Shiomi Nobuyuki, Cui Xiao-Mei, Yamamoto Tadashi, Saito Takashi, Shuler Charles F
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 90033-9062, USA.
Dev Dyn. 2006 Jul;235(7):1785-93. doi: 10.1002/dvdy.20819.
Transforming growth factor (TGF)-beta 3 is known to regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Our previous studies showed that SMAD2, a TGF-beta signaling mediator, was expressed and phosphorylated primarily in the MEE and that SMAD2 phosphorylation in the MEE was temporospatially regulated by TGF-beta 3. The goal of this study was to examine the requirement for SMAD2 to complete the developmental events necessary for palatal fusion. SMAD2 expression was inhibited with Smad2 siRNA transfection into palatal tissues in vitro. The results showed that Smad2 siRNA transfection resulted in the maintenance of MEE cells in the palatal midline. Western blot and immunofluorescence analyses confirmed that the endogenous SMAD2 and phospho-SMAD2 levels were reduced following siRNA transfection. The SMAD3 level was not altered by the Smad2 siRNA transfection. The persistence of the MEE and the decreased SMAD2/phospho-SMAD2 levels were coincident with increased MEE cell proliferation. Addition of exogenous TGF-beta 3 increased p-SMAD2 level but not the total SMAD2 level. Therefore, exogenous TGF-beta 3 was not able to induce p-SMAD2 enough to rescue the palatal phenotype in the Smad2 siRNA group. The results indicated that the endogenous SMAD2 level is crucial in the regulation of disappearance of MEE during palatal fusion.
已知转化生长因子（TGF）-β3在腭融合过程中调节小鼠内侧边缘上皮（MEE）的消失。我们之前的研究表明，TGF-β信号转导介质SMAD2主要在MEE中表达并磷酸化，且MEE中SMAD2的磷酸化受TGF-β3的时空调节。本研究的目的是检测SMAD2对于完成腭融合所需发育事件的必要性。通过体外将Smad2小干扰RNA（siRNA）转染到腭组织中来抑制SMAD2的表达。结果显示，Smad2 siRNA转染导致MEE细胞在腭中线处维持存在。蛋白质免疫印迹和免疫荧光分析证实，siRNA转染后内源性SMAD2和磷酸化SMAD2水平降低。Smad2 siRNA转染未改变SMAD3水平。MEE的持续存在以及SMAD2/磷酸化SMAD2水平的降低与MEE细胞增殖增加同时出现。添加外源性TGF-β3可提高磷酸化SMAD2水平，但不提高总SMAD2水平。因此，外源性TGF-β3不足以诱导磷酸化SMAD2以挽救Smad2 siRNA组中的腭部表型。结果表明，内源性SMAD2水平在腭融合过程中MEE消失的调节中至关重要。
