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

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

Functional impairment of Tax-specific but not cytomegalovirus-specific CD8+ T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers

<p>Abstract</p> <p>Background</p> <p>Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8⁺T-cells in various clinical status of HTLV-1 infection.</p> <p>Results</p> <p>By using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8⁺T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8⁺T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8⁺T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8⁺T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8⁺T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8⁺T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8⁺T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8⁺T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8⁺T-cells.</p> <p>Conclusions</p> <p>These findings indicated that Tax-specific CD8⁺T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8⁺T-cells in early stages.</p

Up-regulation of stanniocalcin 1 expression by 1,25-dihydroxy vitamin D3 and parathyroid hormone in renal proximal tubular cells

Stanniocalcin 1 and stanniocalcin 2 are two glycoprotein hormones, which act as calcium phosphate-regulating factor on intestine and kidney. We have previously reported that stanniocalcin 2 expression is positively and negatively controlled by 1,25(OH)2D3 and parathyroid hormone in renal proximal tubular cells. However, it has been unclear whether they regulate the stanniocalcin 1 gene expression. In this study, we identified the opossum stanniocalcin 1 cDNA sequence. The opossum stanniocalcin 1 amino acid sequence had 83% homology with human stanniocalcin 1, and has a conserved putative N-linked glycosylation site. Real-time PCR analysis using opossum kidney proximal tubular (OK-P) cells revealed that the mRNA levels of stanniocalcin 1 gene is up-regulated by both 1,25(OH)2D3 and parathyroid hormone in dose-dependent and time-dependent manners. We also demonstrated that the stanniocalcin 1 expression was increased in parathyroid hormone injected rat kidney. Furthermore, the mRNA expression of stanniocalcin 1 and stanniocalcin 2 were oppositely regulated by phorbol 12,13-myristic acetate, a specific PKC activator. Interestingly, the up-regulation of stanniocalcin 1 gene by 1,25(OH)2D3 and phorbol 12,13-myristic acetate were not prevented in the presence of actinomycin D, an RNA synthesis inhibitor. These results suggest that the stanniocalcin 1 gene expression is up-regulated by 1,25(OH)2D3 and parathyroid hormone through mRNA stabilization in renal proximal tubular cells

Identification and Modulation of the Key Amino Acid Residue Responsible for the pH Sensitivity of Neoculin, a Taste-Modifying Protein

Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor–taste substance in particular

Murine breast cancers disorganize the liver transcriptome in a zonated manner

がんが宿主の臓器に及ぼす悪影響を捉えた --がんをもつ個体における「肝機能の空間的制御」の破綻--. 京都大学プレスリリース. 2023-02-01.The spatially organized gene expression program within the liver specifies hepatocyte functions according to their relative distances to the bloodstream (i.e., zonation), contributing to liver homeostasis. Despite the knowledge that solid cancers remotely disrupt liver homeostasis, it remains unexplored whether solid cancers affect liver zonation. Here, using spatial transcriptomics, we thoroughly investigate the abundance and zonation of hepatic genes in cancer-bearing mice. We find that breast cancers affect liver zonation in various distinct manners depending on biological pathways. Aspartate metabolism and triglyceride catabolic processes retain relatively intact zonation patterns, but the zonation of xenobiotic catabolic process genes exhibits a strong disruption. The acute phase response is induced in zonated manners. Furthermore, we demonstrate that breast cancers activate innate immune cells in particular neutrophils in distinct zonated manners, rather than in a uniform fashion within the liver. Collectively, breast cancers disorganize hepatic transcriptomes in zonated manners, thereby disrupting zonated functions of the liver

上部消化管出血における血液凝固第XIII因子活性の低下

We report on blood coagulation factor XⅢ（F１３）activity in upper gastrointestinal bleeding （UGIB）due to peptic ulcer（PU）. 【Methods】From January ２０１１ to December ２０１６, 42 patients who had UGIB with PU, performed endoscopic hemostasis（EH）, and measured F１３ activity （normal range：７０‐１４０％）, were retrospectively studied. The clinical signs, peripheral blood, biochemistry, coagulation, F１３ activity, and endoscopic findings were examined by logistic regression analysis（LRA）in３２patients with successful hemostasis and１０patients with rebleeding. 【Results】There were２５elderly patients aged７０ and over. The average F１３ activity was ６５．７±２２．５％, ６８．３±２２．１％ in the case of successful hemostasis, below the lower limit of normal, and further decreased to ５７．５±１８．１％ in the case of rebleeding. LRA showed association with rebleeding in Forrest classification and F１３activity. 【Conclusion】F１３activity decreased in many cases of UGIB due to PU, and associated with rebleeding after EH