Department of Physiology, University of Tennessee Health Science Center, Memphis, 38163, USA.
Am J Physiol Gastrointest Liver Physiol. 2011 Jun;300(6):G988-97. doi: 10.1152/ajpgi.00409.2010. Epub 2011 Mar 3.
Integrin binding to the extracellular matrix (ECM) activated Rho GTPases, Src, and focal adhesion kinase in intestinal epithelial cells (IEC)-6. Polyamine depletion inhibited activities of Rac1, RhoA, and Cdc42 and thereby migration. However, constitutively active (CA) Rac1 expression abolished the inhibitory effect of polyamine depletion, indicating that polyamines are involved in a process upstream of Rac1. In the present study, we examined the role of polyamines in the regulation of the guanine nucleotide exchange factor, diffuse B-cell lymphoma (Dbl), for Rho GTPases. Polyamine depletion decreased the level as well as the activation of Dbl protein. Dbl knockdown by siRNA altered cytoskeletal structure and decreased Rac1 activity and migration. Cells expressing CA-Dbl increased migration, Rac1 activity, and proliferation. CA-Dbl restored migration in polyamine-depleted cells by activating RhoA, Rac1, and Cdc42. CA-Dbl caused extensive reorganization of the F-actin cortex into stress fibers. Inhibition of Rac1 by NSC23766 significantly decreased migration of vector-transfected cells and CA-Dbl-transfected cells. However, the inhibition of migration was significantly higher in the vector-transfected cells compared with that seen in the CA-Dbl-transfected cells. Dbl localized in the perinuclear region in polyamine-depleted cells, whereas it localized with the stress fibers in control cells. CA-Dbl localized with stress fibers in both the control and polyamine-depleted cells. These results suggest that polyamines regulate the activation of Dbl, a membrane-proximal process upstream of Rac1.
整合素与细胞外基质（ECM）的结合激活了肠道上皮细胞（IEC）-6 中的 Rho GTPases、Src 和 focal adhesion kinase。多胺耗竭抑制了 Rac1、RhoA 和 Cdc42 的活性，从而抑制了迁移。然而，组成型激活（CA）Rac1 的表达消除了多胺耗竭的抑制作用，表明多胺参与了 Rac1 上游的过程。在本研究中，我们研究了多胺在调节 Rho GTPases 的鸟嘌呤核苷酸交换因子弥漫性 B 细胞淋巴瘤（Dbl）中的作用。多胺耗竭降低了 Dbl 蛋白的水平和激活。Dbl 的 siRNA 敲低改变了细胞骨架结构并降低了 Rac1 活性和迁移。表达 CA-Dbl 的细胞增加了迁移、Rac1 活性和增殖。CA-Dbl 通过激活 RhoA、Rac1 和 Cdc42 恢复了多胺耗竭细胞的迁移。CA-Dbl 导致 F-肌动蛋白皮质广泛重组为应力纤维。NSC23766 抑制 Rac1 显著降低了载体转染细胞和 CA-Dbl 转染细胞的迁移。然而，与 CA-Dbl 转染细胞相比，载体转染细胞的迁移抑制更为显著。多胺耗竭细胞中 Dbl 定位于核周区，而对照细胞中 Dbl 定位于应力纤维。CA-Dbl 在对照和多胺耗竭细胞中均与应力纤维定位。这些结果表明，多胺调节 Rac1 上游的膜近端过程 Dbl 的激活。
