31 research outputs found
シングルセルRNAシーケンシングによる1,25-ジヒドロキシビタミンD3反応性Fgf23発現骨細胞の同定
Fibroblast growth factor 23 (FGF23), a hormone, mainly produced by osteocytes, regulates phosphate and vitamin D metabolism. By contrast, 1,25-dihydroxyvitamin D3, the active form of vitamin D, has been shown to enhance FGF23 production. While it is likely that osteocytes are heterogenous in terms of gene expression profiles, specific subpopulations of Fgf23-expressing osteocytes have not been identified. Single-cell RNA sequencing (scRNA-seq) technology can characterize the transcriptome of an individual cell. Recently, scRNA-seq has been used for bone tissue analysis. However, owing to technical difficulties associated with isolation of osteocytes, studies using scRNA-seq analysis to characterize FGF23-producing osteocytes are lacking. In this study, we characterized osteocytes secreting FGF23 from murine femurs in response to calcitriol (1,25-dihydroxyvitamin D3) using scRNA-seq. We first detected Dmp1, Mepe, and Phex expression in murine osteocytes by in situ hybridization and used these as marker genes of osteocytes. After decalcification, enzyme digestion, and removal of CD45+ cells, femoral bone cells were subjected to scRNA-seq. We identified cell clusters containing osteocytes using marker gene expression. While Fgf23 expression was observed in some osteocytes isolated from femurs of calcitriol-injected mice, no Fgf23 expression was detected in untreated mice. In addition, the expression of several genes which are known to be changed after 1,25-dihydroxyvitamin D3 treatment such as Ccnd2, Fn1, Igfbp7, Pdgfa, and Timp1 was also affected by calcitriol treatment in Fgf23-expressing osteocytes, but not in those lacking Fgf23 expression, even after calcitriol administration. Furthermore, box-and-whisker plots indicated that Fgf23 expression was observed in osteocytes with higher expression levels of the Fam20c, Dmp1, and Phex genes, whose inactivating mutations have been shown to cause FGF23-related hypophosphatemic diseases. These results indicate that osteocytes are heterogeneous with respect to their responsiveness to 1,25-dihydroxyvitamin D3, and sensitivity to 1,25-dihydroxyvitamin D3 is one of the characteristics of osteocytes with Fgf23 expression. It is likely that there is a subpopulation of osteocytes expressing several genes, including Fgf23, involved in phosphate metabolism
Lactisole: an inhibitor of the glucose-sensing receptor T1R3 expressed in pancreatic B-cells
Glucose activates the glucose-sensing receptor T1R3 and facilitates its own metabolism in pancreatic beta-cells. An inhibitor of this receptor would be helpful in elucidating the physiological function of the glucose-sensing receptor. The present study was conducted to examine whether or not lactisole can be used as an inhibitor of the glucose-sensing receptor. In MIN6 cells, in a dose-dependent manner, lactisole inhibited insulin secretion induced by sweeteners, acesulfame-K, sucralose and glycyrrhizin. The IC50 was similar to 4 mmol/l. Lactisole attenuated the elevation of cytoplasmic Ca2+ concentration ([Ca2+](c)) evoked by sucralose and acesulfame-K but did not affect the elevation of intracellular cAMP concentration ([cAMP](c)) induced by these sweeteners. Lactisole also inhibited the action of glucose in MIN6 cells. Thus, lactisole significantly reduced elevations of intracellular [NADH] and intracellular [ATP] induced by glucose, and also inhibited glucose-induced insulin secretion. To further examine the effect of lactisole on T1R3, we prepared HEK293 cells stably expressing mouse T1R3. In these cells, sucralose elevated both [Ca2+](c) and [cAMP](c). Lactisole attenuated the sucralose-induced increase in [Ca2+](c) but did not affect the elevation of [cAMP](c). Finally, lactisole inhibited insulin secretion induced by a high concentration of glucose in mouse islets. These results indicate that the mouse glucose-sensing receptor was inhibited by lactisole. Lactisole may be useful in assessing the role of the glucose-sensing receptor in mouse pancreatic beta-cells
Single-cell RNA sequencing identifies Fgf23-expressing osteocytes in response to 1,25-dihydroxyvitamin D3 treatment
Fibroblast growth factor 23 (FGF23), a hormone, mainly produced by osteocytes, regulates phosphate and vitamin D metabolism. By contrast, 1,25-dihydroxyvitamin D3, the active form of vitamin D, has been shown to enhance FGF23 production. While it is likely that osteocytes are heterogenous in terms of gene expression profiles, specific subpopulations of Fgf23-expressing osteocytes have not been identified. Single-cell RNA sequencing (scRNA-seq) technology can characterize the transcriptome of an individual cell. Recently, scRNA-seq has been used for bone tissue analysis. However, owing to technical difficulties associated with isolation of osteocytes, studies using scRNA-seq analysis to characterize FGF23-producing osteocytes are lacking. In this study, we