IKK Inhibitor Research Articles

P27 kip1 haplo-insufficiency improves cardiac function in early-stages of myocardial infarction by protecting myocardium and increasing angiogenesis by promoting IKK activation.

p27kip1 (p27) is widely known as a potent cell cycle inhibitor in several organs, especially in the heart. However, its role has not been fully defined during the early phase of myocardial infarction (MI). In this study, we investigated the relationships between p27, vascular endothelial growth factor/hepatocyte growth factor (VEGF/HGF) and NF-κB in post-MI cardiac function repair both in vivo and in the hypoxia/ischemia-induced rat myocardiocyte model. In vivo, haplo-insufficiency of p27 improved cardiac function, diminished the infarct zone, protected myocardiocytes and increased angiogenesis by enhancing the production of VEGF/HGF. In vitro, the presence of conditioned medium from hypoxia/ischemia-induced p27 knockdown myocardiocytes reduced the injury caused by hypoxia/ischemia in myocardiocytes, and this effect was reversed by VEGF/HGF neutralizing antibodies, consistent with the cardioprotection being due to VEGF/HGF secretion. We also observed that p27 bound to IKK and that p27 haplo-insufficiency promoted IKK/p65 activation both in vivo and in vitro, thereby inducing the NF-κB downstream regulator, VEGF/HGF. Furthermore, IKKi and IKK inhibitor negated the effect of VEGF/HGF. Therefore, we conclude that p27 haplo-insufficiency protects against heart injury by VEGF/HGF mediated cardioprotection and increased angiogenesis through promoting IKK activation.

A screen for transcription factor targets of glycogen synthase kinase-3 highlights an inverse correlation of NFκB and androgen receptor signaling in prostate cancer.

Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer.

Sirtuin-1 (SIRT1) Is Required for Promoting Chondrogenic Differentiation of Mesenchymal Stem Cells

Sirtuin-1 (SIRT1), NAD(+)-dependent deacetylase, has been linked to anabolic effects in cartilage, although the mechanisms of SIRT1 signaling during differentiation of mesenchymal stem cells (MSCs) to chondrocytes are poorly understood. Therefore, we investigated the role of SIRT1-mediated signaling during chondrogenic differentiation of MSCs in vitro. High density and alginate cultures of MSCs were treated with chondrogenic induction medium with/without the SIRT1 inhibitor nicotinamide, antisense oligonucleotides against SIRT1 (SIRT1-ASO), IL-1β, and/or resveratrol. Transient transfection of MSCs with SIRT1-antisense oligonucleotides, nicotinamide, and IL-1β inhibited chondrogenesis-induced down-regulation of cartilage-specific proteins, cartilage-specific transcription factor Sox9, and enhanced NF-κB-regulated gene products involved in the inflammatory and degradative processes in cartilage (MMP-9, COX-2, and caspase-3), and NF-κB phosphorylation, acetylation, and activation of IκBα kinase. In contrast, the SIRT1 activator resveratrol or BMS-345541 (inhibitor of IKK) inhibited IL-1β- and NAM-induced suppression of cartilage-specific proteins, Sox9, and up-regulation of NF-κB-regulated gene products. Moreover, SIRT1 was found to interact directly with NF-κB and resveratrol-suppressed IL-1β and NAM but not SIRT1-ASO-induced NF-κB phosphorylation, acetylation, and activation of IκBα kinase. Knockdown of SIRT1 by mRNA abolished the inhibitory effects of resveratrol on inflammatory and apoptotic signaling and Sox9 expression, suggesting the essential role of this enzyme. Finally, the modulatory effects of resveratrol were found to be mediated at least in part by the association between SIRT1 and Sox9. These results indicate for the first time that SIRT1 supports chondrogenic development of MSCs at least in part through inhibition/deacetylation of NF-κB and activation of Sox9.

