In a previous study, we demonstrated that sodium salicylate (NaSal) selectively inhibits tumor necrosis factor (TNF)-induced activation of the p42 and p44 mitogenactivated protein kinases (MAPKs) (known as extracellular signal-regulated kinases). Here we show that in normal human FS-4 fibroblasts NaSal inhibits TNF-induced activation of another member of the MAPK family, the c-Jun N-terminal kinase͞stress-activated protein kinase. c-Jun N-terminal kinase activation induced by interleukin 1 or epidermal growth factor was less strongly inhibited by NaSal. Unexpectedly, treatment of FS-4 cells with NaSal alone produced a strong activation of p38 MAPK and cell death by apoptosis. NaSalinduced apoptosis was blocked by the selective p38 MAPK inhibitor SB-203580, indicating that p38 MAPK serves as a mediator of NaSal-induced apoptosis in human fibroblasts. Activation of p38 MAPK and the resulting induction of apoptosis may be important in the demonstrated antineoplastic actions of nonsteroidal anti-inf lammatory drugs.

Many actions of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) on gene expression are mediated by the transcription factor NF-κB. Activation of NF-κB by TNF and IL-1 is initiated by the phosphorylation of the inhibitory subunit, IκB, which targets IκB for degradation and leads to the release of active NF-κB. The nonsteroidal anti-inflammatory drug sodium salicylate (NaSal) interferes with TNF-induced NF-κB activation by inhibiting phosphorylation and subsequent degradation of the IκBα protein. Recent evidence indicated that NaSal activates the p38 mitogen-activated protein kinase (MAPK), raising the possibility that inhibition of NF-κB activation by NaSal is mediated by p38 MAPK. We now show that inhibition of TNF-induced IκBα phosphorylation and degradation by NaSal is prevented by treatment of cells with SB203580, a highly specific p38 MAPK inhibitor. Both p38 activation and inhibition of TNF-induced IκBα degradation were seen after only 30 s to 1 min of NaSal treatment. Induction of p38 MAPK activation and inhibition of TNF-induced IκBα degradation were demonstrated with pharmacologically achievable doses of NaSal. These findings provide evidence for a role of NaSal-induced p38 MAPK activation in the inhibition of TNF signaling and suggest a possible role for the p38 MAPK in the anti-inflammatory actions of salicylates. In addition, these results implicate the p38 MAPK as a possible negative regulator of TNF signaling that leads to NF-κB activation.

Tumor necrosis factor (TNF) exerts many actions through activation of the transcription factor NF-B. NF-B is sequestered in the cytosol by an inhibitory subunit IB, which is inducibly phosphorylated by an IB kinase complex and subsequently degraded. Sodium salicylate (NaSal) can block NF-B activation by inhibiting IB␣ phosphorylation. Recently, we used the specific p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 to demonstrate that inhibition of TNFinduced IB␣ phosphorylation requires NaSal-induced p38 activation. We demonstrate that NaSal similarly inhibits TNF-induced IB␤ degradation in a p38-dependent manner. To further examine the role of p38, we determined whether other agents that activate p38 can block TNF-induced IB phosphorylation and degradation. Association of the inflammatory cytokines tumor necrosis factor (TNF) 1 and interleukin-1 (IL-1) with their respective cell surface receptors triggers intracellular signal transduction cascades that may culminate in gene activation (1-3). Among the events leading to enhanced gene expression by these cytokines is activation of the transcription factor NF-B, important in the regulation of genes involved in immune and inflammatory responses. NF-B exists as a homo-or heterodimer of members of the Rel family of proteins, consisting of p50/p105, p52/p100, p65/RelA, c-Rel, and RelB. These proteins contain an N-terminal ϳ300-amino acid region known as the Rel homology domain that is responsible for dimerization, DNA binding, and interaction with an inhibitory IB subunit, such as IB␣, IB␤, or IB⑀ (4). In most cell types, an IB protein sequesters NF-B in the cytoplasm. IB may be inducibly phosphorylated on two serine residues at its N terminus and degraded by the ubiquitin-proteasome pathway, leading to the release of NF-B and its translocation to the nucleus. The pathway by which TNF leads to NF-B activation involves the TNF-induced oligomerization of the type 1 TNF receptor, allowing for the recruitment of cytoplasmic proteins, including TNF receptor-associated death domain (TRADD), TNF receptor-associated factor 2 (TRAF2), and receptor-interacting protein (RIP), and activation of kinases, such as MAP ERK kinase kinase 1 (MEKK1) and the NF-B-inducing kinase NIK (5, 6). IL-1 activates NF-B by recruiting the IL-1 receptor accessory protein (IL-1RAcP) to the IL-1 receptor, enabling the association of MyD88 and the serine/threonine kinases IL-1 receptor-associated kinase (IRAK) and 8). The IRAKs interact with TRAF6, which associates with and leads to activation of NIK (9 -12). Thus, NIK is a point of convergence for TNF and IL-1 signaling leading to NF-B activation. Both NIK and MEKK1 have been shown to activate a multiprotein IB kinase (IKK) complex of 700 -900 kDa containing the catalytic subunits . IKK␣ and IKK␤ directly phosphorylate the appropriate N-terminal serines of IB␣ and IB␤ (17,18).Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine kinases that are important mediators of cellular responses to a variety of stimuli. The ...

