Inhibitor Of κB Kinase Research Articles

Abstract 4634: Inhibitory-κB kinase alpha as a biomarker and therapeutic target in colorectal cancer

Abstract Introduction/Aims: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality. The canonical NF-κB pathway driven by IKKβ has been implicated in CRC development and progression, however less research has focused on non-canonical NF-κB signaling, regulated by inhibitory-κB kinase alpha (IKKα). This project aimed to assess IKKα, phospho-IKKα (pIKKαs176) and IKKβ expression in a retrospective CRC cohort to establish association with survival outcomes. Subsequently, this project aimed to determine the effect of abrogating IKKα activity without perturbing the canonical NF-κB pathway controlled by IKKβ using a first-in-class selective IKKα inhibitor in vitro/ex vivo. Methods: Immunohistochemical staining for IKKα/pIKKα s176/IKKβ was performed using tissue microarrays from a CRC patient cohort (n=787). QuPath® software was used to semi-quantitively assess expression levels and scores were analyzed for association with cancer-specific survival (CSS). CRC cell lines (n=3), patient-derived organoids (PDOs; n=5) and patient-derived explants (n=5) were treated with novel IKKα inhibitor SU1644 and assessed for cell viability and proliferation. Results: Positive staining for IKKα, pIKKαs176 and IKKβ was detected in tumor specimens. High cytoplasmic expression of IKKα within tumor cells was associated with reduced CSS in patients with right-sided colon cancer (HR=3.091, 95%CI; 1.128-9.467, log rank p=0.021). Conversely, high IKKβ expression was a marker of good prognosis (HR=0.627, 95%CI; 0.457-0.861, log rank p=0.004). A high ratio of IKKα to IKKβ was associated with reduced CSS in the full cohort (HR=1.404, 95%CI; 1.050-1.879, log rank p=0.021) and this was potentiated in right-sided colon cases (HR=1.772, 95%CI; 1.107-2.837, log rank p=0.016). Selective inhibition of IKKα using 1µM SU1644 in vitro caused a significant reduction in HT29 colony formation (p=0.012) and cell viability (p=0.039). PDOs showed altered cell morphology, reduced cell viability and proliferation when treated with SU1644. In the patient-derived explant model, Ki67 expression was reduced in tumor explants treated with SU1644. Conclusions: These data establish high cytoplasmic IKKα expression within tumor cells as a marker of poor prognosis in CRC and conversely high IKKβ expression as a marker of good prognosis. This highlights the importance of development of selective IKKα inhibitor SU1644. IKKα inhibition using SU1644 demonstrated anti-cancer activity in recapitulative CRC disease models. Citation Format: Kathryn A. Pennel, Molly McKenzie, Guang-Yu Lian, Jean A. Quinn, Sara Samir Foad Al-Badran, Campbell S. Roxburgh, Simon P. Mackay, Joanna Birch, Joanne Edwards. Inhibitory-κB kinase alpha as a biomarker and therapeutic target in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4634.

Mechanism of Fushen Granules treat peritoneal dialysis-related peritonitis by regulating TLR4/NF-κB pathway:based on network pharmacology and animal experiments

