Jun Udagawa

Strategic compartmentalization of Toll-like receptor 4 in the mouse gut.

Pattern recognition receptors (PRRs), which include the Toll-like receptors (TLRs), are involved in the innate immune response to infection. TLR4 is a model for the TLR family and is the main LPS receptor. We wanted to determine the expression of TLR4 and compare it with that of TLR2 and CD14 along the gastrointestinal mucosa of normal and colitic BALB/c mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS). Mucosa from seven segments of the digestive tract (stomach, small intestine in three parts, and colon in three parts) was isolated by two different methods. Mucosal TLR4, CD14, TLR2, MyD88, and IL-1β mRNA were semiquantified by Northern blotting. TLR4 protein was determined by Western blotting. TLR4/MD-2 complex and CD14 were evaluated by immunohistochemistry. PRR genes were constitutively expressed and were especially stronger in colon. TLR4 and CD14 mRNA were increased in the distal colon, but TLR2 mRNA was expressed more strongly in the proximal colon, and MyD88 had a uniform expression throughout the gut. Accordingly, TLR4 and CD14 protein levels were higher in the distal colon. TLR4/MD-2 and CD14 were localized at crypt bottom epithelial cells. TLR4/MD2, but not CD14, was found in mucosal mononuclear cells. Finally, DSS-induced inflammation was localized in the distal colon. All genes studied were up-regulated during DSS-induced inflammation, but the normal colon-stressed gut distribution was preserved. Our findings demonstrate that TLR4, CD14, and TLR2 are expressed in a compartmentalized manner in the mouse gut and provide novel information about the in vivo localization of PRRs.

Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization

Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulin-induced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c-/- mice compared with that in Munc18c+/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated GLUT4 externalization, without affecting GLUT4 translocation to the cell periphery, in Munc18c+/+ adipocytes, but it did not affect GLUT4 externalization in Munc18c-/- cells. Phosphatidylinositol 3-phosphate, which induced GLUT4 translocation to the cell periphery without externalization in Munc18c+/+ cells, elicited GLUT4 externalization in Munc18c-/- cells. These findings demonstrate that Munc18c inhibits insulin-stimulated externalization of GLUT4 in a wortmannin-sensitive manner, and they suggest that disruption of the interaction between syntaxin4 and Munc18c in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.

Roles of leptin in prenatal and perinatal brain development

ABSTRACT  Leptin is a hormone that reduces food intake and increases energy expenditure by acting on the arcuate nucleus in the hypothalamus. Recent studies indicated that the neuronal circuit related to food intake in the hypothalamus is formed in the neonatal period and that leptin is necessary for the formation of this circuit. Our studies have further suggested that leptin may act on the fetal cerebral cortex, including the cingulate cortex, which is involved in motor and cognitive processes, and that leptin may affect maintenance and differentiation of neural stem cells, glial‐restricted progenitor cells and/or neuronal lineage cells. These recent studies showed that leptin not only has homeostatic functions in adults, but also regulates brain development in the prenatal and neonatal periods. These findings suggest that leptin is related to formation of the normal brain structure and regenerative potency of neural cells as well as the predisposition to homeostatic dysfunction, low locomotor activity or impairment of cognitive function.

Expression of Angiopoietin-1, Angiopoietin-2, and Tie2 Genes in Normal Ovary with Corpus luteum and in Ovarian Cancer

