Department of Clinical Biochemistry, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen, Denmark.
Neuropeptides. 2009 Oct;43(5):387-96. doi: 10.1016/j.npep.2009.08.002. Epub 2009 Aug 26.
Ganglia expressing the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) innervate vasoactive intestinal peptide (VIP) containing neurons suggesting a role of PACAP in regulating VIP expression. Human NB-1 neuroblastoma cells were applied to study PACAP regulated VIP gene expression aiming to identify the receptor and the signaling proteins involved. The PACAP receptor subtype PAC1 induced VIP gene expression as (i) PACAP and the PAC1 receptor agonist maxadilan were equally efficient and approximately 200-fold more potent than VIP, and (ii) PACAP6-38 and PG99-465, antagonists of PAC1 and VPAC2 receptors, respectively, abolished and did not affect the PACAP-induced VIP mRNA expression, respectively. A pivotal role of PKA was implicated in addition to partial involvement of PKC and ERK1/2 in PACAP-induced VIP gene expression as H-89, Bisindolylmaleimide I (BIS), Gö6976 and U0126 attenuated the VIP mRNA expression by 93%, 58%, 58% and 40%, respectively. PACAP modulated the phosphorylation of ERK1/2 (pERK1/2) and CREB/ATF-1 (pCREB/ATF-1) concomitant with a translocation of PKA to the nucleus. Inhibition of conventional PKC isoforms and MEK1/2 completely abolished pERK1/2 without affecting PACAP induced pCREB/ATF-1. In contrast, inhibiting PKA attenuated PACAP induced pCREB/ATF-1. PACAP also enhanced the FOS gene expression and individual presence of H-89, BIS, Gö6976 and U0126 partially attenuated the PACAP induced FOS mRNA expression. Combining the kinase inhibitors completely suppressed the PACAP induced FOS mRNA expression. Immunoblotting confirmed expression of FOS protein upon addition of PACAP, which was diminished by impairment of PKC, ERK1/2 and PKA activities. The resemblance of the signaling pathways involving concomitant activities of PKC, ERK1/2 and PKA in PACAP regulation of the FOS and VIP gene expressions suggest for the first time a role of FOS in PACAP-induced VIP gene expression in human NB-1 neuroblastoma cells.
神经节细胞表达神经肽垂体腺苷酸环化酶激活肽 (PACAP) 支配含有血管活性肠肽 (VIP) 的神经元，提示 PACAP 在调节 VIP 表达中的作用。应用人 NB-1 神经母细胞瘤细胞研究 PACAP 调节 VIP 基因表达，旨在鉴定相关的受体和信号蛋白。PACAP 受体亚型 PAC1 诱导 VIP 基因表达，如 (i) PACAP 和 PAC1 受体激动剂 maxadilan 同样有效，效力约比 VIP 高 200 倍，和 (ii) PACAP6-38 和 PG99-465 分别是 PAC1 和 VPAC2 受体的拮抗剂，分别消除和不影响 PACAP 诱导的 VIP mRNA 表达。除了 PKC 和 ERK1/2 的部分参与外，PKA 的关键作用也暗示在 PACAP 诱导的 VIP 基因表达中。H-89、Bisindolylmaleimide I (BIS)、Gö6976 和 U0126 分别减弱 VIP mRNA 表达 93%、58%、58%和 40%。PACAP 调节 ERK1/2 (pERK1/2) 和 CREB/ATF-1 (pCREB/ATF-1) 的磷酸化，同时 PKA 向核转位。抑制常规 PKC 同工型和 MEK1/2 完全消除了 pERK1/2，而不影响 PACAP 诱导的 pCREB/ATF-1。相反，抑制 PKA 减弱了 PACAP 诱导的 pCREB/ATF-1。PACAP 还增强了 FOS 基因表达，H-89、BIS、Gö6976 和 U0126 的单独存在部分减弱了 PACAP 诱导的 FOS mRNA 表达。联合激酶抑制剂完全抑制了 PACAP 诱导的 FOS mRNA 表达。免疫印迹证实了 FOS 蛋白的表达，加入 PACAP 后，PKC、ERK1/2 和 PKA 活性的损伤降低了 FOS 蛋白的表达。涉及 PKC、ERK1/2 和 PKA 同时活性的信号通路的相似性表明，FOS 蛋白在 PACAP 诱导的人 NB-1 神经母细胞瘤细胞 VIP 基因表达中首次发挥作用。
