Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence

Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 μg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo.     

Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation

LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70–1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.     

Altered exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury

Background

The NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles. Evidence suggests that exosomes can act as intercellular communicators by transporting active proteins to remote cells and may play a role in regulating inflammatory processes. This study aimed to profile the exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury.

Results

To investigate the potential changes in protein expression mediated by NF-κB in secreted exosomes in the serum following I/R injury, the levels of circulating exosomal proteomes in C57BL/6 and NF-κB^−/− mice were compared using two dimensional differential in-gel electrophoresis (2-DE), liquid chromatography tandem mass spectrometry (LC-MS/MS), and proteomic analysis. In C57BL/6 mice, the levels of circulating exosomal proteins, including complement component C3 prepropeptide, PK-120 precursor, alpha-amylase one precursor, beta-enolase isoform 1, and adenylosuccinate synthetase isozyme 1, increased following I/R injury. However, in the NF-κB^−/− mice, the expression of the following was upregulated in the exosomes: protease, serine 1; glyceraldehyde-3-phosphate dehydrogenase-like isoform 1; glyceraldehyde-3-phosphate dehydrogenase; and pregnancy zone protein. In contrast, the expression of apolipoprotein B, complement component C3 prepropeptide, and immunoglobulin kappa light chain variable region was downregulated in NF-κB^−/− mice. Bioinformatic annotation using the Protein Analysis Through Evolutionary Relationships (PANTHER) database revealed that the expression of the exosomal proteins that participate in metabolic processes and in biological regulation was lower in NF-κB^−/− mice than in C57BL/6 mice, whereas the expression of proteins that participate in the response to stimuli, in cellular processes, and in the immune system was higher.

Conclusions

The data presented in this study suggest that NF-κB might regulate exosomal protein expression at a remote site via circulation following I/R injury.     

Sulforaphane Attenuates Muscle Inflammation in Dystrophin-deficient mdx Mice via NF-E2-related Factor 2 (Nrf2)-mediated Inhibition of NF-κB Signaling Pathway

Inflammation is widely distributed in patients with Duchenne muscular dystrophy and ultimately leads to progressive deterioration of muscle function with chronic muscle damage, oxidative stress, and reduced oxidative capacity. NF-E2-related factor 2 (Nrf2) plays a critical role in defending against inflammation in different tissues via activation of phase II enzyme heme oxygenase-1 and inhibition of the NF-κB signaling pathway. However, the role of Nrf2 in the inflammation of dystrophic muscle remains unknown. To determine whether Nrf2 may counteract inflammation in dystrophic muscle, we treated 4-week-old male mdx mice with the Nrf2 activator sulforaphane (SFN) by gavage (2 mg/kg of body weight/day) for 4 weeks. The experimental results demonstrated that SFN treatment increased the expression of muscle phase II enzyme heme oxygenase-1 in an Nrf2-dependent manner. Inflammation in mice was reduced by SFN treatment as indicated by decreased infiltration of immune cells and expression of the inflammatory cytokine CD45 and proinflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the skeletal muscles of mdx mice. In addition, SFN treatment also decreased the expression of NF-κB(p65) and phosphorylated IκB kinase-α as well as increased inhibitor of κB-α expression in mdx mice in an Nrf2-dependent manner. Collectively, these results show that SFN-induced Nrf2 can alleviate muscle inflammation in mdx mice by inhibiting the NF-κB signaling pathway.     