characterized osteocytes secreting FGF23 from murine femurs in response to calcitriol (1,25-dihydroxyvitamin D3) using scRNA-seq. We first detected Dmp1, Mepe, and Phex expression in murine osteocytes by in situ hybridization and used these as marker genes of osteocytes. After decalcification, enzyme digestion, and removal of CD45+ cells, femoral bone cells were subjected to scRNA-seq. We identified cell clusters containing osteocytes using marker gene expression. While Fgf23 expression was observed in some osteocytes isolated from femurs of calcitriol-injected mice, no Fgf23 expression was detected in untreated mice. In addition, the expression of several genes which are known to be changed after 1,25-dihydroxyvitamin D3 treatment such as Ccnd2, Fn1, Igfbp7, Pdgfa, and Timp1 was also affected by calcitriol treatment in Fgf23-expressing osteocytes, but not in those lacking Fgf23 expression, even after calcitriol administration. Furthermore, box-and-whisker plots indicated that Fgf23 expression was observed in osteocytes with higher expression levels of the Fam20c, Dmp1, and Phex genes, whose inactivating mutations have been shown to cause FGF23-related hypophosphatemic diseases. These results indicate that osteocytes are heterogeneous with respect to their responsiveness to 1,25-dihydroxyvitamin D3, and sensitivity to 1,25-dihydroxyvitamin D3 is one of the characteristics of osteocytes with Fgf23 expression. It is likely that there is a subpopulation of osteocytes expressing several genes, including Fgf23, involved in phosphate metabolism
RUNX1 transactivates BCR-ABL1 expression in Philadelphia chromosome positive acute lymphoblastic leukemia
The emergence of tyrosine kinase inhibitors as part of a front-line treatment has greatly improved the clinical outcome of the patients with Ph⁺ acute lymphoblastic leukemia (ALL). However, a portion of them still become refractory to the therapy mainly through acquiring mutations in the BCR-ABL1 gene, necessitating a novel strategy to treat tyrosine kinase inhibitor (TKI)-resistant Ph⁺ ALL cases. In this report, we show evidence that RUNX1 transcription factor stringently controls the expression of BCR-ABL1, which can strategically be targeted by our novel RUNX inhibitor, Chb-M'. Through a series of in vitro experiments, we identified that RUNX1 binds to the promoter of BCR and directly transactivates BCR-ABL1 expression in Ph⁺ ALL cell lines. These cells showed significantly reduced expression of BCR-ABL1 with suppressed proliferation upon RUNX1 knockdown. Moreover, treatment with Chb-M' consistently downregulated the expression of BCR-ABL1 in these cells and this drug was highly effective even in an imatinib-resistant Ph⁺ ALL cell line. In good agreement with these findings, forced expression of BCR-ABL1 in these cells conferred relative resistance to Chb-M'. In addition, in vivo experiments with the Ph⁺ ALL patient-derived xenograft cells showed similar results. In summary, targeting RUNX1 therapeutically in Ph⁺ ALL cells may lead to overcoming TKI resistance through the transcriptional regulation of BCR-ABL1. Chb-M' could be a novel drug for patients with TKI-resistant refractory Ph⁺ ALL
Identification and Characterization of a Ferritin Gene and Its Product from the Multicellular Green Alga Ulva pertusa
Magnetic metal-complex-conducting copolymer core–shell nanoassemblies for a single-drug anticancer platform
Magnetic metal-complex-conducting copolymer core–shell nanoassemblies for a single-drug anticancer platform
Nanoparticulate agents for magnetic drug delivery systems (DDSs) have extensive applications in targeted drug delivery, contrast imaging and therapeutics. However, no simple synthetic method for magnetic DDS agents has been developed without the need to add magnetic nanoparticles. Here, we describe the one-step fabrication of ‘all-in-one’ magneto-assemblies using an ‘inorganic-metal-salt-free’ method, involving spontaneous self-assembly of the water-insoluble prodrug μ-oxo-bis(N,N′-ethylenebis(salicylideniminato)iron) [Fe(salen)] (magnetic core) with polypyrrole (PPy)-b-polycaprolactone (PCL) smart diblock copolymers. In the system, PCL serves as a heat-responsive core scaffold, and PPy serves as an electronic core-size controller and pH-responsive shell. This core–shell nanocomposite has a high-loading capacity (~90%), and the core size is tunable by incorporating albumin or gum arabic as bio-coating agents, which also provide colloidal stability, biocompatibility and thermo-stability. Fe(salen), which has intrinsic antitumor activity, also has ubiquitous magnetic properties, which are dramatically enhanced in these molecular assemblies with magnetic coupling. Moreover, these multifunctional nanoassemblies can be delivered magnetically, can serve as magnetic resonance imaging contrast agents, can generate magneto-hyperthermal effects and can enable magnetic field-triggered release of Fe(salen) molecules under acidic conditions