Browsing by Author "Beck Eichler-Jonsson, Claudia"

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    Aspects of mitogen-activated protein kinase cascade activation by epidermal growth factor (EGF): kinetics and crosstalk mechanism with tumor necrosis factor alpha (TNFalpha)
    (2002) Beck Eichler-Jonsson, Claudia; Pfizenmaier, Klaus (Prof. Dr.)
    Using a mathematical/computational model developed on known components of epidermal growth factor (EGF) signaling pathway (Schoeberl, 2002), several aspects of the EGF signaling pathway could be evaluated and validated when compared to experimental data. The model provided insight into signaling response relationships between the ligand binding to the EGF receptor at the cell surface and the activation of downstream responses, for example, the phosphorylation of ERK-1/2 and expression of the target gene, c-fos. Concentration dependent-inhibition of MEK1 kinase reflected on inhibition of EGF-induced ERK-1/2 phosphorylation and the experimental data correlated well with mathematical model simulation. TNF was able to induce ERK-1/2 phosphorylation in a manner dependent on MEK-1 activation but, unlike EGF-mediated MAPK activation, it seemed independent of Ras, Raf or MEKK1. Despite ERK activation, induction of Elk-1 transcriptional activity and c-fos expression were not observed following TNF stimulation in contrast to EGF-treated cells. These data indicate, first, the existence of an alternative route to ERK phosphorylation by TNF other than the classical Ras-Raf pathway and, second, the existence of transcriptional response specificity which may depend on the amplitude and duration of ERK activation induced by diverse stimuli. Besides activating fairly known specific signaling transduction pathways which lead among others to NF-kB and JNK activation, TNF has been shown to be involved in crosstalk with other receptors, a phenomenon much less understood. In the work presented here the features of crosstalk mechanisms between EGF and TNF signaling pathways were investigated. Short time TNF pre-incubation (3 min) prior to EGF stimulation resulted in additive ERK-1/2 phosphorylation whereas 30 minutes pre-incubation induced decrease on EGF-mediated ERK-1/2 phosphorylation as well as on Raf-1 kinase activation of HeLa cells. However, no apparent differences were observed in the levels of EGF-induced EGFR phosphorylation. Moreover, confocal microscopy showed no physical interaction between the two receptors. Although ceramide formation has been regarded as an important mediator of some TNF-dependent responses, it seems not to play any role in the inhibitory effect in the EGF response of HeLa cells observed after 30 min TNF pre-treatment as no increase in cellular ceramide was detected during this time. In addition, inhibitors of the de novo pathway of ceramide formation did not revert TNF-mediated inhibition of EGF-induced Raf kinase activation and ERK-1/2 phosphorylation in HeLa cells. Taken together, these results suggest that TNF negatively regulates EGF-induced ERK-1/2 activation at the level of c-Raf kinase, in a manner independent of ceramide formation.