A novel Hoxd13 mutation causes synpolydactyly and promotes osteoclast differentiation by regulating pSmad5/p65/c-Fos/Rank axis

Abstract

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.

Fig. 1. A novel missense mutation of Hoxd13 was identified in a Chinese family with synpolydactyly (SPD).

A The pedigree of a four-generation Chinese family with SPD. Arrow represented the proband. Circles and squares represented female and male, respectively. Blank and black represented the unaffected and affected members, respectively. B The clinical characteristics of affected members. C Whole-exon sequencing and Sanger sequencing were performed to identify the mutation of Hoxd13 in the affected members.

Fig. 2. The Hoxd13 mutation caused the SPD phenotypes in mice.

A The sequence alignment of Hoxd13 from human and mouse. B Agarose gel electrophoresis and Sanger sequencing were performed to analyze the genotypes of mice. C The images of mice limbs were captured by micro-CT. All experiments were repeated three times.

Fig. 3. The Hoxd13 mutation promoted the osteoclast differentiation.

A The effect of Hoxd13 mutation on the expression of HOXD13 protein. B The effect of Hoxd13 mutation on osteoclast differentiation analyzed using TRAP staining. ***p < 0.001. C The effect of Hoxd13 mutation on the mRNA expression of osteoclast-associated genes. **p < 0.01; ***p < 0.001. D The effect of Hoxd13 mutation on the expression of osteoclast-associated proteins. *p < 0.05; **p < 0.01. All experiments were repeated at least three times.

Fig. 4. The Hoxd13 mutation caused bone loss in vivo.

A The effect of Hoxd13 mutation on the osteoclast differentiation in bone tissues. B In vivo imaging of the tibia and femur from mice carrying wild and mutant Hoxd13 by micro-CT. C The BV/TV, BS/BV, Tb.Sp, and Tb.Th obtained using micro-CT. *p < 0.05; **p < 0.01. D Immunohistochemistry assay was used to determine the expression of Rank, p65, and c-Fos in bone tissues. *p < 0.05. All experiments were repeated three times.

Fig. 5. The Hoxd13 mutation increased the expression of Rank, c-Fos, and phosphorylated p65 by releasing pSmad5.

A The effect of Hoxd13 mutation on the expression and phosphorylation of Smad5 (pSmad5). **p < 0.01. B The effect of Hoxd13 mutation on the interaction between HOXD13 and pSmad5. *p < 0.05. C The bone marrow monocytes from wild and mutant mice were treated with 1200 nM dorsomorphin, and were then exposed to M-CSF and RANKL for 5 days. Western blot was performed to determine the expression of pSmad5, Rank, p65, and c-Fos. *p < 0.05; **p < 0.01; ***p < 0.001; ^##p < 0.01 vs MT groups; ^###p < 0.001 vs MT groups. D TRAP staining was performed to determine the osteoclast differentiation. **p < 0.01; ^##p < 0.01 vs MT groups. All experiments were repeated three times.

Fig. 6. The Hoxd13 mutation regulated the transcription of Rank via p65 and c-Fos.

A The probable binding site of p65 in the promoter of Rank was predicted by JASPAR, and was confirmed by ChIP assay. B The probable binding site of c-Fos in the promoter of Rank was predicted by JASPAR, and was confirmed by ChIP assay. All experiments were repeated three times.

Fig. 7. The Hoxd13 mutation promoted osteoclast differentiation by regulating pSmad5/p65/c-Fos/Rank axis.

Schematic diagram described the possible functional mechanism of the Hoxd13 mutation in the osteoclast differentiation.