A semi-synthetic derivative of artemisinin, artesunate inhibits prostaglandin E2 production in LPS/IFNγ-activated BV2 microglia

Artesunate is a semi-synthetic derivative of artemisinin used to treat malaria, and has been shown to possess anti-inflammatory activity. In this study, we have investigated the effect of artesunate on PGE2 production/COX-2 protein expression in LPS+IFNγ-activated BV2 microglia. To further understand the mechanism of action of this compound, we investigated its interference with NF-κB and p38 MAPK signalling pathways. PGE2 production was determined using EIA, while protein expressions of inflammatory targets like COX-2, mPGES-1, IκB, p38 and MAPKAPK2 were evaluated using western blot. An NF-κB-bearing luciferase reporter gene assay was used to test the effect of artesunate on NF-κB-mediated pro-inflammatory gene expression in HEK293 cells stimulated with TNFα (1ng/ml). Artesunate (2 and 4μM), significantly (p <0.01) suppressed PGE2 production in LPS+IFNγ-activated BV2 microglia. This effect was found to be mediated via reduction in COX-2 and mPGES-1 proteins. Artesunate also produced significant inhibition of TNFα and IL-6 production in activated BV2 microglia. Further investigations showed that artesunate (0.5–4μM) significantly (p <0.001) reduced NF-κB-driven luciferase expression, and inhibited IκB phosphorylation and degradation, through inhibition of IKK. Artesunate inhibited phosphorylation of p38 MAPK and its substrate MAPKAPK2 following stimulation of microglia with LPS+IFNγ. Taken together, we have shown that artesunate prevents neuroinflammation in BV2 microglia by interfering with NF-κB and p38 MAPK signalling.

Estrogen as Jekyll and Hyde: regulation of cell death.

Estrogen has profound effects on growth, differentiation and function in male and female reproductive systems, and it is important for bone density, brain function and cholesterol mobilization. Despite beneficial estrogen functions, sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary. This opinion article touches upon the dual role estrogen played in cancer and asks whether the use of estrogen in combination with other targeted therapy would be possible, considering the newly identified crosstalk pathway which can switch the effects of estrogen.

Inhibition of IκB kinase (IKK) protects against peripheral nerve dysfunction of experimental diabetes.

Nuclear factor-κB (NF-κB) has been reported as a critical component of signalling mechanisms involved in the pathogenesis of a number of inflammatory conditions. Previous reports have shown that anti-inflammatory agents have a protective role in experimental diabetic neuropathy. Here, we assessed whether the inhibition of NF-κB cascade via IκB kinase (IKK) exerts any neuroprotective effect in experimental diabetic neuropathy. IKK inhibitor SC-514 (1 and 3 mg/kg) was administered daily for 2 weeks starting after 6 weeks of streptozotocin-induced diabetes. Nerve conduction and blood flow were determined by Powerlab and LASER Doppler system, respectively. We evaluated the changes in NF-κB, iNOS, and COX-2 expression by Western blotting in sciatic nerve. We found that IKK inhibition with SC-514 increased nerve blood flow and conduction velocity and improved pain threshold in diabetic animals. SC-514 also reduced the expression of NF-κB and phosphorylation of IKKβ in the sciatic nerve. Treatment with SC-514 reduced the elevated levels of pro-inflammatory cytokines (TNF-α and IL-6), iNOS, and COX-2. SC-514 reduces the expression of NF-κB and its downstream inflammatory components which may be involved in the improvement in nerve functions and pain perception in diabetic neuropathy. From the data of the present study, we suggest that diminution in IKK can be exploited as a drug target to significantly reduce the development of long-term complications of diabetes, particularly neuropathy.