Network pharmacology was employed to probe into the mechanism of Fushen Granules in treating peritoneal dialysis-rela-ted peritonitis(PDRP) in rats. The main active components of Fushen Granules were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and their targets were predicted. PDRP-related targets were retrieved from DisGeNET and other databases. The common targets shared by the drug and the disease were identified by the online tool, and protein-protein interaction(PPI) network of the common targets. The obtained 276 common targets were imported into DAVID for GO function enrichment and KEGG pathway enrichment. The main signaling pathway of Fushen Granules in the treatment of PDRP was predicted as Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB. The rat model of uremia was induced by 5/6 nephrectomy. From two weeks after operation, the rat model of peritoneal dialysis(PD) was established by intraperitoneal injection of 20 mL dialysate with 1.25% glucose every day. The sham operation group and model group received 2 mL normal saline by gavage every day. The rats in Fushen Gra-nules groups were administrated with 2 mL solutions of low-(0.54 g·kg~(-1)), medium-(1.08 g·kg~(-1)) and high-dose(2.16 g·kg~(-1)) Fushen Granules every day. The bifico group received 2 mL(113.4 mg·kg~(-1)) of bifico solution every day. At the end of the 8th week, the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) in each group were measured. The serum levels of hypersensitive C reactive protein(hs-CRP), tumor necrosis factor(TNF)-α, and interleukin(IL)-6 were measured, and the pathological changes in the colon tissue were observed by hematoxylin-eosin(HE) staining. The serum levels of lipopolysaccharide(LPS) and lipopolysaccharide-binding protein(LBP) of rats were measured, and the expression levels of LBP, TLR4, NF-κB p65, inhibitor of κB kinase α(IκBα), TNF-α, and IL-1β in the colon tissue were determined. Compared with sham operation group, the model group had abnormal structure of all layers of colon tissue, sparse and shorter intestinal villi, visible edema in mucosal layer, wider gap, obvious local inflammatory cell infiltration, significantly decreased body weight(P<0.01), and significantly increased kidney function index(Scr, BUN) content(P<0.01). Serum levels of inflammatory cytokines(hs-CRP, TNF-α, IL-6), LPS and LBP were significantly increased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1β were significantly increased(P<0.01), and protein expressions of IκBα were significantly decreased(P<0.01). Compared with model group, intestinal villi damage in colonic tissue of rats in low-, medium-and high-dose Fushen Granules groups and bifico group were alleviated to different degrees, edema in submucosa was alleviated, space was narrowed, and inflammatory cell infiltration in lamina propria was reduced. The contents of renal function index(Scr, BUN) and serum inflammatory factors(hs-CRP, TNF-α, IL-6) were significantly decreased(P<0.05 or P<0.01) in medium-and high-dose Fushen Granules groups and bifico group(P<0.05 or P<0.01). Serum LPS and LBP contents in Fushen Granules group and bifico group were significantly decreased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1β in Fushen Granules group were significantly decreased(P<0.05 or P<0.01), and protein expressions of IκBα were significantly increased(P<0.01). The expression of LBP protein in bifico group was significantly decreased(P<0.01). The results suggest that Fushen Granules can protect the residual renal function of PD rats, reduce the inflammatory response, and protect the colon tissue. Based on network pharmacology, TLR4/NF-κB pathway may be the main signaling pathway of Fushen granule in the treatment of PDRP. The results showed that Fushen Granules could improve intestinal inflammation and protect intestinal barrier to prevent PDRP by regulating the expression of key factors in TLR4/NF-κB pathway in colon of PD rats.

CD147 Sparks Atherosclerosis by Driving M1 Phenotype and Impairing Efferocytosis.

Atherosclerosis is a globally prevalent chronic inflammatory disease with high morbidity and mortality. The development of atherosclerotic lesions is determined by macrophages. This study aimed to investigate the specific role of myeloid-derived CD147 (cluster of differentiation 147) in atherosclerosis and its translational significance. We generated mice with a myeloid-specific knockout of CD147 and mice with restricted CD147 overexpression, both in an apoE-deficient (ApoE-/-) background. Here, the myeloid-specific deletion of CD147 ameliorated atherosclerosis and inflammation. Consistent with our in vivo data, macrophages isolated from myeloid-specific CD147 knockout mice exhibited a phenotype shift from proinflammatory to anti-inflammatory macrophage polarization in response to lipopolysaccharide/IFN (interferon)-γ. These macrophages demonstrated a weakened proinflammatory macrophage phenotype, characterized by reduced production of NO and reactive nitrogen species derived from iNOS (inducible NO synthase). Mechanistically, the TRAF6 (tumor necrosis factor receptor-associated factor 6)-IKK (inhibitor of κB kinase)-IRF5 (IFN regulatory factor 5) signaling pathway was essential for the effect of CD147 on proinflammatory responses. Consistent with the reduced size of the necrotic core, myeloid-specific CD147 deficiency diminished the susceptibility of iNOS-mediated late apoptosis, accompanied by enhanced efferocytotic capacity mediated by increased secretion of GAS6 (growth arrest-specific 6) in proinflammatory macrophages. These findings were consistent in a mouse model with myeloid-restricted overexpression of CD147. Furthermore, we developed a new atherosclerosis model in ApoE-/- mice with humanized CD147 transgenic expression and demonstrated that the administration of an anti-human CD147 antibody effectively suppressed atherosclerosis by targeting inflammation and efferocytosis. Myeloid CD147 plays a crucial role in the growth of plaques by promoting inflammation in a TRAF6-IKK-IRF5-dependent manner and inhibiting efferocytosis by suppressing GAS6 during proinflammatory conditions. Consequently, the use of anti-human CD147 antibodies presents a complementary therapeutic approach to the existing lipid-lowering strategies for treating atherosclerotic diseases.

