A distinct element involved in lipopolysaccharide activation of the tumor necrosis factor-α promoter in monocytes

To delineate the functional role of the tumor necrosis factor-alpha (TNF-alpha) activator protein-1 (AP-1)/cAMP-responsive element (CRE)-like binding element (TAC), we transfected the TNF-alpha promoter lacking TAC into THP-1 monocytic cells and stimulated with lipopolysaccharide (LPS). Chloramphenicol acetyltransferase (CAT) activity was reduced by 22-fold, suggesting that TAC plays a role in LPS induction of the TNF-alpha promoter. Exposure to LPS resulted in the maximum release of soluble TNF-alpha by 2 h, Electrophoretic mobility shift assays (EMSA) using the TAC element as a probe showed a unique pattern for LPS-activated cells: the disappearance of the upper band of a doublet seen in untreated and all-trans retinoic acid (ATRA)-treated cells. Supershift analysis identified c-Jun and activating transcription factor-2 (ATF-2) as components of the LPS-stimulated binding complex, Jun N-terminal kinase (JNK), a known phosphorylator of c-Jun and ATF-2, increased in activity in LPS-stimulated monocytes, ATRA, on the contrary, did not activate JNK activity up to 72 h, Nuclear extracts from LPS-stimulated cells showed an increase in phosphorylated c-Jun by immunoblotting, Likewise, phosphorylated c-Jun bound to the TAC element, suggesting that c-Jun is activated by JNK to transactivate the TNF-alpha promoter in LPS-treated monocytes, Thus, phosphorylated c-Jun and ATF-2 play a role in activating the TAC element of the TNF-alpha promoter.