Nilsson M, Ericson L E
Institute of Anatomy and Cell Biology, University of Göteborg, Sweden.
Exp Cell Res. 1995 Aug;219(2):626-39. doi: 10.1006/excr.1995.1273.
The effects of epidermal growth factor (EGF) and phorbol ester (tetra-O-decanoylphorbol-16-acetate; TPA) on thyroid epithelial integrity were studied in filter-cultured monolayers of porcine thyrocytes, which before experiments were growth-arrested and had a high transepithelial resistance (RTE > 6.10(3) omega.cm2) and polarized, thyroid-specific functions. Both EGF and TPA stimulated dose-dependently the cellular incorporation of [3H]thymidine, which maximally (at 10 ng/ml EGF for 48 h) corresponded to a 65% increase of the DNA content. The EGF-treated cells proliferated mainly within the original monolayer, which became folded due to the increased cell number; clusters of epithelial cells also assembled between the monolayer and the filter. Although the transepithelial potential difference was reduced, from 15-30 mV in controls to 2-10 mV, the epithelial barrier function was maintained (RTE 1-3.10(3) omega.cm2; impermeability to [3H]inulin). EGF did not change the ultrastructural polarity of the plasma membrane or the distinct distribution of ZO-1 and cadherin immunoreactivities to junctions, but cytoplasmic cadherin present in controls disappeared after EGF. In cultures acutely depleted of extracellular Ca2+ EGF pretreatment for 48 h antagonized the preventive effect of thyrotropin on paracellular leakage and loss of cell-cell adhesion. TPA (0.1 microM) induced a temporary barrier dysfunction (maximal after 24 h) accompanied by pronounced alterations of cell shape and actin-based cytoskeleton, dissociation of junctional cadherin, and shedding of cells into the apical medium. In long-term (2-5 days) TPA-treated cultures the epithelial morphotype and barrier function recovered. The combined stimulation with EGF and TPA caused a persistent derangement of the cell layer including attenuation of ZO-1 at cell-cell contacts, paracellular leakage of [3H]inulin, and cell detachment. We conclude that EGF is able to release porcine thyroid epithelial cells from contact inhibition of growth along with intact cell polarity and tight junctions. Yet, when acting together with phorbol ester EGF provokes a lasting morphological transformation. Impaired positive control of Ca(2+)-dependent cell-cell adhesion in EGF-treated cultures suggests a latent defect with possible transforming potential in the cadherin-based regulation of the junctional complex.
在猪甲状腺细胞的滤器培养单层中研究了表皮生长因子（EGF）和佛波酯（四 - O - 癸酰佛波醇 - 16 - 乙酸酯；TPA）对甲状腺上皮完整性的影响。实验前，这些细胞处于生长停滞状态，具有高跨上皮电阻（RTE>6×10³Ω·cm²）以及极化的甲状腺特异性功能。EGF和TPA均剂量依赖性地刺激[³H]胸腺嘧啶核苷的细胞掺入，最大掺入量（10 ng/ml EGF作用48小时）相当于DNA含量增加65%。经EGF处理的细胞主要在原始单层内增殖，由于细胞数量增加，单层变得折叠；上皮细胞簇也在单层与滤器之间聚集。尽管跨上皮电位差从对照中的15 - 30 mV降至2 - 10 mV，但上皮屏障功能得以维持（RTE为1 - 3×10³Ω·cm²；对[³H]菊粉不渗透）。EGF未改变质膜的超微结构极性或ZO - 1和钙黏蛋白免疫反应性在连接处的独特分布，但对照中存在的细胞质钙黏蛋白在EGF处理后消失。在急性耗尽细胞外Ca²⁺的培养物中，EGF预处理48小时可拮抗促甲状腺激素对细胞旁渗漏和细胞间黏附丧失的预防作用。TPA（0.1 μM）诱导暂时屏障功能障碍（24小时后最大），伴有细胞形状和基于肌动蛋白的细胞骨架的明显改变、连接钙黏蛋白的解离以及细胞向顶端培养基的脱落。在长期（2 - 5天）TPA处理的培养物中，上皮形态型和屏障功能得以恢复。EGF和TPA联合刺激导致细胞层持续紊乱，包括细胞间接触处ZO - 1减弱、[³H]菊粉的细胞旁渗漏以及细胞脱离。我们得出结论，EGF能够使猪甲状腺上皮细胞从生长接触抑制中释放出来，同时保持完整的细胞极性和紧密连接。然而，当与佛波酯共同作用时，EGF会引发持久的形态学转变。EGF处理的培养物中Ca²⁺依赖性细胞间黏附的阳性控制受损，提示在基于钙黏蛋白的连接复合体调节中存在可能具有转化潜力的潜在缺陷。
