Hariri Hadla, Addison William N, St-Arnaud René
Research Centre, Shriners Hospital for Children - Canada, 1003 Decarie Boulevard, Montreal, QC, H4A 0A9, Canada.
Department of Human Genetics, McGill University, Montreal, QC, Canada.
Sci Rep. 2021 Apr 19;11(1):8418. doi: 10.1038/s41598-021-87608-x.
We have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (- 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.
我们之前已经表明，甲状旁腺激素（PTH）可诱导DNA结合蛋白新生多肽相关复合物及共调节因子α（NACA）磷酸化，导致NACA核转位并激活靶基因。通过针对NACA的染色质免疫沉淀测序（ChIP-Seq）与RNA测序并行，我们报告鉴定出泛素特异性肽酶53（Usp53）是成骨细胞中PTH激活的NACA的靶基因。通过电泳迁移率变动分析证实了Usp53启动子的ChIP片段内存在NACA结合位点。Usp53启动子（-2325 / + 238 bp）的活性受JUN-CREB复合物调控，这种激活依赖于激活的蛋白激酶A（PKA）和NACA的存在。在ST2基质细胞中敲低Usp53可刺激成骨细胞标志物骨钙蛋白（Bglap2）和碱性磷酸酶（Alpl）的表达，并抑制脂肪生成标志物过氧化物酶体增殖物激活受体γ（Pparg）和CCAAT增强子结合蛋白α（Cebpa）的表达。敲低Naca时也观察到类似效果。在成骨细胞生成过程中，敲低Usp53对PTH反应的影响因细胞成熟阶段而异。在免疫缺陷小鼠体内植入敲低Usp53基因的骨髓基质细胞，可使成骨细胞数量增加，脂肪细胞数量减少。我们的数据表明，Usp53通过影响谱系选择来调节间充质细胞的命运。
