Functional characterization of the mouse lymphotoxin-beta receptor promoter

The lymphotoxin beta-receptor (LT betaR), 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 betaR gene expression on a molecular level, we isolated about 1 kb of the 5' flanking region of the LT betaR 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 betaR 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 betaR 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 betaR gene. This effect was abolished by using the specific glucocorticoid receptor inhibitor RU486, indicating an increased transcriptional activity of the LT betaR promoter after glucocorticoid stimulation.