Involvement of the Constitutive Photomorphogenesis 9 Signalosome Subunit 5 With Programmed Cell Death Protein 1 Ligand in Asthma.

The constitutive photomorphogenesis 9 signalosome (CSN) is a highly conserved protein complex comprised of eight subunits, each of which play crucial roles in diverse cellular processes, such as signal transduction, gene transcription, angiogenesis, and cell proliferation. In the context of asthma, a potential emerging target is the programmed death-ligand 1 (PD-L1)-mediated pathway, which serves as a significant immune checkpoint inhibitor in this condition. However, the precise involvement of CSN subunit 5 (CSN5) in bronchial asthma and the interplay between CSN5 and PD-L1 in asthma remain poorly understood. The potential association between CSN5 and bronchial asthma was explored in a mouse model of ovalbumin (OVA)-induced asthma. Samples were obtained from human lung microvascular endothelial cell (HMVEC-L) treated with Dermatophagoides pteronyssinus (Der p 1) and CSN5 small interfering RNA. The expression of nuclear factor (NF)-κB, IκBα, inhibitor of κB kinase β (IKKβ), PD-L1, and CSN5 was assessed. Additionally, plasma CSN5 levels in asthma patients, both in stable and exacerbated states, were examined. Plasma levels of CSN5 were elevated in patients with exacerbated asthma (n = 19) compared to both healthy controls (n = 10) and patients with stable asthma (n = 19). The CSN5 level demonstrated a correlation with lung function in individuals with asthma. Silencing CSN5 in HMVEC-L led to a reduction in NF-κB protein levels at 4 hours and PD-L1 levels at 4, 8, and 24 hours after Der p 1 treatment. In OVA-sensitized/challenged mice, goblet cell hyperplasia, lung fibrosis, and the levels of CSN5, PD-L1, interleukin-13, interferon-γ, phospho (p)-NF-κB, p-IκBα, and p-IKKβ proteins increased at 33 and 80 days compared to control mice. However, these changes were mitigated by treatment with a PD-L1 inhibitor. These findings suggest that CSN5, along with PD-L1, could serve as a promising target for the treatment of asthma.

Mechanism of ligusticum cycloprolactam against neuroinflammation based on network pharmacology and experimental verification.

Ligustilide, a natural phthalide mainly derived from chuanxiong rhizomes and Angelica Sinensis roots, possesses anti-inflammatory activity, particularly in the context of the nervous system. However, its application is limited because of its unstable chemical properties. To overcome this limitation, ligusticum cycloprolactam (LIGc) was synthesized through structural modification of ligustilide. In this study, we combined network pharmacological methods with experimental verification to investigate the anti-neuroinflammatory effects and mechanisms of ligustilide and LIGc. Based on our network pharmacology analysis, we identified four key targets of ligustilide involved in exerting an anti-inflammatory effect, with the nuclear factor (NF)-κB signal pathway suggested as the main signalling pathway. To verify these results, we examined the expression of inflammatory cytokines and inflammation-related proteins, analysed the phosphorylation level of NF-κB, inhibitor of κBα (IκBα) and inhibitor of κB kinase α and β (IKKα+β), and evaluated the effect of BV2 cell-conditioned medium on HT22 cells in vitro. Our results, demonstrate for the first time that LIGc can downregulate the activation of the NF-κB signal pathway in BV2 cells induced by lipopolysaccharide, suppress the production of inflammatory cytokines and reduce nerve injury in HT22 cells mediated by BV2 cells. These findings suggest that LIGc inhibits the neuroinflammatory response mediated by BV2 cells, providing strong scientific support for the development of anti-inflammatory drugs based on natural ligustilide or its derivatives. However, there are some limitations to our current study. In the future, further experiments using in vivo models may provide additional evidence to support our findings.

