Department of Oral Histology, Matsumoto Dental University, 1780 Hirooka-gobara, Shiojiri, Nagano 399-0781, Japan.
Bone. 2012 May;50(5):1092-9. doi: 10.1016/j.bone.2012.02.008. Epub 2012 Feb 17.
SUMO (small ubiquitin-related modifier) modification (SUMOylation) has been reported to regulate various biological events such as cell-cycle progression, proliferation, and survival. Bone morphogenetic proteins (BMPs) play an important role in osteoblast differentiation and maturation. Although Smad4, which acts as a transcriptional factor in the BMP signaling, is a target of SUMOylation, the involvement of SUMOylation in osteoblast differentiation remains unclear. In this report, we demonstrated spatial expression patterns of SUMO proteins and Ubc9 (ubiquitin conjugating enzyme 9), which is a unique E2-SUMOylation enzyme, in mouse tibia. Furthermore, siRNA knockdown of Ubc9 enhanced osteoblastic differentiation induced by BMP2 in C2C12 mouse myoblasts and ST2 mouse bone-marrow derived stromal cells. Ubc9 knockdown elevated the BMP signaling transduction and reduced the expression of muscle-related genes in cooperation with BMP2. Finally, a luciferase assay using an Id1 (target gene of BMP signaling) reporter revealed that Smad4 mutants prevented from SUMOylation at their Lys158 possessed more potent transcriptional activity than wild-type Smad4. Taken together, these findings suggest that Ubc9 negatively regulates osteoblastic differentiation induced by BMP via, at least in part, SUMOylation of Smad4.
SUMO（小泛素相关修饰物）修饰（SUMOylation）已被报道调节多种生物事件，如细胞周期进程、增殖和存活。骨形态发生蛋白（BMPs）在成骨细胞分化和成熟中发挥重要作用。虽然 Smad4 作为 BMP 信号转导中的转录因子，是 SUMOylation 的靶标，但 SUMOylation 在成骨细胞分化中的参与仍不清楚。在本报告中，我们展示了 SUMO 蛋白和 Ubc9（泛素连接酶 9）在小鼠胫骨中的空间表达模式，Ubc9 是一种独特的 E2-SUMOylation 酶。此外，siRNA 敲低 Ubc9 增强了 BMP2 在 C2C12 小鼠成肌细胞和 ST2 小鼠骨髓基质细胞中诱导的成骨细胞分化。Ubc9 敲低增强了 BMP 信号转导，并与 BMP2 一起降低了肌肉相关基因的表达。最后，使用 Id1（BMP 信号靶基因）报告基因的荧光素酶测定显示，不能在 Lys158 处进行 SUMOylation 的 Smad4 突变体比野生型 Smad4 具有更强的转录活性。总之，这些发现表明 Ubc9 通过至少部分地通过 Smad4 的 SUMOylation 负调控 BMP 诱导的成骨细胞分化。