IKK regulates the deubiquitinase CYLD at the postsynaptic density

K63-linked polyubiquitination of proteins regulates their trafficking into specific cellular pathways such as endocytosis and autophagy. CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is present in high quantities at the postsynaptic density (PSD). It was previously shown that, under excitatory conditions, CaMKII activates CYLD in a Ca2+-dependent manner. The observation that CYLD can also be phosphorylated in the absence of Ca2+ in isolated PSDs led us to further explore the regulation of CYLD under basal conditions. A possible involvement of the autonomous form of CaMKII and IKK, both kinases known to be localized at the PSD, was examined. A CaMKII inhibitor CN21 had no effect on CYLD phosphorylation in the absence of Ca2+, but two different IKK inhibitors, IKK16 and tatNEMO, inhibited its phosphorylation. Immuno-electron microscopy on hippocampal cultures, using an antibody for CYLD phosphorylated at S-418, revealed that the phosphorylated form of CYLD is present at the PSD under basal conditions. Phosphorylation of CYLD under basal conditions was inhibited by IKK16. NMDA treatment further promoted phosphorylation of CYLD at the PSD, but IKK16 failed to block the NMDA-induced effect. In vitro experiments using purified proteins demonstrated direct phosphorylation and activation of CYLD by the beta catalytic subunit of IKK. Activation of IKK in isolated PSDs also promoted phosphorylation of CYLD and an increase in endogenous deubiquitinase activity for K63-linked polyubiquitins. Altogether, the results suggest that in the absence of excitatory conditions, constitutive IKK activity at the PSD regulates CYLD and maintains basal levels of K63-linkage specific deubiquitination at the synapse.

Central role for protein kinase C in oxytocin and epidermal growth factor stimulated cyclooxygenase 2 expression in human myometrial cells.

BackgroundProstaglandins are important mediators of uterine contractility and cervical ripening during labour. Cyclooxygenase-2 (COX-2), also known as prostaglandin-endoperoxide synthase 2, is a rate limiting enzyme involved in the conversion of arachidonic acid into prostaglandins at parturition. In this paper, the pathways underlying agonist-induced cyclooxygenase-2 expression in human myometrial cells were studied.ResultsMyometrial cells were stimulated with different agonists: oxytocin (OXT), epidermal growth factor (EGF), interleukin-1β (IL1β), and phorbol-12-myristate-13-acetate (PMA) alone and in the presence of specific signalling pathway inhibitors. The nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) pathway was inhibited by means of the IKK-2 inhibitor TPCA-1. Signalling through extracellular signal-regulated kinases (ERK) was inhibited using the MEK1/2 inhibitor PD-184352. Bisindolylmaleimide-I was used to inhibit protein kinase C (PKC) signalling. COX-2 expression and ERK phosphorylation were measured using immunoblotting.OXT induced COX-2 expression by activating PKC and ERK. EGF increased COX-2 expression via stimulation of PKC, ERK and NFKB. As expected, the pro-inflammatory cytokine IL1β induced COX-2 expression by activating PKC- and NFKB-dependent pathways. Stimulation of PKC directly with PMA provoked strong COX-2 expression.ConclusionsPKC plays a central role in OXT and EGF induced COX-2 expression in human myometrial cells. However, other pathways, notably ERK and NFKB are also involved to an extent which depends on the type of agonist used.

Disruption of interleukin-1β autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1β. We have previously shown that IL-1β, at low doses (∼30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-κB signaling. However, at higher doses (>2.5 ng/ml, ∼150 pM), IL-1β inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1β in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1β (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-κB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1β blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ∼50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1β, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels.

Improvement of ventilation-induced lung injury in a rodent model by inhibition of inhibitory κB kinase

Inhibition of nuclear factor κB (NF-κB) activation is a well-know strategy to ameliorate ventilation-induced lung injury (VILI). Inhibitory κB kinase (IKK) plays a key role in the regulation of NF-κB activation. In this study, we determined whether inhibition of IKK by an IKK inhibitor exerts lung protection in a rat model of VILI. Anesthetized and mechanically ventilated Sprague-Dawley rats were randomly assigned to a standard (tidal volume, 8 mL/kg) or high-tidal volume (tidal volume, 25 mL/kg) ventilation group. An IKK inhibitor (IKK 16) or vehicle was administrated 1 hour before the induction of VILI. All groups were ventilated and observed for 5 hours. High-pressure ventilation caused activation of NF-κB, increased pulmonary inflammatory mediator levels, lung edema, and impairment of gas exchange. The IKK inhibitor treatment significantly reduced these changes and increased interleukin 10 levels, heme oxygenase 1 activity, protein kinase B (Akt) phosphorylation levels, and nuclear amounts of nuclear factor E2-related factor 2 protein. IKK may be a therapeutic target for VILI. An IKK inhibitor, IKK 16, can dampen VILI in rats. The beneficial effect of the IKK 16 may be mediated through the inhibition of NF-κB pathway and up-regulation of nuclear factor E2-related factor 2-regulated heme oxygenase 1 through the activation of the phosphatidylinositol 3 kinase/Akt.

