Abstract：
Runt-related transcription factor 2 (Runx2) is a key regulator of osteoblast differentiation and bone formation. In Runx2-deficient embryos, skeletal development ceases at the cartilage anlage stage. These embryos die of respiratory failure upon birth and display a complete absence of bone and cartilage mineralization. Here, we identified Hakai, a type of E3 ubiquitin ligase as a potential Runx2 interacting partner through affinity pulldown-based proteomic approach. Subsequently, we observed that similar to Runx2, Hakai was downregulated in osteopenic ovariectomized rats, suggesting its involvement in bone formation. Consistent with this observation, Hakai overexpression significantly enhanced osteoblast differentiation in mesenchyme-like C3H10T1/2 as well as primary rat calvaria osteoblast (RCO) cells in vitro. Conversely, overexpression of a catalytically inactive Hakai mutant (C109A) exhibited minimal to no effect, whereas Hakai depletion markedly reduced endogenous Runx2 levels and impaired osteogenic differentiation in both C3H10T1/2 and RCOs. Mechanistically, Hakai physically interacts with Runx2 and enhances its protein turnover by rescuing it from Smad ubiquitination regulatory factor 2 (Smurf2)-mediated proteasome degradation. Wild-type Hakai but not Hakai-C109A inhibited Smurf2 protein levels through proteasome-mediated degradation. These findings underscore Hakai\'s functional role in bone formation, primarily through its positive modulation of Runx2 protein turnover by protecting it from Smurf2-mediated ubiquitin-proteasomal degradation. Collectively, our results demonstrate Hakai as a promising novel therapeutic target for osteoporosis.
摘要：
Runt相关转录因子2（Runx2）是成骨细胞分化和骨形成的关键调节因子。在Runx2缺陷的胚胎中，骨骼发育在软骨解剖阶段停止。这些胚胎在出生时死于呼吸衰竭，并完全没有骨骼和软骨矿化。这里,我们确认了Hakai,一种E3泛素连接酶作为潜在的Runx2相互作用伴侣，通过基于亲和力下拉的蛋白质组学方法。随后，我们观察到，与Runx2相似，Hakai在骨质减少的卵巢切除大鼠中下调，表明它参与了骨形成。与这一观察一致，Hakai过表达显着增强了间质样C3H10T1/2以及体外原代大鼠颅骨成骨细胞（RCO）细胞中的成骨细胞分化。相反，无催化活性的Hakai突变体（C109A）的过表达表现出最小甚至没有影响，而Hakai耗竭显着降低了C3H10T1/2和RCO的内源性Runx2水平并损害了成骨分化。机械上,Hakai与Runx2物理相互作用，并通过将其从Smad泛素化调节因子2（Smurf2）介导的蛋白酶体降解中解救来增强其蛋白质周转。野生型Hakai而非Hakai-C109A通过蛋白酶体介导的降解抑制Smurf2蛋白水平。这些发现强调了Hakai在骨形成中的功能作用，主要是通过保护Runx2蛋白免受Smurf2介导的泛素-蛋白酶体降解来正向调节Runx2蛋白的周转。总的来说，我们的研究结果证明Hakai是治疗骨质疏松症的一个有前景的新靶点.
