McCormack S A, Blanner P M, Zimmerman B J, Ray R, Poppleton H M, Patel T B, Johnson L R
Department of Physiology and Biophysics, College of Medicine, University of Tennessee, Memphis 38163, USA.
Am J Physiol. 1998 Jan;274(1):C192-205. doi: 10.1152/ajpcell.1998.274.1.C192.
Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.
细胞生长和迁移是肠道上皮细胞分化、维持和修复的重要过程。表皮生长因子（EGF）是这两个过程所需细胞骨架重组中的一个重要因子。由于我们之前发现多胺缺乏时细胞骨架会发生显著变化，因此了解这些变化是否会阻止EGF刺激生长和迁移很有意义。用α-二氟甲基鸟氨酸（DFMO）处理IEC-6细胞可中断多胺生物合成，DFMO是鸟氨酸脱羧酶（多胺生物合成的主要限速酶）的特异性抑制剂。DFMO使细胞增殖停止并抑制细胞迁移，且这两种功能均不能被EGF正常刺激。转铁蛋白受体（用作内吞途径的标志物）的免疫细胞化学显示，在结合EGF 10分钟后，EGF受体（EGFR）分布异常。多胺缺乏使细胞内微丝减少，肌动蛋白皮质增厚，并阻止EGF结合的EGFR与肌动蛋白迅速结合。EGF刺激的170 kDa蛋白酪氨酸磷酸化以及纯化膜EGFR的激酶活性降低了50%。然而，多胺缺乏并未降低免疫沉淀的EGFR蛋白浓度。补充腐胺可防止所有这些变化。细胞骨架破坏、EGFR磷酸化和激酶活性降低、细胞内EGFR分布异常以及与肌动蛋白丝的结合延迟，部分解释了上皮细胞生长和迁移对多胺的依赖性。
