Regulation of TNF expression by multiple mitogen-activated protein kinase pathways

Stimulating macrophages with bacterial endotoxin (LPS) activates numerous i ntracellular signaling pathways that lead to the production of TNF, In this study, we show that four mitogen-activated protein (MAP) kinase pathways a re activated in LPS-stimulated macrophages: the extracellular signal-regula ted kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase, p38, and Big MAP kinase (BMK)/ERK5 pathways. Although specific activation o f a single MAP kinase pathway produces only a modest effect on TNF promoter activation, activation of each MAP kinase pathway is important for full in duction of the TNF gene. Interestingly, a dramatic induction of TNF promote r-driven gene expression was observed when all of the four MAP kinase pathw ays were activated simultaneously, suggesting a cooperative effect among th ese kinases, Unexpectedly, cis elements known to be targeted by MAP kinases do not play a major role in multiple MAP kinase-induced TNF gene expressio n. Rather, a 40-bp sequence harboring the TATA box, is responsible for the gene up-regulation induced by MAP kinases, The proximity of the MAP kinase- responsive element to the transcriptional initiation site suggested that MA P kinases regulate the transcriptional initiation complex. Utiliizing alpha -amanitin-resistant RNA polymerase II mutants with or without a C-terminal domain (CTD) deletion, we found that deleting the CTD to 31 tandem repeats (Delta 31) led to >90% reduction in MAP kinase-mediated TNF production. Thu s, our. data demonstrate coordination of multiple MAP kinase pathways in TN F production and suggest that the CTD of RNA polymerase II is required to e xecute MAP kinase signaling in TNF expression.