Ungefroren H, Krull N B
Institute of Anatomy, University of Hamburg, 20246 Hamburg, Federal Republic of Germany.
J Biol Chem. 1996 Jun 28;271(26):15787-95. doi: 10.1074/jbc.271.26.15787.
The small leucine-rich proteoglycan biglycan is involved in several physiological and pathophysiological processes through the ability of its core protein to interact with other extracellular matrix molecules and transforming growth factor-beta (TGF-beta). To learn more about the regulation of biglycan core protein expression, we have cloned and sequenced 1218 base pairs from the 5'-flanking region of the human biglycan gene, demonstrated functional promoter activity, and investigated the molecular mechanisms through which various agents modulate its transcriptional activity. Sequencing revealed the presence of several cis-acting elements including multiple AP-2 sites and interleukin-6 response elements, a NF-kappaB site, a TGF-beta negative element, and an E-box. The TATA and CAAT box-lacking promoter possesses many features of a growth-related gene, e.g. a GC-rich immediate 5' region, many Sp1 sites, and the use of multiple transcriptional start sites. Transient transfections of the tumor cell lines MG-63, SK-UT-1, and T47D with various biglycan 5'-flanking region-luciferase reporter gene constructs showed that the proximal 78 base pairs are sufficient for full promoter activity. Several agents among them interleukin-6, and tumor necrosis factor-alpha. were capable of altering biglycan promoter activity. However, in MG-63 cells, TGF-beta1 failed to increase either activity of the biglycan promoter constructs or specific transcription from the endogenous biglycan gene. Since TGF-beta1 also did not alter the stability of cytoplasmic biglycan mRNA as determined from Northern analysis after inhibition of transcription with 5,6-dichloro-1beta-D-ribofuranosylbenzimidazole, an as yet unidentified nuclear post-transcriptional mechanism was considered responsible for the TGF-beta effect in this cell type. These results might help to elucidate the molecular pathways leading to pathological alterations of biglycan expression observed in atherosclerosis, glomerulonephritis, and fibrosis.
富含亮氨酸的小分子蛋白聚糖双糖链蛋白聚糖，通过其核心蛋白与其他细胞外基质分子及转化生长因子-β（TGF-β）相互作用的能力，参与多种生理和病理生理过程。为了更深入了解双糖链蛋白聚糖核心蛋白表达的调控机制，我们从人双糖链蛋白聚糖基因的5'侧翼区克隆并测序了1218个碱基对，证实了其启动子功能活性，并研究了各种因子调节其转录活性的分子机制。测序结果显示存在多个顺式作用元件，包括多个AP-2位点、白细胞介素-6反应元件、一个核因子κB位点、一个TGF-β负性元件和一个E盒。缺乏TATA盒和CAAT盒的启动子具有许多与生长相关基因的特征，例如富含GC的紧邻5'区域、多个Sp1位点以及多个转录起始位点的使用。用各种双糖链蛋白聚糖5'侧翼区-荧光素酶报告基因构建体对肿瘤细胞系MG-63、SK-UT-1和T47D进行瞬时转染，结果表明近端78个碱基对足以实现完全的启动子活性。其中几种因子，包括白细胞介素-6和肿瘤坏死因子-α，能够改变双糖链蛋白聚糖启动子活性。然而，在MG-63细胞中，TGF-β1既未能增加双糖链蛋白聚糖启动子构建体的活性，也未能增加内源性双糖链蛋白聚糖基因的特异性转录。由于在用5,6-二氯-1-β-D-呋喃核糖基苯并咪唑抑制转录后通过Northern分析确定TGF-β1也未改变细胞质双糖链蛋白聚糖mRNA的稳定性，因此认为一种尚未明确的核转录后机制负责该细胞类型中的TGF-β效应。这些结果可能有助于阐明在动脉粥样硬化、肾小球肾炎和纤维化中观察到的双糖链蛋白聚糖表达病理改变的分子途径。
