NF-κB-inducing Kinase Expression Research Articles

NIK Stabilization in Osteoclasts Results in Osteoporosis and Enhanced Inflammatory Osteolysis

BackgroundMaintenance of healthy bone requires the balanced activities of osteoclasts (OCs), which resorb bone, and osteoblasts, which build bone. Disproportionate action of OCs is responsible for the bone loss associated with postmenopausal osteoporosis and rheumatoid arthritis. NF-κB inducing kinase (NIK) controls activation of the alternative NF-κB pathway, a critical pathway for OC differentiation. Under basal conditions, TRAF3-mediated NIK degradation prevents downstream signaling, and disruption of the NIK:TRAF3 interaction stabilizes NIK leading to constitutive activation of the alternative NF-κB pathway.Methodology/Principal FindingsUsing transgenic mice with OC-lineage expression of NIK lacking its TRAF3 binding domain (NT3), we now find that alternative NF-κB activation enhances not only OC differentiation but also OC function. Activating NT3 with either lysozyme M Cre or cathepsinK Cre causes high turnover osteoporosis with increased activity of OCs and osteoblasts. In vitro, NT3-expressing precursors form OCs more quickly and at lower doses of RANKL. When cultured on bone, they exhibit larger actin rings and increased resorptive activity. OC-specific NT3 transgenic mice also have an exaggerated osteolytic response to the serum transfer model of arthritis.ConclusionsConstitutive activation of NIK drives enhanced osteoclastogenesis and bone resorption, both in basal conditions and in response to inflammatory stimuli.

Tumor Necrosis Factor Receptor-associated Factor-6 and Ribosomal S6 Kinase Intracellular Pathways Link the Angiotensin II AT1 Receptor to the Phosphorylation and Activation of the IκB Kinase Complex in Vascular Smooth Muscle Cells

Activation of NF-κB transcription factors by locally produced angiotensin II (Ang II) is proposed to be involved in chronic inflammatory reactions leading to atherosclerosis development. However, a clear understanding of the signaling cascades coupling the Ang II AT1 receptors to the activation of NF-κB transcription factors is still lacking. Using primary cultured aortic vascular smooth muscle cells, we show that activation of the IKK complex and NF-κB transcription factors by Ang II is regulated by phosphorylation of the catalytic subunit IKKβ on serine residues 177 and 181 in the activation T-loop. The use of pharmacological inhibitors against conventional protein kinases C (PKCs), mitogen-activated/extracellular signal-regulated kinase (MEK) 1/2, ribosomal S6 kinase (RSK), and silencing RNA technology targeting PKCα, IKKβ subunit, tumor growth factor β-activating kinase-1 (TAK1), the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor-6 (TRAF6), and RSK isoforms, demonstrates the requirement of two distinct signaling pathway for the phosphorylation of IKKβ and the activation of the IKK complex by Ang II. Rapid phosphorylation of IKKβ requires a second messenger-dependent pathway composed of PKCα-TRAF6-TAK1, whereas sustained phosphorylation and activation of IKKβ requires the MEK1/2-ERK1/2-RSK pathway. Importantly, simultaneously targeting components of these two pathways completely blunts the phosphorylation of IKKβ and the proinflammatory effect of the octapeptide. This is the first report demonstrating activation of TAK1 by the AT1R. We propose a model whereby TRAF6-TAK1 and ERK-RSK intracellular pathways independently and sequentially converge to the T-loop phosphorylation for full activation of IKKβ, which is an essential step in the proinflammatory activity of Ang II.

Epigenetic alteration of the NF‐κB‐inducing kinase (NIK) gene is involved in enhanced NIK expression in basal‐like breast cancer

