FUNCTIONAL-ANALYSIS OF THE LYMPHOTOXIN-BETA PROMOTER - SEQUENCE REQUIREMENTS FOR PMA ACTIVATION

Membrane lymphotoxin (LT) complex is a trimer composed of two subunits , LT-alpha and LT-beta, of which the latter is a 33-kDa transmembrane protein. The LT-beta gene is expressed in lymphoid cells and organs, b ut little is known about its inducible regulation, Previously, the sur face expression of LT-beta in jurkat cells has been shown to increase in response to PMA. In this report, we used this model to study the tr anscriptional control of the human and murine LT-beta genes, PMA stron gly induced the expression of LT-beta mRNA, and the level of induction was not changed markedly by cycloheximide (CHX) treatment, The LT-bet a promoter region contains conserved Egr-1, nuclear factor (NF)-kappa B, and Ets binding sites, and PMA-inducible factors bound to these sit es were detected by the gel-retardation technique (electrophoretic mob ility shift assay (EMSA)). To identify sequences involved in transcrip tional control, sets of human and mouse promoter-chloramphenicol acety ltransferase (CAT) constructs were generated and assayed by transient transfections. The PMA response was lost after deletion of the distal Ets binding site at -110, Mutations at either the Ets or NF-kappa B si tes that prevented factor binding dramatically reduced PMA-inducible p romoter activity, suggesting cooperative interaction between correspon ding transcription factors in PMA activation, Mutation at the Egr-1 si te also resulted insubstantial loss of promoter activity, and the resi dual activity may be attributed to binding of constitutively expressed Sp-1 to the same site, We propose that the interaction between the me mbers of NF-kappa B and Ets families of transcription factors and thei r cognate sites in the promoter is the major determinant of inducible expression of the LT-beta gene in Jurkat cells.