193 A Novel First in Class IKK Alpha Inhibitor Abrogates Endothelial Cell Nuclear Factor Kappa B Signalling and Inflammatory Protein Expression

IntroductionUp-regulation of nuclear factor kappa B (NF–B), has been strongly implicated in cardiovascular disease.1 Two main pathways operate to regulate the activation of different NF–B species; the canonical or classical...

Exploiting the Differential Reactivities of Halogen Atoms: Development of a Scalable Route to IKK2 Inhibitor AZD3264

An efficient and scalable synthesis of AZD3264 is described in which the differential reactivities of various halogen atoms have been employed. The process involves five linear chemical steps with three isolated stages starting from commercially available fragments.

A phase I clinical trial of systemically delivered NEMO binding domain peptide in dogs with spontaneous activated B-cell like diffuse large B-cell lymphoma.

Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL.

Sulforaphane inhibits phorbol ester-stimulated IKK-NF-κB signaling and COX-2 expression in human mammary epithelial cells by targeting NF-κB activating kinase and ERK

Sulforaphane, an isothiocyanate present in cruciferous vegetables, has been reported to possess anti-inflammatory and cancer chemopreventive properties. However, the molecular mechanisms by which sulforaphane suppresses inflammation and carcinogenesis are yet to be fully elucidated. Since the aberrant expression of cyclooxygenase-2 (COX-2) links inflammation and cancer, the present study was aimed to elucidate the mechanisms by which sulforaphane modulates COX-2 overexpression in human mammary epithelial (MCF-10A) cells stimulated with a prototypic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of MCF-10A cells with sulforaphane significantly inhibited TPA-induced expression of COX-2 protein and its mRNA transcript. Transient transfection of cells with deletion mutant constructs of COX-2 promoter revealed that the transcription factor nuclear factor-kappaB (NF-κB) plays a key role in TPA-induced COX-2 expression in MCF-10A cells. Pretreatment with sulforaphane significantly attenuated nuclear localization, DNA binding and the transcriptional activity of NF-κB through inhibition of phosphorylation and subsequent degradation of IκBα in MCF-10A cells stimulated with TPA. Sulforaphane also attenuated TPA-induced activation of IκB kinases (IKK), NF-κB-activating kinase (NAK) and extracellular signal-regulated kinase-1/2 (ERK1/2). Pharmacological inhibition of IKK or transient transfection of cells with dominant-negative mutant forms of this kinase abrogated TPA-induced NF-κB activation and COX-2 expression. In addition, the blockade of ERK1/2 activation negated the catalytic activity of IKKα, but not that of IKKβ, whereas silencing NAK by specific siRNA abrogated the IKKβ activity in TPA-treated cells. Taken together, sulforaphane inhibits TPA-induced NF-κB activation and COX-2 expression in MCF-10A cells by blocking two distinct signaling pathways mediated by ERK1/2-IKKα and NAK–IKKβ.

CaMKIIδ splice variants exert differential effects on heart failure development and myocardial ischemia/reperfusion injury (652.14)

Mice that lack CaMKIIδ (KO) are significantly protected from the development of myocardial damage following ischemia reperfusion (I/R). Cardioprotection in KO animals was associated with the loss of NF‐κB‐mediated inflammatory responses. CaMKIIδ is alternatively spliced in the heart to generate the CaMKIIδB and δC subtypes. In vitro data also suggest that δC is deleterious while δB may be cardioprotective. Furthermore, mice expressing δC rapidly and spontaneously progress to heart failure and death while those expressing δB have a milder phenotype. We postulated that CaMKIIδB and δC play opposing roles in response to I/R injury. Mice expressing CaMKIIδB and δC were subjected to global ischemia in the Langendorff mode for 25 minutes followed by reperfusion for various times. CaMKII activation and substrate phosphorylation were assessed by western blot. Subcellular fractionation was performed to determine compartment‐specific CaMKII activation. Infarct size was used as a measure of myocardial injury. I/R led to activation of CaMKIIδC but not δB in the cytosolic compartment. IκB‐Kinase (IKK) phosphorylation was increased in mice expressing CaMKIIδC but not in those expressing δB. Mice expressing δC showed large infarcts compared to WT, while mice expressing CaMKIIδB show greatly diminished infarct formation following I/R. The observation that pharmacological inhibition of IKK mitigated the deleterious effects of δC overexpression suggests that selective activation of CaMKIIδC in the cytosol and subsequent activation of inflammatory signaling contributes to I/R injury.Grant Funding Source: Supported by NIH Grant HL080101

