Department of Hand and Foot Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021, China.
Department of Central Laboratory, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, China.
Cell Death Dis. 2023 Feb 20;14(2):145. doi: 10.1038/s41419-023-05681-8.
The mutations of HOXD13 gene have been involved in synpolydactyly (SPD), and the polyalanine extension mutation of Hoxd13 gene could lead to SPD in mice. In this study, a novel missense mutation of Hoxd13 (NM_000523: exon2: c.G917T: p.R306L) was identified in a Chinese family with SPD. The mice carrying the corresponding Hoxd13mutation were generated. The results showed that the homozygous mutation of Hoxd13 also caused SPD, but heterozygous mutation did not affect limbs development, which was different from that of SPD patients. With the increasing generation, the mice with homozygous Hoxd13 mutation presented more severe syndactyly. Western blotting showed that this mutation did not affect the protein expression of Hoxd13, suggesting that this mutation did not result in haploinsufficiency. Further analysis demonstrated that this homozygous Hoxd13mutation promoted osteoclast differentiation and bone loss, and enhanced the mRNA and protein expression of osteoclast-related genes Rank, c-Fos, and p65. Meanwhile, this homozygous Hoxd13 mutation elevated the level of phosphorylated Smad5 (pSmad5). Co-immunoprecipitation verified that this mutation attenuated the interaction between pSmad5 and HOXD13, suggesting that this mutation released more pSmad5. Inhibition of pSmad5 reduced the expression of Rank, c-Fos, and p65 despite in the mutation group. In addition, inhibition of pSmad5 repressed the osteoclast differentiation. ChIP assay confirmed that p65 and c-Fos could bind to the promoter of Rank. These results suggested that this novel Hoxd13 mutation promoted osteoclast differentiation by regulating Smad5/p65/c-Fos/Rank axis, which might provide a new insight into SPD development.
HOXD13 基因突变与并指症（SPD）有关，Hoxd13 基因的多聚丙氨酸延伸突变可导致小鼠发生 SPD。本研究在一个中国 SPD 家系中发现了 Hoxd13 的一个新错义突变（NM_000523：exon2：c.G917T：p.R306L）。构建了携带相应 Hoxd13 突变的小鼠。结果表明，Hoxd13 纯合突变也导致 SPD，但杂合突变不影响肢体发育，这与 SPD 患者不同。随着世代的增加，携带 Hoxd13 纯合突变的小鼠表现出更严重的并指。Western blot 显示该突变不影响 Hoxd13 蛋白的表达，提示该突变不导致单倍不足。进一步分析表明，该 Hoxd13 纯合突变促进破骨细胞分化和骨丢失，并增强破骨细胞相关基因 Rank、c-Fos 和 p65 的 mRNA 和蛋白表达。同时，该 Hoxd13 纯合突变增加了磷酸化 Smad5（pSmad5）的水平。共免疫沉淀验证了该突变削弱了 pSmad5 与 HOXD13 的相互作用，提示该突变释放了更多的 pSmad5。尽管在突变组中，pSmad5 的抑制降低了 Rank、c-Fos 和 p65 的表达。此外，pSmad5 的抑制抑制了破骨细胞分化。ChIP 分析证实 p65 和 c-Fos 可以结合到 Rank 的启动子上。这些结果表明，该新的 Hoxd13 突变通过调节 Smad5/p65/c-Fos/Rank 轴促进破骨细胞分化，这可能为 SPD 的发展提供新的见解。
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