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Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) are a promising cell source for regenerative medicine and drug discovery. However, the use of animal models for studying human cardiomyocytes derived from hiPSCs in vivo is limited and challenging. Given the shared properties between humans and zebrafish, their ethical advantages over mammalian models, and their immature immune system that is rejection-free against xenografted human cells, zebrafish provide a suitable alternative model for xenograft studies. We microinjected fluorescence-labeled cardiac lineage cells derived from hiPSCs, specifically mesoderm or cardiac mesoderm cells, into the yolk and the area proximal to the outflow tract of the linear heart at 24 hours post-fertilization (hpf). The cells injected into the yolk survived and did not migrate to other tissues. In contrast, the cells injected contiguous with the outflow tract of the linear heart migrated into the pericardial cavity and heart. After 1 day post injection (1 dpi, 22-24 hpi), the injected cells migrated into the pericardial cavity and heart. Importantly, we observed heartbeat-like movements of some injected cells in the zebrafish heart after 1 dpi. These results suggested successful xenografting of hiPSC-derived cardiac lineage cells into the zebrafish embryo heart. Thus, we developed a valuable tool using zebrafish embryos as a model organism for investigating the molecular and cellular mechanisms involved in the grafting process. This is essential in developing cell transplantation-based cardiac therapeutics as well as for drug testing, notably contributing to advancements in the field of cardio-medicine., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Kiyoshi Ohnuma reports financial support was provided by Japan Society for the Promotion of Science. Chinmoy Patra reports financial support was provided by Japan Society for the Promotion of Science. Mika Takahi reports financial support was provided by Nagaoka University of Technology Development Education Research Promotion Association., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Malignant pleural mesothelioma (MPM) is an aggressive malignancy arising from mesothelial lining of pleura. It is associated with a poor prognosis, partly due to the lack of a precise understanding of the molecular mechanisms associated with its malignant behavior. In the present study, we expanded on our previous studies on cell cycle control of MPM cells by targeting CD26 molecule with humanized anti-CD26 monoclonal antibody (HuCD26mAb), focusing particularly on ubiquitin-specific protease 22 (USP22). We showed that USP22 protein expression is detected in clinical specimens of MPM and that USP22 knockdown, as well as CD26 knockdown, significantly inhibits the growth and proliferation of MPM cells in vitro and in vivo. Moreover, depletion of both USP22 and CD26 suppresses MPM cell proliferation even more profoundly. Furthermore, expression levels of USP22 correlate with those of CD26. HuCD26mAb treatment induces a decrease in USP22 level through its interaction with the CD26 molecule, leading to increased levels of ubiquitinated histone H2A and p21. By demonstrating a CD26-related linkage with USP22 in MPM cell inhibition induced by HuCD26mAb, our present study hence characterizes USP22 as a novel target molecule while concurrently suggesting a new therapeutic strategy for MPM., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We previously reported that inhibition of dipeptidyl peptidase (DPP)-4, the catalytic site of CD26, prevents atherosclerosis in animal models through suppression of inflammation; however, the underlying molecular mechanisms have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae located on the surface of the cellular membrane, has been reported to modulate inflammatory responses by binding to CD26 in T cells. In this study, we investigated the role of Cav-1 in the suppression of inflammation mediated by the DPP-4 inhibitor, teneligliptin, using mouse and human macrophages. Mouse peritoneal macrophages were isolated from Cav-1 +/+ and Cav-1 -/- mice after stimulation with 3% thioglycolate. Inflammation was induced by the toll-like receptor (TLR)4 agonist, lipopolysaccharide (LPS), isolated from Escherichia coli. The expression of pro-inflammatory cytokines was determined using reverse