Ravni Aurélia, Vaudry David, Gerdin Matthew J, Eiden Maribeth V, Falluel-Morel Anthony, Gonzalez Bruno J, Vaudry Hubert, Eiden Lee E
Section on Molecular Neuroscience, National Institute of Mental Health, Bethesda, MD 20892, USA.
Mol Pharmacol. 2008 Jun;73(6):1688-708. doi: 10.1124/mol.107.044792. Epub 2008 Mar 24.
The neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) elevates cAMP in PC12 cells. Forskolin and dibutyryl cAMP mimic PACAP's neuritogenic and cell morphological effects, suggesting that they are driven by cAMP. Comparison of microarray expression profiles after exposure of PC12 cells to either forskolin, dibutyryl cAMP, or PACAP revealed a small group of cAMP-dependent target genes. Neuritogenesis induced by all three agents is protein kinase A (PKA)-independent [not blocked by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89)] and extracellular signal-regulated kinase (ERK)-dependent [blocked by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126)], and therefore cAMP-dependent target genes potentially mediating neuritogenesis were selected for further analysis based on the pharmacological profile of their induction by PACAP (i.e., mimicking that of neuritogenesis). Small interfering RNA (siRNA) targeting one of these genes, Egr1, blocked PACAP-induced neuritogenesis, and siRNA targeting another, Vil2, blocked a component of the cell size increase elicited by PACAP. Neither siRNA blocked PACAP's PKA-dependent antiproliferative effects. PACAP signaling to neuritogenesis was also impaired by dominant-negative Rap1 expression but was not affected by inhibition of protein kinase C (PKC), indicating a G-protein-coupled receptor-mediated differentiation pathway distinct from the one activated by receptor tyrosine kinase ligands such as nerve growth factor (NGF), that involves both Rap1 and PKC. We have thus identified a cAMP-dependent, PKA-independent pathway proceeding through ERK that functions to up-regulate the transcription of two genes, Egr1 and Vil2, required for PACAP-dependent neuritogenesis and increased cell size, respectively. Dominant-negative Rap1 expression impairs both PACAP-induced neuritogenesis and Egr1 activation by PACAP, suggesting that cAMP elevation and ERK activation by PACAP are linked through Rap1.
神经营养肽垂体腺苷酸环化酶激活多肽（PACAP）可提高PC12细胞中的环磷酸腺苷（cAMP）水平。福斯高林和二丁酰环磷腺苷模仿PACAP的神经突生长和细胞形态学效应，表明这些效应是由cAMP驱动的。将PC12细胞分别暴露于福斯高林、二丁酰环磷腺苷或PACAP后，对其微阵列表达谱进行比较，发现了一小群cAMP依赖性靶基因。这三种物质诱导的神经突生长均不依赖蛋白激酶A（PKA）[不受N-[2-(4-溴肉桂氨基)乙基]-5-异喹啉（H89）阻断]，而是依赖细胞外信号调节激酶（ERK）[受1,4-二氨基-2,3-二氰基-1,4-双（甲硫基）丁二烯（U0126）阻断]，因此，根据PACAP诱导这些基因的药理学特征（即模仿神经突生长的特征），选择可能介导神经突生长的cAMP依赖性靶基因进行进一步分析。靶向其中一个基因Egr1的小干扰RNA（siRNA）阻断了PACAP诱导的神经突生长，靶向另一个基因Vil2的siRNA阻断了PACAP引起的细胞大小增加的一部分。两种siRNA均未阻断PACAP的PKA依赖性抗增殖作用。显性负性Rap1的表达也损害了PACAP向神经突生长的信号传导，但不受蛋白激酶C（PKC）抑制的影响，这表明存在一条与神经生长因子（NGF）等受体酪氨酸激酶配体激活的途径不同的G蛋白偶联受体介导的分化途径，该途径涉及Rap1和PKC。因此，我们确定了一条通过ERK的cAMP依赖性、PKA非依赖性途径，该途径分别上调PACAP依赖性神经突生长和细胞大小增加所需的两个基因Egr1和Vil2的转录。显性负性Rap1的表达损害了PACAP诱导的神经突生长以及PACAP对Egr1的激活，这表明PACAP引起的cAMP升高和ERK激活通过Rap1相连。
