Surfactant protein D gene regulation - Interactions among the conserved CCAAT/enhancer-binding protein elements
Surfactant protein D gene regulation - Interactions among the conserved CCAAT/enhancer-binding protein elements
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DOI：
10.1074/jbc.m201126200
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发表时间：
2002-05-31
影响因子：
4.8
通讯作者：
Crouch, E
中科院分区：
文献类型：
--
作者：
He, YC;Crouch, E
表面活性物质蛋白D （SP-D）参与肺宿主防御和表面活性物质稳态，在急性肺损伤后增加。鉴于CCAAT/增强子结合蛋白（C/EBP）结合元件在系统性急性期反应和肺发育以及肺上皮细胞C/EBP亚型表达中的重要性，我们假设近远端和近端启动子中保守的C/EBP基序有助于C/EBP对SP-D表达的调节。5个SP-D基序（-432、-340、-319、-140和-90）与C/EBP一致序列同源，在凝胶移位试验中特异性结合C/EBP， 5个位点中的4个（-432、-340、-319和-90）有效地竞争C/EBPalpha、C/EBPbeta或C/EBPdelta与一致低聚物的结合。在H441肺腺癌细胞中共转染C/ ebppalpha、C/EBPbeta或C/EBPdelta cDNA可显著提高含有698上游序列（SS698）的野生型SP-D启动子构建体的荧光素酶活性。转染C/EBP后，H441细胞内源性SP-D mRNA水平升高。通过删除-205上游的序列，取消了报告结构的转激活。-432、-340和-319位点的独立靶向突变使C/ ebp介导的激活降低了近50%，-432位点与-340和-319串联位点的突变阻断了激活。C/ ebp对SS698的转激活不需要-109的保守AP-1元件，而需要最大的启动子活性。因此，近远端启动子中C/EBP元件之间的相互作用可以调节SP-D的启动子活性。
Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following acute lung injury. Given the importance of CCAAT/enhancer-binding protein (C/EBP)binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoforms by lung epithelial cells, we hypothesized that conserved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expression by C/EBPs. Five SP-D motifs (-432, -340, -319, -140, and -90) homologous to the C/EBP consensus sequence specifically bound to C/EBPs in gel shift assays, and four of the five sites (-432, -340, -319, and -90) efficiently competed for the binding of C/EBPalpha, C/EBPbeta, or C/EBPdelta to consensus oligomers. Cotransfection of C/EBPalpha, C/EBPbeta, or C/EBPdelta cDNA in H441 lung adenocarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containing 698 by of upstream sequence (SS698). Transfection of C/EBP also increased the level of endogenous SP-D mRNA in H441 cells. Transactivation of the reporter construct was abrogated by deletion of sequences upstream of -205. Independent site-directed mutagenesis of the sites at -432, -340, and -319 reduced C/EBP-mediated activation by similar to50%, and mutagenesis of the site at -432 in combination with either of the tandem sites at -340 and -319 blocked activation. The conserved AP-1 element at -109 was required for maximal promoter activity, but not for the transactivation of SS698 by C/EBPs. Thus, interactions among C/EBP elements in the near-distal promoter can modulate the promoter activity of SP-D.