Rip1 mediates the Trif-dependent toll-like receptor 3- and 4-induced NF-{kappa}B activation but does not contribute to interferon regulatory factor 3 activation

Rip1 is required for IkappaB kinase activation in response to tumor necrosis factor alpha (TNF-alpha) and has been implicated in the Toll-like receptor 3 (TLR3) response to double-stranded RNA. Cytokine production is impaired when rip1-/- cells are treated with TNF-alpha, poly(I-C), or lipopolysaccharide, implicating Rip1 in the Trif-dependent TLR3 and TLR4 pathways. To examine the role of Rip1 in the Trif-dependent TLR4 pathway, we generated rip1-/- MyD88-/- cells. Lipopolysaccharide failed to stimulate NF-kappaB activation in rip1-/-MyD88-/- cells, revealing that Rip1 is also required for the Trif-dependent TLR4-induced NF-kappaB pathway. In addition to activating NF-kappaB, TLR3/4 pathways also stimulate interferon regulatory factor 3 activation. However, we find that Rip1 expression stimulates NF-kappaB but not interferon regulatory factor 3 activity. In the TNF-alpha pathway, Rip1 interacts with the E3 ubiquitin ligase Traf2 and is modified by polyubiquitin chains. Upon TLR3 activation, Rip1 is also modified by polyubiquitin chains and is recruited to TLR3 along with Traf6 and the ubiquitin-activated kinase Tak1. These studies suggest that Rip1 uses a similar, ubiquitin-dependent mechanism to activate IkappaB kinase-beta in response to TNF-alpha and TLR3 ligands.     

The role of p38 MAP kinase in the synergistic cytotoxic action of calcitriol and TNF-alpha in human breast cancer cells

Calcitriol, the hormonal form of Vitamin D, potentiates the activity of some agents of the anti-cancer immune system including tumor necrosis factor-alpha (TNF-alpha). Different signaling pathways activated by TNF-alpha may be targets for calcitriol action. Activation of p38 MAP kinase was shown to have both pro- and anti-apoptotic actions in TNF-alpha-induced programmed cell death depending on cell context. Treatment of MCF-7 breast cancer cells with TNF-alpha resulted in activation of p38 MAP kinase that persisted for at least 24h. Whereas calcitriol had no effect on the earlier phase of p38 MAP kinase activation (up to 1h), it inhibited the activation of this pathway between one and 24h after exposure to TNF-alpha. Both calcitriol and the p38 MAP kinase inhibitor SB203580 enhanced TNF-alpha-induced cytotoxicity and drop in mitochondrial membrane potential, but their combined effect was sub-additive. Taken together, these findings suggest that p38 MAP kinase plays an anti-apoptotic role in TNF-alpha-induced cytotoxicity in MCF-7 cells and that the synergistic interaction between TNF-alpha and calcitriol, leading to mitochondrial damage and subsequent cell death, is partially due to modulation of this signaling pathway.     

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.     

Ebselen inhibits tumor necrosis factor-alpha-induced c-Jun N-terminal kinase activation and adhesion molecule expression in endothelial cells

Tumor necrosis factor-alpha (TNF-alpha) stimulates expression of endothelial cell (EC) genes that may promote atherosclerosis in part by an activation of mitogen-activated protein (MAP) kinases. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a selenoorganic compound, is effective for acute ischemic stroke; however, its effect on EC has not yet been elucidated. We examined the effect of ebselen on TNF-alpha-induced MAP kinase activation and adhesion molecule expression in cultured human umbilical vein endothelial cells (HUVEC). Extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 were rapidly and significantly activated by TNF-alpha in HUVEC. TNF-alpha-induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 were not affected. Apoptosis signal-regulated kinase 1 (ASK1) was suggested to be involved in TNF-alpha-induced JNK activation because transfection of kinase-inactive ASK1 inhibited TNF-alpha-induced JNK activation. Ebselen inhibited TNF-alpha-induced TNF receptor-associated factor 2 (TRAF2)-ASK1 complex formation and phosphorylation of stress-activated protein kinase ERK kinase 1 (SEK1), which is an upstream signaling molecule of JNK. Finally, TNF-alpha-induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) activation and resultant intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions were inhibited by ebselen. Specific inhibitors for JNK and NF-kappaB also inhibited TNF-alpha-induced ICAM-1 and VCAM-1 expressions in HUVEC. These findings suggest that ebselen prevents TNF-alpha-induced EC activation through the inhibition of TRAF2-ASK1-SEK1 signaling pathway, which leads to JNK activation. Inhibition of JNK by ebselen may imply its usefulness for the prevention of atherosclerosis relevant to EC activation.     

