanger in migration and contraction of rat cultured tendon fibroblasts

In response to injury and inflammation of tendons, tendon fibroblasts are activated, migrate to the wound, and eventually induce contraction of the extracellular matrices to repair the tissue. Under such conditions, Ca2+ signalling is involved in motility and contractility of tendon fibroblasts. Using cultured tendon fibroblasts isolated from rat Achilles tendons, we investigated functional expression of Na+/Ca2+ exchangers (NCX). The fluorometric study showed that the intracellular Ca2+ concentration ([Ca2+]i) was increased by reducing extracellular Na+ concentration ([Na+]o) in tendon fibroblasts. Selective NCX inhibitors, KB‐R7943 and SEA0400, both attenuated [Na+]o‐dependent [Ca2+]i elevation and the resting [Ca2+]i in tendon fibroblasts. RT‐PCR, Western blots and sequence analyses revealed that NCX1.3 and NCX1.7 were expressed in cultured tendon fibroblasts. NCX2 mRNA was undetected. NCX3 expression was negligibly low. Immunofluorescence microscopy indicated that NCX1 protein localized in the plasma membrane especially at the microspikes of tendon fibroblasts. In the wound‐healing scratch assay, the cells migrated toward the space created by a scratch and almost completely filled the space within 48 h. This phenomenon was significantly suppressed by KB‐R7943 and SEA0400. Furthermore, the NCX inhibitors abrogated the tendon fibroblast‐mediated collagen‐matrix contractions. Two types of siRNAs for NCX1 also suppressed the migration and contraction of tendon fibroblasts. We conclude that NCX is expressed and mediates Ca2+ influx in cultured tendon fibroblasts. Since the pharmacological inhibitors and siRNA for NCX1 suppressed motility and contractility of tendon fibroblasts, NCX may play an important role in the function of tendon fibroblasts in the wound healing.

FAM83B is a novel biomarker for diagnosis and prognosis of lung squamous cell carcinoma

Personalized therapy for non-small cell lung cancer (NSCLC), particularly lung adenocarcinoma, has recently been significantly improved by the discovery of various molecular targets. However, this has not been the case for lung squamous cell carcinoma (SCC). In the present study, we identified the family with sequence similarity 83, member B (FAM83B) as a candidate marker for SCC through a comprehensive gene expression analysis and examined its correlations with various clinicopathological factors. The subjects of this study consisted of 215 patients with NSCLC who underwent complete resection from 2005 to 2011 at the Fukushima Medical University Hospital (Fukushima, Japan). They included 102 patients with adenocarcinoma and 113 with SCC. FAM83B expression was first examined in some of the samples by gene expression analysis and western blotting, and then all clinical specimens were evaluated by immunohistochemistry (IHC). The relationship between the quantitative values for IHC and clinicopathological factors was statistically analyzed. The results showed that FAM83B mRNA expression was significantly higher in SCC than in normal lung or adenocarcinoma (P<0.0001). Immunoblot analysis also confirmed this trend. Specimens containing >10% positive area for FAM83B were judged as ‘positive’; 94.3% (107/113) of SCC and 14.7% (15/102) of adenocarcinoma were positive. Patients were divided into two subgroups according to expression (54 high-expression and 53 low-expression patients); the high-expression group was associated with a better disease-free survival (DFS) rate (P=0.042, log-rank test). In conclusion, FAM83B may be a reliable diagnostic and prognostic biomarker for SCC. Detailed analyses of FAM83B function in lung cancer are required to understand how its expression is associated with better prognosis in SCC.

