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1.
Cell Biol Toxicol ; 39(4): 1677-1696, 2023 08.
Article in English | MEDLINE | ID: mdl-36163569

ABSTRACT

The activation of receptor-interacting protein kinase 1 (RIPK1) by death-inducing signaling complex (DISC) formation is essential for triggering the necroptotic mode of cell death under apoptosis-deficient conditions. Thus, targeting the induction of necroptosis by modulating RIPK1 activity could be an effective strategy to bypass apoptosis resistance in certain types of cancer. In this study, we screened a series of arborinane triterpenoids purified from Rubia philippinesis and identified rubiarbonol B (Ru-B) as a potent caspase-8 activator that induces DISC-mediated apoptosis in multiple types of cancer cells. However, in RIPK3-expressing human colorectal cancer (CRC) cells, the pharmacological or genetic inhibition of caspase-8 shifted the mode of cell death by Ru-B from apoptosis to necroptosis though upregulation of RIPK1 phosphorylation. Conversely, Ru-B-induced cell death was almost completely abrogated by RIPK1 deficiency. The enhanced RIPK1 phosphorylation and necroptosis triggered by Ru-B treatment occurred independently of tumor necrosis factor receptor signaling and was mediated by the production of reactive oxygen species via NADPH oxidase 1 in CRC cells. Thus, we propose Ru-B as a novel anticancer agent that activates RIPK1-dependent cell death via ROS production, and suggest its potential as a novel necroptosis-targeting compound in apoptosis-resistant CRC.


Subject(s)
Apoptosis , Necroptosis , Humans , Reactive Oxygen Species/metabolism , Caspase 8/metabolism , Caspase 8/pharmacology , Cell Death , Necrosis , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , NADPH Oxidase 1/metabolism , NADPH Oxidase 1/pharmacology
2.
Biochem Pharmacol ; 192: 114733, 2021 10.
Article in English | MEDLINE | ID: mdl-34411570

ABSTRACT

In tumor necrosis factor (TNF) signaling, IκB kinase (IKK) complex-mediated activation of NF-κB is a well-known protective mechanism against cell death via transcriptional induction of pro-survival genes occurring as a late checkpoint. However, recent belief holds that IKK functions as an early cell death checkpoint to suppress the death-inducing signaling complex by regulating receptor interacting protein kinase1 (RIPK1) phosphorylation. In this study, we propose that two major gernaylated 7-hydroxy coumarins, 6-geranyl-7-hydroxycoumarin (ostruthin) and 8-geranyl-7-hydroxycoumarin (8-geranylumbelliferone, 8-GU) isolated from Paramignya timera, facilitate RIPK1-dependent dual modes of apoptosis and necroptosis by targeting IKKß upon TNF receptor1 (TNFR1) ligation. Analysis of events upstream of NF-κB revealed that 8-GU and ostruthin drastically inhibited TNF-induced IKK phosphorylation, while having no effect on TAK1 phosphorylation and TNFR1 complex-I formation. Interestingly, 8-GU did not affect the cell death induced by Fas ligand or TNF-related apoptosis-inducing ligand or that induced by DNA-damaging agents, indicating that 8-GU sensitizes TNF-induced cell death exclusively. Moreover, 8-GU accelerated TNF-driven necroptosis by up-regulating necrosome formation in FADD deficient cancer cells harboring RIPK3. Thus, the present study provides new insights into the molecular mechanism underlying geranylated 7-hydroxy coumarin-mediated control of the RIPK1-dependent early cell death checkpoint and suggests that 8-GU is a potential anti-cancer therapeutic via an alternative apoptosis-independent strategy to overcome TNF resistance.


Subject(s)
Apoptosis/drug effects , Plant Extracts/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Umbelliferones/pharmacology , Animals , Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/physiology , Coumarins/isolation & purification , Coumarins/pharmacology , HEK293 Cells , HT29 Cells , HeLa Cells , Humans , MCF-7 Cells , Mice , Mice, Knockout , Plant Extracts/isolation & purification , RAW 264.7 Cells , Umbelliferones/isolation & purification
3.
J Nat Prod ; 84(5): 1598-1606, 2021 05 28.
Article in English | MEDLINE | ID: mdl-33939429