Adenophora Stricta Root Extract Alleviates Airway Inflammation in Mice with Ovalbumin-Induced Allergic Asthma.

Adenophora stricta Miq. (Campanulaceae family) is a traditional herb used for relieving cough and phlegm in East Asia. This study explored the effects of A. stricta root extract (AsE) in ovalbumin (OVA)-induced allergic asthma and lipopolysaccharide (LPS)-stimulated macrophages. Administration of 100-400 mg/kg AsE dose-dependently decreased pulmonary congestion and suppressed the reduction of alveolar surface area in mice with OVA-mediated allergic asthma. Histopathological analysis of lung tissue and cytological analysis of bronchioalveolar lavage fluid showed that AsE administration significantly attenuated inflammatory cell infiltration into the lungs. In addition, AsE also alleviated OVA-specific immunoglobulin E, interleukin (IL)-4, and IL-5 production, which are essential for OVA-dependent activation of T helper 2 lymphocytes. In Raw264.7 macrophage cells, AsE significantly blocked nitric oxide, tumor necrosis factor-α, IL-1β, IL-6, and monocyte chemoattractant factor-1 production in response to LPS. Results from an immunoblot assay revealed that AsE inhibited the phosphorylation of c-jun N-terminal kinase, inhibitory-κB kinase α/β, and p65 in LPS-stimulated cells. Furthermore, 2-furoic acid, 5-hydroxymethylfurfural, and vanillic acid 4-β-D-glucopyranoside in AsE were shown to inhibit the production of proinflammatory mediators by LPS. Taken together, the present results suggest that A. stricta root will be a useful herb for relieving allergic asthma through managing airway inflammation.

Doublecortin-like kinase 1 activates NF-κB to induce inflammatory responses by binding directly to IKKβ.

Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein kinase, is involved in neurogenesis, and its levels are elevated in various human cancers. Recent studies suggest that DCLK1 may relate to inflammatory responses in the mouse model of colitis. However, cellular pathways engaged by DCLK1, and potential substrates of the kinase remain undefined. To understand how DCLK1 regulates inflammatory responses, we utilized the well-established lipopolysaccharide (LPS)-stimulated macrophages and mouse model. Through a range of macrophage-based and cell-free platforms, we discovered that DCLK1 binds directly with the inhibitor of κB kinase β (IKKβ) and induces IKKβ phosphorylation on Ser177/181 to initiate nuclear factor-κB (NF-κB) pathway. Deficiency in DCLK1, achieved by silencing or through pharmacological inhibition, prevented LPS-induced NF-κB activation and cytokine production in macrophages. We further show that mice with myeloid-specific DCLK1 knockout or DCLK1 inhibitor treatment are protected against LPS-induced acute lung injury and septic death. Our studies report a novel functional role of macrophage DCLK1 as a direct IKKβ regulator in inflammatory signaling and suggest targeted therapy against DCLK1 for inflammatory diseases.

Essential Oil from Glossogyne tenuifolia Inhibits Lipopolysaccharide-Induced Inflammation-Associated Genes in Macro-Phage Cells via Suppression of NF-κB Signaling Pathway.

