Pathology Department, School of Medicine, University of St Andrews, St Andrews, UK.
Mol Biol Rep. 2022 Sep;49(9):9113-9119. doi: 10.1007/s11033-022-07726-1. Epub 2022 Jul 6.
Two clinical case reports of humans with mutations in Itch reported distinct morphological defects such as stunted growth, macrocephaly, and dysmorphic features indicating a role for Itch in bone remodelling. Studies in mice have found that the encoded E3 ubiquitin ligase acts as a negative regulator of osteoclastogenesis, however no studies have investigated whether this is translatable to a human model.
Human peripheral blood monocytes were separated from whole blood and grown in M-CSF containing media. Media was later supplemented with RANKL to promote osteoclast differentiation. Transient siRNA-mediated Itch knockdown (si-Itch) in monocytes was verified by qPCR and western blot to confirm reduction in both Itch mRNA and protein respectively. Monocytes were aliquoted onto 96-well plates where confluence and osteoclast formation were analysed using automated cytometry analysis before and after staining for tartrate resistant acid phosphatase activity (TRAP). Cells were also stained with Hoechst33342 to look for multinucleate cells.
Cells treated with si-Itch showed an 80% knockdown in Itch mRNA and > 75% reduction in protein. Following the 7-day differentiation period, si-Itch caused a 47% increase in multinucleate cells and a 17% increase in numbers of large cellular bodies and, indicating an overall increase in mature osteoclast formation.
Our preliminary data shows silencing Itch expression increases the potential of primary human monocytes to differentiate into osteoclast-like cells in vitro.
两份关于人类基因突变病例报告显示，ITCH 基因突变会导致明显的形态缺陷，如生长迟缓、大头畸形和发育异常，这表明 ITCH 在骨重塑中起作用。在小鼠中的研究发现，编码的 E3 泛素连接酶作为破骨细胞生成的负调节剂，但尚无研究调查这是否适用于人类模型。
从全血中分离出人外周血单核细胞，并在含有 M-CSF 的培养基中培养。随后用 RANKL 补充培养基以促进破骨细胞分化。通过 qPCR 和 Western blot 验证单核细胞中的瞬时 siRNA 介导的 ITCH 敲低（si-ITCH），以分别确认 ITCH mRNA 和蛋白质的减少。将单核细胞分配到 96 孔板中，在使用抗酒石酸酸性磷酸酶活性（TRAP）染色之前和之后使用自动细胞计数分析来分析细胞融合和破骨细胞形成。还使用 Hoechst33342 染色寻找多核细胞。
用 si-ITCH 处理的细胞显示 ITCH mRNA 降低了 80%，蛋白质降低了超过 75%。在 7 天的分化期后，si-ITCH 导致多核细胞增加了 47%，大细胞体的数量增加了 17%，表明成熟破骨细胞形成的总体增加。
我们的初步数据表明，沉默 ITCH 表达可增加原代人单核细胞体外分化为破骨样细胞的潜力。
