McNulty S, Ross A W, Barrett P, Hastings M H, Morgan P J
Department of Anatomy, University of Cambridge, UK.
J Neuroendocrinol. 1994 Oct;6(5):523-32. doi: 10.1111/j.1365-2826.1994.tb00615.x.
This study investigated whether melatonin could modulate the phosphorylation of the calcium/cyclic AMP response-element binding-protein (CREB) within primary cell cultures of ovine pars tuberalis (oPT) and pars distalis (oPD). Gel shift assays confirmed the presence of nuclear factors able to alter the electrophoretic mobility of a 32P-labelled CRE oligonucleotide. Two shifted bands were observed probably due to monomer and dimer binding to the CRE. Each band was supershifted by antisera directed against both CREB and the phosphorylated form of CREB (P-CREB), consistent with a specific role of CREB proteins in transcriptional regulation. To study the physiological role of CREB, the nuclear immunoreactivity for P-CREB was followed in primary cultures of oPT given different pharmacological treatments. Cells stimulated with forskolin responded with a robust time- and dose-dependent increase in nuclear phospho-CREB immunoreactivity (P-CREB-ir), confirming that activation of this transcription factor occurred through the cyclic AMP-PKA pathway. Maximal stimulation was achieved within 15 min and persisted for up to 1 h. Treatment with melatonin alone did not alter basal P-CREB-ir levels, yet melatonin inhibited the forskolin-induced increase in P-CREB-ir in a dose-dependent manner (IC50 of between 10(-10) M and 10(-8) M melatonin when tested against 1 microM forskolin). In contrast, in primary cultures of oPD, melatonin failed to block forskolin-stimulated increases in either the content of cyclic AMP or the intensity of nuclear P-CREB-ir, confirming that the action of melatonin upon P-CREB-ir is tissue specific. These results demonstrate that, consistent with its inhibitory effect on the activation of PKA within oPT, melatonin prevents or reverses the phosphorylation of CREB induced by activation of the cyclic AMP signal transduction pathway. Therefore melatonin has the potential to regulate gene expression in the oPT by acting upon the CREB transcription factor. However, this paper also shows that 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates PKC also leads to the phosphorylation of CREB in oPT cells, suggesting the potential involvement of other signal transduction pathways in the transcriptional regulation of these cells.
本研究调查了褪黑素是否能够调节绵羊结节部（oPT）和远侧部（oPD）原代细胞培养物中钙/环磷酸腺苷反应元件结合蛋白（CREB）的磷酸化。凝胶迁移试验证实存在能够改变32P标记的CRE寡核苷酸电泳迁移率的核因子。观察到两条迁移带，可能是由于单体和二聚体与CRE结合所致。每条带都被针对CREB和磷酸化形式的CREB（P-CREB）的抗血清超迁移，这与CREB蛋白在转录调控中的特定作用一致。为了研究CREB的生理作用，在给予不同药理学处理的oPT原代培养物中跟踪P-CREB的核免疫反应性。用福斯可林刺激的细胞，其核磷酸化CREB免疫反应性（P-CREB-ir）呈现出强烈的时间和剂量依赖性增加，证实该转录因子的激活是通过环磷酸腺苷-PKA途径发生的。在15分钟内达到最大刺激，并持续长达1小时。单独用褪黑素处理不会改变基础P-CREB-ir水平，但褪黑素以剂量依赖性方式抑制福斯可林诱导的P-CREB-ir增加（当针对1微摩尔福斯可林进行测试时，褪黑素的IC50在10^(-10) M至10^(-8) M之间）。相反，在oPD原代培养物中，褪黑素未能阻断福斯可林刺激的环磷酸腺苷含量增加或核P-CREB-ir强度增加，证实褪黑素对P-CREB-ir的作用具有组织特异性。这些结果表明，与其对oPT内PKA激活的抑制作用一致，褪黑素可预防或逆转由环磷酸腺苷信号转导途径激活诱导的CREB磷酸化。因此，褪黑素有可能通过作用于CREB转录因子来调节oPT中的基因表达。然而，本文还表明，激活PKC的12-O-十四烷酰佛波醇-13-乙酸酯（TPA）也会导致oPT细胞中CREB的磷酸化，这表明其他信号转导途径可能参与这些细胞的转录调控。
