Miloso Mariarosaria, Villa Daniela, Crimi Marco, Galbiati Stefania, Donzelli Elisabetta, Nicolini Gabriella, Tredici Giovanni
Dipartimento di Neuroscienze e Tecnologie Biomediche, Università degli Studi di Milano-Bicocca, Monza, Italy.
Centro Dino Ferrari, Dipartimento di Scienze Neurologiche, Universita' di Milano, IRCCS Ospedale Maggiore Policlinico, Milano, Italy.
J Neurosci Res. 2004 Jan 15;75(2):241-252. doi: 10.1002/jnr.10848.
Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis.
视黄酸（RA）是维生素A的一种活性代谢产物，是参与神经系统发育和分化的天然形态发生素。为了阐明RA诱导神经突生成所涉及的信号传导机制，我们使用人神经母细胞瘤SH-SY5Y细胞（一种用于研究RA作用的成熟体外模型）来研究细胞外信号调节激酶（ERK）1和2在RA诱导的神经突生成和细胞存活中的作用。通过免疫印迹实验，我们观察到RA诱导ERK1和ERK2磷酸化延迟但持续（直至96小时），特异性丝裂原活化蛋白激酶（MAPK）/ERK激酶（MEK）抑制剂U0126可显著降低这种磷酸化。在后续研究中，我们选择24小时作为参考时间。抑制ERK激活不影响RA诱导的神经突生成（有神经突细胞的百分比和神经突长度），但显著降低细胞存活率。此外，我们分析了介导ERK激活的信号通路。我们的结果表明，RA诱导的ERK磷酸化不遵循经典的Raf激酶依赖性途径。蛋白激酶C（PKC）和磷脂酰肌醇3激酶（PI 3-K）可能是参与ERK信号通路的替代激酶。事实上，在存在特异性PKC抑制剂GF 109203X或特异性PI 3-K抑制剂渥曼青霉素的情况下；我们观察到ERK磷酸化显著剂量依赖性降低。GF 109203X以浓度依赖性方式降低RA诱导的神经突生成和细胞存活。这些结果表明，在RA诱导神经突生成的早期阶段，起重要作用的是PKC而非ERK1和ERK2。