Basal-like breast cancers are triple-negative (estrogen receptor negative, progesterone receptor negative, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) negative) tumors with an aggressive clinical behavior that lacks effective molecular targets for therapy. We reported previously that the basal-like subtype cell lines display high constitutive nuclear factor (NF)-κB activation, whose inhibition in the basal-like subtypes suppressed their proliferation. Moreover, NF-κB-inducing kinase (NIK) is involved in the constitutive NF-κB activation. Here, we report that enhanced NIK expression, which is exclusively observed in the basal-like subtype rather than the luminal-like subtype or non-tumorigenic mammary epithelial cells, is caused by epigenetic alteration of the NIK gene. The stability of NIK mRNA and transcriptional activity driven by the NIK promoter are similar in the basal-like and luminal-like subtypes. However, histone H3 acetylation levels were up-regulated in the basal-like subtype. Furthermore, treatment of the luminal-like subtype with a histone deacetylase inhibitor, valproic acid, significantly increased NIK expression. Although DNA methylation of the NIK locus was not detected, NIK expression also increased when the luminal-like subtype was treated with 5-azacytidine, which inhibits histone H3-Lys-9 dimethylation in addition to DNA methylation. Taken together, these results suggest that the closed chromatin structure mediated by histone H3 methylation and deacetylation suppresses NIK expression in the luminal-like subtype, whereas disruption of these suppression mechanisms leads to enhanced NIK expression and the constitutive NF-κB activation in the basal-like subtype. Thus, NIK and genes induced by the NIK-mediated constitutive NF-κB activation could be therapeutic targets of basal-like breast cancer.

Epigenetic alteration of the NF-κB inducing kinase (NIK) gene is involved in enhanced NIK expression in basal-like breast cancer

Epigenetic alteration of the NF-κB inducing kinase (NIK) gene is involved in enhanced NIK expression in basal-like breast cancer

NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.

Overexpressed NF-κB–inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and Hodgkin Reed-Sternberg cells

AbstractThe nuclear factor-κB (NF-κB) transcription factors play important roles in cancer development by preventing apoptosis and facilitating the tumor cell growth. However, the precise mechanisms by which NF-κB is constitutively activated in specific cancer cells remain largely unknown. In our current study, we now report that NF-κB–inducing kinase (NIK) is overexpressed at the pretranslational level in adult T-cell leukemia (ATL) and Hodgkin Reed-Sternberg cells (H-RS) that do not express viral regulatory proteins. The overexpression of NIK causes cell transformation in rat fibroblasts, which is abolished by a super-repressor form of IκBα. Notably, depletion of NIK in ATL cells by RNA interference reduces the DNA-binding activity of NF-κB and NF-κB–dependent transcriptional activity, and efficiently suppresses tumor growth in NOD/SCID/γcnull mice. These results indicate that the deregulated expression of NIK plays a critical role in constitutive NF-κB activation in ATL and H-RS cells, and suggest also that NIK is an attractive molecular target for cancer therapy.

Specificity of TRAF3 in Its Negative Regulation of the Noncanonical NF-κB Pathway

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are critical signaling adaptors downstream of many receptors in the TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Whereas TRAF2, 5, and 6 are activators of the canonical NF-kappaB signaling pathway, TRAF3 is an inhibitor of the noncanonical NF-kappaB pathway. The contribution of the different domains in TRAFs to their respective functions remains unclear. To elucidate the structural and functional specificities of TRAF3, we reconstituted TRAF3-deficient cells with a series of TRAF3 mutants and assessed their abilities to restore TRAF3-mediated inhibition of the noncanonical NF-kappaB pathway as measured by NF-kappaB-inducing kinase (NIK) protein levels and processing of p100 to p52. We found that a structurally intact RING finger domain of TRAF3 is required for inhibition of the noncanonical NF-kappaB pathway. In addition, the three N-terminal domains, but not the C-terminal TRAF domain, of the highly homologous TRAF5 can functionally replace the corresponding domains of TRAF3 in suppression of the noncanonical NF-kappaB pathway. This functional specificity correlates with the specific binding of TRAF3, but not TRAF5, to the previously reported TRAF3 binding motif in NIK. Our studies suggest that both the RING finger domain activity and the specific binding of the TRAF domain to NIK are two critical components of TRAF3 suppression of NIK protein levels and the processing of p100 to p52.

Magnolol suppresses NF-κB activation and NF-κB regulated gene expression through inhibition of IkappaB kinase activation