Objective: The recent discovery of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) has provided novel and important insights into the molecular mechanisms of blood vessel formation. Ang1 and Ang2 bind with similar affinity to the endothelial cell tyrosine kinase receptor Tie2. Our purpose was to assess the potential role of the Ang/Tie2 system in physiological and pathological angiogenesis in the ovary. Methods:Ang1, Ang2, and Tie2 gene expression in 14 normal ovaries with corpus luteum (CL) and in 19 cases of ovarian cancer were analyzed by polymerase chain reaction of RNA after reverse transcription. The level of each gene expression was presented by the relative yield of each gene to the β2-microglobulin gene, respectively. Furthermore, cellular distribution of Ang1 and Ang2 mRNA was examined by in situ hybridization, and localization of Tie2 was studied by immunochemistry. Results: The Ang1, Ang2, and Tie2 gene expression in normal ovary with CL ranged from 0.18 to 1.06 (median 0.54), 0.31–2.64 (median 1.01), and 0.10–0.47 (median 0.20), respectively. The expression of these same genes in ovarian cancer ranged from 0.06 to 0.75 (median 0.14), 0.69–1.59 (median 1.12), and 0.04–0.35 (median 0.15), respectively. Ang1 gene expression in normal ovary with CL was significantly higher than that in ovarian cancer (p = 0.0004). The gene expression levels of Ang2 and Tie2 were statistically the same in both groups. There was a significant correlation between Ang1 gene expression and Tie2 gene expression in normal ovary with CL (r = 0.619, p = 0.018). No such significant correlation was found in ovarian cancer. Moreover, Ang2 gene expression showed no significant correlation with the Tie2 gene expression either in normal ovary with CL or in ovarian cancer. Transcripts for Ang1 were observed in CL cells and endothelial cells around CL, and in tumor cells and endothelial cells at the periphery of tumor invasion. Ang2 transcripts were expressed in the same patterns. Tie2 expression was positive primarily in the endothelial cells around CL and in those at the periphery of tumor invasion. Conclusion: Our results indicate that there is a difference in the Ang/Tie2 gene expression between physiological and pathological angiogenesis in the ovary. This finding may aid in the development of new therapeutic interventions for ovarian cancer.

Expression of the long form of leptin receptor (Ob-Rb) mRNA in the brain of mouse embryos and newborn mice

The long form of the leptin receptor (Ob-Rb) has a cytoplasmic domain which activates the JAK-STAT signal transduction pathway. It is related to appetite and energy expenditure and is expressed in various parts of the brain in adults. In embryos, however, the detailed distribution of Ob-Rb expression sites and the function of the leptin-Ob-Rb system remain unclear, although leptin is detected in human cord plasma and leptin mRNA is detected in mouse embryos. In this study, we investigated the Ob-Rb mRNA expression pattern in the brains of mouse embryos and newborn mice by RT-PCR and in situ hybridization. At embryonic day 10.5 (E10.5), Ob-Rb mRNA was already detected in the brain by RT-PCR. By in situ hybridization, Ob-Rb mRNA was observed in the ventricular zone of the rhombencephalon at E11.5. At E12.5, it was also expressed in the ventricular zone of the telencephalon, mesencephalon and cerebellar primordium. From E14. 5 it was expressed in the cortical plate of the telencephalon and the ventricular zone of the thalamus. At E16.5, it was expressed in the premamillary hypothalamic nucleus, superficial gray matter of the superior colliculus, external germinal and Purkinje cell layers of the cerebellum, and facial nucleus. At E18.5, it was expressed in the arcuate nucleus and ventromedial hypothalamic nucleus. These results suggest that the leptin-Ob-Rb system is related to brain development.

Ror2 is Required for Midgut Elongation During Mouse Development

The receptor tyrosine kinase Ror2 acts as a receptor for Wnt5a to mediate noncanonical Wnt signaling, and it plays essential roles in morphogenesis. Ror2−/− embryos exhibit phenotypes similar to, albeit generally milder than, those of Wnt5a−/− embryos. During mouse embryogenesis, Ror2 is expressed in various organs and regions, although little is known about its expression pattern and roles in the developing gut, while Wnt5a is expressed in the developing gut, where its absence causes abnormal phenotypes. Here, we demonstrated that Ror2 was strongly and differentially expressed in the rostral and middle midgut endoderm from embryonic day (E) 10.5 through embryonic day (E) 12.5. At E11.5, Ror2−/− embryos exhibited a shorter middle midgut with a larger diameter and more accumulation of epithelial cells in the middle midgut than control embryos, while the total cell numbers remained unaltered. These findings suggest that Ror2 plays important roles in midgut elongation by means of an epithelial convergent extension mechanism. Developmental Dynamics 239:941–953, 2010.

Expression of cytoplasmic TFF2 is a marker of tumor metastasis and negative prognostic factor in gastric cancer.

