Tumor-necrosis factor-alpha modulates mitogen-activated protein kinase activity of epidermal-growth-factor-stimulated...

Tumor-necrosis factor(TNF)-alpha inhibited in a dose-dependent fashion the proliferation of epidermal-growth-factor(EGF)-stimulated MCF-7 breast cancer cells with an IC50 of 0.25 nM. A comparable TNF-alpha-mediated inhibition of p42/44 mitogen-activated protein (MAP) kinase activity was observed in 10 nM EGF-stimulated cells. The MAP kinase activity dropped 50% within 3 min of TNF-alpha (1 nM) addition to EGF-stimulated MCF-7 cells. EGF and TNF-alpha, when added independently, led to a transient stimulation of MAP kinase activity with maximal activations within 6-8 min and 1-2 min, respectively. These observations suggest that MAP kinase activity in EGF-stimulated MCF-7 cells is modulated by the growth-inhibitory receptor pathways of TNF-alpha. Phosphorylation measurements on western blots determined the involvement of several individual MAP kinases, namely p42/44 MAP kinases, p38 MAP kinase and c-Jun N2-terminal kinase 1 (JNK1), in EGF and TNF-alpha-induced signalling. Phosphorylation of p42 and p38 MAP kinases only was observed after treatment with either TNF-alpha or EGF. A combination of both ligands inhibited p42 and p38 MAP kinase phosphorylation in MCF-7 cells. In contrast, no JNK1 phosphorylation was detected in these cells. Simultaneous addition of okadaic acid, a potent inhibitor of phosphatases 1 and 2A, blocked the decay of EGF-stimulated MAP kinase activity over 40 min. TNF-alpha added to EGF-stimulated and okadaic-acid-treated cells increased the MAP kinase activity twofold within 1 min. Similarly, okadaic acid treatment partly reverted the TNF-alpha-inhibited growth of MCF-7 cells. These experiments suggest that phosphatases are involved in the rapid shut-down by TNF-alpha of p42 MAP kinase activity.