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In 16HBE14o- human bronchial epithelial cells, maximal tumor necrosis factor (TNF)-α-induced interleukin (IL)-8 expression depends on the activation of two distinct signaling pathways, one constituted in part by activator protein (AP)-1 and the other by nuclear factor (NF)-κB. We examined the upstream signaling intermediates responsible for IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in this system, hypothesizing that p21 Ras and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase (MEKK)-1 function as common upstream activators of both the AP-1 and NF-κB pathways. TNF-α treatment induced both Ras and MEKK1 activation. Dominant-negative forms of Ras (N17Ras) and MEKK1 (MEKK1-KM) each inhibited TNF-α-induced transcription from IL-8 and GM-CSF promoters. Ras was required for maximal activation of extracellular signal-regulated kinase (ERK) and Jun amino terminal kinase (JNK) as well as AP-1 and NF-κB transcriptional activities, but not for activation of IκB kinase (IKK)-β, an upstream activator of NF-κB. MEKK1 was required for maximal activation of ERK, JNK, and IKK, as well as for maximal AP-1 and NF-κB transcriptional activities. We conclude that Ras regulates TNF-α-induced chemokine expression by activating the AP-1 pathway and enhancing transcriptional function of NF-κB, whereas MEKK1 activates both the AP-1 and NF-κB pathways.