McCarthy T L, Thomas M J, Centrella M, Rotwein P
Section of Plastic Surgery, Yale University School of Medicine, New Haven, Connecticut 06520-8041, USA.
Endocrinology. 1995 Sep;136(9):3901-8. doi: 10.1210/endo.136.9.7649098.
Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of IGF-I exon 1 as being essential for this hormonal regulation.
胰岛素样生长因子I（IGF-I）是一种在局部合成的骨合成代谢生长因子。原代胎鼠成骨细胞（Ob）合成IGF-I受增加细胞内cAMP浓度的物质刺激，包括前列腺素E2（PGE2）。先前对Ob培养物的研究表明，PGE2通过选择性使用IGF-I启动子1增强IGF-I转录，对IGF-I信使RNA半衰期影响很小。用一系列启动子1-荧光素酶报告基因融合构建体对Ob培养物进行瞬时转染，现已将负责cAMP刺激基因表达的潜在顺式作用启动子元件定位到IGF-I外显子1的5'-非翻译区（5'-UTR），该区域内缺乏一致的cAMP反应元件。我们的证据来自三个主要观察结果：1）仅包含122个核苷酸（nt）的启动子1和328 nt的5'-UTR的转染构建体保留了PGE2刺激的完整报告基因表达；2）在IGF-I启动子1的背景下进行测试时，最大PGE2驱动的报告基因表达需要外显子1的nt 196至328存在；3）将IGF-I启动子-荧光素酶-报告基因构建体与编码小鼠cAMP依赖性蛋白激酶（PKA）催化亚基α-异构体的质粒共转染，产生的结果与PGE2处理所见结果相当，而与编码不能结合cAMP的PKA突变调节亚基的质粒共转染则阻断了PGE2诱导的报告基因表达。对外显子1的5'-UTR进行脱氧核糖核酸酶I足迹分析，结果显示在HS3A、HS3B和HS3D处有受保护序列，这是先前用大鼠肝核提取物鉴定的六个DNA-蛋白质结合位点中的三个。在这三个区域中，只有HS3D结合位点位于功能上确定的IGF-I外显子1的激素反应片段内。这些结果直接表明PKA参与PGE2对IGF-I基因转录的调控，并确定IGF-I外显子1的一个片段对这种激素调节至关重要。