MAP4K4 gene silencing in human skeletal muscle prevents tumor necrosis factor-alpha-induced insulin resistance

Tumor necrosis factor-alpha (TNF-alpha) induces skeletal muscle insulin resistance by impairing insulin signaling events involved in GLUT4 translocation. We tested whether mitogenic-activated protein kinase kinase kinase kinase isoform 4 (MAP4K4) causes the TNF-alpha-induced negative regulation of extracellular signal-regulated kinase-1/2 (ERK-1/2), c-Jun NH2-terminal kinase (JNK), and the insulin receptor substrate-1 (IRS-1) on the insulin signaling pathway governing glucose metabolism. Using small interfering RNA (siRNA) to suppress the expression of MAP4K4 protein 85% in primary human skeletal muscle cells, we provide evidence that TNF-alpha-induced insulin resistance on glucose uptake was completely prevented. MAP4K4 silencing inhibited TNF-alpha-induced negative signaling inputs by preventing excessive JNK and ERK-1/2 phosphorylation, as well as IRS-1 serine phosphorylation. These results highlight the MAPK4K4/JNK/ERK/IRS module in the negative regulation of insulin signaling to glucose transport in response to TNF-alpha. Depletion of MAP4K4 also prevented TNF-alpha-induced insulin resistance on Akt and the Akt substrate 160 (AS160), providing evidence that appropriate insulin signaling inputs for glucose metabolism were rescued. Silencing of MAP2K1 and MAP2K4, signaling proteins downstream of MAP4K4, recapitulated the effect of MAP4K4 siRNA in TNF-alpha-treated cells. Thus, strategies to inhibit MAP4K4 may be efficacious in the prevention of TNF-alpha-induced inhibitory signals that cause skeletal muscle insulin resistance on glucose metabolism in humans. Moreover, in myotubes from insulin-resistant type II diabetic patients, siRNA against MAP4K4, MAP2K4, or MAP2K1 restored insulin action on glucose uptake to levels observed in healthy subjects. Collectively, our results demonstrate that MAP4K4 silencing prevents insulin resistance in human skeletal muscle and restores appropriate signaling inputs to enhance glucose uptake.     

Participation of Rip2 in lipopolysaccharide signaling is independent of its kinase activity

Rip2 (Rick, Cardiak, CCK2, and CARD3) is a serine/threonine kinase containing a caspase recruitment domain (CARD) at the C terminus. Previous reports have shown that Rip2 is involved in multiple receptor signaling pathways that are important for innate and adaptive immune responses. However, it is not known whether Rip2 kinase activity is required for its function. Here we confirm that Rip2 participates in lipopolysaccharide (LPS)/Toll-like receptor (TLR4) signaling and demonstrate that its kinase activity is not required. Upon LPS stimulation, Rip2 was transiently recruited to the TLR4 receptor complex and associated with key TLR signaling mediators IRAK1 and TRAF6. Furthermore, Rip2 kinase activity was induced by LPS treatment. These data indicate that Rip2 is directly involved in the LPS/TLR4 signaling. Whereas macrophages from Rip2-deficient mice showed impaired NF-kappaB and p38 mitogen-activated protein kinase activation and reduced cytokine production in response to LPS stimulation, LPS signaling was intact in macrophages from mice that express Rip2 kinase-dead mutant. These results demonstrate that Rip2-mediated LPS signaling is independent of its kinase activity. Our findings strongly suggest that Rip2 functions as an adaptor molecule in transducing signals from immune receptors.     

TRAF2 phosphorylation modulates tumor necrosis factor alpha-induced gene expression and cell resistance to apoptosis

TRAF2 is an adaptor protein that regulates the activation of the c-Jun N-terminal kinase (JNK) and IkappaB kinase (IKK) signaling cascades in response to tumor necrosis factor alpha (TNF-alpha) stimulation. Although the downstream events in TNF-alpha signaling are better understood, the membrane-proximal events are still elusive. Here, we demonstrate that TNF-alpha and cellular stresses induce TRAF2 phosphorylation at serine 11 and that this phosphorylation is required for the expression of a subset of NF-kappaB target genes. Although TRAF2 phosphorylation had a minimal effect on the TNF-alpha-induced rapid and transient IKK activation, it was essential for secondary and prolonged IKK activation. Consistent with this, TRAF2 phosphorylation is not required for its recruitment to the TNFR1 complex in response to TNF-alpha stimulation but is required for its association with a cytoplasmic complex containing RIP1 and IKK. In addition, TRAF2 phosphorylation was essential for the full TNF-alpha-induced activation of JNK. Notably, TRAF2 phosphorylation increased both basal and inducible c-Jun and NF-kappaB activities and rendered cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some lymphomas. These results unveil a new, finely tuned mechanism for TNF-alpha-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation contributes to elevated levels of basal NF-kappaB activity in certain human cancers.     

