Department of Physiology, University of Tennessee Health Science Center, Memphis, 38163, USA.
Am J Physiol Cell Physiol. 2011 Jan;300(1):C164-75. doi: 10.1152/ajpcell.00247.2010. Epub 2010 Nov 10.
Cell migration is important to the integrity of the gastrointestinal tract for the normal movement of cells from crypt to villi and the healing of wounds. Polyamines are essential to cell migration, mucosal restitution, and, hence, healing. Polyamine depletion by α-difluoromethyl ornithine (DFMO) inhibited migration by decreasing lamellipodia and stress fiber formation and preventing the activation of Rho-GTPases. Polyamine depletion increased the association of the thick F-actin cortex with phosphorylated myosin regulatory light chain (pMRLC). In this study, we determined why MRLC is constitutively phosphorylated as part of the actin cortex. Inhibition of myosin light chain kinase (MLCK) decreased RhoA and Rac1 activities and significantly inhibited migration. Polyamine depletion increased phosphorylation of MRLC (Thr18/Ser19) and stabilized the actin cortex and focal adhesions. The Rho-kinase inhibitor Y27632 increased spreading and migration by decreasing the phosphorylation of MRLC, remodeling focal adhesions, and by activating Rho-GTPases. Thus phosphorylation of MRLC appears to be the rate-limiting step during the migration of IEC-6 cells. In addition, increased localization of RhoA with the actin cortex in polyamine-depleted cells appears to activate Rho-kinase. In the absence of polyamines, activated Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) at serine-668 leading to its inactivation and preventing the recruitment of phosphatase (protein phosphastase, PP1cδ) to the actomyosin cortex. In this condition, MRLC is constitutively phosphorylated and cycling does not occur. Thus activated myosin binds F-actin stress fibers and prevents focal adhesion turnover, Rho-GTPase activation, and the remodeling of the cytoskeleton required for migration.
细胞迁移对于胃肠道的完整性至关重要，因为它能使细胞从隐窝正常迁移到绒毛，并促进伤口愈合。多胺对于细胞迁移、黏膜修复以及因此而实现的愈合是必不可少的。α-二氟甲基鸟氨酸（DFMO）通过减少片状伪足和应力纤维的形成并阻止 Rho-GTPases 的激活来抑制细胞迁移，从而耗竭多胺。多胺耗竭增加了厚的 F-肌动蛋白皮质与磷酸化肌球蛋白调节轻链（pMRLC）的关联。在这项研究中，我们确定了为什么 MRLC 作为肌动蛋白皮质的一部分被持续磷酸化。肌球蛋白轻链激酶（MLCK）的抑制降低了 RhoA 和 Rac1 的活性，并显著抑制了迁移。多胺耗竭增加了 MRLC（Thr18/Ser19）的磷酸化，稳定了肌动蛋白皮质和焦点粘连。Rho-激酶抑制剂 Y27632 通过降低 MRLC 的磷酸化、重塑焦点粘连和激活 Rho-GTPases，增加了扩散和迁移。因此，MRLC 的磷酸化似乎是 IEC-6 细胞迁移的限速步骤。此外，在多胺耗竭的细胞中，RhoA 与肌动蛋白皮质的定位增加似乎会激活 Rho-激酶。在没有多胺的情况下，激活的 Rho-激酶在丝氨酸 668 位点磷酸化肌球蛋白磷酸酶靶标亚单位 1（MYPT1），导致其失活，并阻止磷酸酶（蛋白磷酸酶，PP1cδ）募集到肌动球蛋白皮质。在这种情况下，MRLC 持续磷酸化，循环不会发生。因此，激活的肌球蛋白与 F-肌动蛋白应力纤维结合，并阻止焦点粘连的转化、Rho-GTPase 的激活以及迁移所需的细胞骨架的重塑。
