Department of Orthopedic Surgery, Musculoskeletal Research Center, Washington University School of Medicine, St. Louis, MO, USA.
Department of Orthopedic Surgery, The Second Xiangya Hospital, Central South University, Changsha, China.
J Bone Miner Res. 2022 Feb;37(2):349-368. doi: 10.1002/jbmr.4473. Epub 2021 Dec 14.
Ectopic calcification is an osteogenic process that leads to the formation of inappropriate bone within intra-articular soft tissues, often in response to injury or surgery. The molecular mechanisms governing this phenotype have yet to be determined. Using a population genetics approach, we identified an association of the kinesin superfamily member 26b (Kif26b) with injury-induced ectopic calcification through quantitative trait locus analysis of recombinant inbred mouse strains, consistent with a genomewide association study that identified KIF26B as a severity locus for ectopic calcification in patients with hip osteoarthritis. Despite these associations of KIF26B with ectopic calcification, its mechanistic role and functional implications have not yet been fully elucidated. Here, we aim to decipher the functional role of KIF26B in osseous and chondrogenic transdifferentiation of human and murine progenitor/stem cells and in a murine model of non-invasive injury-induced intra-articular ectopic calcification. We found that KIF26B ablation via lentivirus-mediated shRNA significantly arrested osteogenesis of progenitor/stem cells and suppressed the expression of typical osteogenic marker genes. Conversely, KIF26B loss-of-function increased chondrogenesis as demonstrated by enhanced Safranin-O staining and by the elevated expression of chondrogenic marker genes. Furthermore, cell function analysis revealed that KIF26B knockdown significantly decreased cell viability and proliferation and induced cellular apoptosis. Mechanistically, loss of osteogenesis was reverted by the addition of a Wnt agonist, SKL2001, demonstrating a role of KIF26B in canonical Wnt/β-catenin signaling. Finally, intra-articular delivery of Kif26b shRNA in B6-129SF2/J mice significantly hampered the development of intra-articular ectopic calcification at 8 weeks after injury compared with mice treated with non-target scrambled shRNA. In summary, these observations highlight that KIF26B plays a crucial role in ectopic bone formation by repressing osteogenesis, but not chondrogenesis, potentially via modulating Wnt/β-catenin signaling. These findings establish KIF26B as a critical determinant of the osteogenic process in pathologic endochondral bone formation and an actionable target for pharmacotherapy to mitigate ectopic calcification (and heterotopic ossification). © 2021 American Society for Bone and Mineral Research (ASBMR).
异位钙化是一种成骨过程，导致关节内软组织中形成不合适的骨骼，通常是对损伤或手术的反应。控制这种表型的分子机制尚未确定。通过对重组近交系小鼠的数量性状基因座分析，我们使用群体遗传学方法鉴定了驱动蛋白超家族成员 26b（Kif26b）与损伤诱导的异位钙化之间的关联，这与全基因组关联研究一致，该研究确定 KIF26B 是髋关节骨关节炎患者异位钙化严重程度的位点。尽管 KIF26B 与异位钙化有关，但它的机械作用和功能意义尚未完全阐明。在这里，我们旨在阐明 KIF26B 在人源和鼠源祖细胞/干细胞的成骨和成软骨分化以及非侵入性损伤诱导的关节内异位钙化的小鼠模型中的功能作用。我们发现，通过慢病毒介导的 shRNA 进行 KIF26B 基因敲除可显著抑制祖细胞/干细胞的成骨作用，并抑制典型成骨标记基因的表达。相反，KIF26B 功能丧失增加了软骨生成，表现为番红 O 染色增强和软骨生成标记基因的表达升高。此外，细胞功能分析表明，KIF26B 敲低显著降低细胞活力和增殖，并诱导细胞凋亡。在机制上，通过添加 Wnt 激动剂 SKL2001 逆转了成骨作用的丧失，表明 KIF26B 在经典 Wnt/β-catenin 信号通路中发挥作用。最后，与用非靶向对照 shRNA 治疗的小鼠相比，在 B6-129SF2/J 小鼠的关节内递送 Kif26b shRNA 可显著阻止损伤后 8 周时关节内异位钙化的发展。总之，这些观察结果表明，KIF26B 通过抑制成骨作用（但不抑制软骨生成）在异位骨形成中发挥关键作用，这可能是通过调节 Wnt/β-catenin 信号通路实现的。这些发现确立了 KIF26B 作为病理性软骨内骨形成中成骨过程的关键决定因素，以及用于减轻异位钙化（和异位骨化）的药物治疗的可行靶点。
