Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.
J Bone Miner Res. 2013 Sep;28(9):1925-35. doi: 10.1002/jbmr.1938.
Ubiquitin E3 ligase-mediated protein degradation promotes proteasomal degradation of key positive regulators of osteoblast functions. For example, the E3 ligases--SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1), Itch, and WW domain-containing E3 ubiquitin protein ligase 1 (Wwp1)--promote degradation of Runt-related transcription factor 2 (Runx2), transcription factor jun-B (JunB), and chemokine (C-X-C) receptor type 4 (CXCR-4) proteins to inhibit their functions. However, the role of E3 ligases in age-associated bone loss is unknown. We found that the expression level of Wwp1, but not Smurf1 or Itch, was significantly increased in CD45-negative (CD45(-)) bone marrow-derived mesenchymal stem cells from 6-month-old and 12-month-old wild-type (WT) mice. Wwp1 knockout (Wwp1(-/-)) mice developed increased bone mass as they aged, associated with increased bone formation rates and normal bone resorption parameters. Bone marrow stromal cells (BMSCs) from Wwp1(-/-) mice formed increased numbers and areas of alkaline phosphatase(+) and Alizarin red(+) nodules and had increased migration potential toward chemokine (C-X-C motif) ligand 12 (CXCL12) gradients. Runx2, JunB, and CXCR-4 protein levels were significantly increased in Wwp1(-/-) BMSCs. Wwp1(-/-) BMSCs had increased amount of ubiquitinated JunB protein, but Runx2 ubiquitination was no change. Knocking down JunB in Wwp1(-/-) BMSCs returned Runx2 protein levels to that in WT cells. Thus, Wwp1 negatively regulates osteoblast functions by affecting both their migration and differentiation. Mechanisms designed to decrease Wwp1 levels in BMSCs may represent a new approach to prevent the decrease in osteoblastic bone formation associated with aging.
泛素 E3 连接酶介导的蛋白质降解促进了成骨细胞功能的关键正调控因子的蛋白酶体降解。例如，E3 连接酶——SMAD 特异性 E3 泛素蛋白连接酶 1（Smurf1）、Itch 和含有 WW 结构域的 E3 泛素蛋白连接酶 1（Wwp1）——促进 Runt 相关转录因子 2（Runx2）、转录因子 jun-B（JunB）和趋化因子（C-X-C）受体 4 型（CXCR-4）蛋白的降解，从而抑制其功能。然而，E3 连接酶在与年龄相关的骨丢失中的作用尚不清楚。我们发现，在 6 个月和 12 个月龄的野生型（WT）小鼠的 CD45 阴性（CD45(-))骨髓间充质干细胞中，Wwp1 的表达水平显著增加，而 Smurf1 或 Itch 的表达水平则没有显著增加。Wwp1 敲除（Wwp1(-/-)) 小鼠随着年龄的增长，骨量增加，与骨形成率增加和正常骨吸收参数有关。Wwp1(-/-) 小鼠的骨髓基质细胞（BMSCs）形成的碱性磷酸酶（+)和茜素红（+)结节数量和面积增加，对趋化因子（C-X-C 基序）配体 12（CXCL12）梯度的迁移潜力增加。Wwp1(-/-) BMSCs 中的 Runx2、JunB 和 CXCR-4 蛋白水平显著增加。Wwp1(-/-) BMSCs 中有更多的泛素化 JunB 蛋白，但 Runx2 的泛素化没有变化。在 Wwp1(-/-) BMSCs 中敲低 JunB 可使 Runx2 蛋白水平恢复至 WT 细胞水平。因此，Wwp1 通过影响 BMSCs 的迁移和分化来负调控成骨细胞的功能。旨在降低 BMSCs 中 Wwp1 水平的机制可能代表了一种防止与衰老相关的成骨细胞骨形成减少的新方法。
