Characterization of Osterix protein stability and physiological role in osteoblast differentiation.
Characterization of Osterix protein stability and physiological role in osteoblast differentiation.
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DOI：
10.1371/journal.pone.0056451
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Ma C
中科院分区：
文献类型：
--
作者：
Peng Y;Shi K;Wang L;Lu J;Li H;Pan S;Ma C
Osterix (Osx/SP7) 是 SP 基因家族的一种含有 C2H2 锌指的转录因子。 Osx 基因敲除小鼠表明该基因在成骨细胞分化和骨形成中发挥重要作用。然而，人们对 Osx 调控机制仍知之甚少。在这里，我们报告了一种在哺乳动物细胞中调节 Osx 的新翻译后机制。我们发现放线菌酮处理后内源性和外源性Osx的稳定性降低。在用蛋白酶体抑制剂 MG-132 或乳胞素处理的细胞中，内源性和外源性 Osx 蛋白表达均以时间依赖性方式增加。免疫共沉淀 (Co-IP) 检测显示内源性和外源性 Osx 均被泛素化。通过构建点突变质粒和荧光素酶报告基因测定，Osx 的 6 个赖氨酸残基被鉴定为候选泛素化位点。此外，我们通过 Co-IP 分析和蛋白质稳定性分析证实 K58 和 K230 是 Osx 的泛素化位点。此外，Osx K58R和K230R突变促进了成骨细胞分化标志物（碱性磷酸酶、胶原蛋白I和骨钙素）的表达，并增强了C2C12细胞的成骨分化。综上所述，我们的数据表明Osx是一种不稳定的蛋白质，泛素-蛋白酶体途径参与Osx的调节，从而调节成骨细胞的分化。
Osterix (Osx/SP7) is a C2H2 zinc finger-containing transcription factor of the SP gene family. Osx knockout mice indicate that the gene plays an essential role in osteoblast differentiation and bone formation. However, the mechanisms involved in the regulation of Osx are still poorly understood. Here, we report a novel post-translational mechanism for the regulation of Osx in mammalian cells. We found that the stability of endogenous and exogenous Osx reduced after cycloheximide treatment. In cells treated with the proteasome inhibitors MG-132 or lactacystin, both endogenous and exogenous Osx protein expression increased in a time-dependent manner. Co-immunoprecipitation (Co-IP) assays showed that both endogenous and exogenous Osx were ubiquitinated. Six lysine residues of Osx were identified as candidate ubiquitination sites by construction of point mutant plasmids and luciferase reporter assays. Furthermore, we confirmed that K58 and K230 are the ubiquitination sites of Osx by Co-IP assays and protein stability assays. Moreover, the Osx K58R and K230R mutations promoted the expression of osteoblast differentiation markers (alkaline phosphatase, collagen I and osteocalcin) and enhanced osteogenic differentiation in C2C12 cells. Taken together, our data indicate that Osx is an unstable protein, and that the ubiquitin-proteasome pathway is involved in the regulation of Osx and thereby regulates osteoblast differentiation.
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