transcription-polymerase chain reaction. Co-expression of Cav-1 and CD26 was detected using immunohistochemistry in both mouse and human macrophages. Teneligliptin treatment (10 nmol/L) suppressed the LPS-induced expression of interleukin (IL)-6 (70%) and tumor necrosis factor-α (37%) in peritoneal macrophages isolated from Cav-1 +/+ mice. However, teneligliptin did not have any effect on the macrophages from Cav-1 -/- mice. In human monocyte/macrophage U937 cells, teneligliptin treatment suppressed LPS-induced expression of pro-inflammatory cytokines in a dose-dependent manner (1-10 nmol/L). These anti-inflammatory effects of teneligliptin were mimicked by gene knockdown of Cav-1 or CD26 using small interfering RNA transfection. Furthermore, neutralization of these molecules using an antibody against CD26 or Cav-1 also showed similar suppression. Teneligliptin treatment specifically inhibited TLR4 and TLR5 agonist-mediated inflammatory responses, and suppressed LPS-induced phosphorylation of IL-1 receptor-associated kinase 4, a downstream molecule of TLR4. Next, we determined whether teneligliptin could directly inhibit the physical interaction between Cav-1 and CD26 using the Biacore system. Binding of CD26 to Cav-1 protein was detected. Unexpectedly, teneligliptin also bound to Cav-1, but did not interfere with CD26-Cav-1 binding, suggesting that teneligliptin competes with CD26 for binding to Cav-1. In conclusion, we demonstrated that Cav-1 is a target molecule for DPP-4 inhibitors in the suppression of TLR4-mediated inflammation in mouse and human macrophages., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulating the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cas-L/NEDD9 is a cytoplasmic docking protein downstream of β1 integrin-mediated signaling pathway and is essential for cellular migration and β1 integrin-mediated costimulation of T cells. We previously found that increased number of Cas-L positive leukocytes migrated into the inflamed joints of HTLV-I tax transgenic mice which spontaneously develop polyarthritis, suggesting a role of Cas-L in rheumatoid arthritis (RA) pathophysiology. Our current study expanded these findings on the role of Cas-L/NEDD9 in the development of RA by analyzing the pathophysiological changes in a Nedd9(-/-) mouse collagen-induced arthritis (CIA) model. Nedd9(-/-) mice exhibited a decrease in arthritis severity as compared to Nedd9(+/+) mice. In addition, as being conducted bone marrow transplantation experiments with a CIA model, Nedd9(-/-)→Nedd9(+/+) transplant showed a decrease in the incidence and severity score of arthritis, compared to those of Nedd9(+/+)→Nedd9(-/-) transplant. For analysis of serum levels of various cytokines, IL-1β, IL-6, IL-17, TNF-α, IFN-γ and anti-collagen antibody were decreased, while IL-4 and IL-10 levels were increased, in Nedd9(-/-) mice as compared to those in Nedd9(+/+) mice. Furthermore, collagen-mediated cellular responses of lymphocytes isolated from spleen or affected lymph nodes of Nedd9(-/-) mice were reduced. Our results strongly suggest that Cas-L/NEDD9 plays a pivotal role in the pathophysiology of CIA, and that Cas-L/NEDD9 may be a potential molecular target for the treatment of RA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Malignant pleural mesothelioma (MPM) is an aggressive malignancy arising from mesothelial lining of pleura. It is generally associated with a history of asbestos exposure and has a very poor prognosis, partly due to the lack of a precise understanding of the molecular mechanisms associated with its malignant behavior. In the present study, we expanded on our previous studies on the enhanced motility and increased CD26 expression in MPM cells, with a particular focus on integrin adhesion molecules. We found that expression of CD26 upregulates periostin secretion by MPM cells, leading to enhanced MPM cell migratory and invasive activity. Moreover, we showed that upregulation of periostin expression results from the nuclear translocation of the basic helix-loop-helix transcription factor Twist1, a process that is mediated by CD26-associated activation of Src phosphorylation. While providing new and profound insights into the molecular mechanisms involved in MPM biology, these findings may also lead to the development of novel therapeutic strategies for MPM., (Copyright © 2014 Elsevier Inc. All rights reserved.)