Ibrutinib inhibits BTK-driven NF-κB p65 activity to overcome bortezomib-resistance in multiple myeloma

Multiple Myeloma (MM) is a haematologic malignancy characterized by the accumulation of clonal plasma cells in the bone marrow. Over the last 10–15 y the introduction of the proteasome-inhibitor bortezomib has improved MM prognosis, however relapse due to bortezomib-resistance is inevitable and the disease, at present, remains incurable. To model bortezomib-resistant MM we generated bortezomib-resistant MM cell lines (n = 4 ) and utilised primary malignant plasma cells from patients relapsing after bortezomib treatment (n = 6 ). We identified enhanced Bruton''s tyrosine kinase (BTK) activity in bortezomib-resistant MM cells and found that inhibition of BTK, either pharmacologically with ibrutinib (0.5 μM) or via lenti-viral miRNA-targeted BTK interference, re-sensitized previously bortezomib-resistant MM cells to further bortezomib therapy at a physiologically relevant concentration (5 nM). Further analysis of pro-survival signaling revealed a role for the NF-κB p65 subunit in MM bortezomib-resistance, thus a combination of BTK and NF-κB p65 inhibition, either pharmacologically or via further lenti-viral miRNA NF-κB p65 interference, also restored sensitivity to bortezomib, significantly reducing cell viability (37.5 ± 6 .9 %, ANOVA P ≤ 0 .001). Accordingly, we propose the clinical evaluation of a bortezomib/ibrutinib combination therapy, including in patients resistant to single-agent bortezomib.     

Pancreatic Acinar Cell Nuclear Factor κB Activation Because of Bile Acid Exposure Is Dependent on Calcineurin

Biliary pancreatitis is the most common etiology of acute pancreatitis, accounting for 30–60% of cases. A dominant theory for the development of biliary pancreatitis is the reflux of bile into the pancreatic duct and subsequent exposure to pancreatic acinar cells. Bile acids are known to induce aberrant Ca²⁺ signals in acinar cells as well as nuclear translocation of NF-κB. In this study, we examined the role of the downstream Ca²⁺ target calcineurin on NF-κB translocation. Freshly isolated mouse acinar cells were infected for 24 h with an adenovirus expressing an NF-κB luciferase reporter. The bile acid taurolithocholic acid-3-sulfate caused NF-κB activation at concentrations (500 μm) that were associated with cell injury. We show that the NF-κB inhibitor Bay 11-7082 (1 μm) blocked translocation and injury. Pretreatment with the Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, the calcineurin inhibitors FK506 and cyclosporine A, or use of acinar cells from calcineurin Aβ-deficient mice each led to reduced NF-κB activation with taurolithocholic acid-3-sulfate. Importantly, these manipulations did not affect LPS-induced NF-κB activation. A critical upstream regulator of NF-κB activation is protein kinase C, which translocates to the membranes of various organelles in the active state. We demonstrate that pharmacologic and genetic inhibition of calcineurin blocks translocation of the PKC-δ isoform. In summary, bile-induced NF-κB activation and acinar cell injury are mediated by calcineurin, and a mechanism for this important early inflammatory response appears to be upstream at the level of PKC translocation.     

Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKβ subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKβ, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the “NEMO-binding domain” at the C terminus of IKK2/IKKβ. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKβ through this secondary interaction in vitro and for full activation of IKK2/IKKβ in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKβ within its scaffold-dimerization domain proximal to the kinase domain–Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture–based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKβ for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling.     

Opposite Action of Peroxisome Proliferator-activated Receptor-γ in Regulating Renal Inflammation: FUNCTIONAL SWITCH BY ITS LIGAND*

Peroxisome proliferator-activated receptor-γ (PPARγ) agonists, a new class of antidiabetic agents, have been shown to possess antiinflammatory activity. In this study, we investigated the molecular mechanism by which PPARγ agonists inhibit proinflammatory cytokine expression in rat glomerular mesangial cells. Both natural and synthetic PPARγ agonists potently inhibited RANTES (regulated upon activation, normal T cell expressed and secreted) and monocyte chemoattractant protein-1 expression induced by TNF-α in mesangial cells, which was dependent on NF-κB signaling. However, PPARγ agonists had little effect on TNF-α-triggered IκBα phosphorylation and its subsequent degradation, p65 phosphorylation, and nuclear translocation. In the absence of PPARγ ligand, TNF-α induced a physical interaction between nuclear p65 and PPARγ, as demonstrated by co-immunoprecipitation. Such an interaction was mediated by the C-terminal region of p65. Activation of PPARγ by its agonist prevented PPARγ·p65 complex formation. Chromatin immunoprecipitation assay revealed that TNF-α induced p65 binding to the cis-acting κB elements in rat RANTES promoter, whereas disruption of PPARγ·p65 by its agonist blocked p65 interaction with its cognate κB sites. Knockdown of PPARγ via siRNA strategy completely abolished TNF-α-mediated p65 binding to κB sites and negated RANTES induction, suggesting that unliganded PPARγ is obligatory for NF-κB signaling. Consistently, overexpression of PPARγ in the absence of its ligand sensitized mesangial cells to TNF-α stimulation. These results uncover a paradoxical action of the unliganded and ligand-activated PPARγ in regulating NF-κB signaling and demonstrate PPARγ ligand as a molecular switch that controls its ability to modulate inflammatory responses in opposite directions.     

Cell shape and the microenvironment regulate nuclear translocation of NF-κB in breast epithelial and tumor cells

Although a great deal is known about the signaling events that promote nuclear translocation of NF-κB, how cellular biophysics and the microenvironment might regulate the dynamics of this pathway is poorly understood. In this study, we used high-content image analysis and Bayesian network modeling to ask whether cell shape and context features influence NF-κB activation using the inherent variability present in unperturbed populations of breast tumor and non-tumor cell lines. Cell–cell contact, cell and nuclear area, and protrusiveness all contributed to variability in NF-κB localization in the absence and presence of TNFα. Higher levels of nuclear NF-κB were associated with mesenchymal-like versus epithelial-like morphologies, and RhoA-ROCK-myosin II signaling was critical for mediating shape-based differences in NF-κB localization and oscillations. Thus, mechanical factors such as cell shape and the microenvironment can influence NF-κB signaling and may in part explain how different phenotypic outcomes can arise from the same chemical cues.     

Hyaluronic Acid Receptor for Endocytosis (HARE)-mediated Endocytosis of Hyaluronan,Heparin, Dermatan Sulfate,and Acetylated Low Density Lipoprotein (AcLDL), but Not Chondroitin Sulfate Types A,C, D,or E,Activates NF-κB-regulated Gene Expression

The hyaluronan (HA) receptor for endocytosis (HARE; Stab2) clears 14 systemic ligands, including HA and heparin. Here, we used NF-κB promoter-driven luciferase reporter assays to test HARE-mediated intracellular signaling during the uptake of eight ligands, whose binding sites in the HARE ectodomain were mapped by competition studies (Harris, E. N., and Weigel, P. H. (2008) Glycobiology 18, 638–648). Unique intermediate size Select-HA^TM, heparin, dermatan sulfate, and acetylated LDL stimulated dose-dependent HARE-mediated NF-κB activation of luciferase expression, with half-maximal values of 10–25 nm. In contrast, chondroitin sulfate types A, C, D, and E did not stimulate NF-κB activation. Moreover, degradation of endogenous IkB-α (an NF-κB inhibitor) was stimulated only by the signaling ligands. The stimulatory activities of pairwise combinations of the four signaling ligands were additive. The four nonstimulatory chondroitin sulfate types, which compete for HA binding, also effectively blocked HA-stimulated signaling. Clathrin siRNA decreased clathrin expression by ∼50% and completely eliminated NF-κB-mediated signaling by all four ligands, indicating that activation of signaling complexes occurs after endocytosis. These results indicate that HARE not only binds and clears extracellular matrix degradation products (e.g. released normally or during infection, injury, tumorigenesis, or other stress situations) but that a subset of ligands also serves as signaling indicator ligands. HARE may be part of a systemic tissue-stress sensor feedback system that responds to abnormal tissue turnover or damage as a danger signal; the signaling indicator ligands would reflect the homeostatic status, whether normal or pathological, of tissue cells and biomatrix components.