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 G i␣ link TNF to p42/p44 MAPK activation, because pretreatment of FS-4 cells with phorbol ester to downregulate protein kinase C or pretreatment with pertussis toxin to block G i␣ 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-B 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.Tumor necrosis factor (TNF), 1 a cytokine originally described as a mediator of endotoxin-induced hemorrhagic necrosis of tumors, possesses potent immunomodulatory capacities and is believed to play key roles in inflammation, septic shock, and cachexia (1, 2). Two TNF receptors (TNFRs) of 55 kDa (p55) and 75 kDa (p75) are expressed on many types of cells and transduce the TNF signal (3, 4). The extracellular portions of the receptors possess structural features also present in the extracellular domain of the nerve growth factor receptor and other receptors comprising the TNFR superfamily (3). Although the intracellular domains of the p55 and the p75 TNFRs do not display significant homology to each other, the cytoplasmic portion of the p55 TNFR contains a death domain also found in the Fas antigen, necessary for signaling cell death (5). Recent studies have identified several proteins associated with the cytoplasmic domains of the p55 (6 -8) and p75 (9) TNFRs. The initial event in TNF signal transduction involves association of a trimeric TNF molecule with its receptor and subsequent receptor oligomerization. There is evidence for the involvement of several receptor-distal elements in the propagation of the TNF signal, including GTP-binding proteins (10, 11), protein kinase A (12), and protein kinase C (...

Salicylates inhibit signaling by tumor necrosis factor (TNF), including TNF-induced activation of mitogen-activated protein kinases (MAPKs). On the other hand, we recently showed that in normal human diploid fibroblasts sodium salicylate (NaSal) elicits activation of p38 MAPK but not activation of c-Jun N-terminal kinase (JNK). Here we show that NaSal treatment of COS-1 or HT-29 cells produced a sustained c-Jun N-terminal kinase (JNK) activation. Activation of JNK or p38 MAPK by NaSal (or aspirin) was not due to a nonspecific hyperosmotic effect because much higher molar concentrations of sorbitol or NaCl were required to produce a similar activation. Three structurally unrelated nonsteroidal antiinflammatory drugs (ibuprofen, acetaminophen, and indomethacin) failed to induce significant activation of JNK or p38 MAPK, suggesting that cyclooxygenase inhibition is not the underlying mechanism whereby salicylates induce p38 MAPK and JNK activation. Activation of JNK and p38 MAPKs may be relevant for some antiinflammatory actions of salicylates.

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Salicylates inhibit signaling by tumor necrosis factor (TNF), including TNF-induced activation of mitogen-activated protein kinases (MAPKs). On the other hand, we recently showed that in normal human diploid fibroblasts sodium salicylate (NaSal) elicits activation of p38 MAPK but not activation of c-Jun N-terminal kinase (JNK). Here we show that NaSal treatment of COS-1 or HT-29 cells produced a sustained c-Jun N-terminal kinase (JNK) activation. Activation of JNK or p38 MAPK by NaSal (or aspirin) was not due to a nonspecific hyperosmotic effect because much higher molar concentrations of sorbitol or NaCl were required to produce a similar activation. Three structurally unrelated nonsteroidal antiinflammatory drugs (ibuprofen, acetaminophen, and indomethacin) failed to induce significant activation of JNK or p38 MAPK, suggesting that cyclooxygenase inhibition is not the underlying mechanism whereby salicylates induce p38 MAPK and JNK activation. Activation of JNK and p38 MAPKs may be relevant for some antiinflammatory actions of salicylates.