The mis-regulation of nuclear factor-kappa B (NF-κB) signal pathway is involved in a variety of inflammatory diseases that leds to the production of inflammatory mediators. Our studies using human U937 promonocytes cells suggested that magnolol, a low molecular weight lignan isolated from the medicinal plant Magnolia officinalis, differentially down-regulated the pharmacologically induced expression of NF-κB-regulated inflammatory gene products MMP-9, IL-8, MCP-1, MIP-1α, TNF-α. Pre-treatment of magnolol blocked TNF-α-induced NF-κB activation in different cell types as evidenced by EMSA. Magnolol did not directly affect the binding of p65/p50 heterodimer to DNA. Immunoblot analysis demonstrated that magnolol inhibited the TNF-α-stimulated phosphorylation and degradation of the cytosolic NF-κB inhibitor IκBα and the effects were dose-dependent. Mechanistically, a non-radioactive IκB kinases (IKK) assay using immunoprecipitated IKKs protein demonstrated that magnolol inhibited both intrinsic and TNF-α-stimulated IKK activity, thus suggesting a critical role of magnolol in abrogating the phosphorylation and degradation of IκBα. The involvement of IKK was further verified in a HeLa cell NF-κB-dependent luciferase reporter system. In this system magnolol suppressed luciferase expression stimulated by TNF-α and by the transient transfection and expression of NIK (NF-κB-inducing kinase), wild type IKKβ, constitutively active IKKα and IKKβ, or the p65 subunit. Magnolol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-κB. In line with the observation that NF-κB activation may up-regulate anti-apoptotic genes, it was shown in U937 cells that magnolol enhanced TNF-α-induced apoptotic cell death. Our results suggest that magnolol or its derivatives may have potential anti-inflammatory actions through IKK inactivation.

IFN INDUCES NIK/TRAF‐DEPENDENT NF‐kB ACTIVATION

Type I interferons (IFNs) play critical roles in the host defense by modulating the expression of various genes via the IFN-dependent activation of STAT (signal transducers and activators of transcription) and NF-κB (nuclear factor kappa B) transcription factors. Previous studies established that IFNα/β activates NF-κB to promote cell survival through a PI-3K/Akt pathway, which involves serine phosphorylation and degradation of IκBα. We now describe a second pathway by which IFNs activate NF-κB that is independent of IκB degradation. This pathway involves NF-κB-inducing kinase (NIK) and the TNF-receptor associated factor-2 (TRAF-2), and results in IFNα/β induced processing of the p100/NF-κB2 precursor into p52. IFNα/β stimulates NF-κB DNA binding and NF-κB-dependent transcription. While expression of NIK and TRAF2 constructs causes NF-κB activation, expression of dominant negative NIK and TRAF2 constructs blocks IFN-promoted NF-κB activation and IFN-stimulated κB-dependent transcription, and IFNα/β induced processing of the p100/NF-κB2 precursor into p52. In contrast, PI-3K does not mediate IFNα/β induced p100 processing although PI3K is involved in the pathway resulting in IκBα degradation. Moreover, while IFN promotes cell survival in lymphoblastoid cells, expression of dominant negative NIK and TRAF2 constructs enhances IFN-induced apoptosis. Our results place for the first time NIK and TRAF2, previously shown to function in TNF signaling, within the IFN signal transduction pathway. Thus, IFN induces NF-κB activation to mediate IFN-dependent cell survival signals through a “canonical” pathway of IκBα proteolysis mediated by PI-3K/Akt and a “noncanonical” pathway of p100 processing mediated by NIK/TRAF.

Stabilization of Basally Translated NF-κB-inducing Kinase (NIK) Protein Functions as a Molecular Switch of Processing of NF-κB2 p100

The non-canonical pathway based on processing of NF-kappaB2 precursor protein p100 to generate p52 plays a critical role in controlling B cell function and lymphoid organogenesis. Activation of this unique pathway by extracellular stimuli requires NF-kappaB-inducing kinase (NIK) and de novo protein synthesis. However, how NIK is regulated is largely unknown. Here, we systematically analyzed NIK expression at different levels in the presence or absence of different NF-kappaB stimuli. We found that NIK mRNA is relatively abundant and undergoes constitutive protein synthesis in resting B cells. However, NIK protein is undetectable. Interestingly, protein expression of NIK is steadily induced by B cell-activating factor or CD40 ligand, two major physiological inducers of p100 processing, but not by mitogen phorbol 12-myristate 13-acetate/ionomycin or cytokine tumor necrosis factor alpha, two well known inducers of the canonical NF-kappaB signaling. Remarkably, both B cell-activating factor and CD40 ligand do not significantly induce expression of NIK at translational or transcriptional level but rather rescue the basally translated NIK protein from undergoing degradation. Furthermore, overexpressed or purified NIK protein triggers p100 processing in the presence of protein synthesis inhibitor. Taken together, these studies define one important mechanism of NIK regulation and the central role of NIK stabilization in the induction of p100 processing. These studies also provide the first evidence explaining why activation of the non-canonical NF-kappaB signaling is delayed and can be inhibited by protein synthesis inhibitor as well as why most classical NF-kappaB stimuli, including mitogens and tumor necrosis factor alpha, fail to induce p100 processing.

