Department of Bone and Mineral Research, Research Institute, Osaka Women's and Children's Hospital, Osaka Prefectural Hospital Organization, Izumi, Osaka 594-1101, Japan; Department of Pediatrics, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
Department of Bone and Mineral Research, Research Institute, Osaka Women's and Children's Hospital, Osaka Prefectural Hospital Organization, Izumi, Osaka 594-1101, Japan.
Bone. 2024 Apr;181:117044. doi: 10.1016/j.bone.2024.117044. Epub 2024 Feb 6.
X-linked hypophosphatemia (XLH) is caused by inactivating variants of the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Although the overproduction of fibroblast growth factor 23 (FGF23) is responsible for hypophosphatemia and impaired vitamin D metabolism, the pathogenesis of XLH remains unclear. We herein generated PHEX-knockout (KO) human induced pluripotent stem (iPS) cells by applying CRISPR/Cas9-mediated gene ablation to an iPS clone derived from a healthy male, and analyzed PHEX-KO iPS cells with deletions extending from exons 1 to 3 and frameshifts by inducing them to differentiate into the osteoblast lineage. We confirmed the increased production of FGF23 in osteoblast lineage cells differentiated from PHEX-KO iPS cells. In vitro mineralization was enhanced in osteoblast lineage cells from PHEX-KO iPS cells than in those from isogenic control iPS cells, which reminded us of high bone mineral density and enthesopathy in patients with XLH. The extracellular level of pyrophosphate (PPi), an inhibitor of mineralization, was elevated, and this increase appeared to be partly due to the reduced activity of tissue non-specific alkaline phosphatase (TNSALP). Osteoblast lineage cells derived from PHEX-KO iPS cells also showed the increased expression of multiple molecules such as dentine matrix protein 1, osteopontin, RUNX2, FGF receptor 1 and early growth response 1. This gene dysregulation was similar to that in the osteoblasts/osteocytes of Phex-deficient Hyp mice, suggesting that common pathogenic mechanisms are shared between human XLH and Hyp mice. Moreover, we found that the phosphorylation of CREB was markedly enhanced in osteoblast lineage cells derived from PHEX-KO iPS cells, which appeared to be associated with the up-regulation of the parathyroid hormone related protein gene. PHEX deficiency also affected the response of the ALPL gene encoding TNSALP to extracellular Pi. Collectively, these results indicate that complex intrinsic abnormalities in osteoblasts/osteocytes underlie the pathogenesis of human XLH.
X 连锁低磷血症（XLH）是由磷酸盐调节内肽酶同源物 X 连锁（PHEX）基因的失活变异引起的。尽管成纤维细胞生长因子 23（FGF23）的过度产生导致低磷血症和维生素 D 代谢受损，但 XLH 的发病机制仍不清楚。我们通过应用 CRISPR/Cas9 介导的基因敲除，对源自健康男性的 iPS 克隆进行基因敲除，从而生成了 PHEX 敲除（KO）人诱导多能干细胞（iPS），并分析了从外显子 1 到 3 缺失和移码的 PHEX-KO iPS 细胞，诱导它们分化为成骨细胞谱系。我们证实了 PHEX-KO iPS 细胞分化的成骨细胞谱系细胞中 FGF23 的产量增加。与源自同基因对照 iPS 细胞的成骨细胞谱系细胞相比，源自 PHEX-KO iPS 细胞的成骨细胞谱系细胞的体外矿化增强，这让我们想起了 XLH 患者的高骨密度和肌腱病。细胞外焦磷酸盐（PPi）水平升高，而这种增加似乎部分是由于组织非特异性碱性磷酸酶（TNSALP）活性降低所致。源自 PHEX-KO iPS 细胞的成骨细胞谱系细胞还表现出多个分子的表达增加，例如牙本质基质蛋白 1、骨桥蛋白、RUNX2、FGF 受体 1 和早期生长反应 1。这种基因失调类似于 Phex 缺陷 Hyp 小鼠的成骨细胞/骨细胞，表明人类 XLH 和 Hyp 小鼠之间存在共同的发病机制。此外，我们发现源自 PHEX-KO iPS 细胞的成骨细胞谱系细胞中 CREB 的磷酸化明显增强，这似乎与甲状旁腺激素相关蛋白基因的上调有关。PHEX 缺乏还影响了编码 TNSALP 的 ALPL 基因对细胞外 Pi 的反应。总之，这些结果表明，人类 XLH 发病机制的基础是成骨细胞/骨细胞中复杂的内在异常。