CD26/DPP-4 is abundantly expressed on capillary of inflamed lesion as well as effector T cells. Recently, CD26/dipeptidyl peptidase 4 (DPP-4) inhibition has been used as a novel oral therapeutic approach for patients with type 2 diabetes. While accumulating data indicate that vascular inflammation is a key feature of both micro- and macro-vascular complications in diabetes, the direct role of CD26/DPP-4 in endothelial biology is to be elucidated. We herein showed that proinflammatory cytokines such as tumor necrosis factor or interleukin-1 reduce expression of CD26 on microvascular endothelial cells, and that genetical or pharmacological inhibition of CD26/DPP-4 enhances endothelial growth both in vitro and in vivo. With DPP-4 inhibitors being used widely in the treatment of type 2 diabetes, our data strongly suggest that DPP-4 inhibition plays a pivotal role in endothelial growth and may have a potential role in the recovery of local circulation following diabetic vascular complications., (Copyright © 2010. Published by Elsevier Inc.)

Motile cilia play crucial roles in the maintenance of homeostasis in vivo. Defects in the biosynthesis of cilia cause immotile cilia syndrome, also known as primary ciliary dyskinesia (PCD), which is associated with a variety of complex diseases. In this study, we found that inhibitory Smad proteins, Smad7 and Smad6, significantly promoted the differentiation of mouse embryonic stem (ES) cells into ciliated cells. Moreover, these Smad proteins specifically induced morphologically distinct Musashi1-positive ciliated cells. These results suggest that inhibitory Smad proteins could be important regulators not only for the regulation of ciliated cell differentiation, but also for the subtype specification of ciliated cells during differentiation from mouse ES cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development., (Copyright © 2010 Elsevier Inc. All rights reserved.)

CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

During vertebrate somitogenesis, various transcriptional factors function coordinately to determine the position of the somite boundary. Previously, we reported on the signaling crosstalk that occurs between two major transcription factors involved in somitogenesis, Tbx6 and mespb/mesp2. These factors synergistically activated the expression of a downstream gene, bowline/Ripply2, which is essential for precise formation of the somite boundary. However, the molecular mechanism underlying this synergistic effect remains unclear. In this report, we found that the Tbx6 and mespb proteins interacted physically with each other. Pulldown assays with various deletion mutants of these proteins identified the essential domains for this physical interaction. Finally, we found that interference with the physical interaction by a dominant-negative form of mespb, mespbDeltaDBD, abrogated the expression of the bowline gene during Xenopus somitogenesis. These results indicate that the appropriate expression of bowline/Ripply2 is regulated by a direct interaction between the Tbx6 and mespb proteins during Xenopus somitogenesis.

We previously reported that HEXIM1 (hexamethylene bisacetamide-inducible protein 1), which suppresses transcription elongation via sequestration of positive transcription elongation factor b (P-TEFb) using 7SK RNA as a scaffold, directly associates with glucocorticoid receptor (GR) to suppress glucocorticoid-inducible gene activation. Here, we revealed that the hinge region of GR is essential for its interaction with HEXIM1, and that oxosteroid receptors including GR show sequence homology in their hinge region and interact with HEXIM1, whereas the other members of nuclear receptors do not. We also showed that HEXIM1 suppresses GR-mediated transcription in two ways: sequestration of P-TEFb by HEXIM1 and direct interaction between GR and HEXIM1. In contrast, peroxisome proliferator-activated receptor gamma-dependent gene expression is negatively modulated by HEXIM1 solely via sequestration of P-TEFb. We, therefore, conclude that HEXIM1 may act as a gene-selective transcriptional regulator via direct interaction with certain transcriptional regulators including GR and contribute to fine-tuning of, for example, glucocorticoid-mediated biological responses.

We identified human decapping enzyme 2 (hDCP2) as a binding protein with Ro52, being colocalized in processing bodies (p-bodies). We also showed that the N-terminus and C-terminus of Ro52 bound to hDCP2. Moreover, Ro52 enhanced decapping activity of hDCP2 in a dose-dependent manner. Our data support the novel notion of the association between Ro52 with hDCP2 protein in cytoplasmic p-bodies, playing a role in mRNA metabolism in response to cellular stimulation.

Cardiomyocytes are known to differentiate spontaneously from embryonic stem (ES) cells when they formed aggregates, so called "embryoid bodies", in the presence of serum. In this study, we explored the induction of cardiomyocytes from mouse ES cells in chemically defined serum-free medium by using a mesoderm-inducing factor, BMP4. Comparing the different inductive methods, we found a candidate cell surface marker, N-cadherin, for cardiomyocyte progenitors from ES cells. N-cadherin-positive cells highly expressed cardiogenic markers, Nkx2.5, Tbx5, and Isl1, and showed a high differentiation rate into cardiomyocyte lineage. These results indicate that N-cadherin can be a useful cell surface marker for the progenitors of cardiomyocyte differentiated from ES cells in the serum-free culture.