Trefoil factor family 2 (TFF2) is a small peptide constitutively expressed in the gastric mucosa, where it plays a protective role in restitution of gastric mucosa. TFF2 has also been shown to be expressed in some gastric cancers, but its role in tumor metastasis and patient prognosis has not been examined. In this study, we examined TFF2 expression at both the mRNA and protein levels and correlated these results with the clinicopathologic characteristics and prognosis of gastric cancer patients. Among the 144 curatively resected samples, 43 (30%) were positive for TFF2. TFF2 expression was preferentially observed in the infiltrating tumor cells sparing the superficial cells. Significantly increased expression of TFF2 was noted in large tumors of the diffuse type. An increased prevalence of TFF2 expression was also found in tumors with advanced T and N stage and in patients with lymphatic and venous invasion. Accordingly, patients with TFF2-expressing tumors had a significantly worse disease-free survival, and in multivariate analysis, this finding remained significant as an independent prognostic factor. Taken together, our results suggest that TFF2 expression may play a role in gastric cancer invasion and as such could be a useful target for therapeutic intervention.

Reg I-knockout mice reveal its role in regulation of cell growth that is required in generation and maintenance of the villous structure of small intestine

Reg I (regenerating gene product I) is a growth factor that plays a central role in the generation and regeneration of the gastric mucosal architecture. On the other hand, mouse Reg I mRNA is expressed at the highest levels in the small intestine among the gastrointestinal tissues. In the current study, with the aim to clarify the role of Reg I protein in the small intestine, the temporal and spatial pattern of Reg I expression and the phenotype of Reg I-knockout mice in the tissue were examined. In the wild-type mice, immunohistochemistry localized Reg I protein expression in absorptive cells located in the lower half of the intestinal villi. Reg I expression was undetectable until embryonic day 13 (E13), when the fetal intestine still lacks villous structure; however, it dramatically increased at E17 along with the formation and maturation of the fetal intestinal villi. In the small intestine of the adult Reg I-knockout mice, less densely packed, round-shaped aberrant morphology of the absorptive cells was observed light microscopically, and electron microscopical examination revealed a strikingly loose connection of these cells to the basement membrane. Antiproliferating cell nuclear antigen staining and anti-Ki67 staining demonstrated the marked decrease in the number of proliferating cells in the small intestinal mucosa of the knockout mice. The cell migration speed visualized by one shot labeling of 5-bromodeoxyuridine was significantly slower in the knockout mice. These phenotypes of Reg I-knockout mice emerged, in accordance with the temporal pattern of Reg I expression described above, from E17. Reg I was considered to be a regulator of cell growth that is required to generate and maintain the villous structure of the small intestine.

Fetal Jaw Movement Affects Condylar Cartilage Development

Using a mouse exo utero system to examine the effects of fetal jaw movement on the development of condylar cartilage, we assessed the effects of restraint of the animals’ mouths from opening, by suture, at embryonic day (E)15.5. We hypothesized that pre-natal jaw movement is an important mechanical factor in endochondral bone formation of the mandibular condyle. Condylar cartilage was reduced in size, and the bone-cartilage margin was ill-defined in the sutured group at E18.5. Volume, total number of cells, and number of 5-bromo-2′-deoxyuridine-positive cells in the mesenchymal zone were lower in the sutured group than in the non-sutured group at E16.5 and E18.5. Hypertrophic chondrocytes were larger, whereas fewer apoptotic chondrocytes and osteoclasts were observed in the hypertrophic zone in the sutured group at E18.5. Analysis of our data revealed that restricted fetal TMJ movement influences the process of endochondral bone formation of condylar cartilage.

The role of leptin in the development of the cortical neuron in mouse embryos

Leptin is an obese gene product that decreases appetite and raises energy expenditure in adults. We previously reported that leptin was detected in the sera of mouse embryos and leptin receptors were expressed in the mouse embryonic cerebrum, suggesting that leptin plays a role in cerebral development. In this study, we injected leptin into the lateral ventricle of the cerebrum in leptin-deficient ob/ob mouse embryos to investigate the function of leptin in cerebral development. When leptin was injected on embryonic day (E) 14, the ratio of the number of cells in the cortical plate (CP) to that in the intermediate zone (IZ) was higher in leptin-injected than in vehicle-injected ob/ob embryos on E16, although there was no significant difference in the number of cells in the ventricular zone (VZ), IZ, or CP between these groups. The number of postmitotic BrdU-positive CP cells was larger in leptin-injected than in vehicle-injected ob/ob embryos on E16 and E17 when BrdU labeling and leptin injection were performed on E14. By in situ hybridization, NPY mRNA expression in CP neurons on E18 was weaker in leptin-injected than in vehicle-injected ob/ob embryos when leptin was injected on E16. These results suggest that leptin promotes the migration of neuronal lineage cells to CP and that the leptin-NPY axis in neurons works in the cerebral cortex on E16.