Rossant C J, Pinnock R D, Hughes J, Hall M D, McNulty S
Parke-Davis Neuroscience Research Center, Cambridge, United Kingdom.
Endocrinology. 1999 Apr;140(4):1525-36. doi: 10.1210/endo.140.4.6656.
CRF exerts a key neuroregulatory control on the function of the hypothalamic-pituitary-adrenal axis. These effects are thought to be mediated primarily through activation of Gs-coupled plasma membrane receptors. In the present study, we investigated the effects of activation of CRF receptors by sauvagine on signaling pathways that converge on phosphorylation of the transcription factor calcium/cAMP response element-binding protein (CREB). Studies were undertaken using CHO cell lines transfected with either rat CRF-1 or CRF-2alpha receptors. Signaling pathways were investigated using immunocytochemical, Western blot, and imaging techniques. Treatment with sauvagine increased phosphorylation of p42/p44, but not of p38 or stress-activated protein kinase (SAPK)/JUN N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases correlating with increased p42/p44 MAP kinase activity. Mobilization of intracellular Ca2+ stores was observed in cells treated with high concentrations (100 nM, 1 microM) of sauvagine. A time- and dose-dependent increase in phosphorylation of the transcription factor CREB was observed in cultures treated with sauvagine. Phosphorylation of CREB occurred at lower concentrations of sauvagine than those required to mobilize intracellular calcium stores, and phosphorylation was not blocked by the mitogen-activated protein kinase kinase inhibitor PD98059 at a concentration (1 microM) that fully inhibited phosphorylation of MAP kinase. Cotreatment of cultures with the protein kinase A inhibitor H89 (10 microM) blocked fully the stimulatory actions of sauvagine (0.1 nM, 1 nM) on phosphorylation of CREB, but not those on phosphorylation of MAP kinase. Phosphorylation of MAP kinase was partially blocked by the phosphoinositide 3-kinase inhibitor LY294002 (5 microM) and by the phosphoinositide-phospholipase C inhibitor U73122 (10 microM). These data demonstrate that cAMP-, Ca2+-, and MAP kinase-dependent signaling pathways are activated by stimulation of CRF-1 and CRF-2alpha receptors. However, in these cells, only protein kinase A-dependent pathways contribute significantly to enhanced phosphorylation of CREB. These represent the first reported observations of CRF receptor-mediated phosphorylation of the transcription factor CREB and activation of MAP kinase signal transduction pathways.
促肾上腺皮质激素释放因子（CRF）对下丘脑 - 垂体 - 肾上腺轴的功能发挥关键的神经调节控制作用。这些作用被认为主要是通过激活与Gs偶联的质膜受体介导的。在本研究中，我们研究了蛙皮素激活CRF受体对汇聚于转录因子钙/ cAMP反应元件结合蛋白（CREB）磷酸化的信号通路的影响。研究使用转染了大鼠CRF - 1或CRF - 2α受体的CHO细胞系进行。使用免疫细胞化学、蛋白质印迹和成像技术研究信号通路。用蛙皮素处理可增加p42 / p44的磷酸化，但不增加p38或应激激活蛋白激酶（SAPK）/ JUN N末端激酶（JNK）丝裂原活化蛋白（MAP）激酶的磷酸化，这与p42 / p44 MAP激酶活性增加相关。在用高浓度（100 nM，1 microM）蛙皮素处理的细胞中观察到细胞内Ca2 +储存的动员。在用蛙皮素处理的培养物中观察到转录因子CREB磷酸化呈时间和剂量依赖性增加。CREB的磷酸化发生在比动员细胞内钙储存所需浓度更低的蛙皮素浓度下，并且在完全抑制MAP激酶磷酸化的浓度（1 microM）下，丝裂原活化蛋白激酶激酶抑制剂PD98059并未阻断磷酸化。用蛋白激酶A抑制剂H89（10 microM）与培养物共同处理可完全阻断蛙皮素（0.1 nM，1 nM）对CREB磷酸化的刺激作用，但不阻断对MAP激酶磷酸化的刺激作用。MAP激酶的磷酸化被磷酸肌醇3 - 激酶抑制剂LY294002（5 microM）和磷酸肌醇 - 磷脂酶C抑制剂U73122（10 microM）部分阻断。这些数据表明，cAMP -、Ca2 + - 和MAP激酶依赖性信号通路通过刺激CRF - 1和CRF - 2α受体而被激活。然而，在这些细胞中，只有蛋白激酶A依赖性通路对CREB磷酸化增强有显著贡献。这些代表了首次报道的CRF受体介导的转录因子CREB磷酸化和MAP激酶信号转导通路激活的观察结果。
