Mutation of cysteine 46 in IKK-beta increases inflammatory responses.

Activation of IκB kinase ß (IKK-ß) and nuclear factor (NF)-κB signaling contributes to cancer pathogenesis and inflammatory disease; therefore, the IKK-ß-NF-κB signaling pathway is a potential therapeutic target. Current drug design strategies focus on blocking NF-κB signaling by binding to specific cysteine residues on IKK-ß. However, mutations in IKK-ß have been found in patients who may eventually develop drug resistance. For these patients, a new generation of IKK-ß inhibitors are required to provide novel treatment options. We demonstrate in vitro that cysteine-46 (Cys-46) is an essential residue for IKK-ß kinase activity. We then validate the role of Cys-46 in the pathogenesis of inflammation using delayed-type hypersensitivity (DTH) and an IKK-ß C46A transgenic mouse model. We show that a novel IKK-ß inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-ß C46A transgenic mice. These findings reveal the role of Cys-46 in the promotion of inflammatory responses, and suggest that Cys-46 is a novel drug-binding site for the inhibition of IKK-ß.

A potential link between obesity and neural stem cell dysfunction.

Given the important role of the hypothalamus in regulating feeding and metabolism, there has been considerable interest in a possible function for hypothalamic stem cells in modulating body weight in health and disease. Mice given a high-fat diet develop inflammation in the hypothalamus and lose key types of neurons. It now appears that another effect of a high-fat diet is to reduce neural stem cell numbers, as well as their ability to make new neurons--effects that are associated with activation of the IKKß/NF-κB pathway--thereby exacerbating the primary loss of neurons and resulting in altered feeding behaviour and obesity.

IKKß/NF-κB disrupts adult hypothalamic neural stem cells to mediate a neurodegenerative mechanism of dietary obesity and pre-diabetes.

Adult neural stem cells (NSCs) are known to exist in a few regions of the brain; however, the entity and physiological/disease relevance of adult hypothalamic NSCs (htNSCs) remain unclear. This work shows that adult htNSCs are multipotent and predominantly present in the mediobasal hypothalamus of adult mice. Chronic high-fat-diet feeding led to not only depletion but also neurogenic impairment of htNSCs associated with IKKß/NF-κB activation. In vitro htNSC models demonstrated that their survival and neurogenesis markedly decreased on IKKß/NF-κB activation but increased on IKKß/NF-κB inhibition, mechanistically mediated by IKKß/NF-κB-controlled apoptosis and Notch signalling. Mouse studies revealed that htNSC-specific IKKß/NF-κB activation led to depletion and impaired neuronal differentiation of htNSCs, and ultimately the development of obesity and pre-diabetes. In conclusion, adult htNSCs are important for the central regulation of metabolic physiology, and IKKß/NF-κB-mediated impairment of adult htNSCs is a critical neurodegenerative mechanism for obesity and related diabetes.

Uncoupling the mechanisms of obesity and hypertension by targeting hypothalamic IKK-ß and NF-κB.

Obesity-related hypertension has become an epidemic health problem and a major risk factor for the development of cardiovascular disease (CVD). Recent research on the pathophysiology of obesity has implicated a role for the hypothalamus in the pathogenesis of this condition. However, it remains unknown whether the often-seen coupling of hypertension with obesity can also be explained by hypothalamic dysfunction, despite the emerging appreciation that many forms of hypertension are neurogenic in origin. Our studies here revealed that acute activation of the proinflammatory protein nuclear factor κB (NF-κB) and its upstream activator IκB kinase-ß (IKK-ß, encoded by Ikbkb) in the mediobasal hypothalamus rapidly elevated blood pressure in mice independently of obesity. This form of hypothalamic inflammation-induced hypertension involved the sympathetic upregulation of hemodynamics and was reversed by sympathetic suppression. Loss-of-function studies further showed that NF-κB inhibition in the mediobasal hypothalamus counteracted obesity-related hypertension in a manner that was dissociable from changes in body weight. In addition, we found that pro-opiomelanocortin (POMC) neurons were crucial for the hypertensive effects of the activation of hypothalamic IKK-ß and NF-κB, which underlie obesity-related hypertension. In conclusion, obesity-associated activation of IKK-ß and NF-κB in the mediobasal hypothalamus--particularly in the hypothalamic POMC neurons--is a primary pathogenic link between obesity and hypertension. Breaking this pathogenic link may represent an avenue for controlling obesity-related hypertension and CVD without requiring obesity control.

Oncoprotein Bmi-1 renders apoptotic resistance to glioma cells through activation of the IKK-nuclear factor-kappaB Pathway.

