Regulation of TNF-alpha expression in normal macrophages: The role of C/EBP beta

C/EBP beta is present in monocytes and macrophages, binds to the proximal r egion of the TNF-alpha promoter, and contributes to its regulation. This st udy was performed to characterize the ability of C/EBP beta to regulate the TNF-alpha gene in myelomonocytic cells and primary macrophages, In transie nt transfection assays, overexpression of wild type C/EBP beta resulted in a 3-4-fold activation of a 120 base pair TNF-alpha promoter-reporter constr uct, while overexpression of a dominant negative (DN) C/EBP beta inhibited LPS-induced activation. In vitro monocyte-differentiated macrophages, infec ted with an adenoviral vector expressing the DN C/EBP beta (AdDNC/EBP beta) or the control Ad beta gal, expressed their transgenes weakly, however exp ression was greatly enhanced in the presence of PMA, Infection with AdDNC/E BP beta resulted in 60% suppression of LPS induced TNF alpha secretion comp ared to Ad beta gal infection (P<0.001) in PMA-treated macrophages. Norther n blot analysis demonstrated approximately a 40% reduction of the TNF-alpha mRNA in the presence of the DN C/EBP beta, suggesting that the effect of t he DN C/EBP beta was at the transcriptional level. In contrast, AdDNC/EBP b eta infection did not result in inhibition of LPS-induced TNF-alpha secreti on in the absence of PMA, Further, DN versions of both C/EBP beta and c-Jun , but not NF-kappa B p65, suppressed PMA-induced TNF-alpha secretion in mac rophages. These observations demonstrate that, C/EBP beta and c-Jun contrib ute to the regulation of the TNF-alpha gene in normal macrophages following treatment with PMA. (C) 2000 Academic Press.