A novel missense mutation of HoxD13 was identified in a Chinese family with SPD
The pedigree of the Chinese family with SPD was shown in Fig. 1A. In this family, this phenotype affected four successive generations composed of 48 members, among of which contained 19 affected members. The proband (No. 45) was a 1.5-year-old boy with SPD. SPD of the middle and ring fingers was observed in both hands. The distal phalanx at the end of the middle and ring fingers was bony connected, and there was excess phalanx between the two fingers (Fig. 1B). The middle and end phalanx of the index and little fingers were deformed with deflection (Fig. 1B). SPD of the second and third toes was observed in both feet. In this study, 28 members were enrolled, including 15 affected members and 13 unaffected family members. Whole-exome sequencing was performed to identify the DNA mutations in affected members compared to the healthy controls. The results showed that a novel missense mutation in HoxD13 (nm_000523: exon2: c.g917t: p.r306l) was observed in all 15 affected members, but not in all 13 unaffected members (Fig. 1C), which were confirmed by Sanger sequencing.
The HoxD13 mutation caused the SPD phenotype in mice
We next constructed transgenic mice carrying HoxD13 mutation (G905T) according to sequence alignment (Fig. 2A). F1 generation mice (No. 116, 119, 121, 123, 126, 127, and 129) were identified as heterozygous mutant mice by PCR and sequencing (Fig. 2B). Interestingly, in F2 generation mice, only homozygous HoxD13 mutation caused obvious SPD phenotype, while did not lead to bone fusion visualized by micro CT (Fig. 2C). As increasing generations, homozygous HoxD13 mutation significantly caused skeletal syndactylia (Fig. 2C), similar to the characters of patients with SPD.
The HoxD13 mutation promoted osteoclast differentiation and bone loss
We further examined the differential expression of HOXD13 between wild and mutant HoxD13 mice. The results showed that the HoxD13 mutation did not affected the expression of HOXD13 (Fig. 3A), suggesting that the HoxD13 mutation did not cause haploinsufficiency. Bone marrow monocyte (BMM) was isolated from wild and mutant HoxD13 mice, and was exposed to m-CSF and RANKL for 5 days. As shown in Fig. 3B, the HoxD13 mutation significantly promoted osteoclast differentiation (Fig. 3B). To confirm the results, we tested the expression of osteoclast-associated proteins, including RANK, p65 and c-Fos. The results indicated that the HoxD13 mutation elevated both mRNA and protein expression of RANK, p65 and c-Fos (Fig. 3C and D). The femur and tibia from wild and mutant HoxD13 mice were analyzed by TRAP staining. The results showed that the HoxD13 mutation notably increased osteoclast differentiation and rarefaction of bone (Fig. 4A). The in vivo imaging of bones from mice carrying wild and mutant HoxD13 was performed using micro-CT. The results demonstrated that this HoxD13 mutation caused bone loss (Fig. 4B). The BV/TV and Tb.Th were decreased in HoxD13 mutant mice compared to wild mice, while the BS/BV and Tb.Sp were increased in HoxD13 mutant mice (Fig. 4C). In addition, the expression of RANK, p65 and c-Fos were upregulated in bone tissues from HoxD13 mutant mice using immunohistochemistry analysis (Fig. 4D). These results suggested that the HoxD13 mutation promoted osteoclast differentiation and bone loss by elevating the expression of RANK, c-Fos and p-p65.
The HoxD13 mutation increased the expression of RANK, c-Fos and phosphorylated p65 by releasing pSMAD5
It is necessary to explore the regulatory mechanism of HOXD13 in the expression of RANK, c-Fos and p65. The results demonstrated that the HoxD13 mutation did not affect the expression of SMAD5, but promoted the phosphorylation of SMAD5 (pSMAD5) (Fig. 4A). Co-Immunoprecipitation assay confirmed that the HoxD13 mutation reduced the interaction between HOXD13 and pSMAD5 (Fig. 4B), suggesting that the HoxD13 mutation increased more free pSMAD5. Inhibition of SMAD5 phosphorylation simultaneously restrained the expression of RANK, c-Fos and p-p65, indicating that pSMAD5 participated in the induction of RANK, c-Fos and p-p65. TRAP staining verified that pSMAD5 inhibition blocked the HoxD13 mutation-induced osteoclast differentiation. These results suggested that the HoxD13 mutation increased the expression of RANK, c-Fos and p-p65 by releasing pSMAD5.
c-Fos and p65 regulated the transcription of RANK
Considering that c-Fos and p65 serve as transcriptional factors in the multiple biological processes, we predicted the likely binding sites of c-Fos and p65 in the promoter of RANK using JASPAR. The functions of c-Fos and p65 in the transcription of RANK were determined by ChIP assay. The results revealed that p65 and c-Fos could bind to the promoter of RANK (Fig. 5A and B). These results suggested that p65 and c-Fos might regulate the transcription of RANK.