State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China.
Int Endod J. 2011 Jan;44(1):2-8. doi: 10.1111/j.1365-2591.2010.01773.x. Epub 2010 Nov 15.
To investigate the effects of lysophosphatidic acid (LPA) and the Rho/Rho-associated kinase (ROCK) pathway on adhesion of dental pulp cells (DPCs).
Human DPCs were cultured ex vivo. After treatment of LPA and Y-27632, a specific ROCK inhibitor, changes in focal contacts (FCs) were examined by immunofluorescent staining. Activation of FCs proteins was examined by measuring tyrosine 397 phosphorylation of focal adhesion kinase (FAK) and paxillin using immunoblotting. The data were analysed by Student's t-test.
The immunofluorescent staining indicated LPA stimulation induced larger focal adhesion in the cell periphery, compared with the control. Inhibition of ROCK by Y-27632 decreased the formation of FCs markedly, even in the LPA-stimulated cells. LPA also increased the level of tyrosine phosphorylation of paxillin at 30min (P<0.05) and FAK at 5 and 30min (P<0.05). Furthermore, p-paxillin levels declined immediately after Y-27632 treatment and remained low at 5, 30, 60min. Y-27632 also suppressed the effects of LPA on p-paxillin and p-FAK at 5 and 30min (P<0.05).
LPA activated Rho and then subsequently activated ROCK, suggesting that LPA influences the FCs of DPCs by modulating tyrosine phosphorylation of FAK and paxillin via the Rho/ROCK pathway.
研究溶血磷脂酸（LPA）和 Rho/Rho 相关激酶（ROCK）通路对牙髓细胞（DPC）黏附的影响。
体外培养人牙髓细胞。用 LPA 和 ROCK 特异性抑制剂 Y-27632 处理后，通过免疫荧光染色观察焦点接触（FCs）的变化。用免疫印迹法测量粘着斑激酶（FAK）和桩蛋白酪氨酸 397 磷酸化（pY397FAK 和 pY118paxillin）来检测 FCs 蛋白的激活。采用 Student's t 检验进行数据分析。
免疫荧光染色表明，与对照组相比，LPA 刺激可在细胞边缘诱导更大的焦点附着。ROCK 的抑制作用通过 Y-27632 明显减少了 FCs 的形成，即使在 LPA 刺激的细胞中也是如此。LPA 还可增加 30min 时 paxillin 的酪氨酸磷酸化水平（P<0.05）和 5min 和 30min 时 FAK 的酪氨酸磷酸化水平（P<0.05）。此外，Y-27632 处理后 p-paxillin 水平立即下降，并在 5、30、60min 时保持较低水平。Y-27632 还抑制了 LPA 在 5 和 30min 时对 p-paxillin 和 p-FAK 的影响（P<0.05）。
LPA 激活 Rho，然后激活 ROCK，表明 LPA 通过调节 FAK 和桩蛋白的酪氨酸磷酸化，通过 Rho/ROCK 通路影响 DPC 的 FCs。