One of the features of malignant gliomas is their deviant resistance to cellular apoptosis induced by cytotoxic reagents. Bmi-1, an oncoprotein, has been linked to oncogenesis and cancer progression in various types of human cancers including gliomas. However, the mechanisms underlying Bmi-1 antiapoptotic function remain largely unknown. In this study, we report that Bmi-1 renders apoptotic resistance to glioma cells through nuclear factor-kappaB (NF-kappaB). In glioma cells, ectopic expression of Bmi-1 significantly inhibits doxorubicin-, BCNU-, or UV irradiation- induced apoptosis through reduction of activated caspase-3 and PARP, and induction of Bcl-X(L). Cellular depletion of Bmi-1 enhances the sensitivity of glioma cells to apoptosis induced by doxorubicin, BCNU, or UV irradiation. Bmi-1 activates NF-kappaB through stimulation of IkappaB phosphorylation, nuclear translocation, and transcriptional activity of NF-kappaB and expression of downstream genes of NF-kappaB including caspase-3, PARP, Bcl-X(L), and c-Myc. Inhibition of the IKK-NF-kappaB pathway abrogates the antiapoptotic effect of Bmi-1 on glioma cells. In high-grade gliomas, Bmi-1 and NF-kappaB are co-expressed in the cell nucleus. Up-regulation of Bmi-1 also correlates with tumor progression and poor survival of patients with gliomas. Together, our data demonstrate that Bmi-1 bestows apoptotic resistance to glioma cells through the IKK-NF-kappaB pathway and suggest Bmi-1 as a useful indicator for glioma prognosis.

Synergistic benefit in inflammatory arthritis by targeting I kappaB kinase epsilon and interferon beta.

OBJECTIVES: The I kappaB kinase (IKK)-related kinase IKKepsilon regulates type I interferon expression and responses as well as proinflammatory mediator production. We examined the role of IKKepsilon in arthritis and its ability to enhance the therapeutic response to systemic interferon (IFN) beta therapy in passive murine K/BxN arthritis. METHODS: IKKepsilon(-/-), IFN alpha(approximately)beta R(-/-) and wild type mice were given K/BxN serum and treated with polyinosinic polycytidylic acid (poly(I:C)), IFN beta, or normal saline. Clinical response and histological scores were assessed. Gene expression in the paws was measured by quantitative PCR. Serum interleukin 1a receptor agonist (IL1Ra) and IL10 were measured by ELISA and multiplex bead array. RESULTS: Arthritis was almost completely blocked in wild type mice if arthritogenic K/BxN serum and the Toll-like receptor (TLR)3 ligand, poly(I:C), were coadministered at the onset of the model, but not in established disease. Mice deficient in IFN alpha(approximately)beta R had an accelerated course of arthritis, and did not respond to poly(I:C). IKKepsilon null mice had a modest decrease in clinical arthritis compared with heterozygous mice. Low doses of IFN beta that were ineffective in wild type mice significantly decreased clinical arthritis in IKKepsilon null mice. Articular chemokine gene expression was reduced in the IKKepsilon(-/-) mice with arthritis and secreted IL1Ra (sIL1Ra) mRNA was significantly increased. Serum levels of IL1Ra were increased in low dose IFN beta-treated IKKepsilon(-/-) mice. CONCLUSIONS: Subtherapeutic doses of IFN beta enhance the anti-inflammatory effects of IKKepsilon deficiency, possibly by increasing production of IL1Ra and unmasking the antichemokine effects. Combination therapy with low dose IFN beta and an IKKepsilon inhibitor might improve efficacy of either agent alone and offers a novel approach to RA.

IkappaB kinase beta inhibition induces cell death in Imatinib-resistant and T315I Dasatinib-resistant BCR-ABL+ cells.

Chronic myelogenous leukemia is a malignant disease of the hematopoietic stem cell compartment, which is characterized by expression of the BCR-ABL fusion protein. Expression of BCR-ABL allows myeloid cells to grow in the absence of the growth factors interleukin-3 and granulocyte-macrophage colony-stimulating factor. The tyrosine kinase activity of BCR-ABL constitutively activates signaling pathways associated with Ras and its downstream effectors and with the Jak/STAT pathway. Additionally, we reported previously that BCR-ABL activates the transcription factor nuclear factor-kappaB (NF-kappaB) in a manner dependent on Ras and that inhibition of NF-kappaB by expression of a modified form of IkappaBalpha blocked BCR-ABL-driven tumor growth in a xenograft model. Here, we show that a highly specific inhibitor of IkappaB kinase beta, a key upstream regulator of the NF-kappaB pathway, induces growth suppression and death in cells expressing wild-type, Imatinib-resistant, or the T315I Imatinib/Dasatinib-resistant forms of BCR-ABL. Cell cycle variables were not affected by this compound. These data indicate that blockage of BCR-ABL-induced NF-kappaB activation via IkappaB kinase beta inhibition represents a potential new approach for treatment of Imatinib- or Dasatinib-resistant forms of chronic myelogenous leukemia.