Cheng L, Xu J, Qian Y Y, Pan H Y, Yang H, Shao M Y, Cheng R, Hu T
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Chengdu, Sichuan.
College of Life Sciences, Sichuan University, Chengdu, Sichuan, China.
Int Endod J. 2017 Jan;50(1):15-23. doi: 10.1111/iej.12587. Epub 2015 Dec 23.
To investigate the effects of mammalian homologue of Drosophila diaphanous-1(mDia1) and Rho-associated coiled-coil-containing protein kinase (ROCK) on the migration and adhesion of dental pulp cells (DPCs).
Lysophosphatidic acid (LPA) was used to activate Rho signalling. mDia1 and ROCK were inhibited by short interfering RNA and the specific inhibitor, Y-27632, respectively. The migration of DPCs was assessed using the transwell migration assay and scratch test. Formation of cytoskeleton and focal adhesions(FAs) was observed by confocal laser scanning microscopy. Cell adhesion and spreading assays were performed. Phosphorylation of focal adhesion kinase (FAK) and paxillin was detected by Western blotting, and the bands were analysed using Adobe Photoshop CS5 software. All experiments were performed at least three times, and data were analysed with one-way anova and a post hoc test.
LPA-triggered activation of Rho and inhibition of ROCK significantly increased the cell migration rate. Cell migration was inhibited by silencing mDia1. mDia1 silencing and ROCK inhibition suppressed the LPA-induced formation of the cytoskeleton, FA and phosphorylation of FAK and paxillin. Inhibition of ROCK or mDia1 facilitated early cell adhesion and spreading; by contrast, the combined inhibition of ROCK and mDia1 neutralized these effects.
mDia1 promoted RhoA-induced migration of DPCs, but ROCK had an opposite effect. Both mDia1 and ROCK participated in cytoskeleton formation and adhesion of DPCs. The interactions between mDia1 and ROCK might influence dental pulp repair by determining the migration and adhesion of DPCs.
研究果蝇透明带蛋白1（mDia1）的哺乳动物同源物和Rho相关卷曲螺旋蛋白激酶（ROCK）对牙髓细胞（DPCs）迁移和黏附的影响。
用溶血磷脂酸（LPA）激活Rho信号。分别用短发夹RNA和特异性抑制剂Y-27632抑制mDia1和ROCK。采用Transwell迁移实验和划痕实验评估DPCs的迁移。通过共聚焦激光扫描显微镜观察细胞骨架和黏着斑（FAs）的形成。进行细胞黏附和铺展实验。通过蛋白质免疫印迹法检测黏着斑激酶（FAK）和桩蛋白的磷酸化，并使用Adobe Photoshop CS5软件分析条带。所有实验至少进行3次，数据采用单因素方差分析和事后检验进行分析。
LPA触发的Rho激活和ROCK抑制显著提高了细胞迁移率。沉默mDia1可抑制细胞迁移。沉默mDia1和抑制ROCK可抑制LPA诱导的细胞骨架、FA形成以及FAK和桩蛋白的磷酸化。抑制ROCK或mDia1促进早期细胞黏附和铺展；相反，联合抑制ROCK和mDia1可抵消这些作用。
mDia1促进RhoA诱导的DPCs迁移，但ROCK具有相反的作用。mDia1和ROCK均参与DPCs的细胞骨架形成和黏附。mDia1与ROCK之间的相互作用可能通过决定DPCs的迁移和黏附来影响牙髓修复。
