Sun W, Lee H, Choe Y, Cho S, Kim D H, Kim K
School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
J Neuroendocrinol. 2001 Mar;13(3):249-60. doi: 10.1046/j.1365-2826.2001.00620.x.
Gonadotropin-releasing hormone (GnRH) is a pivotal neuroendocrine regulator controlling reproductive functions. However, the scattered distribution of GnRH neurones in the mammalian brain has hindered studies on the development and differentiation of GnRH neurones. In the present study, we used the immortalized GnRH-producing GT1-1 cells to examine whether activation of protein kinase C (PKC) pathway with 12-O-tetradecanoyl-13-acetate (TPA) induces morphological and functional differentiation of GnRH neurones. TPA induced neurite outgrowth and inhibited proliferation of GT1-1 cells that were specifically antagonized by cotreatment of PKC inhibitor, calphostin C. The functional significance of TPA-induced differentiation of GT1-1 cells was manifested in part by the changes in the effects of gamma-aminobutyric acid (GABA) on intracellular Ca2+ levels. In untreated GT1-1 cells, activation of GABA-A receptor with 10 microM muscimol increased intracellular Ca2+ levels, whereas such stimulatory effects disappeared in GT1-1 cells bearing neurites. Accordingly, muscimol could not stimulate GnRH release in TPA-treated GT1-1 cells. To elucidate the molecular mechanism underlying TPA-induced neurite outgrowth, we performed differential display reverse transcription-polymerase chain reaction. Among several genes that are affected by TPA treatment, we found a significant induction of beta-catenin mRNA expression. Along with the rapid induction of beta-catenin protein levels, we observed that beta-catenin was reallocated from cell-cell adhesion sites to the growth cones within 3 h of TPA treatment. Transient transfection studies with green fluorescent protein as a reporter gene demonstrated that beta-catenin overexpression alone can promote neurite outgrowth in GT1-1 cells. Moreover, TPA was found to increase the transcription-activational roles of beta-catenin. Together, these data provide evidence that beta-catenin is involved in the TPA-induced functional differentiation of immortalized GnRH neurones.
促性腺激素释放激素（GnRH）是控制生殖功能的关键神经内分泌调节因子。然而，GnRH神经元在哺乳动物大脑中的分散分布阻碍了对GnRH神经元发育和分化的研究。在本研究中，我们使用永生化的GnRH产生细胞GT1-1来检测用12-O-十四烷酰佛波醇-13-乙酸酯（TPA）激活蛋白激酶C（PKC）途径是否会诱导GnRH神经元的形态和功能分化。TPA诱导神经突生长并抑制GT1-1细胞的增殖，而PKC抑制剂钙泊三醇C的共同处理可特异性拮抗这种作用。TPA诱导GT1-1细胞分化的功能意义部分体现在γ-氨基丁酸（GABA）对细胞内Ca2+水平影响的变化上。在未处理的GT1-1细胞中，用10μM蝇蕈醇激活GABA-A受体会增加细胞内Ca2+水平，而在有神经突的GT1-1细胞中这种刺激作用消失。因此，蝇蕈醇不能刺激TPA处理的GT1-1细胞释放GnRH。为了阐明TPA诱导神经突生长的分子机制，我们进行了差异显示逆转录-聚合酶链反应。在受TPA处理影响的几个基因中，我们发现β-连环蛋白mRNA表达显著上调。随着β-连环蛋白蛋白水平的快速诱导，我们观察到在TPA处理后3小时内，β-连环蛋白从细胞间粘附位点重新分布到生长锥。以绿色荧光蛋白作为报告基因的瞬时转染研究表明，单独的β-连环蛋白过表达可以促进GT1-1细胞的神经突生长。此外，发现TPA可增强β-连环蛋白的转录激活作用。总之，这些数据提供了证据表明β-连环蛋白参与了TPA诱导的永生化GnRH神经元的功能分化。
