Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts.

Recent evidence indicates that activation of adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and modulator of cellular energy and redox, regulates cell mitosis. However, the underlying molecular mechanisms for AMPKα subunit regulation of chromosome segregation remain poorly understood. This study aimed to ascertain if AMPKα1 deletion contributes to chromosome missegregation by elevating Polo-like kinase 4 (PLK4) expression. Centrosome proteins and aneuploidy were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J) or AMPKα1 homozygous deficient (AMPKα1-/-) mice by Western blotting and metaphase chromosome spread. Deletion of AMPKα1, the predominant AMPKα isoform in immortalized MEFs, led to centrosome amplification and chromosome missegregation, as well as the consequent aneuploidy (34-66%) and micronucleus. Furthermore, AMPKα1 null cells exhibited a significant induction of PLK4. Knockdown of nuclear factor kappa B2/p52 ameliorated the PLK4 elevation in AMPKα1-deleted MEFs. Finally, PLK4 inhibition by Centrinone reversed centrosome amplification of AMPKα1-deleted MEFs. Taken together, our results suggest that AMPKα1 plays a fundamental role in the maintenance of chromosomal integrity through the control of p52-mediated transcription of PLK4, a trigger of centriole biogenesis.

Inactivation of NF-κB2 (p52) restrains hepatic glucagon response via preserving PDE4B induction.

Glucagon promotes hepatic gluconeogenesis and maintains whole-body glucose levels during fasting. The regulatory factors that are involved in fasting glucagon response are not well understood. Here we report a role of p52, a key activator of the noncanonical nuclear factor-kappaB signaling, in hepatic glucagon response. We show that p52 is activated in livers of HFD-fed and glucagon-challenged mice. Knockdown of p52 lowers glucagon-stimulated hyperglycemia, while p52 overexpression augments glucagon response. Mechanistically, p52 binds to phosphodiesterase 4B promoter to inhibit its transcription and promotes cAMP accumulation, thus augmenting the glucagon response through cAMP/PKA signaling. The anti-diabetic drug metformin and ginsenoside Rb1 lower blood glucose at least in part by inhibiting p52 activation. Our findings reveal that p52 mediates glucagon-triggered hepatic gluconeogenesis and suggests that pharmacological intervention to prevent p52 processing is a potential therapeutic strategy for diabetes.

Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells.

BACKGROUND: The intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underlying SMCs. METHODS: For these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0-25 ng/ml ±â€¯5 ng/ml TRAIL) for 72 h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5 ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RESULTS: Treatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-κB/p52 and canonical NF-κB/p65 pathways in both cell types. In RANKL ±â€¯TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-κB/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-κB2 gene (the molecular forerunner to NF-κB/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls. SUMMARY: These in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-κB pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.

Neuroprotective Effects of Echinacoside on Regulating the Stress-Active p38MAPK and NF-κB p52 Signals in the Mice Model of Parkinson's Disease.

Herbal medicines have long been used to treat Parkinson's disease (PD). To systematically analyze the anti-parkinsonian activity of echinacoside (ECH) in a neurotoxic model of PD and provide a future basis for basic and clinical investigations, male C57BL/6 mice were randomized into blank control, PD model and ECH-administration groups. ECH significantly suppressed the dopaminergic neuron loss (P < 0.01) caused by MPTP and maintained dopamine content (P < 0.01) and dopamine metabolite content (P < 0.05) compared with that measured in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage. Additionally, ECH inhibited the activation of microglia and astrocytes in the substantia nigra, which suggested the involvement of neuroinflammation. The relevant cytokines were detected with a Proteome Profiler Array, which confirmed that ECH participated in the regulation of seven cytokines. Given that p38 mitogen-activated protein kinase (p38MAPK) and NF-kappaB (NF-κB) signals are considered to be closely related to neuroninflammation, the gene expression levels of p38MAPK and six NF-κB DNA-binding subunits were assessed. Western blotting analysis showed that both p38MAPK and the NF-κB p52 subunit were upregulated in the MPTP group and that ECH downregulated their expressions. Minocycline was administered as the positive control to inhibit neuroinflammation, and no differences were detected between the minocycline- and ECH-mediated inhibition of the p38MAPK and NF-κB p52 signals. In conclusion, echinacoside is a potential novel orally active compound for regulating neuroinflammation and related signals in Parkinson's disease and may provide a new prospect for clinical treatment.

Antiinflammatory and antioxidant effects of H2O2 generated by natural sources in Il1ß-treated human endothelial cells.

