Qin Chunhua, Samudio Ismael, Ngwenya Sharon, Safe Stephen
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466, USA.
Mol Carcinog. 2004 Jul;40(3):160-70. doi: 10.1002/mc.20030.
17beta-estradiol (E2) induces ornithine decarboxylase (ODC) activity in several E2-responsive tissues/cells, and this study investigated the mechanism of hormone-induced transactivation in MCF-7 human breast cancer cells. E2-induced reporter gene (luciferase) activity in MCF-7 cells transfected with a construct (pODC1) containing the -164 to +29 region of the human ODC gene promoter linked to bacterial luciferase. This promoter sequence contains GC-rich Sp1 binding sites, CAAT, LSF, cAMP response element (CRE), and TATA motifs. Deletion and mutational analysis of the ODC promoter showed that both CAAT and LSF sites were required for hormone-induced transactivation. Gel mobility shift and DNA footprinting assays indicated that NFYA and LSF bound the CAAT and LSF motifs, respectively, and GAL4-NFYA/GAL4-LSF chimeras were also activated by E2, 8-bromo-cAMP, and protein kinase A (PKA) expression plasmid. However, E2-induced transactivation of GAL4-NFYA and GAL4-LSF was blocked by the PKA inhibitor SQ22356 indicating that the mechanism of ODC induction by E2 involves upregulation of cAMP/PKA through nongenomic pathways of estrogen action.
17β-雌二醇（E2）可在多种对E2有反应的组织/细胞中诱导鸟氨酸脱羧酶（ODC）活性，本研究探讨了激素诱导的MCF-7人乳腺癌细胞中转录激活的机制。在转染了包含人ODC基因启动子-164至+29区域并与细菌荧光素酶相连的构建体（pODC1）的MCF-7细胞中，E2诱导了报告基因（荧光素酶）活性。该启动子序列包含富含GC的Sp1结合位点、CAAT、LSF、cAMP反应元件（CRE）和TATA基序。ODC启动子的缺失和突变分析表明，CAAT和LSF位点都是激素诱导的转录激活所必需的。凝胶迁移率变动分析和DNA足迹分析表明，NFYA和LSF分别结合CAAT和LSF基序，并且GAL4-NFYA/GAL4-LSF嵌合体也被E2、8-溴-cAMP和蛋白激酶A（PKA）表达质粒激活。然而，E2诱导的GAL4-NFYA和GAL4-LSF的转录激活被PKA抑制剂SQ22356阻断，这表明E2诱导ODC的机制涉及通过雌激素作用的非基因组途径上调cAMP/PKA。
