Regenerating gene I regulates interleukin-6 production in squamous esophageal cancer cells

Regenerating gene (REG) I plays important roles in cancer cell biology. The purpose of this study was to determine whether REG I affects cytokine production in cancer cells. We transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Iα and Iβ and examined its effects on cytokine expression. We found that transfecting TE-5 and TE-9 cells with REG I Iα and Iβ led to significantly increased expression of interleukin (IL)-6 mRNA and protein, but it had little or no effect on expression of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17A, interferon-γ, tumor necrosis factor-α, granulocyte-colony stimulating factor or transforming growth factor-β1. The elevated IL-6 expression seen in REG Iα transfectants was silenced by small interfering RNA-mediated knockdown. These finding suggest that REG I may act through IL-6 to exert effects on squamous esophageal cancer cell biology.     

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.     

Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8α⁺ conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8⁺ T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3⁺ DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8⁺ T cells through XCR1.     

Application of new semisynthetic aequorins with long half-decay time of luminescence to G-protein-coupled receptor assay

Aequorin is a Ca²⁺-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O₂ to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca²⁺ of these semisynthetic aequorins were characterized. Two semisynthetic aequorins, namely me- and cf3-aequorin, showed a slow decay of the luminescence pattern with less sensitivity to Ca²⁺ and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays.     

��2,6-Sialic Acid on Platelet Endothelial Cell Adhesion Molecule (PECAM) Regulates Its Homophilic Interactions and Downstream Antiapoptotic Signaling

Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that α2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on α2,6-sialic acid. We also found that the absence of α2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis.     

Hedgehog Promotes Neovascularization in Pancreatic Cancers by Regulating Ang-1 and IGF-1 Expression in Bone-Marrow Derived Pro-Angiogenic Cells

Background

The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells.

Methodology/Principal Findings

Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity.

Conclusions/Significance

We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.     

CXCL14 Deficiency in Mice Attenuates Obesity and Inhibits Feeding Behavior in a Novel Environment

Background

CXCL14 is a chemoattractant for macrophages and immature dendritic cells. We recently reported that CXCL14-deficient (CXCL14 ^−/−) female mice in the mixed background are protected from obesity-induced hyperglycemia and insulin resistance. The decreased macrophage infiltration into visceral adipose tissues and the increased insulin sensitivity of skeletal muscle contributed to these phenotypes.

Methodology/Principal Findings

In this study, we performed a comprehensive study for the body weight control of CXCL14 ^−/− mice in the C57BL/6 background. We show that both male and female CXCL14 ^−/− mice have a 7–11% lower body weight compared to CXCL14 ^+/− and CXCL14 ^+/+ mice in adulthood. This is mainly caused by decreased food intake, and not by increased energy expenditure or locomotor activity. Reduced body weight resulting from the CXCL14 deficiency was more pronounced in double mutant CXCL14^−/− ob/ob and CXCL14 ^−/−A^y mice. In the case of CXCL14 ^−/−A^y mice, oxygen consumption was increased compared to CXCL14 ^+/−A^y mice, in addition to the reduced food intake. In CXCL14 ^−/− mice, fasting-induced up-regulation of Npy and Agrp mRNAs in the hypothalamus was blunted. As intracerebroventricular injection of recombinant CXCL14 did not change the food intake of CXCL14 ^−/− mice, CXCL14 could indirectly regulate appetite. Intriguingly, the food intake of CXCL14 ^−/− mice was significantly repressed when mice were transferred to a novel environment.

Conclusions/Significance

We demonstrated that CXCL14 is involved in the body weight control leading to the fully obese phenotype in leptin-deficient or A^y mutant mice. In addition, we obtained evidence indicating that CXCL14 may play an important role in central nervous system regulation of feeding behavior.     

Synthesis and biological activity of 1-phenylsulfonyl-4-phenylsulfonylaminopyrrolidine derivatives as thromboxane a(2) receptor antagonists

The synthesis and biological activity of novel 1-phenylsulfonyl-4- phenylsulfonylaminopyrrolidine analogues are described. All compounds were produced through modification of the substituent formally corresponding to the 1,3-dioxane ring system and the omega-octenol side chain of thromboxane A(2) (TXA(2)), in reference to the structure of Daltroban. Several compounds were found to be potent TXA(2) receptor antagonists. Compound 51a was the most effective inhibitor of 9,11-epoxymethano PGH(2) (U-46619)-induced rat aortic strip contraction (IC(50)=0.48 nM).     

High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis

Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan derivatization with novel, stable isotope-coded derivatization reagents followed by MALDI-TOF(/TOF) analysis. This analysis revealed distinct features of the N-glycosylation profile of epidermis and dermis for the first time. A high abundance of high mannose type oligosaccharides was found to be characteristic of murine epidermis glycoproteins. Based on this observation, we performed high mannose type glycoform-focused proteomics by direct tryptic digestion of protein mixtures and affinity enrichment. We identified 15 glycoproteins with 19 N-glycosylation sites that carry high mannose type glycans by off-line LC-MALDI-TOF/TOF mass spectrometry. Moreover the relative quantity of microheterogeneity of different glycoforms present at each N-glycan binding site was determined. Glycoproteins identified were often contained in lysosomes (e.g. cathepsin L and gamma-glutamyl hydrolase), lamellar granules (e.g. glucosylceramidase and cathepsin D), and desmosomes (e.g. desmocollin 1, desmocollin 3, and desmoglein). Lamellar granules are organelles found in the terminally differentiating cells of keratinizing epithelia, and desmosomes are intercellular junctions in vertebrate epithelial cells, thus indicating that N-glycosylation of tissue-specific glycoproteins may contribute to increase the relative proportion of high mannose glycans. The striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation may also reflect the observed high abundance of high mannose glycans.