Specific reactive oxygen species (ROS) from different sources, might lead to different and even opposite, cellular effects. We studied the production of specific ROS resulting from the exposure of human umbilical veins endothelial cells (HUVEC) to H2O2 derived from the natural antioxidant epigallocathechin gallate (EGCG) or from the exposure to IL-1ß using a fluorogenic probe and flow cytometry, and evaluated by western blot analysis and immunocytochemistry the associated expression of transcription factors sensitive to both inflammatory and oxidative stress, such as NF-κB and Nrf2, and some downstream activated genes such as cyclooxygenase-2 (COX-2) and hemeoxygenase 1 (HO-1). The results obtained showed that exogenously-generated H2O2 induce anti-inflammatory and antioxidant effects in HUVECs counteracting the pro-inflammatory and pro-oxidant effect of IL-1ß related to the production of superoxide anions. The underlying mechanisms resulting from the extracellular production of H2O2, include (1) Nrf2 nuclear translocation and the enhanced expression of antioxidant enzymes such as HO-1, and (2) the previously unreported inhibition of NF-κB and COX-2 expression. Overall, these findings provide evidence that the production of specific reactive oxygen species finely tunes endothelial cell function and might be relevant for the reappraisal of the effects of exogenous antioxidants in the context of cardiovascular diseases.

The restrained expression of NF-kB in renal tissue ameliorates folic acid induced acute kidney injury in mice.

The Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) represent family of structurally-related eukaryotic transcription factors which regulate diverse array of cellular processes including immunological responses, inflammation, apoptosis, growth & development. Increased expression of NF-kB has often been seen in many diverse diseases, suggesting the importance of genomic deregulation to disease pathophysiology. In the present study we focused on acute kidney injury (AKI), which remains one of the major risk factor showing a high rate of mortality and morbidity. The pathology associated with it, however, remains incompletely known though inflammation has been reported to be one of the major risk factor in the disease pathophysiology. The role of NF-kB thus seemed pertinent. In the present study we show that high dose of folic acid (FA) induced acute kidney injury (AKI) characterized by elevation in levels of blood urea nitrogen & serum creatinine together with extensive tubular necrosis, loss of brush border and marked reduction in mitochondria. One of the salient observations of this study was a coupled increase in the expression of renal, relA, NF-kB2, and p53 genes and proteins during folic acid induced AKI (FA AKI). Treatment of mice with NF-kB inhibitor, pyrrolidine dithio-carbamate ammonium (PDTC) lowered the expression of these transcription factors and ameliorated the aberrant renal function by decreasing serum creatinine levels. In conclusion, our results suggested that NF-kB plays a pivotal role in maintaining renal function that also involved regulating p53 levels during FA AKI.

Low-dose decitabine induces MAGE-A expression and inhibits invasion via suppression of NF-κB2 and MMP2 in Eca109 cells.

Decitabine, a demethylating drug, is the first-line treatment for myelodysplastic syndromes and gains better overall survival, which is based on epigenetic mechanism. Activated by promoter demethylation, melanoma-associated antigens-A (MAGE-A), cancer-testis antigens are attractive targets for immunotherapy. Our purpose was to investigate whether decitabine could show anti-tumor effects for esophageal cancer and explore its mechanism. In addition, we aimed to examine its modulation for most MAGE-A members. The results showed the baseline expression were MAGE-A2, -3,-9, and -10 in Eca109 cells and decitabine (0.5 µM) could induce MAGE-A8 and -A4 whereas reduce MAGE-A9 and -A10. Moreover, decitabine (0.5 µM) inhibited cell proliferation, migration and invasive ability by 15%, 34% and 47.2%, respectively and decreased expressions of NF-κB2 and MMP2. Our results demonstrated that low-dose decitabine induced the expression of MAGE-A8 and -A4, and inhibited cell invasion through decreasing expression of MMP2 and NF-κB2, which provides possibilities for combing decitabine with immunotherapy targeting MAGE-A to treat advanced esophageal squamous cell carcinoma.

Inhibitory effects of fucoxanthinol on the viability of human breast cancer cell lines MCF-7 and MDA-MB-231 are correlated with modulation of the NF-kappaB pathway.

Fucoxanthin is a carotenoid present in the chloroplasts of brown seaweeds. When ingested, it is metabolized mainly to fucoxanthinol in the gastrointestinal tract by digestive enzymes. These compounds have been shown to have many beneficial health effects. The present study was designed to evaluate the molecular mechanisms of action of fucoxanthin and/or of its metabolite fucoxanthinol against viability of estrogen-sensitive MCF-7 and estrogen-resistant MDA-MB-231 breast cancer cell lines. Fucoxanthin and fucoxanthinol reduced the viability of MCF-7 and MDA-MB-231 cells in dose- and time-dependent manners as a result of increased apoptosis. Furthermore, fucoxanthinol-induced apoptosis was more potent than that of fucoxanthin and correlated, for MDA-MB-231 cells, with inhibitory actions on members of the NF-κB pathway p65, p50, RelB, and p52. Being overexpressed and regulated by NF-κB in different types of cancers, the transcription factor SOX9 was also decreased at the nuclear level by fucoxanthin and fucoxanthinol in MDA-MB-231. Taken together, the current results suggest that fucoxanthinol and fucoxanthin could be potentially effective for the treatment and/or prevention of different types of cancers, including breast cancer.