Phosphatidylinositol 3-Kinase/Akt Plays a Part in Tumor Necrosis Factor-alpha-induced Interleukin-6 Synthesis in Osteoblasts.

We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha induced the phosphorylation of Akt depending upon time. Akt inhibitor, 1L-6-hydroxymethyl- CHIRO-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate, significantly suppressed the TNF-alpha-stimulated IL-6 synthesis, but the inhibitory effect was partial. The phosphorylation of Akt induced by TNF-alpha was markedly attenuated by LY294002 and wortmannin, inhibitors of PI3-kinase. Wortmannin and LY294002 significantly reduce the TNF-alpha-induced IL-6 synthesis. On the contrary, the suppressive effects of Akt inhibitor, wortmannin or LY294002 on TNF-alpha-induced phosphorylation of p44/p42 MAP kinase were minor. PD98059, a specific inhibitor of MEK, had little effect on the TNF-alpha-induced phosphorylation of Akt. A combination of Akt inhibitor and PD98059 suppressed the TNF-alpha-induced IL-6 synthesis in an additive manner. These results strongly suggest that PI3-kinase/Akt plays a role in the TNF-alpha-stimulated IL-6 synthesis mainly independent of p44/p42 MAP kinase in osteoblasts.     

Tumor necrosis factor-alpha-stimulated cell proliferation is mediated through sphingosine kinase-dependent Akt activation and cyclin D expression

Tumor necrosis factor-alpha (TNF-alpha) has been shown to activate sphingosine kinase (SphK) in a variety of cell types. The extent to which SphK signaling mediates the pleiotropic effects of TNF-alpha is not entirely clear. The current study examined the role of SphK activity in TNF-alpha-stimulated cell proliferation in 1321N1 glioblastoma cells. We first demonstrated that pharmacological inhibitors of SphK markedly decrease TNF-alpha-stimulated DNA synthesis. Signaling mechanisms through which SphK mediated the effect of TNF-alpha on DNA synthesis were then examined. Inhibition of Rho proteins with C3 exoenzyme or of Rho kinase with Y27632 attenuated TNF-alpha-stimulated DNA synthesis. However, RhoA activation by TNF-alpha was not blocked by SphK inhibition. ERK activation was also required for TNF-alpha-stimulated DNA synthesis but likewise TNF-alpha-induced ERK activation was not blocked by inhibition of SphK. Thus, neither RhoA nor ERK activation are the SphK-dependent transducers of TNF-alpha-induced proliferation. In contrast, TNF-alpha-stimulated Akt phosphorylation, which was also required for DNA synthesis, was attenuated by SphK inhibition or SphK1 knockdown by small interfering RNA. Furthermore, cyclin D expression was increased by TNF-alpha in a SphK- and Akt-dependent manner. Additional studies demonstrated that TNF-alpha effects on DNA synthesis, ERK, and Akt phosphorylation are not mediated through cell surface Gi -coupled S1P receptors, because none of these responses were inhibited by pertussis toxin. We conclude that SphK-dependent Akt activation plays a significant role in TNF-alpha-induced cyclin D expression and cell proliferation.     

