Abstract
Tumor necrosis factor (TNF) activates both p42 and p44 mitogen-activated protein kinases (MAPK) in human FS-4 fibroblasts, cells for which TNF is mitogenic. We now show that TNF activates p42 MAPK in two cell lines whose growth is inhibited by TNF. A mutant TNF that binds only to the p55 TNF receptor (TNFR) produced a similar degree of activation as wild-type TNF in FS-4 fibroblasts, indicating that the p55 TNFR is sufficient to mediate p42/p44 MAPK activation. The upstream intracellular signals that couple the TNFR to MAPK activation are still poorly defined. We now show that neither phorbol ester-sensitive protein kinase C nor Gialpha link TNF to p42/p44 MAPK activation, because pretreatment of FS-4 cells with phorbol ester to down-regulate protein kinase C or pretreatment with pertussis toxin to block Gialpha does not inhibit p42/p44 MAPK activation by TNF. To further analyze MAPK activation in FS-4 cells, we compared p42/p44 MAPK activation by TNF and epidermal growth factor (EGF). While tyrosine phosphorylation of p42/p44 MAPK was detected almost immediately (30 s) after stimulating cells with EGF, TNF-induced tyrosine phosphorylation was detected only after a more prolonged time interval (initially detected at 5 min and peaking at 15-30 min). In addition, the anti-inflammatory drug sodium salicylate, previously demonstrated to inhibit NF- kappaB activation by TNF, blocked the activation of p42/p44 MAPK in response to TNF but not in response to EGF. These findings demonstrate that the TNF and EGF receptors utilize distinct signaling molecules to couple to MAPK activation. Elucidation of the mechanism whereby sodium salicylate blocks TNF-induced p42/p44 MAPK activation may help to clarify TNF-activated signaling pathways.
Highlights
The initial event in Tumor necrosis factor (TNF) signal transduction involves association of a trimeric TNF molecule with its receptor and subsequent receptor oligomerization
Activation of p42 mitogen-activated protein kinases (MAPK) Does Not Correlate with Mitogenic Activity of TNF—We have previously shown that TNF activates both p42 and p44 MAPK in normal human FS-4 fibroblasts [21]
Since TNF is mitogenic for FS-4 cells [45], it was of interest to determine whether TNF would induce MAPK activation in cells for which TNF treatment is growth-inhibitory, such as the HeLa and the HT-29 adenocarcinoma cell lines [46]
Summary
The initial event in TNF signal transduction involves association of a trimeric TNF molecule with its receptor and subsequent receptor oligomerization. MAPKs constitute a family of related and evolutionarily conserved serine/threonine kinases that are important in cell growth and differentiation [25,26,27] They become activated by phosphorylation on threonine and tyrosine in response to many external stimuli. While there is evidence for both MEK [39, 40] and MEKK [40] involvement in TNF actions in some cells, it is not clear what other components may participate in the TNFmediated activation of p42/p44 MAPK. Our findings indicate that in addition to activating p42/p44 MAPK with different kinetics, TNF and EGF-activated pathways differ in their susceptibility to inhibition by sodium salicylate.
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