NFκB regulates expression of Polo-like kinase 4.

Activation of the NFκB signaling pathway allows the cell to respond to infection and stress and can affect many cellular processes. As a consequence, NFκB activity must be integrated with a wide variety of parallel signaling pathways. One mechanism through which NFκB can exert widespread effects is through controlling the expression of key regulatory kinases. Here we report that NFκB regulates the expression of genes required for centrosome duplication, and that Polo-like kinase 4 (PLK4) is a direct NFκB target gene. RNA interference, chromatin immunoprecipitation, and analysis of the PLK4 promoter in a luciferase reporter assay revealed that all NFκB subunits participate in its regulation. Moreover, we demonstrate that NFκB regulation of PLK4 expression is seen in multiple cell types. Significantly long-term deletion of the NFκB2 (p100/p52) subunit leads to defects in centrosome structure. This data reveals a new component of cell cycle regulation by NFκB and suggests a mechanism through which deregulated NFκB activity in cancer can lead to increased genomic instability and uncontrolled proliferation.

Diverse patterns of cyclooxygenase-independent metalloproteinase gene regulation in human monocytes.

BACKGROUND AND PURPOSE Matrix metalloproteinase (MMP) production from monocyte/macrophages is implicated in matrix remodelling and modulation of inflammation. However, knowledge of the patterns and mechanisms of gene regulation of MMPs and their endogenous tissue inhibitors (TIMPs) is fragmentary. MMP up-regulation may be a target for cyclooxygenase (COX) and prostaglandin (PG) receptor inhibition, but the extent and mechanisms of COX-independent MMP up-regulation are unclear. EXPERIMENTAL APPROACH We studied MMP mRNA expression and selected protein levels in human peripheral blood monocytes before and after adhesion, upon stimulation with bacterial lipopolysaccharide (LPS), PGE(2) or forskolin and after culturing with monocyte colony-stimulating factor on plastic or human fibronectin for up to 7 days. KEY RESULTS Monocyte adherence for 2 h transiently up-regulated COX-2, MMP-1, MMP-7 and MMP-10 mRNAs, and persistently up-regulated MMP-2, MMP-9, MMP-14 and MMP-19 mRNAs. LPS, PGE(2) or forskolin selectively increased MMP-1, MMP-9, MMP-10, MMP-12 and MMP-14 mRNAs. LPS increased PGE(2) production through COX but up-regulated MMP levels independently of COX. Differential dependence on inhibition of p42/44 and p38 mitogen-activated protein kinases, c-jun N-terminal kinase and inhibitor of κB kinase2 paralleled the diverse patterns of MMP stimulation by LPS. Differentiation on plastic increased mRNA levels of MMP-7, MMP-9, MMP-12 and MMP-14 and TIMP-2 and TIMP-3 independently of COX; fibronectin accelerated MMP but not TIMP up-regulation. CONCLUSIONS AND IMPLICATIONS Adhesion, LPS stimulation and maturation of human monocytes lead to selective, COX-independent MMP and TIMP gene regulation, which is a potential target for selective inhibition by signalling kinase inhibitors.

Inhibition of NF-kappaB1 and NF-kappaB2 activation in prostate cancer cells treated with antibody against the carboxyl terminal domain of GRP78: effect of p53 upregulation.

Ligation of cancer cell surface GRP78 by activated alpha2-macroglobulin (alpha2M*) triggers pro-proliferative and anti-apoptotic signaling pathways. Cancer patients who develop autoantibodies to the alpha2M* binding site in GRP78 have a poor prognosis since these antibodies are receptor agonists. The NF-kappaB family of transcription factors induces expression of genes affecting cell growth and differentiation. NF-kappaB1 plays a major regulatory role in controlling innate immunity and inflammation, whereas NF-kappaB2 plays a greater role in cancer cell proliferation. Here we report that treatment of prostate cancer cells with antibody directed against the carboxyl terminal domain of GRP78 inhibits alpha2M*-induced activation of NF-kappaB2 by approximately 50% while exerting a lesser effect of approximately 20% on NF-kappaB1 activation. Treatment of these cells nearly abolished alpha2M*-induced activation of IKKalpha involved in the activation of NF-kappaB2. This antibody also suppressed alpha2M*-induced phosphorylation of IKKalpha, IKKalpha/beta, IkappaBalpha, and IkappaBbeta as well as levels of NIK. Antibody treatment of cancer cells elevated pro-apoptotic p21WAF and p27kip while reducing cyclin D1 levels. These studies demonstrate that antibody directed against the carboxyl terminal domain of GRP78 inhibits the pro-proliferative NF-kappaB signaling cascade in cancer cells.

Reduced thymic Aire expression and abnormal NF-kappa B2 signaling in a model of systemic autoimmunity.

The thymic stromal niche normally directs the production and export of a self-tolerant T cell repertoire. Many models of spontaneous autoimmunity, however, develop thymic architectural abnormalities before disease onset. Although this is suspected to affect central tolerance induction, creating an autoimmune predisposition, in-depth analysis of the microenvironment within these thymi is lacking, such that the mechanisms and likely direct effects on the T cell repertoire are unknown or speculative. Here we show that NZB mice, the first described model for systemic autoimmunity, demonstrate a complex thymic phenotype, including a lack of the autoimmune regulator (Aire), early defects in thymic epithelial cell (TEC) expansion, and evidence for altered NF-kappaB2 signaling. Analysis of medullary TEC revealed a numerical loss of the Aire-expressing MHC class II(high) (mTEC-high) subset as well reduced Aire protein and mRNA per cell. RelB expression was also reduced, while chemokines CCL19 and CCL21 were increased. Unexpectedly, the proportion of cortex and medulla in the NZB mice was normal from 36 wk, despite worsening architectural abnormalities. These data show that the NZB defect is more complex than previously appreciated, segregating into early numerical TEC deficiencies that correct with age, late degeneration of the niche architecture that does not affect TEC number, and a persistent reduction in Aire and RelB expression per cell acquired upon mTEC-high differentiation.

Negative regulation of TCR signaling by NF-kappaB2/p100.

The positive regulation of the NF-kappaB-signaling pathway in response to TCR stimulation has been well-studied. However, little is known about the negative regulation of this pathway in T cells. This negative regulation is crucial in controlling the duration of TCR signaling and preventing abnormal lymphocyte activation and proliferation. Therefore, understanding the negative regulation of TCR-mediated NF-kappaB signaling is essential in understanding the mechanisms involved in T cell function and homeostasis. TCR stimulation of human CD4+ T cells resulted in an increase in NF-kappaB2/p100 expression with no appreciable increase in p52, its cleavage product. Due to the presence of inhibitory ankyrin repeats in the unprocessed p100, this observation suggests that p100 may function as a negative regulator of the NF-kappaB pathway. Consistent with this hypothesis, ectopic expression of p100 inhibited TCR-mediated NF-kappaB activity and IL-2 production in Jurkat T cells. Conversely, knockdown of p100 expression enhanced NF-kappaB transcriptional activity and IL-2 production upon TCR activation. p100 inhibited the pathway by binding and sequestering Rel transcription factors in the cytoplasm without affecting the activity of the upstream IkappaB kinase. The kinetics and IkappaB kinase gamma/NF-kappaB essential modulator dependency of p100 induction suggest that NF-kappaB2/p100 acts as a late-acting negative-feedback signaling molecule in the TCR-mediated NF-kappaB pathway.

Activation of non-canonical NF-kappaB pathway mediated by STP-A11, an oncoprotein of Herpesvirus saimiri.

Although Saimiri Transforming Protein (STP)-A11, an oncoprotein of Herpesvirus saimiri, has been known to activate NF-kappaB signaling pathway, the detailed mechanism has not been reported yet. We herein report that STP-A11 activates non-canonical NF-kappaB pathway, resulting in p100 processing to p52. In addition, translocation of p52 protein (NF-kappaB2) into the nucleus is observed by the expression of STP-A11. STP-A11-mediated processing of p100 to p52 protein requires proteosome-mediated proteolysis because MG132 treatment clearly blocked p52 production in spite of the expression of STP-A11. Analysis of STP-A11 mutants to activate NF-kappaB2 pathway discloses the requirement of TRAF6-binding site not Src-binding site for STP-A11-mediated NF-kappaB2 pathway. Blockage of STP-A11-mediated p52 production using siRNA against p52 enhanced a chemotherapeutic drug-mediated cell death, suggesting that p52 production induced by the expression of STP-A11 would contribute to cellular transformation, which results from a resistance to cell death.

Specificity of TRAF3 in its negative regulation of the noncanonical NF-kappa B 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.

Rescue of TRAF3-null mice by p100 NF-kappa B deficiency.

Proper activation of nuclear factor (NF)-kappaB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-kappaB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-kappaB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-kappaB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280-288). We demonstrate that loss of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) results in constitutive noncanonical NF-kappaB activity. Importantly, TRAF3-/- B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-kappaB-inducing kinase in TRAF3-/- cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-kappaB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-kappaB pathway and that constitutive activation of the noncanonical NF-kappaB pathway causes the lethal phenotype of TRAF3-deficient mice.