Activation Of Inhibitor kappaB Kinase Research Articles

Nitric Oxide Inhibits NF-κB-mediated Survival Signaling: Possible Role in Overcoming TRAIL Resistance.

Chemoresistance is a major consequence of multicycle chemotherapy and can be attributed to constitutive activation of pro-survival signaling pathways. Nitric oxide is a ubiquitous signaling molecule which has been shown to inhibit several pathways involved with survival signaling in cancer cells. We have previously demonstrated the anti-tumor activity of a nitric oxide-donor, nitrosylcobalamin (NO-Cbl), mediated by increased expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. We also demonstrated that a functional Apo2L/TRAIL receptor is necessary for the induction of cell death by NO-Cbl and the Apo2L/TRAIL death receptor DR4 (TRAIL R1) is S-nitrosylated. The aim of the study was to examine the effects of nitric oxide (NO) on nuclear factor kappa B (NF-κB) and determine whether nitric oxide could sensitize drug-resistant melanomas to Apo2L/TRAIL via inhibition of NF-κB or inhibitor kappa B kinase (IKK). Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by the TUNEL assay. The activation status of NF-κB was established by assaying luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL, but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of nitric oxide to inhibit NF-κB and potentiate the effects of chemotherapeutic agents, such as Apo2L/TRAIL, represents a promising anti-cancer combination based on recent clinical investigations of anti-TRAIL antibodies for cancer treatment strategies.

Concentrated Ambient PM2.5-Induced Inflammation and Endothelial Dysfunction in a Murine Model of Neural IKK2 Deficiency.

Background:Exposure to ambient fine particulate matter () is associated with cardiovascular mortality, but underlying pathophysiologic mechanisms are not fully understood. Hypothalamic inflammation, characterized by the activation of Inhibitor kappaB kinase 2/Nuclear factor kappaB () signaling pathway, may play an important role in the pathogenesis of cardiovascular diseases. We recently demonstrated that hypothalamic inflammation is increased in mice exposed to concentrated ambient (CAP).Objectives:In the present study, we used a neuron-specific IKK2 knockout mouse model to examine the role of neural IKK2 expression and hypothalamic inflammation in the pathophysiologic effects of .Methods: We assessed inflammatory and vascular responses in () and littermate (control) mice after 4 mo of exposure to filtered air (FA) or CAP.Results:CAP exposure was associated with significantly higher tumor necrosis factor- and interleukin (IL)-6 mRNA in the hypothalamus of control mice, but not mice. In addition, CAP exposure–induced increases in bronchoalveolar lavage fluid (BALF) leukocytes, pulmonary macrophage infiltration and IL-6 expression, plasma and levels, adipose macrophage infiltration and expression, and endothelial dysfunction were reduced or absent in mice compared with controls.Conclusions:Our findings support a role of neural IKK2 in CAP exposure–induced local and systemic pro-inflammatory cytokine expression, pulmonary and adipose inflammation, and endothelial dysfunction, thus providing insight into pathophysiologic mechanisms that may mediate effects of exposure. https://doi.org/10.1289/EHP2311

Activation of Nuclear Factor κB by Somatostatin Type 2 Receptor in Pancreatic Acinar AR42J Cells Involves Gα14 and Multiple Signaling Components: A MECHANISM REQUIRING PROTEIN KINASE C, CALMODULIN-DEPENDENT KINASE II, ERK, AND c-Src

Medications targeting the somatostatin type 2 receptor (SSTR2) have been employed for pancreatic inflammations and cancers, possibly via the regulation of the transcription factor nuclear factor kappaB (NFkappaB). Here we demonstrate that in tumoral pancreatic acinar AR42J cells, activation of SSTR2 leads to stimulation of the inhibitor kappaB kinase (IKK)/NFkappaB signaling cascade via pertussis toxin-insensitive G proteins in a time- and dose-dependent manner. The inability of G(q/11) and G(12/13) proteins to activate IKK/NFkappaB by SSTR2 in transfected human embryonic kidney 293 cells and the lack of Galpha(16) in AR42J cells suggested a possible role of Galpha(14) in mediating SSTR2-induced responses. This regulatory role of Galpha(14) was further confirmed by the activation of IKK and NFkappaB in human embryonic kidney 293 cells expressing SSTR2 and Galpha(14) upon induction. The stimulatory effect of Gbeta(1)gamma(2) and the abrogation by overexpressing transducin confirmed the participation of Gbetagamma in SSTR2-mediated IKK/NFkappaB activation. By the application of specific inhibitors and dominant negative mutants, phospholipase Cbeta, protein kinase C, and calmodulin-dependent kinase II were shown to be involved in SSTR2-induced responses. Inhibition of c-Src and numerous intermediates, including Ras, Raf-1 kinase, MEK1/2, along with the extracellular signal-regulated kinase cascade attenuated somatostatin-mediated IKK/NFkappaB activation. Although c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) were also stimulated by SSTR2, suppression of these two MAPKs was ineffective in altering the somatostatin-mediated responses. Similar results were also obtained using AR42J cells. These data suggest that activation of the IKK/NFkappaB signaling cascade by SSTR2 requires a complicated network consisting of Galpha(14) and multiple intermediates.