ABSTRACT

Three new alkamides, tulipiferamides A-C (1-3, respectively), and 30 known compounds (4-33) were obtained from the roots of Liriodendron tulipifera (Magnoliaceae). Dehydrotemisin (4), an elemane sesquiterpene lactone, was isolated for the first time from nature. The structures were deduced by the interpretation of NMR spectroscopic and MS spectral data. The geometries of the double bonds in tulipiferamides A-C (1-3, respectively) were determined on the basis of 1H-1H coupling constants and 13C chemical shifts. The presence of the alkamide type in this plant is reported for the first time. An analysis of the inflammatory response revealed that seven compounds (1, 4, 7, 9, 14, 23, and 27) suppressed the nitric oxide production induced by LPS in RAW264.7 macrophages. Furthermore, tulipiferamide A (1) inhibits NF-κB activation by selectively targeting IKKß, an upstream kinase of NF-κB, resulting in the suppression of inflammatory mediators, including iNOS, COX-2, IL-1ß, TNFα, and IL-6. Our results provide a rationale for the further development of tulipiferamide A as a selective IKKß inhibitor to modulate inflammatory diseases.


Subject(s)
Anti-Inflammatory Agents/pharmacology , I-kappa B Kinase/metabolism , Lactones/pharmacology , Liriodendron/chemistry , Sesquiterpenes/pharmacology , Animals , Anti-Inflammatory Agents/isolation & purification , HEK293 Cells , Humans , Lactones/isolation & purification , Mice , Molecular Structure , Nitric Oxide/biosynthesis , Phosphorylation , Phytochemicals/isolation & purification , Phytochemicals/pharmacology , Plant Roots/chemistry , RAW 264.7 Cells , Republic of Korea , Sesquiterpenes/isolation & purification
4.
FASEB J ; 34(3): 4369-4383, 2020 03.
Article in English | MEDLINE | ID: mdl-32027418

ABSTRACT

In tumor necrosis factor (TNF) signaling, phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) by upstream kinases is an essential checkpoint in the suppression of TNF-induced cell death. Thus, discovery of pharmacological agents targeting RIPK1 may provide new strategies for improving the therapeutic efficacy of TNF. In this study, we found that 3-O-acetylrubianol C (3AR-C), an arborinane triterpenoid isolated from Rubia philippinesis, promoted TNF-induced apoptotic and necroptotic cell death. To identify the molecular mechanism, we found that in mouse embryonic fibroblasts, 3AR-C drastically upregulated RIPK1 kinase activity by selectively inhibiting IKKß. Notably, 3AR-C did not interfere with IKKα or affect the formation of the TNF receptor1 (TNFR1) complex-I. Moreover, in human cancer cells, 3AR-C was only sufficient to sensitize TNF-induced cell death when c-FLIPL expression was downregulated to facilitate the formation of TNFR1 complex-II and necrosome. Taken together, our study identified a novel arborinane triterpenoid 3AR-C as a potent activator of TNF-induced cell death via inhibition of IKKß phosphorylation and promotion of the cytotoxic potential of RIPK1, thus providing a rationale for further development of 3AR-C as a selective IKKß inhibitor to overcome TNF resistance in cancer therpay.


Subject(s)
Apoptosis/physiology , I-kappa B Kinase/metabolism , Programmed Cell Death 1 Receptor/metabolism , Animals , Apoptosis/genetics , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/physiology , Humans , I-kappa B Kinase/genetics , Magnetic Resonance Spectroscopy , Mice , Programmed Cell Death 1 Receptor/genetics , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
5.
Sci Rep ; 9(1): 13505, 2019 09 18.
Article in English | MEDLINE | ID: mdl-31534206

ABSTRACT

Plant-derived lignans have numerous biological effects including anti-tumor and anti-inflammatory activities. Screening of purified constituents of Rubia philippinensis from human glioblastoma cells resistant to TNF-related apoptosis-inducing ligand (TRAIL) has suggested that the lignan pinoresinol was a highly active TRAIL sensitizer. Here we show that treatment with nontoxic doses of pinoresinol in combination with TRAIL induced rapid apoptosis and caspase activation in many types of glioblastoma cells, but not in normal astrocytes. Analyses of apoptotic signaling events revealed that pinoresinol enhanced the formation of TRAIL-mediated death-inducing signaling complex (DISC) and complete processing of procaspase-8 within the DISC in glioblastoma cells, in which caspase-8 was inactivated. Mechanistically, pinoresinol downregulated the expression of cellular FLICE-inhibitory protein (cFLIPL) and survivin through proteasome-mediated degradation, without affecting death receptors or downstream intracellular apoptosis-related proteins. Furthermore, the sensitization of TRAIL-mediated apoptosis by pinoresinol strictly depended on the expression level of cFLIPL, which was regulated through de novo protein synthesis, rather than by NF-κB or p53 signaling. Taken together, our results indicate that pinoresinol facilitates DISC-mediated caspase-8 activation by targeting cFLIPL in an early event in apoptotic signaling, which provides a potential therapeutic module for TRAIL-based chemotherapy.