ArfGAP3 Regulates the Transport of Cation-Independent Mannose 6-Phosphate Receptor in the Post-Golgi Compartment

ArfGAPs are known to be involved in cargo sorting in COPI transport. However, the role of ArfGAPs in post-Golgi membrane traffic has not been defined. To determine the function of ArfGAPs in post-Golgi traffic, we used small interfering RNA to examine each of 25 ArfGAPs for effects on cation-independent mannose 6-phosphate receptor (CIMPR) localization. We found that downregulation of ArfGAP3 resulted in the peripheral localization of CIMPR. The effect was specific for ArfGAP3 and dependent on its GAP activity, because the phenotype was rescued by ArfGAP3 but not by ArfGAP1, ArfGAP2, or the GAP domain mutants of ArfGAP3. ArfGAP3 localized to the trans-Golgi network and early endosomes. In cells with reduced expression of ArfGAP3, Cathepsin D maturation was slowed and its secretion was accelerated. Also retrograde transport from the endosomes to the trans-Golgi network of endogenous CIMPR, but not truncated CIMPR lacking the luminal domain, was perturbed in cells with reduced expression of ArfGAP3. Furthermore the exit of epidermal growth factor receptor (EGFR) from the early endosomes and degradation of EGFR after EGF stimulation was slowed in cells with reduced expression of ArfGAP3. ArfGAP3 associates with Golgi-localized, γ-ear-containing, ADP-ribosylation factor binding proteins (GGAs), and ArfGAP3 knockdown reduces membrane association of GGAs. A possible mechanism explaining our results is that ArfGAP3 regulates transport from early endosomes to late endosomes. We suggest a model in which ArfGAP3 regulates Golgi association of GGA clathrin adaptors.

HMGB1 promotes ductular reaction and tumorigenesis in autophagy-deficient livers

Autophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.

Autocrine and paracrine interactions between multiple myeloma cells and bone marrow stromal cells by growth arrest-specific gene 6 crosstalk with interleukin-6

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant up-regulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 were significantly increased in the MM patients when compared with healthy controls. Gas6 was overexpressed in the human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditional medium from the human BM stromal cell line HS-5 induced cell proliferation and anti-apoptosis of the MM cells with extracellular signal-regulated kinase, Akt, and nuclear factor-κB phosphorylation, which were reversed by the neutralizing antibody to Gas6 or IL-6. The TAM family receptor Mer, which has been identified as a Gas6 receptor, was overexpressed in BM cells of MM patients. The knockdown of Mer by siRNA inhibited cell proliferation, anti-apoptosis, and up-regulation of intercellular cell adhesion molecule-1 (ICAM-1) in MM cells stimulated by an HS-5 cell-conditioned medium. Furthermore, the Gas6-neutralizing antibody reduced the up-regulation of IL-6 and ICAM-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathogenesis of MM, suggesting that Gas6-Mer-related signaling pathways may be a promising novel target for treating MM.

Clinicopathological examination of dipeptidase 1 expression in colorectal cancer

Dipeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is fundamental in glutathione and leukotriene metabolism. DPEP1 was initially considered as a tumor suppressor gene in Wilms tumor and breast cancer. However, it has been reported that DPEP1 is upregulated in colorectal cancers (CRCs) and high DPEP1 expression levels are associated with poorer patient survival. The role of DPEP1 genes in CRC, as well as their expression, requires investigation. Therefore, the present study investigated DPEP1 expression using reverse transcription-quantitative polymerase chain reaction or immunohistochemistry on surgically resected samples from CRC cases, and further examined the biological significance of DPEP1 by comparing the expression of the epithelial to mesenchymal transition (EMT) markers, including epithelial cadherin and Vimentin to clarify the function of DPEP1 in CRC, particularly in metastasis. The level of DPEP1 expression was identified to be significantly increased in tumorous tissue samples compared with that in non-tumorous tissue samples. In addition, increased DPEP1 mRNA expression levels were associated with positive lymph node metastasis in the included cohort. However, no positive correlations were observed between DPEP1 and EMT markers in the cohort. The results indiciates that further investigations into the upregulation of DPEP1 in colorectal carcinogenesis and the role of therapeutic or prognostic biomarkers are required.