Flotillin-1/reggie-2 protein plays dual role in activation of receptor-tyrosine kinase/mitogen-activated protein kinase signaling

Our previous work has shown that the membrane microdomain-associated flotillin proteins are potentially involved in epidermal growth factor (EGF) receptor signaling. Here we show that knockdown of flotillin-1/reggie-2 results in reduced EGF-induced phosphorylation of specific tyrosines in the EGF receptor (EGFR) and in inefficient activation of the downstream mitogen-activated protein (MAP) kinase and Akt signaling. Although flotillin-1 has been implicated in endocytosis, its depletion affects neither the endocytosis nor the ubiquitination of the EGFR. However, EGF-induced clustering of EGFR at the cell surface is altered in cells lacking flotillin-1. Furthermore, we show that flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner. However, knockdown of flotillin-1 appears to affect the activation of the downstream MAP kinase signaling more directly. We here show that flotillin-1 forms a complex with CRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) and that flotillin-1 knockdown leads to a direct inactivation of ERK1/2. Thus, flotillin-1 plays a direct role during both the early phase (activation of the receptor) and late (activation of MAP kinases) phase of growth factor signaling. Our results here unveil a novel role for flotillin-1 as a scaffolding factor in the regulation of classical MAP kinase signaling. Furthermore, our results imply that other receptor-tyrosine kinases may also rely on flotillin-1 upon activation, thus suggesting a general role for flotillin-1 as a novel factor in receptor-tyrosine kinase/MAP kinase signaling.     

Implication of TNF receptor-I-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in growth of AIDS-associated Kaposi's sarcoma cells: a possible role of a novel death domain protein MADD in TNF-alpha-induced ERK1/2 activation in Kaposi's sarcoma cells

TNF-alpha is a key pathogenic mediator of infectious and inflammatory diseases. HIV infection stimulates and dysregulates the immune system, leading to abnormal production of TNF-alpha. Despite its cytotoxic effect on some tumor cell lines, TNF-alpha functions as a growth stimulator for Kaposi's sarcoma (KS), a common malignancy in HIV-infected patients. However, signaling pathways linked to TNF-alpha-induced mitogenic responses are not well understood. We found that extracellular signal-regulated kinases 1 and 2 (ERK1/2) in KS cells were significantly activated by TNF-alpha through tyrosine/threonine phosphorylation. Using neutralizing anti-TNFR-I and TNFR-II mAbs, we have now obtained evidence that TNF-alpha-induced KS cell growth and ERK1/2 activation are mediated exclusively by TNFR-I, not by TNFR-II. A selective inhibitor for ERK1/2 activator kinases, PD98059, profoundly inhibited not only the activation of ERK1/2, but also the TNF-alpha-induced KS cell proliferation. We therefore propose that the TNFR-I-ERK1/2 pathway plays a pivotal role in transmitting to KS cells the mitogenic signals of TNF-alpha. TNFR-I possesses no intrinsic kinase activity, suggesting that TNFR-I-associated proteins may provide a link between TNFR-I and ERK1/2 activation. We found that actinomycin D treatment of KS cells selectively abolished expression of mitogen-activated protein kinase-activating death domain protein (MADD), a novel TNFR-I-associated death domain protein. TNF-alpha failed to induce ERK1/2 activation in the actinomycin D-treated cells. MADD may couple TNFR-I with the ERK1/2 signaling pathway required for KS cell proliferation.     

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.     

Tumor Necrosis Factor-α Induces Stress Fiber Formation through Ceramide Production: Role of Sphingosine Kinase

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.     

Ubiquitination of free cyclin D1 is independent of phosphorylation on threonine 286

Cyclin D1 binds and regulates the activity of cyclin-dependent kinases (CDKs) 4 and 6. Phosphorylation of the retinoblastoma protein by cyclin D1.CDK4/6 complexes during the G(1) phase of the cell cycle promotes entry into S phase. Cyclin D1 protein is ubiquitinated and degraded by the 26 S proteasome. Previous studies have demonstrated that cyclin D1 ubiquitination is dependent on its phosphorylation by glycogen synthase kinase 3beta (GSK-3beta) on threonine 286 and that this phosphorylation event is greatly enhanced by binding to CDK4 (Diehl, J. A., Cheng, M. G., Roussel, M. F., and Sherr, C. J. (1998) Genes Dev. 12, 3499-3511). We now report an additional pathway for the ubiquitination of free cyclin D1 (unbound to CDKs). We show that, when unbound to CDK4, a cyclin D1-T286A mutant is ubiquitinated. Further, we show that a mutant of cyclin D1 that cannot bind to CDK4 (cyclin D1-KE) is also ubiquitinated in vivo. Our results demonstrate that free cyclin D1 is ubiquitinated independently of its phosphorylation on threonine 286 by GSK-3beta, suggesting that, as has been shown for cyclin E, distinct pathways of ubiquitination lead to the degradation of free and CDK-bound cyclin D1. The pathway responsible for ubiquitination of free cyclin D1 may be important in limiting the effects of cyclin D1 overexpression in a variety of cancers.