Muller P, Männel D N, Hehlgans T
Institute of Pathology and Tumor Immunology, University of Regensburg, D-93042 Regensburg, Germany.
Eur Cytokine Netw. 2001 Apr-Jun;12(2):325-30.
The lymphotoxin beta-receptor (LT beta R), a member of the tumor necrosis factor (TNF) receptor family, plays a crucial role in lymphoid organogenesis by signaling through its functional ligand LT alpha(1)beta(2). While the receptor is expressed on a wide range of cell types e.g. fibroblasts and monocytes, the ligand is expressed only on activated T, B and NK cells. Remarkably, no cell type has been identified so far that expresses both the receptor and the ligand. In order to characterize the mouse LT beta R gene expression on a molecular level, we isolated about 1 kb of the 5' flanking region of the LT beta R gene. Primer extension analysis revealed one transcriptional start site located at - 60 upstream of the ATG-containing first exon. Northern blot analysis showed that the LT beta R is abundantly expressed in the mouse fibroblast cell line NIH 3T3, and to a lesser extent, in the mouse macrophage-like cell line RAW 264.7. To determine whether the 5' flanking region exerts functional promoter activity, we generated deletion mutants fused to the luciferase reporter gene. Transfection experiments using these reporter gene constructs showed that the isolated 5' flanking region is transcriptionally active in NIH 3T3 and RAW 264.7 cells, and determined a minimum length required for the transcriptional activity of the LT beta R promoter in these cells. Further sequence analysis of the isolated 5' flanking region identified a number of putative DNA-binding sites for transcription factors. Interestingly, incubation of NIH 3T3 cells with dexamethasone resulted in an elevated mRNA level of the LT beta R gene. This effect was abolished by using the specific glucocorticoid receptor inhibitor RU486, indicating an increased transcriptional activity of the LT beta R promoter after glucocorticoid stimulation.
淋巴毒素β受体（LTβR）是肿瘤坏死因子（TNF）受体家族的成员，通过其功能性配体LTα(1)β(2)进行信号传导，在淋巴器官发生中起关键作用。虽然该受体在多种细胞类型如成纤维细胞和单核细胞上表达，但配体仅在活化的T、B和NK细胞上表达。值得注意的是，迄今为止尚未鉴定出同时表达受体和配体的细胞类型。为了在分子水平上表征小鼠LTβR基因的表达，我们分离了LTβR基因5'侧翼区域约1 kb的片段。引物延伸分析揭示了一个转录起始位点，位于含ATG的第一个外显子上游-60处。Northern印迹分析表明，LTβR在小鼠成纤维细胞系NIH 3T3中大量表达，在小鼠巨噬细胞样细胞系RAW 264.7中表达程度较低。为了确定5'侧翼区域是否发挥功能性启动子活性，我们构建了与荧光素酶报告基因融合 的缺失突变体。使用这些报告基因构建体进行的转染实验表明，分离的5'侧翼区域在NIH 3T3和RAW 264.7细胞中具有转录活性，并确定了这些细胞中LTβR启动子转录活性所需的最小长度。对分离的5'侧翼区域进行进一步的序列分析，鉴定出了许多转录因子的假定DNA结合位点。有趣的是，用 地塞米松处理NIH 3T3细胞会导致LTβR基因 的mRNA水平升高。使用特异性糖皮质激素受体抑制剂RU486可消除这种效应，表明糖皮质激素刺激后LTβR启动子的转录活性增加。
