The death domain kinase RIP1 is essential for tumor necrosis factor alpha signaling to p38 mitogen-activated protein kinase

The cytokine tumor necrosis factor alpha (TNF-alpha) stimulates the NF-kappaB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting RIP1 and TRAF2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that RIP1 links the TNFR1 to the IkappaB kinase (IKK) complex, whereas TRAF2 couples the TNFR1 to the SAPK/JNK cascade. In transfection studies, RIP1 and TRAF2 stimulate p38 MAP kinase activation, and dominant-negative forms of RIP1 and TRAF2 inhibit TNF-alpha-induced p38 MAP kinase activation. We found TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production impaired in rip1(-/-) murine embryonic fibroblasts (MEF) but unaffected in traf2(-/-) MEF. Yet, both rip1(-/-) and traf2(-/-) MEF exhibit a normal p38 MAP kinase response to inducers of osmotic shock or IL-1alpha. Thus, RIP1 is a specific mediator of the p38 MAP kinase response to TNF-alpha. These studies suggest that TNF-alpha-induced activation of p38 MAP kinase and SAPK/JNK pathways bifurcate at the level of RIP1 and TRAF2. Moreover, endogenous RIP1 associates with the MAP kinase kinase kinase (MAP3K) MEKK3 in TNF-alpha-treated cells, and decreased TNF-alpha-induced p38 MAP kinase activation is observed in Mekk3(-/-) cells. Taken together, these studies suggest a mechanism whereby RIP1 may mediate the p38 MAP kinase response to TNF-alpha, by recruiting the MAP3K MEKK3.     

Multiple activation mechanisms of p38alpha mitogen-activated protein kinase

The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38alpha. In addition to activation by upstream kinases, p38alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.     

Protein kinase C epsilon is required for the induction of mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages

LPS induces in bone marrow macrophages the transient expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). Because MKP-1 plays a crucial role in the attenuation of different MAPK cascades, we were interested in the characterization of the signaling mechanisms involved in the control of MKP-1 expression in LPS-stimulated macrophages. The induction of MKP-1 was blocked by genistein, a tyrosine kinase inhibitor, and by two different protein kinase C (PKC) inhibitors (GF109203X and calphostin C). We had previously shown that bone marrow macrophages express the isoforms PKC beta I, epsilon, and zeta. Of all these, only PKC beta I and epsilon are inhibited by GF109203X. The following arguments suggest that PKC epsilon is required selectively for the induction of MKP-1 by LPS. First, in macrophages exposed to prolonged treatment with PMA, MKP-1 induction by LPS correlates with the levels of expression of PKC epsilon but not with that of PKC beta I. Second, G?6976, an inhibitor selective for conventional PKCs, including PKC beta I, does not alter MKP-1 induction by LPS. Last, antisense oligonucleotides that block the expression of PKC epsilon, but not those selective for PKC beta I or PKC zeta, inhibit MKP-1 induction and lead to an increase of extracellular-signal regulated kinase activity during the macrophage response to LPS. Finally, in macrophages stimulated with LPS we observed significant activation of PKC epsilon. In conclusion, our results demonstrate an important role for PKC epsilon in the induction of MKP-1 and the subsequent negative control of MAPK activity in macrophages.     

Involvement of p38alpha mitogen-activated protein kinase in lung metastasis of tumor cells

To study the role of p38 mitogen-activated protein kinase (p38) activity during the process of metastasis, p38alpha(+/-) mice were subjected to an in vivo metastasis assay. The number of lung colonies of tumor cells intravenously injected in p38alpha(+/-) mice was markedly decreased compared with that in wild-type (WT) mice. On the other hand, the time-dependent increase in tumor volume after subcutaneous tumor cells transplantation was comparable between WT and p38alpha(+/-) mice. Platelets of p38alpha(+/-) mice were poorly bound to tumor cells in vitro and in vivo compared with those of WT mice. E- and P-selectin mRNAs were markedly induced in the lung after intravenous injection of tumor cells. However, the induction of these selectin mRNAs in p38alpha(+/-) mice was weaker than that in WT mice. Furthermore, the resting expression levels of E-selectin in lung endothelial cells and P-selectin in platelets of p38alpha(+/-) mice were suppressed compared with those of WT mice. The number of tumor cells attached on lung endothelial cells of p38alpha(+/-) mice was significantly reduced compared with that of WT mice. The transmigrating activity of tumor cells through lung endothelial cells of p38alpha(+/-) mice was similar to that of WT mice. These results suggest that p38alpha plays an important role in extravasation of tumor cells, possibly through regulating the formation of tumor-platelet aggregates and their interaction with the endothelium involved in a step of hematogenous metastasis.     

Delay in oral mucosal ulcer healing by aspirin is linked to the disturbances in p38 mitogen-activated protein kinase activation.

