Mahata S K, Mahata M, Wu H, Parmer R J, O'Connor D T
Department of Medicine and Center for Molecular Genetics, University of California, San Diego, USA.
Neuroscience. 1999 Jan;88(2):405-24. doi: 10.1016/s0306-4522(98)00225-5.
Nerve growth factor differentiates precursor cells into sympathetic neurons. Does acquisition of a "neuronal" phenotype after nerve growth factor involve biosynthesis of chromogranin A, the major soluble protein in chromaffin granule cores? Nerve growth factor activated chromogranin A gene expression 7.6-fold in PC12 pheochromocytoma cells, and similarly activated PC12-transfected mouse, rat or human chromogranin A promoter/reporter constructs. Chromogranin A promoter 5'-deletions narrowed the nerve growth factor response element to a region from - 77 to - 61 bp upstream of the cap site, a region containing the chromogranin A cyclic AMP response element (TGACGTAA). Three different site-directed mutations of the cyclic AMP response element each reduced the nerve growth factor effect by >90%. Transfer of the cyclic AMP response element to a heterologous (thymidine kinase) promoter activated that promoter approximately 5-fold after nerve growth factor, while transfer of a cyclic AMP response element point-gap mutant (TGA-GTAA) to a heterologous promoter abolished the nerve growth factor effect. These findings indicate that the cyclic AMP response element in cis is, at least in part, both necessary and sufficient to activate the chromogranin A gene. Chemical blockade of the nerve growth factor receptor TrkA or the mitogen-activated protein kinase pathway component MEK substantially diminished nerve growth factor-induced expression of chromogranin A. By contrast, the response of chromogranin A to nerve growth factor was not impaired after blockade of phospholipase C-gamma or phosphoinositide-3 kinase. Chemical blockade of TrkA, Ras, MEK or mitogen-activated protein kinase similarly inhibited nerve growth factor activation of chromogranin A. Expression of constitutively activated Ras, Raf or MEK mutants increased chromogranin A promoter activity. Expression of dominant negative (inhibitory) mutants of Sos, Ha-Ras, Rafl, mitogen-activated protein kinase, ribosomal protein S6 serine kinase II (CREB kinase) or CREB (KCREB) each inhibited the nerve growth factor-induced increase in chromogranin A promoter activity. Thus, each component of the mitogen-activated protein kinase pathway is crucially involved in relaying the nerve growth factor signal in trans to the chromogranin A gene, in the following proposed sequence: nerve growth factor --> TrkA --> Shc/Grb2/Sos --> Ras --> Raf --> MEK --> mitogen-activated protein kinase --> ribosomal protein S6 serine kinase II --> CREB cyclic AMP response element.
神经生长因子可将前体细胞分化为交感神经元。神经生长因子作用后获得“神经元”表型是否涉及嗜铬粒蛋白A（嗜铬粒核心中的主要可溶性蛋白）的生物合成？神经生长因子可使PC12嗜铬细胞瘤细胞中的嗜铬粒蛋白A基因表达增加7.6倍，同样也能激活PC12转染的小鼠、大鼠或人嗜铬粒蛋白A启动子/报告基因构建体。嗜铬粒蛋白A启动子的5'端缺失将神经生长因子反应元件缩小至帽位点上游-77至-61 bp的区域，该区域包含嗜铬粒蛋白A环磷酸腺苷反应元件（TGACGTAA）。环磷酸腺苷反应元件的三种不同的定点突变均使神经生长因子的作用降低>90%。将环磷酸腺苷反应元件转移至异源（胸苷激酶）启动子后，神经生长因子作用下该启动子活性增加约5倍，而将环磷酸腺苷反应元件点突变体（TGA-GTAA）转移至异源启动子则消除了神经生长因子的作用。这些发现表明，顺式作用的环磷酸腺苷反应元件至少部分对于激活嗜铬粒蛋白A基因既是必需的也是充分的。对神经生长因子受体TrkA或丝裂原活化蛋白激酶途径组分MEK进行化学阻断，可显著降低神经生长因子诱导的嗜铬粒蛋白A表达。相比之下，阻断磷脂酶C-γ或磷脂酰肌醇-3激酶后，嗜铬粒蛋白A对神经生长因子的反应并未受损。对TrkA、Ras、MEK或丝裂原活化蛋白激酶进行化学阻断同样可抑制神经生长因子对嗜铬粒蛋白A的激活。组成型激活的Ras、Raf或MEK突变体的表达可增加嗜铬粒蛋白A启动子活性。Sos、Ha-Ras、Raf1、丝裂原活化蛋白激酶、核糖体蛋白S6丝氨酸激酶II（CREB激酶）或CREB（KCREB）的显性负性（抑制性）突变体的表达均抑制了神经生长因子诱导的嗜铬粒蛋白A启动子活性增加。因此，丝裂原活化蛋白激酶途径的每个组分都至关重要地参与了将神经生长因子信号反式传递至嗜铬粒蛋白A基因，其顺序如下：神经生长因子-->TrkA-->Shc/Grb2/Sos-->Ras-->Raf-->MEK-->丝裂原活化蛋白激酶-->核糖体蛋白S6丝氨酸激酶II-->CREB环磷酸腺苷反应元件。
