Riuko Ohashi

Expression of pentraxin 3 (PTX3) in human atherosclerotic lesions

Pentraxin 3 (PTX3) and C‐reactive protein (CRP) are members of the pentraxin superfamily. PTX3 expression is induced in response to inflammatory signals, and is produced at sites of inflammation by several types of cell, primarily monocytes/macrophages, dendritic cells (DCs), endothelial cells, smooth muscle cells (SMCs), and fibroblasts, but is not produced by hepatocytes, which are a major source of CRP. The aim of our study was to investigate the expression pattern of PTX3 in human atherosclerotic lesions using a novel monoclonal antibody against PTX3. We examined coronary arterial thrombi containing an atherosclerotic plaque component removed from patients with acute myocardial infarction and human aortic tissues with various degrees of atherosclerosis sampled from autopsy cases. Immunohistochemical study of paraffin and frozen sections indicated that macrophages, mainly foam cells, expressed PTX3 in advanced atherosclerotic lesions. Interestingly, we also clearly observed PTX3‐positive neutrophils infiltrating into atherosclerotic plaques, suggesting that PTX3 derived from neutrophils as well as macrophages plays an important role in atherogenesis. Copyright

PIK3CA mutation and amplification in human lung cancer

To explore the significance of phosphatidylinositol‐3‐kinase, catalytic, alpha (PIK3CA) in the carcinogenesis in human lung, mutations and copy number changes were investigated in 148 Japanese patients with primary cancer of the lung. For biological validation, the effects of exogenously expressed wild‐type and mutated PIK3CA were studied in an immortalized human airway epithelial cell line. Mutations in PIK3CA were found in five (3.6%) of the 139 available patients, and copy number gains were found in 21 (18.3%) of 115 patients, respectively. Overall, mutations or copy number gains were detected in 24 of the 106 patients (22.6%) for whom results in both analyses were available. The prevalence of copy number gains was higher in men, smokers, and in patients with squamous cell carcinoma than in the opposite categories. The copy number changes showed a trend toward higher prevalence in the earlier stages (P = 0.038). Interestingly, the presence of mutations and of copy number alterations were mutually exclusive in the present patients, implying that both entail equivalent oncogenic potential. Over‐expressed wild‐type PIK3CA and its two common mutants, K545E and H1047R, significantly enhanced the anchorage‐independent growth activity and migration activity of immortalized airway epithelium 16HBE14o– cells, but the effects of the K545E and H1047R mutants were more remarkable than those of the wild‐type. The present demonstrates an important role of PIK3CA in human lung carcinogenesis.

Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4α in the pathogenesis of human cancer

Hepatocyte nuclear factor‐4α (HNF4α) exists in multiple isoforms that are generated by alternative promoter (P1 and P2) usage and splicing. Here we establish monoclonal antibodies (mAbs) for detecting P1 and P2 promoter‐driven HNF4α, and evaluate their expression in normal adult human tissues and surgically resected carcinomas of different origins. Using immunohistochemical analysis, we demonstrate that, while P1 promoter‐driven HNF4α is expressed in hepatocytes, small intestine, colon, kidney and epididymis, P2 promoter‐driven HNF4α is expressed in bile duct, pancreas, stomach, small intestine, colon and epididymis. Altered expression patterns of P1 and P2 promoter‐driven HNF4α were observed in gastric, hepatocellular and colorectal carcinomas. HNF4α was expressed in lung metastases from renal cell, hepatocellular and colorectal carcinoma but was not observed in lung tumours. The P1 and P2 promoter‐driven HNF4α expression pattern of tumour metastases correlated with the primary site of origin. P1 promoter‐driven HNF4α was also found in intestinal metaplasia of the stomach. These data provide evidence for the tissue distribution of P1 and P2 promoter‐driven HNF4α at the protein level and suggest that HNF4α may be a novel diagnostic marker for metastases of unknown primary. We propose that the dysregulation of alternative promoter usage of HNF4α is associated with the pathogenesis of certain cancers. Copyright

Tiam1 mediates neurite outgrowth induced by ephrin‐B1 and EphA2

Bidirectional signals mediated by Eph receptor tyrosine kinases and their membrane‐bound ligands, ephrins, play pivotal roles in the formation of neural networks by induction of both collapse and elongation of neurites. However, the downstream molecular modules to deliver these cues are largely unknown. We report here that the interaction of a Rac1‐specific guanine nucleotide‐exchanging factor, Tiam1, with ephrin‐B1 and EphA2 mediates neurite outgrowth. In cells coexpressing Tiam1 and ephrin‐B1, Rac1 is activated by the extracellular stimulation of clustered soluble EphB2 receptors. Similarly, soluble ephrin‐A1 activates Rac1 in cells coexpressing Tiam1 and EphA2. Cortical neurons from the E14 mouse embryos and neuroblastoma cells significantly extend neurites when placed on surfaces coated with the extracellular domain of EphB2 or ephrin‐A1, which were abolished by the forced expression of the dominant‐negative mutant of ephrin‐B1 or EphA2. Furthermore, the introduction of a dominant‐negative form of Tiam1 also inhibits neurite outgrowth induced by the ephrin‐B1 and EphA2 signals. These results indicate that Tiam1 is required for neurite outgrowth induced by both ephrin‐B1‐mediated reverse signaling and EphA2‐mediated forward signaling.

