Modelling Takenouchi-Kosaki syndrome using disease-specific iPSCs.

Takenouchi-Kosaki Syndrome (TKS) is a congenital multi-organ disorder caused by the de novo missense mutation c.191A > G p. Tyr64Cys (Y64C) in the CDC42 gene. We previously elucidated the functional abnormalities and thrombopoietic effects of Y64C using HEK293 and MEG01 cells. In the present study, we used iPSCs derived from TKS patients to model the disease and successfully recapitulated macrothrombocytopenia, a prominent TKS phenotype. The megakaryopoietic differentiation potential of TKS-iPSCs and platelet production capacity were examined using an efficient platelet production method redesigned from existing protocols. The results obtained showed that TKS-iPSCs produced fewer hematopoietic progenitor cells, exhibited defective megakaryopoiesis, and released platelets with an abnormally low count and giant morphology. We herein report the first analysis of TKS-iPSC-derived megakaryocytes and platelets, and currently utilize this model to perform drug evaluations for TKS. Therefore, our simple yet effective differentiation method, which mimics the disease in a dish, is a feasible strategy for studying hematopoiesis and related diseases.

Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells.

Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.

Calponin 3 regulates stress fiber formation in dermal fibroblasts during wound healing.

Skin wound healing is an intricate process involving various cell types and molecules. In granulation tissue, fibroblasts proliferate and differentiate into myofibroblasts and generate mechanical tension for wound closure and contraction. Actin stress fibers formed in these cells, especially those containing α-smooth muscle actin (α-SMA), are the central machinery for contractile force generation. In the present study, calponin 3 (CNN3), which has a phosphorylation-dependent actin-binding property, was identified in the molecular mechanism underlying stress fiber formation. CNN3 was expressed by fibroblasts/myofibroblasts in the proliferation phase of wound healing, and was associated with α-SMA in stress fibers formed by cultured dermal fibroblasts. CNN3 expression was post-transcriptionally regulated by tension, as demonstrated by disruption of actin filament organization under floating culture or blebbistatin treatment. CNN3 knockdown in primary fibroblasts impaired stress fiber formation, resulting in a phenotype of decreased cellular dynamics such as cell motility and contractile ability. These findings indicate that CNN3 participates in actin stress fiber remodeling, which is required for cell motility and contraction of dermal fibroblasts in the wound healing process.

Rock-dependent calponin 3 phosphorylation regulates myoblast fusion.

Myogenesis occurs during embryonic development as well as regeneration following postnatal muscle fiber damage. Herein, we show that acidic calponin or calponin 3 (CNN3) regulates both myoblast cell fusion and muscle-specific gene expressions. Overexpression of CNN3 impaired C2C12 cell fusion, whereas CNN3 gene knockdown promoted skeletal myosin expression and fusion. CNN3 was phosphorylated at Ser293/296 in the C-terminal region. The basal inhibitory property of CNN3 against myoblast differentiation was enhanced by Ser293/296Ala mutation or deletion of the C-terminal region, and this inhibition was reversed by Ser293/296Asp mutation. Ser293/296 phosphorylation was required for CNN3 to bind actin and was dependent on Rho-associated kinases 1/2 (ROCK 1/2). Gene knockdown of ROCK1/2 suppressed CNN3 phosphorylation and impaired myoblast fusion, and these effects were partially attenuated by additional CNN3 overexpression of Ser293/296Asp CNN3. These findings indicated that CNN3 phosphorylation by ROCK blunts CNN3's inhibitory effects on muscle cell differentiation and fusion. In muscle tissues, satellite cells, but not mature myofibrils, expressed CNN3. CNN3 was also expressed and phosphorylated during myotube induction in isolated muscle satellite cells. Taken together, these results indicate that CNN3 is a downstream regulator of the ROCK signaling pathway for myogenesis.

Calponin 3 regulates actin cytoskeleton rearrangement in trophoblastic cell fusion.

Cell-cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent.

Role of neutrophils in matrix metalloproteinase activity in the preimplantation mouse uterus.

Matrix metalloproteinases (MMPs) have been implicated in embryonal implantation processes such as trophoblast invasion and decidualization. The temporal and spatial distributions of MMP bioactivities were analyzed by in situ zymography, which indicated these activities to be markedly increased in the postcoital mouse uterus compared with those in the later implantation stage. Activity was ascribed to proMMP9, which moved from the uterine serosa to the endometrium but was not associated with mRNA up-regulation. The activity was colocalized with infiltrating neutrophils, and neutropenic mice did not exhibit MMP9 expression. Removing the seminal vesicles from male mice abolished the postcoital increase in MMP9 in the female. These results indicate the major MMP activity in the preimplantation uterus to originate in proMMP9-bearing neutrophils attracted by seminal plasma. Considering our results together with those of previous reports of reduced fertility in Mmp9-deficient female mice, we speculate that neutrophil infiltration participates in the extracellular matrix degradation needed to support pregnancy.