Dab2 is pivotal for endothelial cell migration by mediating VEGF expression in cancer cells

Although angiogenesis is crucial for tumor growth and metastasis, the molecular mechanisms controlling this process are not clearly understood. Here, we explore the role of Dab2 in tumor angiogenesis. We found that Dab2 is expressed in several cancer cells, including A549 lung cancer cells, but it is hardly detectable in SW480 colon cancer cells. Migration and Erk phosphorylation were enhanced in human umbilical vein endothelial cells (HUVECs) treated with the conditioned medium obtained from Dab2-overexpressing SW480 stable cells. In addition, vascular endothelial growth factor (VEGF) protein was strongly detected in conditioned medium derived from Dab2-overexpressing SW480 cells, and Erk phosphorylation enhanced by Dab2(+) CM was restored by VEGF inhibition. Moreover, Dab2 depletion in A549 cells led to a decrease in HUVEC migration and Erk phosphorylation. Furthermore, we show that Dab2 is required for the TGFβ-induced gene expression of angiogenic factors such as VEGF and FGF2. Taken together, these results suggest that Dab2, which is expressed in cancer cells, is pivotal for endothelial cell migration by affecting VEGF expression.     

Therapeutic effects of autologous tumor-derived nanovesicles on melanoma growth and metastasis

Cancer vaccines with optimal tumor-associated antigens show promise for anti-tumor immunotherapy. Recently, nano-sized vesicles, such as exosomes derived from tumors, were suggested as potential antigen candidates, although the total yield of exosomes is not sufficient for clinical applications. In the present study, we developed a new vaccine strategy based on nano-sized vesicles derived from primary autologous tumors. Through homogenization and sonication of tumor tissues, we achieved high yields of vesicle-bound antigens. These nanovesicles were enriched with antigenic membrane targets but lacked nuclear autoantigens. Furthermore, these nanovesicles together with adjuvant activated dendritic cells in vitro, and induced effective anti-tumor immune responses in both primary and metastatic melanoma mouse models. Therefore, autologous tumor-derived nanovesicles may represent a novel source of antigens with high-level immunogenicity for use in acellular vaccines without compromising safety. Our strategy is cost-effective and can be applied to patient-specific cancer therapeutic vaccination.     

A single point mutation in Ms44 results in dominant male sterility and improves nitrogen use efficiency in maize

Application of nitrogen fertilizer in the past 50 years has resulted in significant increases in crop yields. However, loss of nitrogen from crop fields has been associated with negative impacts on the environment. Developing maize hybrids with improved nitrogen use efficiency is a cost‐effective strategy for increasing yield sustainably. We report that a dominant male‐sterile mutant Ms44 encodes a lipid transfer protein which is expressed specifically in the tapetum. A single amino acid change from alanine to threonine at the signal peptide cleavage site of the Ms44 protein abolished protein processing and impeded the secretion of protein from tapetal cells into the locule, resulting in dominant male sterility. While the total nitrogen (N) content in plants was not changed, Ms44 male‐sterile plants reduced tassel growth and improved ear growth by partitioning more nitrogen to the ear, resulting in a 9.6% increase in kernel number. Hybrids carrying the Ms44 allele demonstrated a 4%–8.5% yield advantage when N is limiting, 1.7% yield advantage under drought and 0.9% yield advantage under optimal growth conditions relative to the yield of wild type. Furthermore, we have developed an Ms44 maintainer line for fertility restoration, male‐sterile inbred seed increase and hybrid seed production. This study reveals that protein secretion from the tapetum into the locule is critical for pollen development and demonstrates that a reduction in competition between tassel and ear by male sterility improves grain yield under low‐nitrogen conditions in maize.     

Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis

The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC‐MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 ( http://proteomecentral.proteomexchange.org/dataset/PXD001160 ).     

Cutting Edge: Inhibition of the retinoid X receptor (RXR) blocks T helper 2 differentiation and prevents allergic lung inflammation

Among the many factors regulating Th cell differentiation, some nuclear hormone receptors are emerging as important players. The retinoid X receptor (RXR) functions as heterodimerization partner for a variety of nuclear hormone receptors. We show in this study that RXR is critical for Th2-mediated immunity. An RXR antagonist inhibited Th2 differentiation, resulting in reduced production of IL-4, IL-10, and IL-13, whereas IFN-gamma production was enhanced. This effect was dependent on the presence of APCs. In addition, IL-5 production was blocked directly in Th cells. In vivo, inhibition of RXR prevented experimentally induced allergic lung inflammation. Th1-mediated inflammation was not affected. Its specific role in Th2-mediated inflammation makes RXR a promising target for the development of therapies against diseases such as allergic asthma and atopic dermatitis.     