Honokiol inhibits TNF-α-stimulated NF-κB activation and NF-κB-regulated gene expression through suppression of IKK activation

Honokiol, a small molecular weight lignan originally isolated from Magnolia officinalis, shows anti-angiogenic, anti-invasive and anti-proliferative activities in a variety of cancers. In this study, we investigated whether honokiol affects the transcription factor nuclear factor-kappa B (NF-κB) which controls a large number of genes involved in angiogenesis, metastasis and cell survival. We observed that the tumor necrosis factor-alpha (TNF-α)-induced NF-κB activation was blocked by honokiol in four different cancer cell lines as evidenced by EMSA. Honokiol did not directly affect the NF-κB-DNA binding. Immunoblot experiments demonstrated that honokiol inhibited the TNF-α-stimulated phosphorylation and degradation of the cytosolic NF-κB inhibitor IκBα. Furthermore, honokiol suppressed the intrinsic and TNF-α-stimulated upstream IκB kinases (IKKs) activities measured by a non-radioactive kinase assay using immunoprecipitated IKKs, suggesting a critical role of honokiol in abrogating the phosphorylation and degradation of IκBα. In a HeLa cell NF-κB-dependent luciferase reporter system, honokiol suppressed luciferase expression stimulated by TNF-α and by the transient transfection and expression of NIK (NF-κB-inducing kinase), wild type IKKβ, constitutively active IKKα and IKKβ, or the p65 subunit. Honokiol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-κB. RT-PCR results showed that honokiol suppressed NF-κB-regulated inflammatory and carcinogenic gene products including MMP-9, TNF-α, IL-8, ICAM-1 and MCP-1. In line with the observation that NF-κB activation may up-regulate anti-apoptotic genes, it was shown that honokiol enhanced TNF-α-induced apoptotic cell death. In summary, our results demonstrate that honokiol suppresses NF-κB activation and NF-κB-regulated gene expression through the inhibition of IKKs, which provides a possible mechanism for its anti-tumor actions.

Interferon Induces NF-κB-inducing Kinase/Tumor Necrosis Factor Receptor-associated Factor-dependent NF-κB Activation to Promote Cell Survival

Type I interferons (IFNs) play critical roles in the host defense by modulating the expression of various genes via the IFN-dependent activation of signal transducers and activators of transcription and NF-kappaB (nuclear factor kappa B) transcription factors. Previous studies established that IFNalpha/beta activates NF-kappaB to promote cell survival through a phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which involves serine phosphorylation and degradation of IkappaB alpha. We now describe a second pathway by which IFNs activate NF-kappaB that is independent of IkappaB degradation. This pathway involves NF-kappaB-inducing kinase (NIK) and the tumor necrosis factor receptor-associated factor-2 (TRAF2) and results in IFNalpha/beta-induced processing of the p100/NF-kappaB2 precursor into p52. IFNalpha/beta stimulates NF-kappaB DNA binding and NF-kappaB-dependent transcription. Whereas expression of NIK and TRAF2 constructs causes NF-kappaB activation, expression of dominant negative NIK and TRAF2 constructs blocks IFN-promoted NF-kappaB activation and IFN-stimulated kappaB-dependent transcription and IFNalpha/beta-induced processing of the p100/NF-kappaB2 precursor into p52. In contrast, PI3K does not mediate IFNalpha/beta-induced p100 processing, although PI3K is involved in the pathway resulting in IkappaB alpha degradation. Moreover, whereas IFN promotes cell survival in lymphoblastoid cells, expression of dominant negative NIK and TRAF2 constructs enhances IFN-induced apoptosis. Our results for the first time place NIK and TRAF2, previously shown to function in TNF signaling, within the IFN signal transduction pathway. Thus, IFN induces NF-kappaB activation to mediate IFN-dependent cell survival signals through a "canonical" pathway of IkappaB alpha proteolysis mediated by PI3K/Akt and a "noncanonical" pathway of p100 processing mediated by NIK/TRAF.