Subject(s)
Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Furans/pharmacology , Lignans/pharmacology , Caspase 8/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Furans/metabolism , Glioblastoma/metabolism , Humans , Lignans/metabolism , NF-kappa B/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Rubia/metabolism , Signal Transduction/drug effects , TNF-Related Apoptosis-Inducing Ligand/drug effects , TNF-Related Apoptosis-Inducing Ligand/metabolism
6.
Biochimie ; 165: 108-114, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31336136

ABSTRACT

Oridonin is a diterpenoid isolated from the Rabdosia rubescens and has multiple biological effects, such as anti-inflammation and anti-tumor activities. In present study, we revealed that the sensitizing effect of oridonin on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in several cancer cells, but not in normal cells. Oridonin enhanced death-signaling inducing complexes (DISC) formation and DR5 glycosylation without affecting expression of downstream intracellular apoptosis-related proteins. Oridonin upregulated peptidyl O-glycosyltransferase GALNT14 in a dose- and time-dependent manner. Knockdown of GALNT14 by siRNA and Endo H treatment reduced oridonin-induced DR5 glycosylation. Furthermore, treatment with inhibitor of glycosylation (benzyl-α-GalNAc) blocked oridonin plus TRAIL-induced apoptosis. Collectively, our results suggest that oridonin-induced DR5 glycosylation contributes to TRAIL-induced apoptotic cell death in cancer cells.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis/drug effects , Diterpenes, Kaurane/pharmacology , N-Acetylgalactosaminyltransferases/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/physiology , Cell Line , Glycosylation , Humans , TNF-Related Apoptosis-Inducing Ligand/pharmacology
7.
Biochem Pharmacol ; 158: 243-260, 2018 12.
Article in English | MEDLINE | ID: mdl-30359578

ABSTRACT

Despite recent tremendous progress, targeting of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapy has limited success in many clinical trials, in part due to inactivation of death inducing signaling complex (DISC)-mediated caspase-8 signaling cascade in highly malignant tumors such as glioblastoma. In this study, screening of constituents derived from Astilbe rivularis for TRAIL-sensitizing activity identified C-27-carboxylated oleanolic acid derivatives (C27OAs) including 3ß-hydroxyolean-12-en-27-oic acid (C27OA-1), 3ß,6ß,7α-trihydroxyolean-12-en-27-oic acid (C27OA-2), and 3ß-trans-p-coumaroyloxy-olean-12-en-27-oic acid (C27OA-3) as novel TRAIL sensitizers. Interestingly, these C27OAs did not affect apoptotic cell death induced by either ligation of other death receptor (DR) types, such as TNF and Fas or DNA damaging agents, which suggests that C27OAs effectively and selectively sensitize TRAIL-mediated caspase-8 activation. Mechanistically, C27OAs upregulate the expression of cell surface DR5 and DISC formation without affecting downstream intracellular apoptosis-related proteins. The upregulation of DR5 expression by C27OAs strictly depends on transactivation of C/EBP homology protein, which is regulated through the p38 MAPK pathway, rather than p53 and intracellular reactive oxygen species status. Taken together, our results identify the novel C27OAs as TRAIL sensitizers targeting the upstream DISC assembly of DR5, and provide a rationale for further development of C27OAs for facilitating TRAIL-based chemotherapy in glioblastoma patients.


Subject(s)
Glioblastoma/metabolism , Oleanolic Acid/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/biosynthesis , TNF-Related Apoptosis-Inducing Ligand/biosynthesis , Transcription Factor CHOP/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Humans , Oleanolic Acid/chemistry , Oleanolic Acid/isolation & purification , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , TNF-Related Apoptosis-Inducing Ligand/genetics , Transcription Factor CHOP/genetics , Up-Regulation/drug effects , Up-Regulation/physiology , p38 Mitogen-Activated Protein Kinases/genetics
8.
Mol Brain ; 9(1): 99, 2016 12 16.
Article in English | MEDLINE | ID: mdl-27986089