Glossogyne tenuifolia Cassini (Hsiang-Ju in Chinese) is a perennial herb native to Taiwan. It was used in traditional Chinese medicine (TCM) as an antipyretic, anti-inflammatory, and hepatoprotective agent. Recent studies have shown that extracts of G. tenuifolia possess various bioactivities, including anti-oxidant, anti-inflammatory, immunomodulation, and anti-cancer properties. However, the pharmacological activities of G. tenuifolia essential oils have not been studied. In this study, we extracted essential oil from air-dried G. tenuifolia plants, then investigated the anti-inflammatory potential of G. tenuifolia essential oil (GTEO) on lipopolysaccharide (LPS)-induced inflammation in murine macrophage cells (RAW 264.7) in vitro. Treatment with GTEO (25, 50, and 100 μg/mL) significantly as well as dose-dependently inhibited LPS-induced pro-inflammatory molecules, such as nitric oxide (NO) and prostaglandin E2 (PGE2) production, without causing cytotoxicity. Q-PCR and immunoblotting analysis revealed that the inhibition of NO and PGE2 was caused by downregulation of their corresponding mediator genes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), respectively. Immunofluorescence and luciferase reporter assays revealed that the inhibition of iNOS and COX-2 genes by GTEO was associated with the suppression of nuclear export and transcriptional activation of the redox-sensitive transcription factor, nuclear factor -κB (NF-κB). In addition, GTEO treatment significantly inhibited phosphorylation and proteosomal degradation of the inhibitor of NF-κB (I-κBα), an endogenous repressor of NF-κB. Moreover, treatment with GTEO significantly blocked the LPS-mediated activation of inhibitory κB kinase α (IKKα), an upstream kinase of the I-κBα. Furthermore, p-cymene, β-myrcene, β-cedrene, cis-β-ocimene, α-pinene, and D-limonene were represented as major components of GTEO. We found that treatment with p-cymene, α-pinene, and D-limonene were significantly inhibiting LPS-induced NO production in RAW 264.7 cells. Taken together, these results strongly suggest that GTEO inhibits inflammation through the downregulation of NF-κB-mediated inflammatory genes and pro-inflammatory molecules in macrophage cells.

Targeting IKKβ Activity to Limit Sterile Inflammation in Acetaminophen-Induced Hepatotoxicity in Mice.

The kinase activity of inhibitory κB kinase β (IKKβ) acts as a signal transducer in the activating pathway of nuclear factor-κB (NF-κB), a master regulator of inflammation and cell death in the development of numerous hepatocellular injuries. However, the importance of IKKβ activity on acetaminophen (APAP)-induced hepatotoxicity remains to be defined. Here, a derivative of caffeic acid benzylamide (CABA) inhibited the kinase activity of IKKβ, as did IMD-0354 and sulfasalazine which show therapeutic efficacy against inflammatory diseases through a common mechanism: inhibiting IKKβ activity. To understand the importance of IKKβ activity in sterile inflammation during hepatotoxicity, C57BL/6 mice were treated with CABA, IMD-0354, or sulfasalazine after APAP overdose. These small-molecule inhibitors of IKKβ activity protected the APAP-challenged mice from necrotic injury around the centrilobular zone in the liver, and rescued the mice from hepatic damage-associated lethality. From a molecular perspective, IKKβ inhibitors directly interrupted sterile inflammation in the Kupffer cells of APAP-challenged mice, such as damage-associated molecular pattern (DAMP)-induced activation of NF-κB activity via IKKβ, and NF-κB-regulated expression of cytokines and chemokines. However, CABA did not affect the upstream pathogenic events, including oxidative stress with glutathione depletion in hepatocytes after APAP overdose. N-acetyl cysteine (NAC), the only FDA-approved antidote against APAP overdose, replenishes cellular levels of glutathione, but its limited efficacy is concerning in late-presenting patients who have already undergone oxidative stress in the liver. Taken together, we propose a novel hypothesis that chemical inhibition of IKKβ activity in sterile inflammation could mitigate APAP-induced hepatotoxicity in mice, and have the potential to complement NAC treatment in APAP overdoses.

