Sundaresan S, Colin I M, Pestell R G, Jameson J L
Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Endocrinology. 1996 Jan;137(1):304-11. doi: 10.1210/endo.137.1.8536629.
GnRH regulates gonadotropin biosynthesis and secretion. Multiple intracellular signaling pathways are activated by GnRH, including phosphoinositol turnover, release of intracellular calcium and influx of extracellular calcium, and activation of protein kinase C (PKC), among others. In this study, we investigated whether GnRH stimulates mitogen-activated protein kinase (MAPKs), and whether this pathway plays a role in the transcriptional activation of the gonadotropin alpha-gene. In alpha T3-1 gonadotrope cells, treatment with GnRH caused 4- to 5-fold induction of MAPK activity. Stimulation of MAPK activity was detected within 5 min of GnRH treatment and persisted for 60 min. MAPK activation by GnRH was also seen in primary cultures of rat pituitary cells. Treatment with phorbol 12-myristate 13-acetate (TPA) caused 4- to 5-fold induction of MAPK activity in alpha T3-1 cells. Pretreatment with TPA, however, decreased both GnRH- and TPA-induced MAPK activation, suggesting that PKC is involved in GnRH-mediated activation of MAPK. Western blot analyses of PKC isoforms alpha and epsilon confirmed that they were depleted by chronic treatment with TPA, whereas MAPK protein levels were unaffected. Because transcriptional stimulation of the glycoprotein hormone alpha-gene by GnRH is also inhibited by PKC depletion, additional experiments were performed to explore a potential role for MAPK in alpha-gene expression. Cotransfection of a dominant negative inhibitors of MAPK isoforms (ERK1 and ERK2) suppressed basal expression of the alpha-promoter by 60%, but had less effect on the extent of GnRH stimulation in alpha T3-1 cells. These experiments indicate that GnRH stimulates MAPK activity, probably through a pathway involving PKC. Although PKC depletion inhibits both MAPK- and GnRH-stimulated alpha-gene transcription, pathways other than MAPK are also likely to be involved in mediating the transcriptional effects of GnRH.
促性腺激素释放激素（GnRH）调节促性腺激素的生物合成与分泌。GnRH可激活多种细胞内信号通路，包括磷酸肌醇代谢、细胞内钙释放与细胞外钙内流以及蛋白激酶C（PKC）的激活等。在本研究中，我们调查了GnRH是否刺激丝裂原活化蛋白激酶（MAPKs），以及该通路是否在促性腺激素α基因的转录激活中发挥作用。在αT3 - 1促性腺激素细胞中，用GnRH处理可使MAPK活性诱导增加4至5倍。在GnRH处理后5分钟内即可检测到MAPK活性的刺激，并持续60分钟。在大鼠垂体细胞原代培养物中也观察到GnRH激活MAPK。用佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（TPA）处理可使αT3 - 1细胞中的MAPK活性诱导增加4至5倍。然而，用TPA预处理会降低GnRH和TPA诱导的MAPK激活，表明PKC参与GnRH介导的MAPK激活。对PKC同工型α和ε的蛋白质印迹分析证实，长期用TPA处理会使其耗尽，而MAPK蛋白水平不受影响。由于PKC耗竭也会抑制GnRH对糖蛋白激素α基因的转录刺激，因此进行了额外实验以探索MAPK在α基因表达中的潜在作用。共转染MAPK同工型（ERK1和ERK2）的显性负性抑制剂可使α启动子的基础表达抑制60%，但对αT3 - 1细胞中GnRH刺激的程度影响较小。这些实验表明，GnRH可能通过涉及PKC的途径刺激MAPK活性。虽然PKC耗竭会抑制MAPK和GnRH刺激的α基因转录，但MAPK以外的途径也可能参与介导GnRH的转录作用。
