Ooi G T, Cohen F J, Tseng L Y, Rechler M M, Boisclair Y R
Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Endocrinol. 1997 Jun;11(7):997-1007. doi: 10.1210/mend.11.7.9942.
The growth-promoting activity of GH, the principal hormonal determinant of body size, is mediated by insulin-like growth factor I (IGF-I). Most of the IGF-I in plasma circulates in a 150-kDa complex that contains IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit (ALS). The 150-kDa complex serves as a reservoir of IGF-I and determines its bioavailability to the tissues. Formation of the 150-kDa complex depends upon the synthesis of ALS, which is synthesized primarily in liver and is regulated by GH. The present study demonstrates that GH stimulates ALS gene transcription in rat liver and ALS promoter activity in a rat hepatoma cell line. ALS messenger RNA (mRNA) and ALS nuclear transcripts were decreased to similar extents in the livers of GH-deficient hypophysectomized rats. GH increased hepatic ALS mRNA within 3-4 h to about 65% of the levels seen in sham-operated control rats. To confirm that GH stimulated ALS gene transcription, we transiently transfected an ALS promoter-luciferase reporter gene construct into H4-II-E rat hepatoma cells and primary rat hepatocytes. Recombinant human GH (hGH) stimulated promoter activity about 3-fold. In contrast, basal promoter activity was lower, and GH stimulation was absent when the ALS reporter construct was transfected into GH-responsive 3T3-F442A mouse preadipocyte fibroblasts. GH stimulation of ALS promoter activity in H4-II-E cells was mediated by functional GH receptors; nonprimate (rat and bovine) GH gave identical stimulation to hGH, and stimulation by hGH occurred at physiological concentrations. Reverse transcriptase-PCR analysis indicated that GH receptor mRNA was present in H4-II-E cells at approximately 40% of the level seen in rat liver. GH also induced the expression of the endogenous c-fos gene, indicating that the signaling pathway necessary for the activation of gene expression by GH was intact in H4-II-E cells. Thus, H4-II-E cells are a GH-responsive liver cell line that should provide a useful system in which to study the molecular mechanism of transcriptional regulation by GH of ALS and other hepatic genes.
生长激素（GH）是决定身体大小的主要激素，其促生长活性由胰岛素样生长因子I（IGF-I）介导。血浆中大部分IGF-I以150 kDa复合物的形式循环，该复合物包含IGF结合蛋白-3（IGFBP-3）和酸不稳定亚基（ALS）。150 kDa复合物作为IGF-I的储存库，并决定其对组织的生物利用度。150 kDa复合物的形成取决于ALS的合成，ALS主要在肝脏中合成，并受GH调节。本研究表明，GH刺激大鼠肝脏中的ALS基因转录以及大鼠肝癌细胞系中的ALS启动子活性。在GH缺乏的垂体切除大鼠肝脏中，ALS信使核糖核酸（mRNA）和ALS核转录本下降到相似程度。GH在3 - 4小时内使肝脏ALS mRNA增加到假手术对照大鼠所见水平的约65%。为了证实GH刺激ALS基因转录，我们将ALS启动子 - 荧光素酶报告基因构建体瞬时转染到H4-II-E大鼠肝癌细胞和原代大鼠肝细胞中。重组人生长激素（hGH）刺激启动子活性约3倍。相比之下，当将ALS报告构建体转染到对GH有反应的3T3-F442A小鼠前脂肪细胞成纤维细胞中时，基础启动子活性较低且不存在GH刺激。GH对H4-II-E细胞中ALS启动子活性的刺激由功能性GH受体介导；非灵长类（大鼠和牛）GH对hGH的刺激相同，并且hGH在生理浓度下产生刺激。逆转录酶 - 聚合酶链反应分析表明，H4-II-E细胞中GH受体mRNA的水平约为大鼠肝脏中所见水平的40%。GH还诱导内源性c-fos基因的表达，表明GH激活基因表达所需的信号通路在H4-II-E细胞中是完整的。因此，H4-II-E细胞是一种对GH有反应的肝细胞系，应该为研究GH对ALS和其他肝脏基因转录调控的分子机制提供一个有用的系统。
