Watanabe Jun, Ohba Motoi, Ohno Fusako, Kikuyama Sakae, Nakamura Masahisa, Nakaya Kazuyasu, Arimura Akira, Shioda Seiji, Nakajo Shigeo
Department of Anatomy, School of Medicine, Showa University, Tokyo, Japan.
J Neurosci Res. 2006 Dec;84(8):1645-55. doi: 10.1002/jnr.21065.
We have found previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases the number of astrocytes generated from cultured mouse neural stem cells (NSCs) via a mechanism that is independent of the cyclic AMP/protein kinase A pathway (Ohno et al., 2005). In the present study, the signaling pathway involved in the differentiation process was further investigated. PACAP-induced differentiation was inhibited by the phospholipase C inhibitor, U73122, the protein kinase C (PKC) inhibitor, chelerythrine, and the intracellular calcium chelator, BAPTA-AM, and was mimicked by phorbol 12-myristate 13-acetate (PMA), but not by 4alpha-PMA. These results suggest that the PACAP-generated signal was mediated via the PACAP receptor, PAC1 stimulated heterotrimeric G-protein, resulting in activation of phospholipase C, followed by calcium- and phospholipid-dependent protein kinase C (cPKC). To elucidate the involvement of the different isoforms of cPKC, their gene and protein expression were examined. Embryonic NSCs expressed alpha and betaII PKC, but lacked PKCgamma. When NSCs were exposed to 2 nM PACAP, protein expression levels of the betaII isoform transiently increased two-fold before differentiation, returning to basal levels by Day 4, whereas the level of PKCalpha increased linearly up to Day 6. Overexpression of PKCbetaII with adenovirus vector synergistically enhanced differentiation in the presence of 1 nM PACAP, whereas expression of the dominant-negative mutant of PKCbetaII proved inhibitory. These results indicate that the beta isoform of PKC plays a crucial role in the PACAP-induced differentiation of mouse embryonic NSCs into astrocytes.
我们之前发现，垂体腺苷酸环化酶激活多肽（PACAP）通过一种独立于环磷酸腺苷/蛋白激酶A途径的机制，增加了从小鼠培养神经干细胞（NSCs）产生的星形胶质细胞的数量（Ohno等人，2005年）。在本研究中，进一步研究了参与分化过程的信号通路。PACAP诱导的分化受到磷脂酶C抑制剂U73122、蛋白激酶C（PKC）抑制剂白屈菜红碱和细胞内钙螯合剂BAPTA-AM的抑制，并被佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）模拟，但不受4α-PMA模拟。这些结果表明，PACAP产生的信号是通过PACAP受体PAC1刺激异源三聚体G蛋白介导的，导致磷脂酶C激活，随后是钙和磷脂依赖性蛋白激酶C（cPKC）。为了阐明不同cPKC同工型的参与情况，检测了它们的基因和蛋白表达。胚胎NSCs表达α和βII PKC，但缺乏PKCγ。当NSCs暴露于2 nM PACAP时，βII同工型的蛋白表达水平在分化前短暂增加两倍，到第4天恢复到基础水平，而PKCα的水平在第6天前呈线性增加。用腺病毒载体过表达PKCβII在存在1 nM PACAP的情况下协同增强分化，而PKCβII显性负突变体的表达则具有抑制作用。这些结果表明，PKC的β同工型在PACAP诱导的小鼠胚胎NSCs向星形胶质细胞的分化中起关键作用。