Metastasis of breast cancer cells to the bone, lung, and lymph nodes promotes resistance to ionizing radiation Metastasierung von Brustkrebszellen in Knochen, Lunge und Lymphknoten steigert die Resistenz gegenüber ionisierender Strahlung

BackgroundMetastasis represents the leading cause of breast cancer deaths, necessitating strategies for its treatment. Although radiotherapy is employed for both primary and metastatic breast cancers, the difference in their ionizing radiation response remains incompletely understood. This study is the first to compare the radioresponse of axa0breast cancer cell line with its metastatic variants and report that such metastatic variants are more radioresistant.Materials and methodsAxa0luciferase expressing cell line was established from human basal-like breast adenocarcinoma MDA-MB-231 and underwent in vivo selections, whereby axa0cycle of inoculations into the left cardiac ventricle or the mammary fat pad of athymic nude mice, isolation of metastases to the bone, lung and lymph nodes visualized with bioluminescence imaging, and expansion of obtained cells was repeated twice or three times. The established metastatic cell lines were assessed for cell proliferation, wound healing, invasion, clonogenic survival, and apoptosis.ResultsThe established metastatic cell lines possessed an increased proliferative potential in vivo and were more chemotactic, invasive, and resistant to X‑ray-induced clonogenic inactivation and apoptosis in vitro.ConclusionBreast cancer metastasis to the bone, lung, and lymph nodes promotes radioresistance.ZusammenfassungHintergrundMetastasierung ist die Hauptursache für den tödlichen Verlauf von Brustkrebserkrankungen. Darauf müssen spezifische Behandlungsstrategien ausgerichtet werden. Sowohl primäre als auch metastatische Brustkrebsarten können mit einer Strahlentherapie behandelt werden, allerdings sind die Unterschiede in der Reaktion auf ionisierende Strahlung bis heute nicht vollständig verstanden. In dieser Studie wird zum ersten Mal die Strahlenantwort einer Brustkrebszelllinie mit der ihrer metastatischen Varianten verglichen und die erhöhte Strahlenresistenz der metastatischen Varianten gezeigt.Material und MethodenEine Luciferase-exprimierende Zelllinie wurde aus humanen basaloiden Brustadenokarzinomen MDA-MB-231 etabliert und in zwei oder drei Zyklen inxa0vivo selektiert. Dabei wurden die Zellen entweder in den linken Herzventrikel oder in das Fettgewebe der Brust athymischer Nacktmäuse inokuliert. Die durch Biolumineszenz sichtbar gemachten Metastasen wurden aus Knochen, Lunge und Lymphknoten isoliert und expandiert. Die etablierten metastatischen Zelllinien wurden auf Zellproliferation, Wundheilung, Invasion, Klonüberleben und Apoptose getestet.ErgebnisseDie etablierten metastatischen Zelllinien wiesen inxa0vivo ein gesteigertes proliferatives Potenzial auf. Inxa0vitro zeigten sie erhöhte chemotaktische und invasive Aktivität, zudem besaßen sie eine erhöhte Resistenz gegen röntgenstrahleninduzierte klonogene Inaktivierung und Apoptose.SchlussfolgerungMetastasen in Knochen, Lunge und Lymphknoten erhöhen die Strahlenresistenz von Brustkrebs.

Skull base invasive low-grade meningiomas, a distinct genetic subgroup: A microarray gene expression profile analysis.

Introduction Meningioma is the most common adult primary brain tumor originating from meningeal coverings of the brain and spinal cord. Commonly, World Health Organization (WHO) grade-I meningiomas are slowly growing and surgically curative, some present with clinically aggressive behavior, invading the skull base bone and soft tissues by extending into the extracranial spaces. Methods To detect the genetic background of the Skull Base Invasive Low-grade Meningioma (SBILM), we conducted a comprehensive analysis of gene expression was conducted on 32 meningioma samples. Results The cluster analysis of the gene expression profile demonstrated a distinctive clustering pattern of the SBILM. Based on the clinical behavior and the microarray findings, they might be a distinct subgroup of meningiomas. Conclusion Further studies on characterization of genes specifically expressed by the SBILM could lead to the development of diagnostic tools, differentiating it from other WHO grade-I meningiomas and assist in the appropriate management and follow-up strategy, and open the door for development of pharmacological therapies.