Cherry fruit anthocyanins cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside protect against blue light-induced cytotoxicity in HaCaT cells

Blue light derived from multiple sources, including sunlight, generates reactive oxygen species (ROS) and negatively affects the skin in a manner similar to that of ultraviolet light. Cyanidin-3-O-glucoside (C3OG) and cyanidin-3-O-rutinoside (C3OR) are anthocyanin antioxidants that have protective effects on various tissues and cell types. However, the effects of anthocyanins on blue light-mediated changes remain unconfirmed. In this study, we determined the protective effects of C3OG and C3OR isolated and purified from waste cherry fruits (Prunus serrulata L. var. tomentella Nakai) against the blue light-induced ROS formation and inflammatory responses in HaCaT cells. It is showed that the treatment of C3OG and C3OR significantly reduced the blue light-induced cytotoxicity and ROS production in a dose dependent manner. Furthermore, we found that focal adhesion kinase (FAK) is a major upstream of blue light-induced expression of inflammatory cytokines (TNF-α, IL-6 and IL-8), and these effects were attenuated by C3OG or C3OR treatment. In the initial reaction, blue lights increased the phosphorylation of inhibitory-κB Kinase α (IKKα), c-jun N-terminal kinase (JNK), and p38. The phosphorylation of these intracellular proteins was reduced via FAK inhibitor, NAC (ROS scavenger), and anthocyanin treatments. After 24 h of blue light irradiation, C3OG or C3OR treatment markedly inhibited caspase-3-mediated apoptosis and cleaved-FAK-mediated anoikis, which is cell detachment-induced apoptosis. Therefore, our results indicate that C3OG and C3OR effectively protected human keratinocytes from harmful blue light-induced cytotoxicity and inflammation.

A novel TBK1/IKKϵ is involved in immune response and interacts with MyD88 and MAVS in the scallop Chlamys farreri.

Inhibitor of κB kinase (IKK) family proteins are key signaling molecules in the animal innate immune system and are considered master regulators of inflammation and innate immunity that act by controlling the activation of transcription factors such as NF-κB. However, few functional studies on IKK in invertebrates have been conducted, especially in marine mollusks. In this study, we cloned the IKK gene in the Zhikong scallop Chlamys farreri and named it CfIKK3. CfIKK3 encodes a 773-amino acid-long protein, and phylogenetic analysis showed that CfIKK3 belongs to the invertebrate TBK1/IKKϵ protein family. Quantitative real-time PCR analysis showed that CfIKK3 mRNA is ubiquitously expressed in all tested scallop tissues. The expression of CfIKK3 transcripts was significantly induced after challenge with lipopolysaccharide, peptidoglycan, or poly(I:C). Co-immunoprecipitation (co-IP) assays confirmed the direct interaction of CfIKK3 with MyD88 (the key adaptor in the TLR pathway) and MAVS (the key adaptor in the RLR pathway), suggesting that this IKK protein plays a crucial role in scallop innate immune signal transduction. In addition, the CfIKK3 protein formed homodimers and bound to CfIKK2, which may be a key step in the activation of its own and downstream transcription factors. Finally, in HEK293T cells, dual-luciferase reporter gene experiments showed that overexpression of CfIKK3 protein activated the NF-κB reporter gene in a dose-dependent manner. In conclusion, our experimental results confirmed that CfIKK3 could respond to PAMPs challenge and participate in scallop TLR and RLR pathway signaling, ultimately activating NF-κB. Therefore, as a key signaling molecule and modulator of immune activity, CfIKK3 plays an important role in the innate immune system of scallops.

Dynamic changes of monocytes subsets predict major adverse cardiovascular events and left ventricular function after STEMI

We explored how dynamic changes in monocyte subset counts (as opposed to static values to specific time points), and their phagocytic and NFκB activity relate to major adverse cardiovascular events (MACE) and left ventricular ejection fraction (LVEF) in patients with ST-elevation myocardial infarction (STEMI). Changes in counts, phagocytic activity and intracellular levels of inhibitory κB kinase β (IKKβ) (a marker of NFκB activity) of monocyte subsets (CD14++CD16−CCR2+ [Mon1], CD14++CD16+CCR2+ [Mon2] and CD14+CD16++CCR2− [Mon3]) were measured by flow cytometry in patients with STEMI at baseline, and again after one week, two weeks, and one month. LVEF was measured by echocardiography at baseline and six months after STEMI. Baseline data included 245 patients (mean ± SD age 60 ± 12 years; 22% female), who were followed for a median of 46 (19–61) months. Multivariate Cox regression demonstrated that more prominent dynamic reduction in Mon2 by week 1 (n = 37) was independently associated with fewer MACE (HR 0.06, 95% CI 0.01–0.55, p = 0.01). Also, less prominent reduction in Mon2 at month 1 (n = 24) was independently predictive of 6-month LVEF. None of the other dynamic changes in monocyte subsets were associated with changes in survival from MACE. Neither phagocytic activity nor IKKβ were associated with survival for each monocyte subset. We showed how distinct pattern of dynamic changes in Mon2 are related to both MACE risk and recovery of cardiac contractility. Further research is needed to understand the mechanism of the monocyte effect and possibilities of their pharmacological manipulation.

