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 γ-aminobutyric acid (GABA) on intracellular Ca2+ levels. In untreated GT1-1 cells, activation of GABA-A receptor with 10μM 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 β-catenin mRNA expression. Along with the rapid induction of β-catenin protein levels, we observed that β-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 β-catenin overexpression alone can promote neurite outgrowth in GT1-1 cells. Moreover, TPA was found to increase the transcription-activational roles of β-catenin. Together, these data provide evidence that β-catenin is involved in the TPA-induced functional differentiation of immortalized GnRH neurones.
All Science Journal Classification (ASJC) codes
- Endocrinology, Diabetes and Metabolism
- Endocrine and Autonomic Systems
- Cellular and Molecular Neuroscience
- Neurite outgrowth
- Protein kinase C
- γ-aminobutyric acid