del Castillo-Olivares Antonio, Campos José A, Pandak William M, Gil Gregorio
Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0614, USA.
J Biol Chem. 2004 Apr 16;279(16):16813-21. doi: 10.1074/jbc.M400646200. Epub 2004 Feb 6.
Two key regulatory enzymes in the bile acid biosynthesis pathway are cholesterol 7alpha-hydroxylase/CYP7A1 (7alpha-hydroxylase) and sterol 12alpha-hydroxylase/CYP8B1 (12alpha-hydroxylase). It has been shown previously that hepatocyte nuclear factor-4alpha (HNF-4) and the alpha(1)-fetoprotein transcription factor (FTF) are activators of 7alpha-and 12alpha-hydroxylase transcription and that the small heterodimer partner (SHP) suppresses bile acid biosynthesis by heterodimerizing with FTF. However, the role of FTF in bile acid biosynthesis has been studied only in tissue culture systems. In heterozygous FTF knockout mice, 7alpha- and 12alpha-hydroxylase genes were expressed at 5-7-fold higher levels than in wild-type mice, an apparent direct contradiction to previous in vitro observations. This higher expression of the 7alpha- and 12alpha-hydroxylase genes resulted in a 33% higher bile acid pool in their gallbladders, bile more enriched in cholic acid, and a 13% decrease in plasma cholesterol levels. Adenovirus-mediated FTF overexpression in wild-type mice resulted in 10-fold lower expression of the 7alpha- and 12alpha-hydroxylase genes and up to 8-fold higher SHP expression, highlighting the dual role that FTF plays in different promoters. Shorter overexpression times still resulted in lower 7alpha- and 12alpha-hydroxylase expression, but unchanged SHP expression, suggesting that two different mechanisms are involved in the FTF-mediated suppression of 7alpha- and 12alpha-hydroxylase expression. This FTF-mediated suppression of the expression of two bile acid biosynthesis genes resulted in a 3-fold lower rate of bile acid synthesis in a rat bile fistula animal model. Based on these observations and on protein binding studies performed in vitro and by chromatin immunoprecipitation, we hypothesize that FTF has two synergetic effects that contribute to its role in bile acid biosynthesis: 1) it has the ability to activate the expression of SHP, which in turn heterodimerizes and suppresses FTF transactivation activity; and 2) it occupies the FTF/HNF-4 recognition site within the 7alpha- and 12alpha-hydroxylase promoters, which can otherwise be occupied by a factor (HNF-4) that cannot be suppressed by SHP.
胆汁酸生物合成途径中的两种关键调节酶是胆固醇7α-羟化酶/CYP7A1（7α-羟化酶）和固醇12α-羟化酶/CYP8B1（12α-羟化酶）。先前的研究表明，肝细胞核因子-4α（HNF-4）和甲胎蛋白转录因子（FTF）是7α-和12α-羟化酶转录的激活剂，而小异二聚体伴侣（SHP）通过与FTF异二聚化来抑制胆汁酸的生物合成。然而，FTF在胆汁酸生物合成中的作用仅在组织培养系统中进行过研究。在杂合子FTF基因敲除小鼠中，7α-和12α-羟化酶基因的表达水平比野生型小鼠高5至7倍，这显然与之前的体外观察结果直接矛盾。7α-和12α-羟化酶基因的这种较高表达导致其胆囊中的胆汁酸池增加33%，胆汁中胆酸含量更高，血浆胆固醇水平降低13%。在野生型小鼠中，腺病毒介导的FTF过表达导致7α-和12α-羟化酶基因的表达降低10倍，SHP表达升高8倍，突出了FTF在不同启动子中发挥的双重作用。较短的过表达时间仍会导致7α-和12α-羟化酶表达降低，但SHP表达不变，这表明FTF介导的7α-和12α-羟化酶表达抑制涉及两种不同的机制。在大鼠胆瘘动物模型中，这种FTF介导的两种胆汁酸生物合成基因表达的抑制导致胆汁酸合成速率降低3倍。基于这些观察结果以及体外和染色质免疫沉淀实验进行的蛋白质结合研究，我们推测FTF具有两种协同作用，这有助于其在胆汁酸生物合成中发挥作用：1）它具有激活SHP表达的能力，SHP反过来异二聚化并抑制FTF的反式激活活性；2）它占据7α-和12α-羟化酶启动子内的FTF/HNF-4识别位点，否则该位点会被一种不能被SHP抑制的因子（HNF-4）占据。