2‐Methoxystypandrone inhibits signal transducer and activator of transcription 3 and nuclear factor‐κB signaling by inhibiting Janus kinase 2 and IκB kinase

Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) or the nuclear factor-κB (NF-κB) pathway occurs frequently in cancer cells and contributes to oncogenesis. The activation of Janus kinase 2 (JAK2) and IκB kinase (IKK) are key events in STAT3 and NF-κB signaling, respectively. We have identified 2-methoxystypandrone (2-MS) from a traditional Chinese medicinal herb Polygonum cuspidatum as a novel dual inhibitor of JAK2 and IKK. 2-MS inhibits both interleukin-6-induced and constitutively-activated STAT3, as well as tumor necrosis factor-α-induced NF-κB activation. 2-MS specifically inhibits JAK and IKKβ kinase activities but has little effect on activities of other kinases tested. The inhibitory effects of 2-MS on STAT3 and NF-κB signaling can be eliminated by DTT or glutathione and can last for 4 h after a pulse treatment. Furthermore, 2-MS inhibits growth and induces death of tumor cells, particularly those with constitutively-activated STAT3 or NF-κB signaling. We propose that the natural compound 2-MS, as a potent dual inhibitor of STAT3 and NF-κB pathways, is a promising anticancer drug candidate.

Anti-melanoma activity of Cynanchi atrati Radix is mediated by regulation of NF-kappa B activity and pro-apoptotic proteins

Ethnopharmacological relevanceCynanchi atrati Radix has been traditionally prescribed for patients with inflammatory fever or chronic tumoral disorders. Melanoma is one of the most devastating cancer types, in which overexpression of nuclear factor kappa B (NF-κB) enables the cancer to survive without apoptosis. To identify a potential anti-melanoma candidate, we evaluated the apoptotic activity of an ethanol extract of Cynanchi atrati Radix (CAE) on melanoma and its underlying mechanisms. Materials and methodsSixty C57BL/6N mice with melanoma were orally administrated CAE (100 or 200mg/kg) or distilled water for 10 days. Survival, tumor weight and volume were monitored and measured. Intratumoral apoptotic change was measured using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. To confirm the pro-apoptotic activity of CAE (10, 50 or 100μg/mL) compared to positive drug (10μg/mL of IKK-2 inhibitor IV), cell proliferation, caspase-3/7 activity, flow cytometric analysis, TUNEL and DAPI staining, immunoblotting and gene expression analyses for apoptosis-associated genes were conducted using B16F10 cell line. ResultsCAE administration remarkably improved survivability with a significant reduction in tumor weight (p<0.01) and volume (p<0.01), as well as increased apoptotic bodies in melanoma tissue. The CAE treatment significantly inhibited proliferation of B16F10 cells (p<0.001), but increased caspase-3/7 activity (p<0.01 or 0.001) and apoptotic population. The CAE partially blocked nuclear translocation of NF-κB but activated the p53-associated apoptotic pathway. ConclusionThese results indicate that the CAE has anti-melanoma potential, and the underlying mechanisms involve inhibition of the activities of NF-κB and its target proteins as well as promoting the activities of pro-apoptotic proteins.