A membranous form of ICAM-1 on exosomes efficiently blocks leukocyte adhesion to activated endothelial cells

While intercellular adhesion molecule-1 (ICAM-1) is a transmembrane protein, two types of extracellular ICAM-1 have been detected in cell culture supernatants as well as in the serum: a soluble form of ICAM-1 (sICAM-1) and a membranous form of ICAM-1 (mICAM-1) associated with exosomes. Previous observations have demonstrated that sICAM-1 cannot exert potent immune modulatory activity due to its low affinity for leukocyte function-associated antigen-1 (LFA-1) or membrane attack complex-1. In this report, we initially observed that human cancer cells shed mICAM-1(+)-exosomes but were devoid of vascular cell adhesion molecule-1 and E-selectin. We demonstrate that mICAM-1 on exosomes retained its topology similar to that of cell surface ICAM-1, and could bind to leukocytes. In addition, we show that exosomal mICAM-1 exhibits potent anti-leukocyte adhesion activity to tumor necrosis factor-α-activated endothelial cells compared to that of sICAM-1. Taken together with previous findings, our results indicate that mICAM-1 on exosomes exhibits potent immune modulatory activity.     

The Ral/exocyst effector complex counters c-Jun N-terminal kinase-dependent apoptosis in Drosophila melanogaster

Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK.     

Hydrangenol suppresses VEGF-stimulated angiogenesis by targeting p27KIP1-dependent G1-cell cycle arrest,VEGFR-2-mediated signaling,and MMP-2 expression*

We previously reported that hydrangenol has potent antitumor activity against human bladder cancer EJ cells. Here, we investigated the antiangiogenic activity of hydrangenol using in vitro and ex vivo models. Treatment with hydrangenol significantly inhibited the proliferation of vascular endothelial growth factor (VEGF)-induced HUVECs in a concentration-dependent manner (EC_50?=?10?μM). Flow cytometry analysis revealed that hydrangenol suppressed the VEGF-induced inhibition of G1-cell cycle phase and also decreased cyclin D1, cyclin E, CDK2, and CDK4 levels. Hydrangenol-mediated arrest in the G1-cell cycle phase was associated with p27KIP1 level, but not p21WAF1 or p53 level. Hydrangenol also significantly inhibited VEGFR-2-mediated signaling pathways including ERK1/2, AKT, and endothelial nitric oxide synthase. Interestingly, immunoprecipitation assay demonstrated that the inhibition of VEGFR-2 activation was independent of VEGF binding, thereby suggesting an allosteric regulation of hydrangenol against VEGFR-2. Additionally, hydrangenol inhibited migration, invasion, and capillary-like tubular formation in VEGF-stimulated HUVECs. Zymography and immunoblot analyses revealed that these inhibitory activities were partially owing to the VEGF-induced inhibition of matrix metalloproteinase-2 activity. Finally, VEGF-mediated microvessel sprouting was inhibited in the presence of hydrangenol in ex vivo aortic ring assay. Taken together, hydrangenol possesses a potent antiangiogenesis potential; thus we believe that hydrangenol may be developed as a therapeutic reagent to treat angiogenesis-mediated diseases.     

Gram‐positive bacteria produce membrane vesicles: Proteomics‐based characterization of Staphylococcus aureus‐derived membrane vesicles

Although archaea, Gram‐negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell‐free intercellular communication, this cellular process has been overlooked in Gram‐positive bacteria. Here, we found for the first time that Gram‐positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus‐derived MVs are both similar to those of Gram‐negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus‐derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram‐positive bacteria.     

Control of cell fate choices by lateral signaling in the adult peripheral nervous system of Drosophila melanogaster

The thoracic integument of the adult fruit fly is a relatively simple but highly patterned structure. It is composed of sensory organ cells distributed within a monolayer of epidermal cells. Both cell types are easily detected at the cuticular surface, as each external sense organ forms a sensory bristle and each epidermal cell secretes a small nonsensory hair. Inhibitory cell—cell interactions play a key role in regulating the distribution as well as the formation of the sense organs. This review focuses on the role of these cell—cell interactions in the adoption of alternative cell fates. We also show that Notch, Hairless, and Suppressor of Hairless, three components of this intercellular signaling pathway, exhibit dose-dependent genetic interactions. Finally we address how this intercellular signaling mechanism may be modulated to result in highly reproducible outcomes. © 1996 Wiley-Liss, Inc.