JNKing Revealed

In a recent issue of Molecular Cell, Sabapathy et al. (2004) demonstrate that JNK2 destabilizes c-Jun under nonstimulating conditions, whereas after stress, activated JNK1 phosphorylates c-Jun, resulting in its activation and stabilization, thereby highlighting functional differences between JNK1 and JNK2.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

JNK and tumor necrosis factor-alpha mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes

Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKKbeta, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-alpha, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-alpha signaling with neutralizing antibodies to TNF-alpha or its receptors or with a dominant negative TNF-alpha peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-alpha signaling, whereas the FFA-induced increase in TNF-alpha secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-alpha-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-alpha is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.     

c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, is essential for tumor necrosis factor alpha-induced c-Jun kinase activation and apoptosis

Two ubiquitously expressed isoforms of c-Jun N-terminal protein kinase (JNK), JNK1 and JNK2, have shared functions and different functions. However, the molecular mechanism is unknown. Here we report that JNK1, but not JNK2, is essential for tumor necrosis factor alpha (TNF-alpha)-induced c-Jun kinase activation, c-Jun expression, and apoptosis. Using mouse fibroblasts deficient in either Jnk1 or Jnk2, we found that JNK1 was activated by TNF-alpha, whereas JNK2 activation was negligible. In addition, JNK2 interfered with JNK1 activation via its "futile" phosphorylation by upstream kinases. Consequently, expression and activation of c-Jun, which depends on JNK activity, were impaired in Jnk1 null cells but enhanced in Jnk2 null cells. TNF-alpha-induced apoptosis was also suppressed in Jnk1 null fibroblasts but increased in Jnk2 null cells. Thus, our results provide a molecular mechanism underlying the different biological functions of JNK isoforms.     

Activation of the Drosophila MLK by ceramide reveals TNF-alpha and ceramide as agonists of mammalian MLK3

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity remain unknown. Here, we demonstrate ceramide as the activator of Drosophila MLK (dMLK) and identify ceramide and TNF-alpha as agonists of mammalian MLK3. dMLK and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of dMLK and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-alpha also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of dMLK and MLK3, and MLK3 contributes to JNK activation induced by TNF-alpha.     

Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-Kinase and Syk-mediated pathways

Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.     

Protein phosphatase 4 is involved in tumor necrosis factor-alpha-induced activation of c-Jun N-terminal kinase.

Protein phosphatase 4 (PP4, previously named protein phosphatase X (PPX)), a PP2A-related serine/threonine phosphatase, has been shown to be involved in essential cellular processes, such as microtubule growth and nuclear factor kappa B activation. We provide evidence that PP4 is involved in tumor necrosis factor (TNF)-alpha signaling in human embryonic kidney 293T (HEK293T) cells. Treatment of HEK293T cells with TNF-alpha resulted in time-dependent activation of endogenous PP4, peaking at 10 min, as well as increased serine and threonine phosphorylation of PP4. We also found that PP4 is involved in relaying the TNF-alpha signal to c-Jun N-terminal kinase (JNK) as indicated by the ability of PP4-RL, a dominant-negative PP4 mutant, to block TNF-alpha-induced JNK activation. Moreover, the response of JNK to TNF-alpha was inhibited in HEK293 cells stably expressing PP4-RL in comparison to parental HEK293 cells. The involvement of PP4 in JNK signaling was further demonstrated by the specific activation of JNK, but not p38 and ERK2, by PP4 in transient transfection assays. However, no direct PP4-JNK interaction was detected, suggesting that PP4 exerts its positive regulatory effect on JNK in an indirect manner. Taken together, these data indicate that PP4 is a signaling component of the JNK cascade and involved in relaying the TNF-alpha signal to the JNK pathway.     

Leptin enhances TNF-alpha production via p38 and JNK MAPK in LPS-stimulated Kupffer cells

Leptin is now recognized as a proinflammatory cytokine and thought to be a progressive factor for non-alcoholic steatohepatitis (NASH). Here we showed the effects of leptin on the production of TNF-alpha (tumor necrosis factor-alpha) by Kupffer cells (KCs) with signal transduction. Leptin enhanced TNF-alpha production accompanied by a dose-dependent increase of MAPK activity in lipopolysaccharide (LPS)-stimulated KCs. SB203580 and JNK inhibitor I, specific inhibitors of P38 and JNK, inhibited TNF-alpha production in KCs but PD98059, an inhibitor of the ERK pathway, did not affect TNF-alpha production by KCs. Recombinant constitutively active adenovirus (Ad)-MKK6 and-MKK7 increased TNF-alpha production in KCs with activation of P38 and JNK without any change by Ad-MEK1 delivery. On the other hand, KCs isolated from the Zucker rat (fa/fa), a leptin receptor-deficient rat, showed reduced production of TNF-alpha on stimulation with LPS. The delivery of Ad-MKK6 and-MKK7, but not Ad-MEK1, increased TNF-alpha production in KCs of Zucker rats with activation of P38 and JNK. Addition of leptin to normal rats increased LPS-induced hepatic TNF-alpha production in vivo and leptin receptor-deficient Zucker rats showed reduced hepatic TNF-alpha production on addition of LPS in vivo. These findings indicate that P38 and JNK pathways are involved in the signal transduction of leptin enhancement of LPS-induced TNF-alpha production.     

Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-alpha in IEC-6 cells

Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-alpha (TNF-alpha). TNF-alpha or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-alpha, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with alpha-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-alpha/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-alpha-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.     

Oxygen JNKies: phosphatases overdose on ROS

Proinflammatory cytokine TNFalpha triggers cell death by inducing reactive oxygen species (ROS). These then inflict cytotoxicity through downstream activation of the JNK MAPK cascade. Yet the mechanisms by which ROS trigger JNK signaling have remained elusive. In a recent issue of Cell, Kamata et al. now provide one such mechanism.     

Involvement of p38 and JNK MAPKs pathways in Substance P-induced production of TNF-alpha by peritoneal mast cells

Mast cells play a central role in both inflammation and immediate allergic reactions. We have previously shown that Substance P (SP) stimulates TNF-alpha mRNA and protein expression in rat peritoneal mast cells (PMC). In the present paper, we investigated whether the induction of TNF-alpha production by the mast cells agonist involves MAPKs signalling pathways. We found that as early as 5 min after PMC exposure to SP, phosphorylation of p38 MAPK and JNK was induced. On the contrary, phosphorylation of p42/44 MAPK occurred only after a 30 min exposure to SP and did not correlate with SP-induced TNF-alpha production. The highly specific p38 MAPK inhibitor SB203580 and the blocker of PI-3K wortmannin, abolished SP-induced increase in TNF-alpha mRNA and protein levels and showed to reduce the SP-mediated histamine secretion. In addition, wortmannin reduced SP-mediated JNK phosphorylation. The results reveal that the induction of TNF-alpha expression and histamine exocytosis by exposure of rat PMC to substance P requires the activation of p38 and JNK MAPKs pathways. Moreover, they suggest PI-3K as a possible upstream component of JNK pathway in SP-induced inflammatory reactions.     

Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts.

We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-alpha. Similarly, LY294002 significantly diminished TNF-alpha-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-alpha is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-alpha to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-alpha signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-alpha.     

TNF-alpha promotes cell survival through stimulation of K+ channel and NFkappaB activity in corneal epithelial cells

Tumor necrosis factor (TNF-alpha) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-alpha also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-alpha stimulation induced activation of a voltage-gated K+ channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-alpha on downstream events included NFkappaB nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-alpha induced increases in p21 expression resulting in partial cell cycle attenuation in the G1 phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-alpha-induced K+ channel activity effectively prevented NFkappaB nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-alpha. In conclusion, TNF-alpha promotes survival of HCE cells through sequential stimulation of K+ channel and NFkappaB activities. This response to TNF-alpha is dependent on stimulating K+ channel activity because following suppression of K+ channel activity TNF-alpha failed to activate NFkappaB nuclear translocation and binding to nuclear DNA.     

Differential regulation of JNK activation and MKP-1 expression by peroxovanadium complexes

Bisperoxovanadium complexes have been identified as insulinomimetic agents and protein tyrosine phosphatase inhibitors. The aim of the present study was to examine the effects of the most potent bisperoxovanadium complex, potassium bisperoxo (1,10-phenanthroline) oxovanadate (V) [bpV(phen)], on expression and activation of c-jun N-terminal protein kinases (JNK) and on expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cell lines. We compared the effects of bpV(phen) with the effects of tumor necrosis factor-alpha (TNF-alpha), a known regulator of JNK phosphorylation and inducer of MKP-1. Treatment with bpV(phen) causes significant and sustained down-regulation of MKP-1 expression both in PC12 and HeLa cells. In contrast, TNF-alpha induces MKP-1 expression in PC12 cells and does not alter MKP-1 expression in HeLa cells. Both bpV(phen) and TNF-alpha induce MKP-1 expression in OVCAR-3 cell line but with different dynamics: TNF-alpha causes transient and bpV(phen) sustained induction of MKP-1 expression. Temporal pattern of level of MKP-1 expression correlates with the regulation of JNK phosphorylation by bpV(phen) and TNF-alpha in PC12 cells. However, no detectable phospho-JNK signal is observed in either OVCAR-3 or HeLa cells treated with bpV(phen). In contrast, TNF-alpha causes strong and sustained JNK phosphorylation in OVCAR-3 cell line, and strong but transient JNK activation in HeLa cells. BpV(phen) and TNF-alpha does not alter JNK expression in any of the cell lines studied. We demonstrate that the effect of two stressors, bpV(phen) and TNF-alpha, on MKP-1 expression and JNK phosphorylation are strikingly different, depending on the cell type. These results suggest the possible role of MKP-1 in regulation of JNK phosphorylation in both PC12 and OVCAR-3 cell lines treated with bpV(phen).