The inhibitor of κB kinase β (IKKβ) phosphorylates IκBα twice in a single binding event through a sequential mechanism

Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre-steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32s-1 and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) was 12μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1-54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event.

PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway.

Osteoclast generation from monocyte/macrophage lineage precursor cells needs to be tightly regulated to maintain bone homeostasis and is frequently over-activated in inflammatory conditions. PARK2, a protein associated with Parkinson's disease, plays an important role in mitophagy via its ubiquitin ligase function. In this study, we investigated whether PARK2 is involved in osteoclastogenesis. PARK2 expression was found to be increased during the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. PARK2 gene silencing with siRNA significantly reduced osteoclastogenesis induced by RANKL, LPS (lipopolysaccharide), TNFα (tumor necrosis factor α), and IL-1β (interleukin-1β). On the other hand, overexpression of PARK2 promoted osteoclastogenesis. This regulation of osteoclastogenesis by PARK2 was mediated by IKK (inhibitory κB kinase) and NF-κB activation while MAPK (mitogen-activated protein kinases) activation was not involved. Additionally, administration of PARK2 siRNA significantly reduced osteoclastogenesis and bone loss in an in vivo model of inflammatory bone erosion. Taken together, this study establishes a novel role for PARK2 as a positive regulator in osteoclast differentiation and inflammatory bone destruction.

Salt-inducible kinase 3 protects tumor cells from cytotoxic T-cell attack by promoting TNF-induced NF-κB activation

BackgroundCancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor...

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.

LED Light-Induced ROS Differentially Regulates Focal Adhesion Kinase Activity in HaCaT Cell Viability.

In this study, changes in cell signaling mechanisms in skin cells induced by various wavelengths and intensities of light-emitting diodes (LED) were investigated, focusing on the activity of focal adhesion kinase (FAK) in particular. We examined the effect of LED irradiation on cell survival, the generation of intracellular reactive oxygen species (ROS), and the activity of various cell-signaling proteins. Red LED light increased cell viability at all intensities, whereas strong green and blue LED light reduced cell viability, and this effect was reversed by NAC or DPI treatment. Red LED light caused an increase in ROS formation according to the increase in the intensity of the LED light, and green and blue LED lights led to sharp increases in ROS formation. In the initial reaction to LEDs, red LED light only increased the phosphorylation of FAK and extracellular-signal regulated protein kinase (ERK), whereas green and blue LED lights increased the phosphorylation of inhibitory-κB Kinase α (IKKα), c-jun N-terminal kinase (JNK), and p38. The phosphorylation of these intracellular proteins was reduced via FAK inhibitor, NAC, and DPI treatments. Even after 24 h of LED irradiation, the activity of FAK and ERK appeared in cells treated with red LED light but did not appear in cells treated with green and blue LED lights. Furthermore, the activity of caspase-3 was confirmed along with cell detachment. Therefore, our results suggest that red LED light induced mitogenic effects via low levels of ROS–FAK–ERK, while green and blue LED lights induced cytotoxic effects via cellular stress and apoptosis signaling resulting from high levels of ROS.

IFN-γ Induces IL-15 Trans-Presentation by Epithelial Cells via IRF1.

