The cAMP pathway in combination with BMP2 regulates Phox2a transcription via cAMP response element binding sites
The cAMP pathway in combination with BMP2 regulates Phox2a transcription via cAMP response element binding sites
复制标题
DOI：
10.1074/jbc.m503939200
复制
发表时间：
2006-02-03
影响因子：
4.8
通讯作者：
Andrisani, OM
中科院分区：
文献类型：
--
作者：
Benjanirut, C;Paris, M;Andrisani, OM
在cAMP反应元件（CRE）结合蛋白（CREB）介导的机制中，BMP 2和cAMP信号的组合通过增加前神经转录因子Phox 2a的表达来诱导神经嵴（NC）培养物中的儿茶酚胺能谱系。为了确定CREB是否直接作用于由BMP 2 + cAMP-升高剂IBMX诱导的Phox 2a转录，在禽类NC培养物和鼠儿茶酚胺能CAD细胞中进行hPhox 2a-报告基因构建体的瞬时转染。虽然BMP 2 +IBMX增加内源性Phox 2a表达，但表达荧光素酶或DsRed 1-E5荧光蛋白的7.5-kb hPhox 2a报告基因对BMP 2 +IBMX无反应，但在两种细胞类型中均有活性。细胞分选的荧光阳性NC细胞表达的7.5 kb的hPhox 2a荧光定时器报告分化为相同数量的儿茶酚胺能细胞作为荧光阴性细胞，这表明不适当的转录从转染的hPhox 2a启动子。用组蛋白去乙酰化酶抑制剂BMP 2 +IBMX处理的NC或CAD细胞显示增加的内源性Phox 2a转录和延长的CREB磷酸化，表明Phox 2a染色质重塑与CREB活化有关。采用CREB、CREB结合蛋白和乙酰化H4抗体的染色质免疫沉淀鉴定了鼠Phox 2a启动子中约5.5kb处的两个CRE半位点，其在人启动子中也是保守的。在CRE半位点的近端，在170-bp区域内，是E-box和CCAAT结合位点，在小鼠和人基因中也是保守的。该170-bp启动子区赋予cAMP、BMP 2和增强的BMP 2 +cAMP调节Phox 2a-荧光素酶报告基因。我们得出结论，这些克雷斯是功能性的，CREB直接激活Phox 2a转录。由于E-box结合bHLH蛋白，如在NC细胞中由BMP 2诱导的ASH 1，我们提出这个新的170 bp顺式作用元件是一个复合位点，介导BMP 2 +cAMP对Phox 2a转录的协同调节。
Combined BMP2 and cAMP signaling induces the catecholaminergic lineage in neural crest (NC) cultures by increasing expression of the proneural transcription factor Phox2a, in a cAMP response element (CRE)-binding protein (CREB)-mediated mechanism. To determine whether CREB acts directly on Phox2a transcription induced by BMP2+cAMP-elevating agent IBMX, transient transfections of hPhox2a-reporter constructs were performed in avian NC cultures and murine, catecholaminergic CAD cells. Although BMP2+IBMX increased endogenous Phox2a expression, the 7.5-kb hPhox2a reporters expressing either luciferase or DsRed1-E5 fluorescent protein were unresponsive to BMP2+IBMX, but active in both cell types. Cell sorting of fluorescence-positive NC cells expressing the 7.5-kb hPhox2a fluorescent timer reporter differentiated to equal numbers of catecholaminergic cells as fluorescence-negative cells, suggesting inappropriate transcription from the transfected hPhox2a promoter. NC or CAD cells treated with histone deacetylase inhibitor trichostatin A and BMP2+IBMX display increased endogenous Phox2a transcription and prolonged CREB phosphorylation, indicating Phox2a chromatin remodeling is linked to CREB activation. Chromatin immunoprecipitations employing CREB, CREB-binding protein, and acetylated H4 antibodies identified two CRE half-sites at -5.5 kb in the murine Phox2a promoter, which is also conserved in the human promoter. Proximal to the CRE half-sites, within a 170-bp region, are E-box and CCAAT binding sites, also conserved in mouse and human genes. This 170-bp promoter region confers cAMP, BMP2, and enhanced BMP2+cAMP regulation to Phox2a-luciferase reporters. We conclude these CREs are functional, with CREB directly activating Phox2a transcription. Because the E-box binds bHLH proteins like ASH1 induced in NC cells by BMP2, we propose this novel 170-bp cis-acting element is a composite site, mediating the synergistic regulation by BMP2+cAMP on Phox2a transcription.