BACKGROUND: Among the early manifestations of oral mucosal impairment by nonsteroidal anti-inflammatory drugs is the delay in soft oral tissue repair brought about by the amplification of apoptotic events. In this study, we investigated the effect of a specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB 203580, on the rate of buccal mucosal ulcer healing and the apoptotic processes in rats subjected to intragastric administration of aspirin. METHODS: Groups of rats with experimentally induced buccal mucosal ulcers were administered twice daily for 10 days with SB 203580 (5, 10, and 20 mg/kg) or vehicle followed 30 min later by concomitant administration (twice daily for 10 days) of aspirin at 20 mg/kg. The animals were killed at different periods of treatment and their mucosal tissue subjected to macroscopic assessment of ulcer healing rate, measurement of soluble tumor necrosis factor-alpha (TNF-alpha), and the assay of epithelial cell apoptosis. RESULTS: In the control group the ulcer healed by the tenth day and the rate of healing was not affected by SB 203580 administration, whereas a 54.8% reduction in the ulcer area was attained in the presence of aspirin administration. Moreover, by the tenth day, the delay in ulcer healing caused by aspirin was manifested in a 5.6-fold higher rate of apoptosis and a 5.2-fold higher level of soluble TNF-alpha. Treatment with SB 203580 produced dose-dependent reduction (59.5-74.8%) in aspirin-induced increase in the mucosal level of soluble TNF-alpha, evoked 53.2-69.7% decrease in the rate of epithelial cell apoptosis, and led to a marked reversal (51.8-73.9%) in aspirin-induced delay in ulcer healing. CONCLUSIONS: The results of our findings link the delay in buccal mucosal ulcer healing caused by aspirin ingestion to the disturbances in the p38 MAPK activation.     

p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease

Inflammatory bowel diseases (IBD)--Crohn's disease and ulcerative colitis--are relapsing chronic inflammatory disorders which involve genetic, immunological, and environmental factors. The regulation of TNF-alpha, a key mediator in the inflammatory process in IBD, is interconnected with mitogen-activated protein kinase pathways. The aim of this study was to characterize the activity and expression of the four p38 subtypes (p38alpha-delta), c-Jun N-terminal kinases (JNKs), and the extracellular signal-regulated kinases (ERK)1/2 in the inflamed intestinal mucosa. Western blot analysis revealed that p38alpha, JNKs, and ERK1/2 were significantly activated in IBD, with p38alpha showing the most pronounced increase in kinase activity. Protein expression of p38 and JNK was only moderately altered in IBD patients compared with normal controls, whereas ERK1/2 protein was significantly down-regulated. Immunohistochemical analysis of inflamed mucosal biopsies localized the main expression of p38alpha to lamina propria macrophages and neutrophils. ELISA screening of the supernatants of Crohn's disease mucosal biopsy cultures showed that incubation with the p38 inhibitor SB 203580 significantly reduced secretion of TNF-alpha. In vivo inhibition of TNF-alpha by a single infusion of anti-TNF-alpha Ab (infliximab) resulted in a highly significant transient increase of p38alpha activity during the first 48 h after infusion. A significant infliximab-dependent p38alpha activation was also observed in THP-1 myelomonocytic cells. In human monocytes, infliximab enhanced TNF-alpha gene expression, which could be inhibited by SB 203580. In conclusion, p38alpha signaling is involved in the pathophysiology of IBD.     

Active mutants of the human p38alpha mitogen-activated protein kinase

Mitogen-activated protein (MAP) kinases compose a family of serine/threonine kinases that function in many signal transduction pathways and affect various cellular phenotypes. Despite the abundance of available data, the exact role of each MAP kinase is not completely defined, in part because of the inability to activate MAP kinase molecules individually and specifically. Based on activating mutations found in the yeast MAP kinase p38/Hog1 (Bell, M., Capone, R., Pashtan, I., Levitzki, A., and Engelberg, D. (2001) J. Biol. Chem. 276, 25351-25358), we designed and constructed single and multiple mutants of human MAP kinase p38alpha. Single (p38D176A, p38F327L, and p38F327S) and subsequent double (p38D176A/F327L and p38D176A/F327S) mutants acquired high intrinsic activity independent of any upstream regulation and reached levels of 10 and 25%, respectively, in reference to the dually phosphorylated wild type p38alpha. The active p38 mutants have retained high specificity toward p38 substrates and were inhibited by the specific p38 inhibitors SB-203580 and PD-169316. We also show that similar mutations can render p38gamma active as well. Based on the available structures of p38 and ERK2, we have analyzed the p38 mutants and identified a hydrophobic core stabilized by three aromatic residues, Tyr-69, Phe-327, and Trp-337, in the vicinity of the L16 loop region. Upon activation, a segment of the L16 loop, including Phe-327, becomes disordered. Structural analysis suggests that the active p38 mutants emulate the conformational changes imposed naturally by dual phosphorylation, namely, destabilization of the hydrophobic core. Essentially, the hydrophobic core is an inherent stabilizer that maintains low basal activity level in unphosphorylated p38.     

Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization

Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.     

Interferon-beta protects astrocytes against tumour necrosis factor-induced apoptosis via activation of p38 mitogen-activated protein kinase

Several large clinical trials have demonstrated that interferon-beta (IFN-beta) therapy is effective in the treatment of multiple sclerosis (MS) patients. However, the mechanisms underlying the beneficial effects of IFN-beta are not fully understood. Most of the effort in the study of the relevant mechanisms of IFN-beta has dealt with its immunomodulatory actions. However, the beneficial effects of IFN-beta in MS patients may also depend on non-immune mechanisms, including the modulation of astrocyte function. In the present work, we have found that IFN-beta treatment protects astrocytes against tumour necrosis factor-induced apoptosis via activation of p38 mitogen-activated protein kinase. We propose that this effect may be of importance to protect astrocytes against apoptosis within the demyelinated plaques of the MS.     

Hydrogen sulfide attenuates lipopolysaccharide-induced inflammation by inhibition of p38 mitogen-activated protein kinase in microglia

The present study attempts to investigate the effect of H(2)S on lipopolysaccharide (LPS)-induced inflammation in both primary cultured microglia and immortalized murine BV-2 microglial cells. We found that exogenous application of sodium hydrosulfide (NaHS) (a H(2)S donor, 10-300 micro mol/L) attenuated LPS-stimulated nitric oxide (NO) in a concentration-dependent manner. Stimulating endogenous H(2)S production decreased LPS-stimulated NO production, whereas lowering endogenous H(2)S level increased basal NO production. Western blot analysis showed that both exogenous and endogenous H(2)S significantly attenuated the stimulatory effect of LPS on inducible nitric oxide synthase expression, which is mimicked by SB 203580, a specific p38 mitogen-activated protein kinase (MAPK) inhibitor. Exogenously applied NaHS significantly attenuated LPS-induced p38 MAPK phosphorylation in BV-2 microglial cells. Moreover, both NaHS (300 micro mol/L) and SB 203580 (1 micro mol/L) significantly attenuated LPS-induced tumor necrosis factor-alpha secretion, another inflammatory indicator. In addition, NaHS (10-300 micro mol/L) dose-dependently decreased LPS-stimulated NO production in primary cultured astrocytes, suggesting that the anti-neuroinflammatory effect of H(2)S is not specific to microglial cells alone. Taken together, H(2)S produced an anti-inflammatory effect in LPS-stimulated microglia and astrocytes, which may be due to inhibition of inducible nitric oxide synthase and p38 MAPK signaling pathways. These findings may have important implications in the treatment of neuroinflammation-related diseases.     

Tumour necrosis factor activates the mitogen-activated protein kinases p38alpha and ERK in the synovial membrane in vivo

Tumour necrosis factor (TNF) is considered to be a major factor in chronic synovial inflammation and is an inducer of mitogen-activated protein kinase (MAPK) signalling. In the present study we investigated the ability of TNF to activate MAPKs in the synovial membrane in vivo. We studied human TNF transgenic mice--an in vivo model of TNF-induced arthritis--to examine phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38MAPKalpha in the inflamed joints by means of immunoblot and immunohistochemistry. In addition, the effects of systemic blockade of TNF, IL-1 and receptor activator of nuclear factor-kappaB (RANK) ligand on the activation of MAPKs were assessed. In vivo, overexpression of TNF induced activation of p38MAPKalpha and ERK in the synovial membrane, whereas activation of JNK was less pronounced and rarely observed on immunohistochemical analysis. Activated p38MAPKalpha was predominantly found in synovial macrophages, whereas ERK activation was present in both synovial macrophages and fibroblasts. T and B lymphocytes did not exhibit major activation of any of the three MAPKs. Systemic blockade of TNF reduced activation of p38MAPKalpha and ERK, whereas inhibition of IL-1 only affected p38MAPKalpha and blockade of RANK ligand did not result in any decrease in MAPK activation in the synovial membrane. These data indicate that TNF preferentially activates p38MAPKalpha and ERK in synovial membrane exposed to TNF. This not only suggests that targeted inhibition of p38MAPKalpha and ERK is a feasible strategy for blocking TNF-mediated effects on joints, but it also shows that even currently available methods to block TNF effectively reduce activation of these two MAPKs.     

Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing

We examined the effect of p38 mitogen-activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immunocytochemistry revealed that phosphorylated p38 MAPK-immunoreactive cells were predominantly present in laminae I-IV of the dorsal horn. Double-staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co-localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD-282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo-oxygenase-2 and inflammation-induced appearance of Fos-positive neurons, was blocked by pretreatment, but not post-treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation-induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.     

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.