IL-15 exhibits pleiotropic effects on NK and CD8+ T cells and contributes to host protection or immunopathology during infection. Although both type I IFNs and IFN-γ upregulate IL-15 expression, their effects on IL-15 upregulation and underlying mechanisms have not been compared comprehensively. In addition, little is known about trans-presentation of IL-15 by epithelial cells to lymphocytes. In this study, we analyzed the expression of IL-15 and IL-15Rα in the human hepatocyte-derived Huh-7 cell line after stimulation with IFN-α, IFN-β, or IFN-γ using RT-PCR, flow cytometry, and confocal microscopy. We also performed knockdown experiments to investigate the signaling pathway involved in IL-15 upregulation. IFN-γ more potently upregulated IL-15 expression in Huh-7 cells than IFN-α and IFN-β. Knockdown experiments revealed that IFN-γ- and IFN-β-induced IL-15 expression relied on IFN regulatory factor 1 (IRF1), which is upregulated by STAT1 and IFN-stimulated gene factor 3, respectively. Inhibitor of κB kinase α/β was also involved in IFN-γ-induced upregulation of IL-15. Furthermore, human NK cells were activated by coculture with IFN-γ-treated Huh-7 cells, which was abrogated by knocking down IL-15Rα in IFN-γ-treated Huh-7 cells, indicating that IFN-γ-induced IL-15 on Huh-7 cells activates NK cells via trans-presentation. In summary, our data demonstrate that IFN-γ potently elicits IL-15 trans-presentation by epithelial cells via IRF1. These data also suggest that the IFN-γ-IRF1-IL-15 axis may be a regulatory target for the treatment of diseases with IL-15 dysregulation.

Upgrade of an old drug: Auranofin in innovative cancer therapies to overcome drug resistance and to increase drug effectiveness.

Auranofin is an oral gold(I) compound, initially developed for the treatment of rheumatoid arthritis. Currently, Auranofin is under investigation for oncological application within a drug repurposing plan due to the relevant antineoplastic activity observed both in vitro and in vivo tumor models. In this review, we analysed studies in which Auranofin was used as a single drug or in combination with other molecules to enhance their anticancer activity or to overcome chemoresistance. The analysis of different targets/pathways affected by this drug in different cancer types has allowed us to highlight several interesting targets and effects of Auranofin besides the already well‐known inhibition of thioredoxin reductase. Among these targets, inhibitory‐κB kinase, deubiquitinates, protein kinase C iota have been frequently suggested. To rationalize the effects of Auranofin by a system biology‐like approach, we exploited transcriptomic data obtained from a wide range of cell models, extrapolating the data deposited in the Connectivity Maps website and we attempted to provide a general conclusion and discussed the major points that need further investigation.

Protective and proliferative effect of Aesculus indica extract on stressed human adipose stem cells via downregulation of NF-κB pathway

Inflammatory microenvironment after transplantation affects the proliferation and causes senescence of adipose-derived mesenchymal stem cells (hADMSCs) thus compromising their clinical efficacy. Priming stem cells with herbal extracts is considered very promising to improve their viability in the inflammatory milieu. Aesculus indica (A. indica) is used to treat many inflammatory diseases in Asia for decades. Herein, we explored the protective role of A. indica extract on human adipose-derived Mesenchymal Stem Cells (hADMSCs) against Monosodium Iodoacetate (MIA) induced stress in vitro. A. indica ameliorated the injury as depicted by significantly enhanced proliferation, viability, improved cell migration and superoxide dismutase activity. Furthermore, reduced lactate dehydrogenase activity, reactive oxygen species release, senescent and apoptotic cells were detected in A. indica primed hADMSCs. Downregulation of NF-κB pathway and associated inflammatory genes, NF-κB p65/RelA and p50/NF-κB 1, Interleukin 6 (IL-6), Interleukin 1 (IL-1β), Tumor necrosis factor alpha (TNF-α) and matrix metalloproteinase 13 (MMP-13) were observed in A. indica primed hADMSCs as compared to stressed hADMSCs. Complementary to gene expression, A. indica priming reduced the release of transcription factor p65, inhibitory-κB kinase (IKK) α and β, IL-1β and TNF-α proteins expression. Our data elucidates that A. indica extract preconditioning rescued hADMSCs against oxidative stress and improved their therapeutic potential by relieving inflammation through regulation of NF-κB pathway.