Hanai Ayako, Kawabata Ayako, Nakajima Kenta, Masuda Kazuhiro, Urakawa Itaru, Abe Masahiro, Yamazaki Yuji, Fukumoto Seiji
R&D Division, Kyowa Kirin Co., Ltd., Tokyo, Japan.
Department of Endocrinology, Metabolism and Hematology, Tokushima University Graduate School of Medical Sciences, Tokushima, Japan.
Front Physiol. 2023 Jan 27;14:1102751. doi: 10.3389/fphys.2023.1102751. eCollection 2023.
Fibroblast growth factor 23 (FGF23), a hormone, mainly produced by osteocytes, regulates phosphate and vitamin D metabolism. By contrast, 1,25-dihydroxyvitamin D, 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 -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 D) using scRNA-seq. We first detected , , and expression in murine osteocytes by 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 expression was observed in some osteocytes isolated from femurs of calcitriol-injected mice, no expression was detected in untreated mice. In addition, the expression of several genes which are known to be changed after 1,25-dihydroxyvitamin D treatment such as , , , , and was also affected by calcitriol treatment in -expressing osteocytes, but not in those lacking expression, even after calcitriol administration. Furthermore, box-and-whisker plots indicated that expression was observed in osteocytes with higher expression levels of the , , and 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 D, and sensitivity to 1,25-dihydroxyvitamin D is one of the characteristics of osteocytes with expression. It is likely that there is a subpopulation of osteocytes expressing several genes, including , involved in phosphate metabolism.
成纤维细胞生长因子23（FGF23）是一种主要由骨细胞产生的激素，可调节磷酸盐和维生素D代谢。相比之下，维生素D的活性形式1,25-二羟基维生素D已被证明可增强FGF23的产生。虽然骨细胞在基因表达谱方面可能存在异质性，但尚未鉴定出表达特定基因的骨细胞亚群。单细胞RNA测序（scRNA-seq）技术可以表征单个细胞的转录组。最近，scRNA-seq已用于骨组织分析。然而，由于与骨细胞分离相关的技术困难，缺乏使用scRNA-seq分析来表征产生FGF23的骨细胞的研究。在本研究中，我们使用scRNA-seq对来自小鼠股骨的分泌FGF23的骨细胞进行了表征，以响应骨化三醇（1,25-二羟基维生素D）。我们首先通过原位杂交在小鼠骨细胞中检测到、和的表达，并将这些用作骨细胞的标记基因。脱钙、酶消化和去除CD45细胞后，对股骨细胞进行scRNA-seq。我们使用标记基因表达鉴定了含有骨细胞的细胞簇。虽然在从注射骨化三醇的小鼠股骨中分离的一些骨细胞中观察到表达，但在未处理的小鼠中未检测到表达。此外，在表达的骨细胞中，一些已知在1,25-二羟基维生素D处理后会发生变化的基因，如、、、和的表达也受到骨化三醇处理的影响，但在缺乏表达的骨细胞中，即使在给予骨化三醇后也没有受到影响。此外，箱线图表明，在、和基因表达水平较高的骨细胞中观察到表达，这些基因的失活突变已被证明会导致FGF23相关的低磷血症疾病。这些结果表明，骨细胞对1,25-二羟基维生素D的反应性存在异质性，对1,25-二羟基维生素D的敏感性是表达骨细胞的特征之一。可能存在一个表达包括在内的几种参与磷酸盐代谢基因的骨细胞亚群。
