Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation

CDC42-C末端異常症に於ける炎症病態を解明 --ゴルジ体への異常蓄積がパイリンインフラマソーム形成を過剰促進--. 京都大学プレスリリース. 2022-05-02.Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Effectiveness of IL-1β–blocking therapy indicates that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42[R186C], we found that patient-derived cells secreted larger amounts of IL-1β in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42[R186C] protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42[*192C*24] that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but not that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave the way for elucidating the mechanism of pyrin inflammasome formation

Ccdc85C, a causative protein for hydrocephalus and subcortical heterotopia, is expressed in the systemic epithelia with proliferative activity in rats

. Coiled-coil domain containing 85c (Ccdc85c) is a causative gene for spontaneous mutant mouse with non-obstructive hydrocephalus and subcortical heterotopia. Detailed functions of Ccdc85C protein have not been clarified. To reveal roles of Ccdc85C, we examined the distribution and expression pattern of Ccdc85C in the systemic developing organs in rats. Ccdc85C was expressed in various simple epithelia but not stratified epithelia. In the various epithelia, Ccdc85C was localized at cell-cell junctions and its expression was strong at apical junctions. Furthermore, intense expression was seen at developing period and gradually decreased with advancing development. Distribution of Ccdc85C coincides with that of proliferating epithelial cells. These results suggest that Ccdc85C plays an important role in the proliferative property of simple epithelia

Characterization of rat testicular teratoma and its derived cell lines, with particular reference to possible mesenchymal differentiations

The original tumor, 4 cm in diameter, was found in the left testis of a 2-month old SD rat. The tumor consisted of well-differentiated, mature tissues such as bone, cartilage, adipose tissue, smooth and skeletal muscles, skin, hair, glands (salivary, sebaceous, apocrine and pancreatic exocrine glands) and trachea, as well as nerve tissues. The tumor was diagnosed as a mature type of teratoma, a rare in rat testis. Cloned cell lines (named TSD-B4S and TSD-F9R) were established from the tumor; cellular properties of these cell lines were similar to each other; basically, their cultured cells exhibited vimentin-positive mesenchymal nature with occasional cells reacting to α-smooth muscle actin, glial fibrillary acidic protein and CD163 (a macrophage marker). The cell lines showed tumorigenicity when inoculated into nude mice, being composed of immature mesenchymal cells arranged mainly in a sheet. In TSDB4S cells treated with differentiation factors, we demonstrated mesenchymal differentiations towards adipogenic, osteogenic and myofibrogenic cells. The cell line (TSD-B4S) would become a useful tool for studies on stem cell differentiation, because the teratoma arises from primordial germ cells like embryonic stem cells

Expression of Ccdc85C, a causative protein for murine hydrocephalus, in the mammary gland tumors of dogs

Coiled-coil domain containing 85c (Ccdc85c) is a causative gene for hemorrhagic hydrocephalus mouse which shows hydrocephalus with frequent brain hemorrhage and formation of subcortical band heterotopia. A previous study revealed that Ccdc85C protein is expressed in the systemic simple epithelial cells with proliferative activity in rats and suggested that Ccdc85C expression may be related to the cell proliferation of simple epithelial cells. To reveal the roles of Ccdc85C in the proliferative lesion, we examined the expression patterns of Ccdc85C in the mammary gland tumor of dogs, a common representative tumor derived from simple epithelial cells. In canine mammary gland tumors, Ccdc85C was expressed at the apical junctions of the luminal epithelial cells. Ccdc85C was also distributed throughout the entire cytoplasm of the myoepithelial cells. Ccdc85C expression was observed at the epithelial cells with luminal structures, but was not observed at the epithelial cells forming sheet growth pattern without luminal structure. In carcinomas, Ccdc85C expression in mammary tumor tissue tended to be weaker than that in surrounding normal mammary gland tissue. Ccdc85C is known to cause neurological diseases such as hydrocephalus, and subcortical heterotopia, and the present study is the first to demonstrate Ccdc85C expression in canine mammary tumors and a relationship between Ccdc85C expression and tumor malignanc

Participation of Somatic Stem Cells, Labeled by a Unique Antibody (A3) Recognizing Both N-glycan and Peptide, to Hair Follicle Cycle and Cutaneous Wound Healing in Rats

A monoclonal antibody (A3) was generated by using rat malignant fibrous histiocytoma (MFH) cells as the antigen. Generally, MFH is considered to be a sarcoma derived from undifferentiated mesenchymal cells. Molecular biological analyses using the lysate of rat MFH cells revealed that A3 is a conformation specific antibody recognizing both N-glycan and peptide. A3-labeled cells in bone marrow were regarded as somatic stem cells, because the cells partly coexpressed CD90 and CD105 (both immature mesenchymal markers). In the hair follicle cycle, particularly the anagen, the immature epithelial cells (suprabasal cells) near the bulge and some immature mesenchymal cells in the disassembling dermal papilla and regenerating connective tissue sheath/hair papilla reacted to A3. In the cutaneous wound-healing process, A3-labeled epithelial cells participated in re-epithelialization in the wound bed, and apparently, the labeled cells were derived from the hair bulge; in addition, A3-labeled immature mesenchymal cells in the connective tissue sheath of hair follicles at the wound edge showed the expansion of the A3 immunolabeling. A3-labeled immature epithelial and mesenchymal cells contributed to morphogenesis in the hair cycle and tissue repair after a cutaneous wound. A3 could become a unique antibody to identify somatic stem cells capable of differentiating both epithelial and mesenchymal cells in rat tissues

Rat hair follicle-constituting cells labeled by a newly-developed somatic stem cell-recognizing antibody: a possible marker of hair follicle development

. A3 was generated as an antibody recognizing somatic stem cells in rat tissues. We investigated the distribution of A3-positive cells in developing rat hair follicles by immunolabeling. A3-positive cells began to be seen in the hair germ and peg in fetuses and neonates; the positive cells were epithelial cells above basal cells. Furthermore, A3-positive cells were seen in the outer root sheath adjacent to the bulge in mature hair follicles. Double immunofluorescence revealed that these A3- positive epithelial cells reacted to E-cadherin (for all epithelial elements) but not to CK15 (for basal cells/epithelial stem cells) or to nestin (for stem cells), indicating that A3-positive epithelial cells are suprabasal cells in the developing epidermic hair follicle. Additionally, spindle-shaped mesenchymal cells surrounding the hair peg and mature hair follicle reacted to A3; in double immunofluorescence, the A3-positive cells were located outside collagen type IV-positive glassy membrane, and reacted to vimentin (for mesenchmal cells), Thy-1 (for immature mesenchymal cells), CD34 (for stem cells) and nestin, but not to α- smooth muscle actin (for myofibroblasts); the positive cells were regarded as immature mesenchymal cells with stem cell nature in the connective tissue sheath of developing hair follicles. A3-positive epithelial and mesenchymal cells did not show proliferating activity. Collectively, it is considered that A3-positive cells seen in developing rat hair follicles may be quiescent postprogenitor cells with the potential to differentiate into either highly-differentiated epithelial or mesenchymal cells. A3 would become a useful antibody to know the kinetics of rat hair follicle-constituting cells