A novel HOXD13 mutation causes synpolydactyly and promotes osteoclast differentiation by regulating pSMAD5/p65/c-Fos/RANK axis

Abstract

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 HoxD13 mutation 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 HOXD13 expression, suggesting that this mutation did not result in haploinsufficiency. Further analysis demonstrated that this homozygous HoxD13 mutation promoted osteoclast differentiation and bone loss, and enhanced the 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.