Rao Jaladanki N, Guo Xin, Liu Lan, Zou Tongtong, Murthy Karnam S, Yuan Jason X-J, Wang Jian-Ying
Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Am J Physiol Cell Physiol. 2003 Apr;284(4):C848-59. doi: 10.1152/ajpcell.00371.2002. Epub 2002 Dec 4.
Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca(2+) concentration (Ca(2+)) through controlling voltage-gated K(+) channel expression and membrane potential (E(m)) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated Ca(2+) activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced Ca(2+), by either polyamine depletion or exposure to the Ca(2+)-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of Ca(2+) also inhibited cell migration after wounding. Elevation of Ca(2+) induced by the Ca(2+) ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via Ca(2+) can interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.
多胺是上皮细胞迁移导致的黏膜修复早期阶段所必需的。我们之前的研究表明，在肠道上皮修复过程中，多胺通过控制电压门控钾通道表达和膜电位（E(m)）来提高细胞溶质游离钙（Ca(2+)）浓度（Ca(2+)），从而增加RhoA活性。当前的研究进一步确定，Ca(2+)升高后RhoA增加是否会激活Rho激酶（ROK/ROCK），导致肌球蛋白轻链（MLC）磷酸化。研究在稳定转染Cdx2的肠道上皮细胞（IEC-Cdx2L1）中进行，这些细胞具有高度分化的表型。通过多胺耗竭或暴露于无钙培养基降低Ca(2+)，会降低RhoA蛋白表达，同时GTP结合的RhoA、ROCK-1和ROKalpha蛋白、Rho激酶活性以及MLC磷酸化也会显著降低。Ca(2+)的降低还会抑制损伤后的细胞迁移。钙离子载体离子霉素诱导的Ca(2+)升高会增加GTP结合的RhoA、ROCK-1和ROKalpha蛋白、Rho激酶活性以及MLC磷酸化。显性负性突变体RhoA对RhoA功能的抑制会降低Rho激酶活性，并导致细胞骨架重组。特异性抑制剂Y-27632对ROK/ROCK活性的抑制不仅会降低MLC磷酸化，还会抑制细胞迁移。这些结果表明，多胺通过Ca(2+)使GTP结合的RhoA增加，在肠道上皮修复过程中可与Rho激酶相互作用并激活它。Rho激酶的激活导致MLC磷酸化增加，从而刺激肌球蛋白应力纤维形成和细胞迁移。