ABSTRACT

Apurinic/apyrimidinic endonuclease 1 (APE1), a ubiquitous multipurpose protein, is also known as redox effector factor-1 (Ref-1). It is involved in DNA repair and redox signaling and, in turn, oxidative stress-induced neurodegeneration. Although previous studies have demonstrated that APE1/Ref-1 functions as a negative regulator of inflammatory response via several mechanisms in neuronal cells, little is known about the roles of APE1/Ref-1 in glial cells. In this study, we found that cytoplasmic APE1/Ref-1 expression was upregulated in reactive astrocytes of the kainic acid- or lipopolysaccharide (LPS)-injected hippocampus. Analysis of the inflammatory response induced by extranuclear APE1/Ref-1 (ΔNLS-Ref-1) in cultured primary astrocytes revealed that it markedly suppressed inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor-α (TNF-α) secretion induced by LPS to a similar extent as did wild type APE1/Ref-1 (WT-Ref-1), supporting the concept an anti-inflammatory role of extranuclear APE1/Ref-1 in astrocytes. Additionally, overexpression of WT- and ΔNLS-Ref-1 suppressed the transcriptional activity of nuclear factor-κB (NF-κB), although it effectively enhanced activator protein 1 (AP-1) activity. The blunting effect of APE1/Ref-1 on LPS-induced NF-κB activation was not mediated by IκB kinase (IKK) activity. Instead, APE1/Ref-1 inhibited p300-mediated acetylation of p65 by suppressing intracellular reactive oxygen species (ROS) levels following LPS treatment. Taken together, our results showed that altered expression and/or subcellular distribution of APE1/Ref-1 in activated astrocytes regulated the neuroinflammatory response to excitotoxin and endotoxin insults used in model of neurodegenerative brain diseases.


Subject(s)
Anti-Inflammatory Agents/metabolism , Astrocytes/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Acetylation , Animals , Cells, Cultured , Cytoplasm/enzymology , E1A-Associated p300 Protein/metabolism , Hippocampus/pathology , Humans , I-kappa B Kinase/metabolism , Inflammation/pathology , Kainic Acid , Lipopolysaccharides , Nitric Oxide Synthase Type II/metabolism , Protein Transport , Rats , Reactive Oxygen Species/metabolism , Sequence Deletion , Signal Transduction , Subcellular Fractions/metabolism , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/metabolism
9.
Biochem Pharmacol ; 121: 52-66, 2016 Dec 01.
Article in English | MEDLINE | ID: mdl-27664853

ABSTRACT

A specific small-molecule inhibitor of the TLR4 signaling complex upstream of the IKK would likely provide therapeutic benefit for NF-κB-mediated inflammatory disease. We previously identified brazilin as a selective upstream IKK inhibitor targeting the Myddosome complex. In this study, using a cell-based ubiquitination assay for IRAK1 and a chemical library comprising a series of structural analogues of brazilin, a novel small molecule, 2-hydroxy-5,6-dihydroisoindolo[1,2-a]isoquinoline-3,8-dione (IinQ), was identified as a selective and potent inhibitor of IRAK1-dependent NF-κB activation upon TLR4 ligation. In RAW264.7 macrophages, IinQ drastically suppressed activation of upstream IKK signaling events including membrane-bound IRAK1 ubiquitination and IKK phosphorylation by the TLR4 ligand, resulting in reduced expression of proinflammatory mediators including IL-6, TNF-α, and nitric oxide. Interestingly, IinQ did not suppress NF-κB activation via the TLR3 ligand, DNA damaging agents, or a protein kinase C activator, indicating IinQ is specific for TLR4 signaling. Analysis of upstream signaling events further confirmed that IinQ disrupts the MyD88-IRAK1-TRAF6 complex formation induced by LPS treatment, without affecting TLR4 oligomerization. Moreover, intravenous administration of IinQ significantly reduced lethality and attenuated systemic inflammatory responses in an in vivo mouse model of endotoxin shock following LPS challenge. Thus, IinQ represents a novel class of brazilin analogues with improved potency and specificity toward disruption of Myddosome complex formation in TLR4 signaling, indicating that IinQ may be a promising therapeutic candidate for the treatment of systemic inflammatory diseases.


Subject(s)
I-kappa B Kinase/antagonists & inhibitors , Interleukin-1 Receptor-Associated Kinases/metabolism , Isoindoles/pharmacology , Isoquinolines/pharmacology , Myeloid Differentiation Factor 88/metabolism , Systemic Inflammatory Response Syndrome/drug therapy , TNF Receptor-Associated Factor 6/metabolism , Toll-Like Receptor 4/metabolism , Animals , Cell Line , Female , Isoindoles/chemical synthesis , Isoindoles/chemistry , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Lipopolysaccharides/toxicity , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Signal Transduction/drug effects , Systemic Inflammatory Response Syndrome/metabolism , Ubiquitination/drug effects
10.
Arch Pharm Res ; 39(8): 1075-84, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27287455

