Fuentealba Luis, Schworer Charles, Schroering Allen, Rahmatullah Mohammed, Carey David J
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2601, USA.
Glia. 2004 Feb;45(3):238-48. doi: 10.1002/glia.10325.
Heregulin, a polypeptide growth factor, and forskolin, an adenylyl cyclase activator, synergistically stimulate expression of cyclin D3 and cell division in Schwann cells. Heregulin induces expression in Schwann cells of a luciferase reporter gene linked to the cyclin D3 promoter. Forskolin markedly augments reporter expression in the presence of heregulin. Deletion analysis identified several promoter sites that contribute to high-level reporter expression in heregulin- and forskolin-treated Schwann cells. A promoter fragment that contains 103 bp of 5'-flanking sequence produced significant reporter expression in heregulin- and forskolin-stimulated cells. Deletion of a consensus CCAAT site within this promoter fragment caused a nearly complete loss of reporter expression. Similar results were obtained when CCAAT site mutations were introduced into the promoter. Heregulin and forskolin increased steady-state levels of CCAAT/enhancer binding protein-beta (C/EBPbeta) in Schwann cells. Mobility shift assays identified proteins in Schwann cell nuclear extracts that formed stable complexes with the cyclin D3 CCAAT promoter element and were disrupted by anti-C/EBPbeta antibody. Transfection of Schwann cells with C/EBPbeta cDNA increased cyclin D3 reporter expression. In contrast to these results, mutation of a cAMP response element in the cyclin D3 promoter had only a modest effect on heregulin- and forskolin-stimulated reporter expression. These findings demonstrate that C/EBPbeta plays a key role in the heregulin and cAMP-dependent regulation of cyclin D3 expression in Schwann cells.
神经调节蛋白（一种多肽生长因子）和福斯高林（一种腺苷酸环化酶激活剂）可协同刺激施万细胞中细胞周期蛋白D3的表达和细胞分裂。神经调节蛋白可诱导施万细胞中与细胞周期蛋白D3启动子相连的荧光素酶报告基因的表达。在存在神经调节蛋白的情况下，福斯高林可显著增强报告基因的表达。缺失分析确定了几个启动子位点，这些位点有助于在经神经调节蛋白和福斯高林处理的施万细胞中实现高水平的报告基因表达。一个包含103 bp 5'-侧翼序列的启动子片段在经神经调节蛋白和福斯高林刺激的细胞中产生了显著的报告基因表达。删除该启动子片段内的一个共有CCAAT位点导致报告基因表达几乎完全丧失。当将CCAAT位点突变引入启动子时，也获得了类似的结果。神经调节蛋白和福斯高林可提高施万细胞中CCAAT/增强子结合蛋白β（C/EBPβ）的稳态水平。凝胶迁移试验确定了施万细胞核提取物中的蛋白质，这些蛋白质与细胞周期蛋白D3 CCAAT启动子元件形成稳定复合物，并被抗C/EBPβ抗体破坏。用C/EBPβ cDNA转染施万细胞可增加细胞周期蛋白D3报告基因的表达。与这些结果相反，细胞周期蛋白D3启动子中cAMP反应元件的突变对经神经调节蛋白和福斯高林刺激的报告基因表达只有适度影响。这些发现表明，C/EBPβ在施万细胞中神经调节蛋白和cAMP依赖性的细胞周期蛋白D3表达调控中起关键作用。
