Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

Accumulating evidence suggests that exosomes are heterogeneous in molecular composition and physical properties. Here we examined whether epithelial cells secrete a heterogeneous population of exosomes, and if that is the case, whether epithelial cell polarity affects release of different populations of exosomes, especially that of those carrying Wnt. Sucrose-density ultracentrifugation and molecular marker analysis revealed that different populations of exosomes or exosome-like vesicles were released from MDCK cells depending on the cell polarity. Wnt3a associated with these vesicles were detectable in culture media collected from both apical and basolateral sides of the cells. Basolaterally secreted Wnt3a were co-fractionated with a typical exosomal protein TSG101 in fractions having typical exosome densities. In contrast, most of apically secreted Wnt3a, as well as Wnt11, were co-fractionated with CD63 and Hsp70, which are also common to the most exosomes, but recovered in higher density fractions. Wnt3a exhibiting similar floatation behavior to the apically secreted ones were also detectable in the culture media of Wnt3a-expressing L and HEK293 cells. The lipidation of Wnt3a was required for its basolateral secretion in exosomes but was dispensable for the apical one. Thus, epithelial cells release Wnt via distinct populations of vesicles differing in secretion polarity and lipidation dependency

Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast Differentiation and Induces Osteocyte Apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis

5，6－ヘキソーセン誘導体の簡便な合成法

講演番号：1W1

SHISA6 Confers Resistance to Differentiation-Promoting Wnt/β-Catenin Signaling in Mouse Spermatogenic Stem Cells

In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/β-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1+ cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1+ cells and confers resistance to the Wnt/β-catenin signaling. Shisa6+ cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)+ cells that are found largely overlapped with Shisa6+ cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates

Recent genetic studies in Drosophila identified a novel non-canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements

Intercellular exchange of Wnt ligands reduces cell population heterogeneity during embryogenesis

Neuromesodermal progenitor (NMP) cells produce and receive Wnt ligands. Here, the authors show that mutual intercellular exchange of Wnt ligands reduces heterogeneity between NMP cells and makes NMP populations robust to environmental stress