A Gain-of-Function Mutation in <i>Tnni2</i> Impeded Bone Development through Increasing <i>Hif3a</i> Expression in DA2B Mice

<div><p>Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (<i>TNNI2</i>), which encodes a fast-twitch skeletal muscle protein. <i>Tnni2^K175del</i> mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning <i>TNNI2</i> could not explain the small body phenotype of DA2B mice. We found that <i>Tnni2</i> was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that <i>Hif3a</i> expression was significantly increased in the <i>Tnni2^K175del</i> mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the <i>Hif3a</i> promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate <i>Hif3a</i> than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced <i>Vegf</i> expression in DA2B mice. Together, our data indicated that <i>Tnni2^K175del</i> mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of <i>Tnni2^K175del</i> mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in <i>Tnni2</i> in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder.</p>