A distinct element involved in lipopolysaccharide activation of the tumor necrosis factor-alpha 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.
Diaz
B
B
Department of Bioimmunotherapy, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Lopez-Berestein
G
G
D051698:Activating Transcription Factor 2; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; C497106:Atf2 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D008070:Lipopolysaccharides; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha