Regulation of TNF-Induced Osteoclast Differentiation.

Increased osteoclast (OC) differentiation and activity is the critical event that results in bone loss and joint destruction in common pathological bone conditions, such as osteoporosis and rheumatoid arthritis (RA). RANKL and its decoy receptor, osteoprotegerin (OPG), control OC differentiation and activity. However, there is a specific concern of a rebound effect of denosumab discontinuation in treating osteoporosis. TNFα can induce OC differentiation that is independent of the RANKL/RANK system. In this review, we discuss the factors that negatively and positively regulate TNFα induction of OC formation, and the mechanisms involved to inform the design of new anti-resorptive agents for the treatment of bone conditions with enhanced OC formation. Similar to, and being independent of, RANKL, TNFα recruits TNF receptor-associated factors (TRAFs) to sequentially activate transcriptional factors NF-κB p50 and p52, followed by c-Fos, and then NFATc1 to induce OC differentiation. However, induction of OC formation by TNFα alone is very limited, since it also induces many inhibitory proteins, such as TRAF3, p100, IRF8, and RBP-j. TNFα induction of OC differentiation is, however, versatile, and Interleukin-1 or TGFβ1 can enhance TNFα-induced OC formation through a mechanism which is independent of RANKL, TRAF6, and/or NF-κB. However, TNFα polarized macrophages also produce anabolic factors, including insulin such as 6 peptide and Jagged1, to slow down bone loss in the pathological conditions. Thus, the development of novel approaches targeting TNFα signaling should focus on its downstream molecules that do not affect its anabolic effect

Stimulation of the Pro-Resolving Receptor Fpr2 Reverses Inflammatory Microglial Activity by Suppressing NFκB Activity

Neuroinflammation driven primarily by microglia directly contributes to neuronal death in many neurodegenerative diseases. Classical anti-inflammatory approaches aim to suppress pro-inflammatory mediator production, but exploitation of inflammatory resolution may also be of benefit. A key driver of peripheral inflammatory resolution, formyl peptide receptor 2 (Fpr2), is expressed by microglia, but its therapeutic potential in neurodegeneration remains unclear. Here, we studied whether targeting of Fpr2 could reverse inflammatory microglial activation induced by the potent bacterial inflammogen lipopolysaccharide (LPS). Exposure of murine primary or immortalised BV2 microglia to LPS triggered pro-inflammatory phenotypic change and activation of ROS production, effects significantly attenuated by subsequent treatment with the Fpr2 agonist C43. Mechanistic studies showed C43 to act through p38 MAPK phosphorylation and reduction of LPS-induced NFκB nuclear translocation via prevention of IκBα degradation. Here, we provide proof-of-concept data highlighting Fpr2 as a potential target for control of microglial pro-inflammatory activity, suggesting that it may be a promising therapeutic target for the treatment of neuroinflammatory disease

Melanocortin peptides protect chondrocytes from mechanically induced cartilage injury

Introduction Mechanical injury can greatly influence articular cartilage, propagating inflammation, cell injury and death – risk factors for the development of osteoarthritis. Melanocortin peptides and their receptors mediate anti-inflammatory and pro-resolving mechanisms in chondrocytes. This study aimed to investigate the potential chondroprotective properties of α-MSH and [DTRP8]-γ-MSH in mechanically injured cartilage explants, their ability to inhibit pro-inflammatory and stimulate anti-inflammatory cytokines in in situ and in freshly isolated articular chondrocytes. Methods The effect of melanocortins on in situ chondrocyte viability was investigated using confocal laser scanning microscopy of bovine articular cartilage explants, subjected to a single blunt impact (1.14 N, 6.47 kPa) delivered by a drop tower. Chondroprotective effects of α-MSH, [DTRP8]-γ-MSH and dexamethasone on cytokine release by TNF-α-activated freshly isolated articular chondrocytes/mechanically injured cartilage explants were investigated by ELISA. Results A single impact to cartilage caused discreet areas of chondrocyte death, accompanied by pro-inflammatory cytokine release; both parameters were modulated by α-MSH, [DTRP8]-γ-MSH and dexamethasone. Melanocortin pre-treatment of TNF-α-stimulated freshly isolated chondrocytes resulted in a bell-shaped inhibition in IL-1β, IL-6 and IL-8, and elevation of IL-10 production. The MC3/4 antagonist, SHU9119, abrogated the effect of [DTRP8]-γ-MSH but not α-MSH on cytokine release. Conclusion Melanocortin peptide pre-treatment prevented chondrocyte death following mechanical impact to cartilage and led to a marked reduction of pro-inflammatory cytokines, whilst prompting the production of anti-inflammatory/pro-resolving cytokine IL-10. Development of small molecule agonists towards melanocortin receptors could thus be a viable approach for preventing chondrocyte inflammation and death within cartilage and represent an alternative approach for the treatment of osteoarthritis

Extracellular vesicles and their nucleic acids for biomarker discovery

Extracellular vesicles (EVs) are a heterogenous population of vesicles originate from cells. EVs are found in different biofluids and carry different macromolecules, including proteins, lipids, and nucleic acids, providing a snap shot of the parental cells at the time of release. EVs have the ability to transfer molecular cargoes to other cells and can initiate different physiological and pathological processes. Mounting lines of evidence demonstrated that EVs' cargo and machinery is affected in disease states, positioning EVs as potential sources for the discovery of novel biomarkers. In this review, we demonstrate a conceptual overview of the EV field with particular focus on their nucleic acid cargoes. Current knowledge of EV subtypes, nucleic acid cargo and pathophysiological roles are outlined, with emphasis placed on advantages against competing analytes. We review the utility of EVs and their nucleic acid cargoes as biomarkers and critically assess the newly available advances in the field of EV biomarkers and high throughput technologies. Challenges to achieving the diagnostic potential of EVs, including sample handling, EV isolation, methodological considerations, and bioassay reproducibility are discussed. Future implementation of ‘omics-based technologies and integration of systems biology approaches for the development of EV-based biomarkers and personalized medicine are also considered

Modelling Feedback Excitation, Pacemaker Properties and Sensory Switching of Electrically Coupled Brainstem Neurons Controlling Rhythmic Activity

What cellular and network properties allow reliable neuronal rhythm generation or firing that can be started and stopped by brief synaptic inputs? We investigate rhythmic activity in an electrically-coupled population of brainstem neurons driving swimming locomotion in young frog tadpoles, and how activity is switched on and off by brief sensory stimulation. We build a computational model of 30 electrically-coupled conditional pacemaker neurons on one side of the tadpole hindbrain and spinal cord. Based on experimental estimates for neuron properties, population sizes, synapse strengths and connections, we show that: long-lasting, mutual, glutamatergic excitation between the neurons allows the network to sustain rhythmic pacemaker firing at swimming frequencies following brief synaptic excitation; activity persists but rhythm breaks down without electrical coupling; NMDA voltage-dependency doubles the range of synaptic feedback strengths generating sustained rhythm. The network can be switched on and off at short latency by brief synaptic excitation and inhibition. We demonstrate that a population of generic Hodgkin-Huxley type neurons coupled by glutamatergic excitatory feedback can generate sustained asynchronous firing switched on and off synaptically. We conclude that networks of neurons with NMDAR mediated feedback excitation can generate self-sustained activity following brief synaptic excitation. The frequency of activity is limited by the kinetics of the neuron membrane channels and can be stopped by brief inhibitory input. Network activity can be rhythmic at lower frequencies if the neurons are electrically coupled. Our key finding is that excitatory synaptic feedback within a population of neurons can produce switchable, stable, sustained firing without synaptic inhibition

Mode of Glucocorticoid Actions in Airway Disease

Synthetic glucocorticoids are the most potent anti-inflammatory agents used to treat chronic inflammatory disease, such as asthma. However, a small number (<5%) of asthmatic patients and almost all patients with chronic obstructive pulmonary disease (COPD) do not respond well, or at all, to glucocorticoid therapy. If the molecular mechanism of glucocorticoid insensitivity is uncovered, it may in turn provide insight into the key mechanism of glucocorticoid action and allow a rational way to implement treatment regimens that restore glucocorticoid sensitivity. Glucocorticoids exert their effects by binding to a cytoplasmic glucocorticoid receptor (GR), which is subjected to post-translational modifications. Receptor phosphorylation, acetylation, nitrosylation, ubiquitinylation, and other modifications influence hormone binding, nuclear translocation, and protein half-life. Analysis of GR interactions to other molecules, such as coactivators or corepressors, may explain the genetic specificity of GR action. Priming with inflammatory cytokine or oxidative/nitrative stress is a mechanism for the glucocorticoid resistance observed in chronic inflammatory airway disease via reduction of corepressors or GR modification. Therapies targeting these aspects of the GR activation pathway may reverse glucocorticoid resistance in patients with glucocorticoid-insensitive airway disease and some patients with other inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. KEYWORDS: glucocorticoid, inflammation, asthma, COPD, glucocorticoid receptor, glucocorticoid resistance INTRODUCTION Although glucocorticoids are the most potent anti-inflammatory agents for the treatment of chronic inflammatory disease, such as asthma, a small population of asthmatic patients and almost all patients with chronic obstructive pulmonary disease (COPD) show a poor response to glucocorticoids Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 THE MOLECULAR BASIS OF INFLAMMATION IN ASTHMA AND COPD Lower airways inflammation is a central feature of many lung diseases including asthma and COPD. These diseases always involve recruitment and activation of inflammatory cells with changes in the structural cells of the lung, though the specific characteristics of the inflammatory response and the site of inflammation differ from one disease to another. Inflammation in asthma is associated with increased airway hyperresponsiveness leading to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. The inflammation is present even in those with very mild asthma; T-lymphocytes of the T-helper (Th) type 2 phenotype, eosinophils, macrophages/monocytes, and mast cells infiltrate the airway wall. Airway inflammation is also amplified during exacerbation with an increase in eosinophils and sometimes neutrophils. These conditions are also characterized by an increased expression of components of the inflammatory cascade including chemokines, cytokines, growth factors, enzymes, noxious gas, reactive oxygen, receptors, and adhesion molecules COPD is a chronic inflammatory disease of the lower airways and lung, which is enhanced during exacerbations Increased inflammatory gene transcription seen in inflammatory airway disease is regulated by proinflammatory transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). NF-κB is ubiquitously expressed and is able to not only control the induction of inflammatory genes in its own right, but also enhances the activity of other cell-and signal-specific transcription factors Alterations in the structure of chromatin are critical to the regulation of gene expression Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1752 FIGURE 1. Molecular mechanism of gene activation and repression. Gene activation and repression are regulated by acetylation of core histones. Histone acetylation is mediated by coactivators that have intrinsic HAT activity, opening up the chromatin structure to allow binding of RNA polymerase II and transcription factors that were unable to bind DNA in the closed chromatin configuration. This is reversed by corepressors, which include histone deacetylases (HDACs) and other associated proteins that reverse this acetylation, thereby causing gene silencing. STATs, signal transduction activated transcription factors. transcription is, therefore, associated with an increase in histone acetylation, whereas hypoacetylation is correlated with reduced transcription or gene silencing, which is regulated by histone deacetylase (HDAC) MECHANISMS OF GLUCOCORTICOID RECEPTOR FUNCTION Glucocorticoids exert their effects by binding to a single 777-amino-acid glucocorticoid receptor (GR) that is localized to the cytoplasm of almost all cell types Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1753 FIGURE 2. Structure of GRα and its post-translational modification. The N-terminus represents the constitutive transcriptional activation function (AF-1). The C-terminus contains the LBD, ligand-dependent activation function (AF-2), dimerization domain, and cofactor binding domains, all separated from the DBD by the hinge region (H). GR is post-translationally modified. Su (sumolyation), Ub (ubiquitinylation), PS (serine phosphorylation), PT (threonine phosphorylation), Ac (acetylation). HOW TO SWITCH OFF INFLAMMATORY GENES The major anti-inflammatory effects of glucocorticoids are thought to be due to repression of inflammatory and immune genes Second, the GR represses gene expression via DNA binding. After GR nuclear translocation, GR combines with another GR to form a dimer and it binds to consensus DNA sites termed glucocorticoid response elements (GREs, GGTACAnnnTGTTCT) in the regulating regions of corticosteroid-responsive genes. The GR dimer can bind to a GRE that overlaps the DNA binding site for a proinflammatory transcription factor or at the start site of transcription such as in the genes for IL-6 and osteocalcin, thus blocking gene expression Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1754 FIGURE 3. How glucocorticoids turn off gene transcription. Cytokine, oxidative stress, growth factors, and lipopolysaccharides (LPS) stimulate activation of transcriptional factors, such as NF-κB. Activated NF-κB translocates to nuclei and recruits HAT, and then acetylates histones. Consequently, genes are switched on as shown in the left side. GR exists in the cytoplasm (center of Third, the GR dimer, through the binding to GRE, may suppress inflammation by increasing the synthesis of anti-inflammatory proteins, such as IL-10, MAPK phosphatase-1 (MKP-1), the inhibitor of NF-κB (IκB-α), annexin 1 (and annexin 1 peptides), glucocorticoid-induced leucine zipper (GILZ), and secretory lymphocyte protease inhibitor (SLPI) Fourth, glucocorticoids can increase the levels of cell ribonucleases and mRNA destabilizing proteins, thereby reducing the levels of mRNA Last, GR is also reported to act on the cell membrane nonspecifically or through membrane-bound GR. This is known as nongenomic effects of glucocorticoids, but it is not fully elucidated GLUCOCORTICOID RECEPTOR MULTICOMPLEXES WITH VARYING COFACTORS Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1755 Activation of GR by glucocorticoids is a multistep process that involves hormone binding, hormonedependent structural transformation, translocation into the nucleus, and identification of target genes. There is increasing evidence that each step in this process is controlled by the interaction of GR with cofactors, such as a heat shock protein 90 (hsp90)-containing chaperone complex, kinases, high mobility group (HMG) proteins, mineralocorticoid receptor, and other transcription factors Components of the BRG1(SW1/SNF) complex, an ATP-dependent chromatin remodeling complex, are the well-known coactivators associated with GR CBP and p300 are histone acetylases and act as scaffold molecules that interact stably or transiently with a large number of transcription factors, including GR. CBP/p300 is recruited to both GR and other NRs either directly, through AF-1, or indirectly through coactivators interacting with AF-2 and this is involved in GR transactivation Other well-known GR-associated coactivators are the molecules in the p160 coactivator family Li et al. showed that upon ligand treatment, progesterone receptor (PR) interacted preferentially with SRC-1, which recruited CBP and significantly enhanced acetylation at K5 of histone H4 Very interestingly, the GR coactivators, SRC-1 and GRIP-1 (SRC-2), also act as corepressors of the GR Real corepressors are also recruited to the GR multicomplex, such as SMRT (silencing mediator for retinoid and thyroid-hormone receptors), NCoR (nuclear receptor corepressor), and receptor-interacting protein 140 (RIP140) Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1756 recruitment HDAC2 is the most important corepressor to determine glucocorticoid insensitivity POST-TRANSLATIONAL MODIFICATION Phosphorylation GR is a phosphoprotein containing multiple potential sites for phosphorylation Eight phosphorylation sites (seven for serine, one for threonine) in mouse GR have been identified Bodwell et al. showed that GR response varies during the cell cycle, with cells being less sensitive to corticosteroids during G2/M, and in the G2/M period, GR was highly phosphorylated Acetylation HAT and HDAC, which are associated with GR, are also known as protein acetyltransferase/deacetylase as well as histone acetyltransferase/deacetylase. Previous studies have shown that both the estrogen receptor Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1757 (ER) and the androgen receptor (AR) are acetylated within their hinge/LBDs and that this can modulate hormone-induced gene induction We previously reported that HDAC2 expression and activity is decreased in smokers[81] and patients with COPD Nitrosylation/Nitration Oxidative/nitrative stress is the key factor in pathogenesis of COPD and maybe in severe asthma Galigniana et al. showed that NO donors (S-nitroso-acetyl-DL-penicillamine, S-nitroso-DL-penicillamine, or S-nitroso-glutathione) decreased the number of ligand binding sites and K d for the binding of [ 3 H]-triamcinolone to immunoprecipitated GR from mouse L929 fibroblasts, without any change in GR protein levels Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1758 FIGURE 4. GR acetylation. After ligand binding, GR is acetylated and dimerized. The dimer binds GRE and induces antiinflammatory factors where this binding likely causes side effects. On the other hand, HDAC2 is recruited to the acetylated GR and deacetylates it to allow binding to NF-κB, resulting in repression of NF-κB-dependent gene expression. Ubiquitinylation/Sumoylation The ubiquitin-proteasome-dependent protein degradation pathway (UPP) regulates the turnover of many transcription factors including steroid hormone receptors, such as the ER and PR. Glucocorticoid treatment induced a down-regulation of GR Wallace and Cidlowski indicated that the phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover and phosphorylation is a key signal for ubiquitination and proteasomal catabolism of GR Small ubiquitin-related modifier-1 (SUMO-1) is covalently attached to many cellular targets to regulate protein-protein and protein-DNA interactions, as well as localization and stability of the target protein. GR is reported to be post-translationally modified by SUMO-1 (sumoylated) in a ligand-enhanced fashion Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1759 receptor's transactivation potential MOLECULAR MECHANISMS OF GLUCOCORTICOID RESISTANCE IN AIRWAY DISEASE As mentioned above, an important feature of severe asthma and COPD is glucocorticoid resistance Regarding COPD patients, several large studies suggest that long-term treatment with corticosteroids did not stop the inexorable decline of lung function. This is consistent with the demonstration that inhaled or oral corticosteroids fail to reduce inflammatory cell numbers, cytokines, chemokines, or proteases in induced sputum or bronchial biopsies of patients with COPD At a molecular level, resistance to the anti-inflammatory effects of glucocorticoids can be induced by several mechanisms. The reduction in corticosteroid responsiveness observed in cells from these subjects has been ascribed to reduced number of GR, altered affinity of the ligand for GR, reduced ability of the GR to bind to DNA, or increased expression of inflammatory transcription factors, such as AP-1, that compete for DNA binding Defects in GR Sequence and Pharmacokinetics Unlike familial glucocorticoid resistance where there is a mutation in the LBD of GR and a subsequent resetting of the basal cortisol level, corticosteroid-resistant (CR) patients have normal cortisol levels and are not addisonian It has previously been demonstrated, using whole cell binding assays, that no significant changes exist in monocyte and T-cell binding affinity (K d ), receptor density, and expression of the GR in patients with glucocorticoid-resistant asthma The mechanism of IL-2/IL-4, or IL-13 alone, induced defect of ligand binding characteristics have been explained in two ways. Leung and colleagues have associated these changes with an increased expression of the dominant negative isoform of GR, GRβ[94], although others have been unable to detect enhanced GRβ expression in PBMCs from these glucocorticoid-resistant patients Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1760 Furthermore, Zhou et al. demonstrated at least 7 isoforms of GR by alternative splicing such as GRα, GRβ, GRγ, GR-P, GR-A, and the relative levels of these variants play a role in differential glucocorticoid-induced responsiveness GR Nuclear Translocation and GR/GRE Binding In one subgroup of severe patients, nuclear localization of GR in response to a high concentration (10 -6 M) of dexamethasone is impaired In a separate subgroup of severe asthma patients, GR nuclear translocation is normal, but dexamethasone cannot correctly stimulate histone H4 lysine (K)5 acetylation Cross-Talk with Other Transcription Factors Transcription factors, especially AP-1, were reported to be excessively activated in glucocorticoid-resistant asthma, in addition to a reduced ability of GR to interact and repress AP-1 activity Neutrophilic Inflammation Neutrophils have been implicated in the pathogenesis of many diseases including COPD, severe asthma, psoriasis, and a variety of collagen-vascular diseases Formation of 52-and 30-kD GR fragments due to proteolysis by neutrophil elastase (a 28-kD serine protease) is found in cytosol of leukemia cells Latent Viral Infection Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1761 Epidemiologic studies have implicated childhood respiratory infection as an independent risk factor for the subsequent development of persistent asthma and COPD Reduced HDAC Activity in COPD and Severe Asthma We have demonstrated that in peripheral lung tissue and alveolar macrophages, there is a reduction in HDAC activity and HDAC2 expression in normal smokers, and a striking reduction in patients with COPD Oxidative Stress in COPD Exhaled markers of oxidative stress, such as 8-isoprostane and ethane, are increased in normal smokers with a much greater increase in patients with COPD, even when they have stopped smoking Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1762 FIGURE 5. Proposed mechanism of glucocorticoid insensitivity in COPD patients. In normal alveolar macrophages, NF-κB and other transcription factors are activated following stimulation, and switch on HAT, leading to histone acetylation and, subsequently, to the transcription of genes encoding inflammatory proteins, such as TNF-α, IL-8, and MMP-9. Glucocorticoids reverse this by binding to GR and recruiting HDAC2. This reverses the histone acetylation induced by NF-κB and switches off the activated inflammatory genes. However, in COPD patients, cigarette smoke activates macrophages, but oxidative stress (acting through the formation of peroxynitrite) impairs the activity of HDAC2. This amplifies the inflammatory response to NF-κB activation, but also reduces the anti-inflammatory effect of glucocorticoids, as HDAC2 is now unable to reverse histone acetylation. This figure is adapted from Barnes et al. THERAPEUTIC IMPLICATION Inhaled glucocorticoids are now used as first-line therapy for the treatment of persistent asthma in adults and children in many countries, as they are the most effective treatments for asthma currently available Dissociated Corticosteroids There is an ongoing search for novel glucocorticoids that selectively transrepress without significant transactivation. The major task in developing these drugs is to dissociate the anti-inflammatory effects from the endocrine actions that are associated with side effects. Recently, a novel class of glucocorticoids has been described in which there is potent transrepression with relatively little transactivation. These "dissociated" glucocorticoids, including RU24858, RU40066, and ZK216348, have anti-inflammatory effects in vitro, although there is little or some separation of antiinflammatory effects and systemic side effects in vivo Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1763 Restoration of Glucocorticoid Action As discussed above, oxidative stress is markedly increased in severe asthma and in patients with COPD, and can attenuate GR function via reduction of HDAC2 or excessive activation of transcriptional factors. Since oxidative and nitrative stress may drive the glucocorticoid insensitivity, antioxidants such as N-acetyl cysteine (NAC), fudostein, or inhibitors of inducible NO synthase (NOS2) should reverse glucocorticoid resistance. A couple of clinical studies with NAC in patients with COPD have produced variable results As well as HDAC reduction, defect of GR nuclear translocation is another important impairment of GR action. We recently showed that long-acting β 2 -agonist (LABA) is more effective in these patients. This combination therapy of LABA and glucocorticoid is established as a more effective therapy in asthma and also in COPD Nonsteroidal Anti-Inflammatory Treatments: Glucocorticoid-Sparing Therapy A variety of anti-inflammatory compounds are now being developed. Many of the anti-inflammatory effects of glucocorticoids appear to be mediated via inhibition of the transcriptional effects of NF-κB, and smallmolecule inhibitors of IKK (inhibitor of I-κB kinase-2) that activate NF-κB are in development CONCLUSION Current systemic and inhaled pharmacological treatment of severe asthma and COPD is unsatisfactory as it does not significantly influence the severity of the disease or its natural course apart from the treatment of exacerbations. The identification of an active resistance mechanism suggests that glucocorticoid resistance/insensitivity is potentially reversible, which would have enormous implications for the future therapy of chronic inflammatory diseases. Since oxidative and nitrative stress may inactivate HDAC2, interfere with the action of other HDACs, alter cofactor recruitment, or induce post-translational modification of GR, antioxidants, HDAC activators, and kinase inhibitors are likely to prove effective in restoring corticosteroid sensitivity in these diseases

Regulation of TNF-Induced Osteoclast Differentiation

Increased osteoclast (OC) differentiation and activity is the critical event that results in bone loss and joint destruction in common pathological bone conditions, such as osteoporosis and rheumatoid arthritis (RA). RANKL and its decoy receptor, osteoprotegerin (OPG), control OC differentiation and activity. However, there is a specific concern of a rebound effect of denosumab discontinuation in treating osteoporosis. TNFα can induce OC differentiation that is independent of the RANKL/RANK system. In this review, we discuss the factors that negatively and positively regulate TNFα induction of OC formation, and the mechanisms involved to inform the design of new anti-resorptive agents for the treatment of bone conditions with enhanced OC formation. Similar to, and being independent of, RANKL, TNFα recruits TNF receptor-associated factors (TRAFs) to sequentially activate transcriptional factors NF-κB p50 and p52, followed by c-Fos, and then NFATc1 to induce OC differentiation. However, induction of OC formation by TNFα alone is very limited, since it also induces many inhibitory proteins, such as TRAF3, p100, IRF8, and RBP-j. TNFα induction of OC differentiation is, however, versatile, and Interleukin-1 or TGFβ1 can enhance TNFα-induced OC formation through a mechanism which is independent of RANKL, TRAF6, and/or NF-κB. However, TNFα polarized macrophages also produce anabolic factors, including insulin such as 6 peptide and Jagged1, to slow down bone loss in the pathological conditions. Thus, the development of novel approaches targeting TNFα signaling should focus on its downstream molecules that do not affect its anabolic effect