ABSTRACT

The transcription factor nuclear factor-kappa B (NF-κB) controls a number of essential cellular functions, including the immune response, cell proliferation, and apoptosis. NF-κB signaling must be engaged temporally and spatially and well orchestrated to prevent aberrant activation because loss of normal regulation of NF-κB is a major contributor to a variety of pathological diseases, including inflammatory diseases, autoimmune diseases, and cancers. Thus, understanding the molecular mechanisms controlling NF-κB activation is an important part of treatment of these relevant diseases. Although NF-κB transcriptional activity is largely regulated by nuclear translocation, post-translational modification of NF-κB signaling components, including phosphorylation, ubiquitination, acetylation, and methylation, has emerged as an important mechanism affecting activity. Many proteins have been shown to ubiquitinate and regulate NF-κB activation at the receptor signaling complex in response to a variety of ligands, such as tumor necrosis factor, interleukin-1, and Toll-like receptor ligands. In this review, we discuss our current knowledge of ubiquitination patterns and their functional role in NF-κB regulation.


Subject(s)
NF-kappa B/metabolism , Protein Processing, Post-Translational/physiology , Signal Transduction/physiology , Ubiquitination/physiology , Animals , Humans , NF-kappa B/genetics
11.
Sci Rep ; 6: 25094, 2016 04 27.
Article in English | MEDLINE | ID: mdl-27117478

ABSTRACT

Death receptor (DR) ligation elicits two different modes of cell death (necroptosis and apoptosis) depending on the cellular context. By screening a plant extract library from cells undergoing necroptosis or apoptosis, we identified a water extract of Terminalia chebula (WETC) as a novel and potent dual inhibitor of DR-mediated cell death. Investigation of the underlying mechanisms of its anti-necroptotic and anti-apoptotic action revealed that WETC or its constituents (e.g., gallic acid) protected against tumor necrosis factor-induced necroptosis via the suppression of TNF-induced ROS without affecting the upstream signaling events. Surprisingly, WETC also provided protection against DR-mediated apoptosis by inhibition of the caspase cascade. Furthermore, it activated the autophagy pathway via suppression of mTOR. Of the WETC constituents, punicalagin and geraniin appeared to possess the most potent anti-apoptotic and autophagy activation effect. Importantly, blockage of autophagy with pharmacological inhibitors or genetic silencing of Atg5 selectively abolished the anti-apoptotic function of WETC. These results suggest that WETC protects against dual modes of cell death upon DR ligation. Therefore, WETC might serve as a potential treatment for diseases characterized by aberrantly sensitized apoptotic or non-apoptotic signaling cascades.


Subject(s)
Cell Death/drug effects , Plant Extracts/metabolism , Receptors, Death Domain/metabolism , Terminalia/chemistry , Glucosides/isolation & purification , Glucosides/metabolism , Hydrolyzable Tannins/isolation & purification , Hydrolyzable Tannins/metabolism , Plant Extracts/isolation & purification , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors
12.
PLoS One ; 10(8): e0136122, 2015.
Article in English | MEDLINE | ID: mdl-26295477

ABSTRACT

Brazilin is an active compound of Caesalpinia sappan L. (Leguminosae), which possesses pro-apoptotic and anti-inflammation potentials depending on the specific cell type. However, it is largely unknown whether autophagy is implicated in the mechanism underlying its chemotherapeutic and anti-inflammatory effects in rheumatoid arthritis (RA). Here, we show that treatment of RA fibroblast-like synoviocytes (FLS) with brazilin results in enhanced level of autophagic flux, evidenced by accumulation of autophagosome and increased level of lipidated LC3 (LC3-II), which is mainly mediated by enhanced production of reactive oxygen species (ROS). Interestingly, long-term exposure of brazilin was able to restore cell survival against the cytotoxity, exclusively in RA FLS, but not in normal fibroblast. Importantly, such a restoration from brazilin-induced cytotoxity in RA FLS was completely abrogated after co-treatment with autophagy inhibitors including NH4Cl or chloroquine. Furthermore, we found that the pretreatment of RA FLS with brazilin reduced LPS- or TNF-induced NF-κB activation and the secretion of inflammatory cytokines in parallel with the enhanced autophagic flux. Such anti-NF-κB potentials of brazilin were drastically masked in RA FLS when autophagy was suppressed. These results suggest that brazilin is capable of activating autophagy exclusively in RA FLS, and such inducible autophagy promotes cell survival and limits inflammatory response.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Arthritis, Rheumatoid/drug therapy , Autophagy/drug effects , Benzopyrans/pharmacology , Fibroblasts/drug effects , Fibroblasts/pathology , Synovial Membrane/cytology , Animals , Anti-Inflammatory Agents/chemistry , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Benzopyrans/chemistry , Caesalpinia/chemistry , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/immunology , Humans , Mice , NF-kappa B/immunology , NIH 3T3 Cells , Reactive Oxygen Species/immunology , Synovial Membrane/drug effects , Synovial Membrane/immunology , Synovial Membrane/pathology
13.
J Pharmacol Exp Ther ; 350(2): 301-12, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24898266

ABSTRACT

Increased N-methyl-d-aspartate receptor (NMDAR) activity and phosphorylation in the spinal cord are critically involved in the synaptic plasticity and central sensitization associated with neuropathic pain. However, the mechanisms underlying increased NMDAR activity in neuropathic pain conditions remain poorly understood. Here we show that peripheral nerve injury induces a large GluN2A-mediated increase in NMDAR activity in spinal lamina II, but not lamina I, neurons. However, NMDAR currents in spinal dorsal horn neurons are not significantly altered in rat models of diabetic neuropathic pain and resiniferatoxin-induced painful neuropathy (postherpedic neuralgia). Inhibition of protein tyrosine kinases or protein kinase C has little effect on NMDAR currents potentiated by nerve injury. Strikingly, casein kinase II (CK2) inhibitors normalize increased NMDAR currents of dorsal horn neurons in nerve-injured rats. In addition, inhibition of calcineurin, but not protein phosphatase 1/2A, augments NMDAR currents only in control rats. CK2 inhibition blocks the increase in spinal NMDAR activity by the calcineurin inhibitor in control rats. Furthermore, nerve injury significantly increases CK2α and CK2ß protein levels in the spinal cord. In addition, inhibition of CK2 or CK2ß knockdown at the spinal level substantially reverses pain hypersensitivity induced by nerve injury. Our study indicates that neuropathic pain conditions with different etiologies do not share the same mechanisms, and increased spinal NMDAR activity is distinctly associated with traumatic nerve injury. CK2 plays a prominent role in the potentiation of NMDAR activity in the spinal dorsal horn and may represent a new target for treatments of chronic pain caused by nerve injury.


Subject(s)
Casein Kinase II/physiology , Neuralgia/etiology , Peripheral Nerve Injuries/complications , Receptors, N-Methyl-D-Aspartate/physiology , Spinal Cord/physiology , Animals , Calcineurin/physiology , Casein Kinase II/analysis , Diabetic Neuropathies/etiology , Hyperalgesia/etiology , Male , Protein Kinase C/physiology , Protein-Tyrosine Kinases/physiology , Rats , Rats, Sprague-Dawley
14.
Biochem Pharmacol ; 89(4): 515-25, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24735611

ABSTRACT

The ligation of interleukin-1 receptor (IL-1R) or tumor necrosis factor receptor 1 (TNFR1) induces the recruitment of adaptor proteins and their concomitant ubiquitination to the proximal receptor signaling complex, respectively. Such are upstream signaling events of IKK that play essential roles in NF-κB activation. Thus, the discovery of a substance that would modulate the recruitment of key proximal signaling elements at the upstream level of IKK has been impending in this field of study. Here, we propose that brazilin, an active compound of Caesalpinia sappan L. (Leguminosae), is a potent NF-κB inhibitor that selectively disrupts the formation of the upstream IL-1R signaling complex. Analysis of upstream signaling events revealed that brazilin markedly abolished the IL-1ß-induced polyubiquitination of IRAK1 and its interaction with IKK-γ counterpart. Notably, pretreatment of brazilin drastically interfered the recruitment of the receptor-proximal signaling components including IRAK1/4 and TRAF6 onto MyD88 in IL-1R-triggerd NF-κB activation. Interestingly, brazilin did not affect the TNF-induced RIP1 ubiquitination and the recruitment of RIP1 and TRAF2 to TNFR1, suggesting that brazilin is effective in selectively suppressing the proximal signaling complex formation of IL-1R, but not that of TNFR1. Moreover, our findings suggest that such a disruption of IL-1R-proximal complex formation by brazilin is not mediated by affecting the heterodimerization of IL-1R and IL-1RAcP. Taken together, the results suggest that the anti-IKK activity of brazilin is induced by targeting IKK upstream signaling components and subsequently disrupting proximal IL-1 receptor signaling complex formation.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzopyrans/pharmacology , I-kappa B Kinase/antagonists & inhibitors , Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Toll-Like Receptor 4/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/isolation & purification , Benzopyrans/chemistry , Benzopyrans/isolation & purification , Caesalpinia/chemistry , Ethnopharmacology , Genes, Reporter/drug effects , HEK293 Cells , HeLa Cells , Humans , I-kappa B Kinase/metabolism , I-kappa B Proteins/antagonists & inhibitors , I-kappa B Proteins/genetics , I-kappa B Proteins/metabolism , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-1 Receptor-Associated Kinases/metabolism , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Molecular Structure , NF-kappa B/agonists , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Receptors, Interleukin-1/agonists , Receptors, Interleukin-1/antagonists & inhibitors , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Republic of Korea , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Ubiquitination/drug effects , Wood/chemistry
15.
J Pharmacol Exp Ther ; 347(3): 765-72, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24030012

ABSTRACT

The glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are critically involved in the excitatory synaptic transmission, and blocking AMPARs at the spinal level reverses neuropathic pain. However, little is known about changes in the composition of synaptic AMPARs in the spinal dorsal horn after peripheral nerve injury. AMPARs lacking the GluA2 subunit are permeable to Ca(2+), and their currents show unique inward rectification. We found that AMPAR-mediated excitatory postsynaptic currents (AMPAR-EPSCs) of spinal dorsal horn neurons exhibited a linear current-voltage relationship in control rats, whereas AMPAR-EPSCs of dorsal horn neurons displayed inward rectification in rats with spinal nerve injury. In nerve-injured rats, compared with control rats, the GluA2 protein level was significantly less in the plasma membrane but was greater in the cytosolic vesicle fraction in the dorsal spinal cord. However, the GluA1 protein levels in these fractions did not differ significantly between nerve-injured and control rats. Blocking N-methyl-d-aspartate receptors (NMDARs) abolished inward rectification of AMPAR-EPSCs of dorsal horn neurons in nerve-injured rats. Furthermore, inhibition of calpain or calcineurin, but not protein kinase C, completely blocked nerve injury-induced inward rectification of AMPAR-EPSCs of dorsal horn neurons. In addition, blocking GluA2-lacking AMPARs at the spinal cord level reduced nerve injury-induced pain hypersensitivity. Our study suggests that nerve injury increases GluA2 internalization and the prevalence of GluA2-lacking AMPARs in the spinal dorsal horn to maintain chronic neuropathic pain. Increased prevalence of spinal GluA2-lacking AMPARs in neuropathic pain is mediated by NMDARs and subsequent stimulation of calpain and calcineurin signaling.


Subject(s)
Receptors, AMPA/metabolism , Spinal Cord/metabolism , Trauma, Nervous System/metabolism , Animals , Blotting, Western , Calcineurin/physiology , Calpain/physiology , Electrophysiological Phenomena/genetics , Electrophysiological Phenomena/physiology , Hyperalgesia/physiopathology , In Vitro Techniques , Male , Physical Stimulation , Posterior Horn Cells/physiology , Protein Kinase C/physiology , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology
16.
Nat Commun ; 4: 2062, 2013.
Article in English | MEDLINE | ID: mdl-23797602

ABSTRACT

Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 (PHF20) maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that PHF20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In PHF20 overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between PHF20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that PHF20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies PHF20 as a novel regulator of NF-κB activation and suggests that elevated expression of PHF20 may drive constitutive NF-κB activation in some cancers.


Subject(s)
Antigens, Neoplasm/metabolism , Biomarkers, Tumor/metabolism , Protein Phosphatase 2/metabolism , Signal Transduction , Transcription Factor RelA/metabolism , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , DNA-Binding Proteins , Glioma/metabolism , Glioma/pathology , HEK293 Cells , HeLa Cells , Humans , Immunohistochemistry , Lysine/metabolism , Methylation/drug effects , Models, Biological , Phosphorylation/drug effects , Protein Binding/drug effects , Transcription Factors , Tumor Necrosis Factor-alpha/pharmacology
17.
Rheumatology (Oxford) ; 52(3): 427-37, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23148088

ABSTRACT

OBJECTIVE: Aberrant splicing is one of the most significant components generating functional diversity in many pathological conditions. The objective of this study was to analyse the mutations or aberrant splicing of A20 transcript, the region encompassing the ovarian tumour (OTU) domain [which is functionally important as an inhibitor of nuclear factor (NF)-κB activation] in fibroblast-like synoviocytes (FLSs) from RA patients. METHODS: Alterations in A20 transcripts were determined through sequence analysis of 10 clones of A20 cDNA in FLSs from each of the five RA patients. The levels of aberrant A20 transcript were measured by quantitative real-time RT-PCR with primers to specifically recognize the inserted introns. The functional role of A20 and its aberrant variants were examined by analysing NF-κB luciferase reporter activity and NF-κB-dependent target gene expression. RESULTS: In RA FLSs, we discovered four novel aberrant A20 transcripts, most of which resulted from insertion of partial intron 2, intron 4 and/or deletion of exon 4. In each of these FLSs, sequence analysis revealed that these aberrant insertional sequences were flanked by consensus splice donor and acceptor sequences without nucleotide substitution, suggesting alternative splicing as the likely mutational mechanism. These variants elicited a codon frame shift by creating a premature translational stop codon, and eventually, disruption of the OTU domain (which is functionally important as an inhibitor of NF-κB activation) of A20. The expression level of aberrant A20 transcript was correlated well with persisitently enhanced status of NF-κB signalling, as evident by the phosphorylation of inhibitor of NF-κB (IκB)-α and transcription of NF-κB target genes. CONCLUSION: The results suggest that A20 inactivation by the novel aberrant splicing may contribute to RA progression by inducing persistent NF-κB activation.


Subject(s)
Alternative Splicing , Arthritis, Rheumatoid/genetics , DNA-Binding Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Cells, Cultured , DNA-Binding Proteins/physiology , Humans , Intracellular Signaling Peptides and Proteins/physiology , NF-kappa B/physiology , Nuclear Proteins/physiology , RNA, Messenger/analysis , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNA , Signal Transduction/genetics , Synovial Membrane/cytology , Tumor Necrosis Factor alpha-Induced Protein 3
18.
J Biol Chem ; 287(40): 33853-64, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-22854961

ABSTRACT

Loss of synaptic inhibition by γ-aminobutyric acid and glycine due to potassium chloride cotransporter-2 (KCC2) down-regulation in the spinal cord is a critical mechanism of synaptic plasticity in neuropathic pain. Here we present novel evidence that peripheral nerve injury diminishes glycine-mediated inhibition and induces a depolarizing shift in the reversal potential of glycine-mediated currents (E(glycine)) in spinal dorsal horn neurons. Blocking glutamate N-methyl-D-aspartate (NMDA) receptors normalizes synaptic inhibition, E(glycine), and KCC2 by nerve injury. Strikingly, nerve injury increases calcium-dependent calpain activity in the spinal cord that in turn causes KCC2 cleavage at the C terminus. Inhibiting calpain blocks KCC2 cleavage induced by nerve injury and NMDA, thereby normalizing E(glycine). Furthermore, calpain inhibition or silencing of µ-calpain at the spinal level reduces neuropathic pain. Thus, nerve injury promotes proteolytic cleavage of KCC2 through NMDA receptor-calpain activation, resulting in disruption of chloride homeostasis and diminished synaptic inhibition in the spinal cord. Targeting calpain may represent a new strategy for restoring KCC2 levels and tonic synaptic inhibition and for treating chronic neuropathic pain.


Subject(s)
Calpain/metabolism , Chlorides/chemistry , Neuralgia/metabolism , Receptors, N-Methyl-D-Aspartate/chemistry , Symporters/chemistry , Animals , Biological Transport , Electrophysiology/methods , Glutamic Acid/metabolism , Homeostasis , Male , Neuronal Plasticity , Pain , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Synapses/metabolism , K Cl- Cotransporters
19.
J Neurosci ; 32(25): 8560-8, 2012 Jun 20.
Article in English | MEDLINE | ID: mdl-22723696

ABSTRACT

Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.


Subject(s)
Chlorides/metabolism , Homeostasis/genetics , Hypertension/metabolism , Hypertension/physiopathology , Hypothalamus/metabolism , Neurons/physiology , Sodium-Potassium-Chloride Symporters/biosynthesis , Sympathetic Nervous System/physiopathology , Animals , Blood Pressure/physiology , Blotting, Western , Cell Membrane/metabolism , Electrophysiological Phenomena , Ganglia, Sympathetic/physiology , Ganglionectomy , Glycosylation , Male , Paraventricular Hypothalamic Nucleus/metabolism , Paraventricular Hypothalamic Nucleus/physiopathology , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Solute Carrier Family 12, Member 2 , Synapses/physiology , Up-Regulation , gamma-Aminobutyric Acid/physiology
20.
Autophagy ; 8(4): 559-76, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22441021

ABSTRACT

Targeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspase-independent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.


Subject(s)
Apoptosis/drug effects , Autophagy/drug effects , Caspases/metabolism , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Triterpenes/pharmacology , Autophagy/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic/pathology , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Methylnitronitrosoguanidine/pharmacology , Mitochondria/drug effects , Mitochondria/ultrastructure , Models, Biological , Phenanthridines/metabolism , STAT3 Transcription Factor/metabolism
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