Activation of IKKβ by glucose is necessary and sufficient to impair insulin signaling and nitric oxide production in endothelial cells

Hyperglycemic impairment of nitric oxide (NO) production by endothelial cells is implicated in the effect of diabetes to increase cardiovascular disease risk, but the molecular basis for this effect is unknown. In skeletal muscle, diabetes induces activation of inhibitor kappaB kinase (IKKβ), a key cellular mediator of the response to inflammatory stimuli, and this impairs insulin signal transduction via the insulin receptor substrate-phosphatidylinositol 3-OH kinase (IRS-1/PI3-kinase) pathway. Since activation of endothelial nitric oxide synthase (eNOS) is dependent on IRS-1/PI3-kinase signaling, we hypothesized that activation of IKKβ may contribute to the effect of glucose to impair NO production. Here, we show that exposure of bovine aortic endothelial cells to high glucose (25 mM) for 24 h impaired insulin-mediated tyrosine phosphorylation of IRS-1, serine phosphorylation of Akt, activation of eNOS, and production of NO. High glucose treatment also activated IKKβ, and pretreatment with aspirin, a pharmacological inhibitor of IKKβ, prevented both glucose-induced IKKβ activation and the effect of high glucose to impair insulin-mediated NO production. These adverse responses to glucose were also blocked by selective inhibition of IKKβ signaling via overexpression of a kinase-inactive form of the enzyme. Conversely, overexpression of wild-type IKKβ recapitulated the deleterious effect of high glucose on insulin-mediated activation of eNOS. These data demonstrate that activation of IKKβ plays a critical and novel role to mediate the deleterious effects of high glucose on endothelial cell function.

Inhibitor kappaB kinase is involved in the paracrine crosstalk between human fat and muscle cells.

Adipose tissue is now considered as an endocrine and secretory organ, and some adipocyte factors are thought to play a major role in the induction of insulin resistance in skeletal muscle. Here we tested the hypothesis that the crosstalk between fat and muscle involves activation of inhibitor kappaB Kinase (IKK) in the myocytes. Adipocyte-conditioned culture medium was added to the muscle cells overnight, or human fat and muscle cells were kept in co-culture. Insulin signalling was subsequently analysed in the myocytes. Involvement of IKK was assessed using I229, a highly specific inhibitor of the IKK complex. Adipocyte-conditioned medium strongly inhibited insulin-induced serine phosphorylation of Akt in myocytes with a rapid parallel activation of the nuclear factor kappaB pathway in these cells. Conditioned medium lacking the perturbation of insulin signalling did not activate NF-kappaB. Insulin signalling to Akt was completely abrogated under co-culture conditions. The IKK inhibitor I229 did not affect protein expression of Akt, but fully restored insulin action in myocytes subjected to co-culture. These data show that the release of fat cell factors may rapidly induce insulin resistance in human skeletal muscle cells. This process appears to be mediated by an IKK/NF-kappaB dependent pathway. We suggest that inhibitors of IKK would be of use to counteract the negative crosstalk between fat and muscle.

Inhibition of insulin sensitivity by free fatty acids requires activation of multiple serine kinases in 3T3-L1 adipocytes.

Insulin receptor substrate (IRS) has been suggested as a molecular target of free fatty acids (FFAs) for insulin resistance. However, the signaling pathways by which FFAs lead to the inhibition of IRS function remain to be established. In this study, we explored the FFA-signaling pathway that contributes to serine phosphorylation and degradation of IRS-1 in adipocytes and in dietary obese mice. Linoleic acid, an FFA used in this study, resulted in a reduction in insulin-induced glucose uptake in 3T3-L1 adipocytes. This mimics insulin resistance induced by high-fat diet in C57BL/6J mice. The reduction in glucose uptake is associated with a decrease in IRS-1, but not IRS-2 or GLUT4 protein abundance. Decrease in IRS-1 protein was proceeded by IRS-1 (serine 307) phosphorylation that was catalyzed by serine kinases inhibitor kappaB kinase (IKK) and c-JUN NH2-terminal kinase (JNK). IKK and JNK were activated by linoleic acid and inhibition of the two kinases led to prevention of IRS-1 reduction. We demonstrate that protein kinase C (PKC) theta is expressed in adipocytes. In 3T3-L1 adipocytes and fat tissue, PKCtheta was activated by fatty acids as indicated by its phosphorylation status, and by its protein level, respectively. Activation of PKCtheta contributes to IKK and JNK activation as inhibition of PKCtheta by calphostin C blocked activation of the latter kinases. Inhibition of either PKCtheta or IKK plus JNK by chemical inhibitors resulted in protection of IRS-1 function and insulin sensitivity in 3T3-L1 adipocytes. These data suggest that: 1) activation of PKCtheta contributes to IKK and JNK activation by FFAs; 2) IKK and JNK mediate PKCtheta signals for IRS-1 serine phosphorylation and degradation; and 3) this molecular mechanism may be responsible for insulin resistance associated with hyperlipidemia.

Tumor Necrosis Factor α Produces Insulin Resistance in Skeletal Muscle by Activation of Inhibitor κB Kinase in a p38 MAPK-dependent Manner

Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-alpha caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-alpha could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-alpha. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-alpha was still produced. Moreover, TNF-alpha produced inhibitor kappaB kinase (IKK)-beta activation and inhibitor kappaB-beta and -alpha degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-alpha produced serine phosphorylation of IR and IRS-1 (total and on Ser(307) residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-alpha, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.

Serine Phosphorylation of Insulin Receptor Substrate 1 by Inhibitor κB Kinase Complex

Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor kappaB kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser(307) (equivalent to Ser(312) in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-alpha or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser(312) in response to tumor necrosis factor-alpha was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J(2) as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser(312). Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser(312) (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways.