RESUMEN
The liver is exposed to gut-derived bacterial endotoxin via portal circulation, and recognizes it through toll-like receptor 4 (TLR4). Endotoxin lipopolysaccharide (LPS) stimulates the self-ubiquitination of ubiquitin ligase TRAF6, which is linked to scaffold with protein kinase TAK1 for auto-phosphorylation and subsequent activation. TAK1 activity is a signal transducer in the activating pathways of transcription factors NF-κB and AP-1 for production of various cytokines. Here, we hypothesized that TRAF6-TAK1 axis would be implicated in endotoxin-induced liver disease. Following exposure to endotoxin LPS, TLR4-mediated phosphorylation of TAK1 and transcription of cell-death cytokine TNF-α were triggered in Kupffer cells but not in hepatocytes as well as TNF receptor-mediated and caspase-3-executed apoptosis was occurred in D-galactosamine (GalN)-sensitized hepatocytes under co-culture with Kupffer cells. Treatment with pyridinylmethylene benzothiophene (PMBT) improved endotoxin LPS-induced hepatocyte apoptosis in GalN-sensitized C57BL/6 mice via suppressing NF-κB- and AP-1-regulated expression of TNF-α in Kupffer cells, and rescued the mice from hepatic damage-associated bleeding and death. As a mechanism, PMBT directly inhibited Lys 63-linked ubiquitination of TRAF6, and mitigated scaffold assembly between TRAF6 and the TAK1-activator adaptors TAB1 and TAB2 complex in Kupffer cells. Thereby, PMBT interrupted TRAF6 ubiquitination-induced activation of TAK1 activity in the TLR4-mediated signal cascade leading to TNF-α production. However, PMBT did not directly affect the apoptotic activity of TNF-α on GalN-sensitized hepatocytes. Finally, we propose chemical inhibition of TRAF6-TAK1 axis in Kupffer cells as a strategy for treating liver disease due to gut-derived endotoxin or Gram-negative bacterial infection.
Asunto(s)
Hepatopatías , Factor 6 Asociado a Receptor de TNF , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Caspasa 3/metabolismo , Citocinas/metabolismo , Endotoxinas/toxicidad , Galactosamina/toxicidad , Ligasas/metabolismo , Lipopolisacáridos/toxicidad , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinas/metabolismoRESUMEN
YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/farmacología , Proteína 1 Similar a Quitinasa-3/antagonistas & inhibidores , Fragmentos Fab de Inmunoglobulinas/inmunología , Neoplasias/tratamiento farmacológico , Biblioteca de Péptidos , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Apoptosis , Movimiento Celular , Proliferación Celular , Proteína 1 Similar a Quitinasa-3/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human lysophosphatidic acid receptor 2 (LPA2), a member of the G-protein coupled receptor family, mediates lysophosphatidic acid (LPA)-dependent signaling by recruiting various G proteins. Particularly, it is directly implicated in the progression of colorectal and ovarian cancer through G protein signaling cascades. To investigate the biochemical binding properties of LPA2 against various alpha subunits of G protein (Gα), a functional recombinant LPA2 was overexpressed in E. coli membrane with a P9∗ expression system, and the purified protein was stabilized with an amphipathic polymer that had been synthesized by coupling octylamine, glucosamine, and diethyl aminoproylamine at the carboxylic groups of poly-γ-glutamic acid. The purified LPA2 stabilized with the amphipathic polymer showed selective binding activity to the various Gα proteins as well as agonist-dependent dissociation from Gαi3. Understanding the binding properties of LPA2 against various Gα proteins advances the understanding of downstream signaling cascades of LPA2. The functional LPA2 prepared using a P9∗ expression system and an amphipathic polymer could also facilitate the development of LPA2-targeting drugs.
Asunto(s)
Escherichia coli/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/química , Virus de la Anemia Infecciosa Equina/genética , Receptores del Ácido Lisofosfatídico/química , Receptores del Ácido Lisofosfatídico/fisiología , Sitios de Unión , Clonación Molecular/métodos , Escherichia coli/genética , Humanos , Unión ProteicaRESUMEN
Mice lacking the IL-1R-associated kinase 4 (IRAK4) are completely resistant to LPS-induced endotoxic disorder or the TLR9 agonist CpG DNA plus d-galactosamine-induced acute liver injury (ALI), whereas wild-type strains succumb. However, translational drugs against sepsis or ALI remain elusive. Lonicerae flos extract is undergoing the clinical trial phase I in LPS-injected healthy human volunteers for sepsis treatment. In the current study, chlorogenic acid (CGA), a major anti-inflammatory constituent of lonicerae flos extract, rescued endotoxic mortality of LPS-intoxicated C57BL/6 mice, as well as ameliorated ALI of LPS/d-galactosamine-challenged C57BL/6 mice. As a mechanism, CGA inhibited various TLR agonist-, IL-1α-, or high-mobility group box-1-stimulated autophosphorylation (activation) of IRAK4 in peritoneal macrophages from C57BL/6 or C3H/HeJ mice via directly affecting the kinase activity of IRAK4, a proximal signal transducer in the MyD88-mediated innate immunity that enhances transcriptional activity of NF-κB or AP-1. CGA consequently attenuated protein or mRNA levels of NF-κB/AP-1 target genes encoding TNF-α, IL-1α, IL-6, and high-mobility group box-1 in vivo under endotoxemia or ALI. Finally, this study suggests IRAK4 as a molecular target of CGA in the treatment of innate immunity-related shock and organ dysfunction following insult of various TLR pathogens from bacteria and viruses.
Asunto(s)
Antiinflamatorios/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Ácido Clorogénico/farmacología , Inmunidad Innata/genética , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Choque Séptico/inmunología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Citocinas/sangre , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/inmunología , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/antagonistas & inhibidores , Choque Séptico/genética , Choque Séptico/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/antagonistas & inhibidoresRESUMEN
Lonicerae flos extract (HS-23) is a clinical candidate currently undergoing Phase I trial in lipopolysaccharide (LPS)-injected healthy human volunteers, but its molecular basis remains to be defined. Here, we investigated protective effects of HS-23 or its major constituents on Escherichia coli LPS-induced septic mortality in mice. Intravenous treatment with HS-23 rescued LPS-intoxicated C57BL/6J mice under septic conditions, and decreased the levels of cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and high-mobility group box-1 (HMGB-1) in the blood. Chlorogenic acid (CGA) and its isomers were assigned as major constituents of HS-23 in the protection against endotoxemia. As a molecular mechanism, HS-23 or CGA isomers inhibited endotoxin LPS-induced autophosphorylation of the IL-1 receptor-associated kinase 4 (IRAK-4) in mouse peritoneal macrophages as well as the kinase activity of IRAK-4 in cell-free reactions. HS-23 consequently suppressed downstream pathways critical for LPS-induced activation of nuclear factor (NF)-κB or activating protein 1 (AP-1) in the peritoneal macrophages. HS-23 also inhibited various toll-like receptor agonists-induced nitric oxide (NO) production, and down-regulated LPS-induced expression of NF-κB/AP-1-target inflammatory genes in the cells. Taken together, HS-23 or CGA isomers exhibited anti-inflammatory therapy against LPS-induced septic mortality in mice, at least in part, mediated through the inhibition of IRAK-4.