Cooperative Interaction between Hepatocyte Nuclear Factor 4α and GATA Transcription Factors Regulates ATP-Binding Cassette Sterol Transporters ABCG5 and ABCG8

ABSTRACT Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4α (HNF4α) in the ABCG5/ABCG8 intergenic promoter, through which HNF4α strongly activated the expression of a reporter gene in both directions. The HNF4α-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4α. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4α leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4α and GATA were essential for maximal synergism. We also show that HNF4α, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4α and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4α acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.

SOX6 attenuates glucose stimulated insulin secretion by repressing PDX1 transcriptional activity and is down-regulated in hyperinsulinemic obese mice

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing β-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca2+ mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in β-cells may contribute to the β-cell adaptation in obesity-related insulin resistance.

Long pentraxin 3 (PTX3) expression and release by neutrophils in vitro and in ulcerative colitis

Pentraxin 3 (PTX3) is the first identified long pentraxin, and it is rapidly produced and released by several cell types in response to proinflammatory signals. The aim of this study was to investigate the behavior of neutrophils to produce PTX3 protein in response to proinflammatory cytokine IL‐8 in vitro, as well as identify the expression pattern of PTX3 in human ulcerative colitis lesions.

The Wnt/Planar Cell Polarity Pathway Component Vangl2 Induces Synapse Formation through Direct Control of N-Cadherin

Although regulators of the Wnt/planar cell polarity (PCP) pathway are widely expressed in vertebrate nervous systems, their roles at synapses are unknown. Here, we show that Vangl2 is a postsynaptic factor crucial for synaptogenesis and that it coprecipitates with N-cadherin and PSD-95 from synapse-rich brain extracts. Vangl2 directly binds N-cadherin and enhances its internalization in a Rab5-dependent manner. This physical and functional interaction is suppressed by β-catenin, which binds the same intracellular region of N-cadherin as Vangl2. In hippocampal neurons expressing reduced Vangl2 levels, dendritic spine formation as well as synaptic marker clustering is significantly impaired. Furthermore, Prickle2, another postsynaptic PCP component, inhibits the N-cadherin-Vangl2 interaction and is required for normal spine formation. These results demonstrate direct control of classic cadherin by PCP factors; this control may play a central role in the precise formation and maturation of cell-cell adhesions at the synapse.

The proteomic profile of circulating pentraxin 3 (PTX3) complex in sepsis demonstrates the interaction with azurocidin 1 and other components of neutrophil extracellular traps

Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (∼200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (KD = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.

Phosphatidylinositol 3-Phosphatase Myotubularin-related Protein 6 (MTMR6) Is Regulated by Small GTPase Rab1B in the Early Secretory and Autophagic Pathways

Background: The activity of myotubularin-related protein 6 (MTMR6) is regulated by an unknown mechanism. Results: The cellular localization of MTMR6 was regulated by Rab1B via the GRAM domain. They were functionally related in the early secretory and autophagic pathways. Conclusion: MTMR6 is regulated by Rab1B via the conserved GRAM domain. Significance: Our results reveal a novel regulatory mechanism of MTM phosphatases. A large family of myotubularin phosphatases dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, which are known to play important roles in vesicular trafficking and autophagy. The family is composed of 16 members, and understanding their regulatory mechanisms is important to understand their functions and related genetic diseases. We prepared anti-myotubularin-related protein 6 (MTMR6) monoclonal antibody and used it to study the regulatory mechanism of MTMR6. Endogenous MTMR6 was present in the cytoplasm and was condensed in the perinuclear region in a microtubule-dependent manner. MTMR6 preferentially interacted with GDP-bound Rab1B via the GRAM domain and partly overlapped with Rab1B in the pericentrosomal and peri-Golgi regions in normal rat kidney cells. Overexpression of GDP-bound Rab1B and the reduction of Rab1B disrupted the localization of MTMR6, suggesting that Rab1B regulates the localization of MTMR6. The reduction of MTMR6 accelerated the transport of vesicular stomatitis virus glycoprotein in which Rab1B is involved. Furthermore, reduction of MTMR6 or Rab1B inhibited the formation of the tubular omegasome that is induced by overexpression of DFCP1 in autophagy. Our results indicate that the cellular localization of MTMR6 is regulated by Rab1B in the early secretory and autophagic pathways. We propose a new regulatory mechanism of myotubularin phosphatase by the small GTPase Rab1B.