Functional characterization of the mouse lymphotoxin-beta receptor promoter

The lymphotoxin beta-receptor (LT betaR), a member of the tumor necrosis fa ctor (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 mo nocytes, the ligand is expressed only on activated T,B and NK cells, Remark ably, no cell type has been identified so far that expresses both the recep tor 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 gen e. Primer extension analysis revealed one transcriptional start site locate d at - 60 upstream of the ATG-containing first exon, Northern blot analysis showed that the LT betaR is abundantly expressed in the mouse fibroblast c ell 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 functio nal promoter activity, we generated deletion mutants fused to the luciferas e reporter gene. Transfection experiments using these reporter gene constru cts showed that the isolated 5' flanking region is transcriptionally active in NIH 3T3 and RAW 264.7 cells, and determined a minimum length required f or the transcriptional activity of the LT betaR promoter in these cells, Fu rther sequence analysis of the isolated 5' flanking region identified a num ber of putative DNA-binding sites for transcription factors. Interestingly, incubation of NIH 3T3 cells with dexamethasone resulted in a n elevated mRNA level of the LT betaR gene. This effect was abolished by us ing the specific glucocorticoid receptor inhibitor RU486, indicating an inc reased transcriptional activity of the LT betaR promoter after glucocortico id stimulation.