Soluble Calreticulin Induces Tumor Necrosis Factor-α (TNF-α) and Interleukin (IL)-6 Production by Macrophages through Mitogen-Activated Protein Kinase (MAPK) and NFκB Signaling Pathways

We have recently reported that soluble calreticulin (CRT) accumulates in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus. Moreover, following self-oligomerization, soluble recombinant CRT (rCRT) polypeptides exhibit potent immunostimulatory activities including macrophage activation in vitro and antibody induction in vivo. This study was designed to further investigate the underlying molecular mechanisms for soluble CRT-induced macrophage activation. Treatment of murine macrophages with oligomerized rCRT (OrCRT) led to (i) TNF-α and IL-6 transcription and protein expression without affecting intracellular mRNA stability; and (ii) IκBα degradation, NFκB phosphorylation and sustained MAPK phosphorylation in cells. Inhibition of IKK and JNK in macrophages substantially abrogated production of TNF-α and IL-6 induced by OrCRT, while ERK suppression only reduced IL-6 expression in parallel experiments. In vitro, fucoidan, a scavenger receptor A (SRA)-specific ligand, significantly reduced the uptake of FITC-labeled OrCRT by macrophages and subsequent MAPK and NFκB activation, thereby suggesting SRA as one of the potential cell surface receptors for soluble CRT. Together, these data indicate that soluble CRT in oligomerized form could play a pathogenic role in autoimmune diseases through induction of pro-inflammatory cytokines (e.g., TNF-α and IL-6) by macrophages via MAPK-NFκB signaling pathway.

A novel IKKα inhibitor, noraristeromycin, blocks the chronic inflammation associated with collagen-induced arthritis in mice

Objectives. To evaluate the therapeutic efficacy of a novel inhibitor for IκB kinase alpha (IKKα), noraristeromycin (NAM), for murine experimental model of rheumatoid arthritis, collagen- induced arthritis (CIA).Methods. NAM has been chemically synthesized as reported earlier. CIA was induced in DBA/1JNCrlj mice by intradermal inoculation of bovine type II collagen (col II) together with Freund Complete Adjuvant. Following the Day 21 booster injection of col II with Freund Incomplete Adjuvant, the animals were monitored for the development of arthritis and clinically evaluated. NAM was administered orally at different doses prior to induction (prophylactic protocol) or after the emergence of definitive arthritis (therapeutic protocol).Results. Here we demonstrate the experimental evidence that oral administration of NAM could completely prevent the occurrence of experimental arthritis in CIA mouse model at 0.3 mg/kg with ED50 value of approximately 0.1 mg/kg twice daily. Moreover, twice daily oral therapeutic dosage of 1 mg/kg of NAM significantly inhibited the paw swelling and disease progression even after the occurrence of experimental CIA. In addition, NAM exhibited an excellent pharmacokinetics in mice and oral administration of NAM could suppress the production of TNFα elicited by lipopolysaccharide (LPS) in a dose-dependent manner.Conclusions. These results indicated that IKKα inhibition is an effective novel therapy for the treatment of chronic inflammatory processes such as those associated with RA and other related conditions.

IKK2 Inhibition Attenuates Laser-Induced Choroidal Neovascularization

Choroidal neovascularization (CNV) is aberrant angiogenesis associated with exudative age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Inflammation has been suggested as a risk factor for AMD. The IKK2/NF-κB pathway plays a key role in the inflammatory response through regulation of the transcription of cytokines, chemokines, growth factors and angiogenic factors. We investigated the functional role of IKK2 in development of the laser-induced CNV using either Ikk2 conditional knockout mice or an IKK2 inhibitor. The retinal neuronal tissue and RPE deletion of IKK2 was generated by breeding Ikk2−/flox mice with Nestin-Cre mice. Deletion of Ikk2 in the retina caused no obvious defect in retinal development or function, but resulted in a significant reduction in laser-induced CNV. In addition, intravitreal or retrobulbar injection of an IKK2 specific chemical inhibitor, TPCA-1, also showed similar inhibition of CNV. Furthermore, in vitro inhibition of IKK2 in ARPE-19 cells significantly reduced heat shock-induced expression of NFKBIA, IL1B, CCL2, VEGFA, PDGFA, HIF1A, and MMP-2, suggesting that IKK2 may regulate multiple molecular pathways involved in laser-induced CNV. The in vivo laser-induced expression of VEGFA, and HIF1A in RPE and choroidal tissue was also blocked by TPCA-1 treatment. Thus, IKK2/NF-κB signaling appears responsible for production of pro-inflammatory and pro-angiogenic factors in laser-induced CNV, suggesting that this intracellular pathway may serve as an important therapeutic target for aberrant angiogenesis in exudative AMD.