Growing evidence supports the role of transcription factor activation in the pathophysiology of inflammatory disorders, sepsis, ARDS, SIRS, and shock. Kinase mediated phosphorylation of IκBα is a crucial step in the NFκB activation pathway. We investigated IκBα phosphorylation in murine liver and lung extracts after cecal ligation and puncture (CLP) in the presence and absence of a glucan ligand. ICR mice were subjected to CLP. Unoperated and sham-operated mice served as the controls. Glucan phosphate (50 mg/kg) was administered 1 h before or 15 min after CLP. CLP increased hepatic and pulmonary levels of phospho-IκBα by 48-192%. Pre-or post-treatment with glucan phosphate decreased (P < 0.05) tissue phospho-IκBα levels in CLP mice. Phospho-IκBα in the glucan-CLP group were not significantly different from the unoperated controls. To investigate mechanisms we examined IKKβ kinase activity, IκBα phosphorylation and degradation, and NFκB activity in a murine macrophage cell line, J774a.1, treated with LPS (1 μg/mL) and/or glucan phosphate (1 μg/mL) for up to 120 min. The glucan ligand blunted LPS-induced IKKβ kinase activity, phosphorylation and degradation of IκBα, and NFκB nuclear binding activity. The data indicate that one mechanism by which (1→3)-β-D-glucan may alter the response to endotoxin or polymicrobial sepsis involves modulation of IKKβ kinase activity with subsequent decreases in IκBα phosphorylation and NFκB activation.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19972号 / 薬科博第63号 / 新制||薬科||7(附属図書館) / 33068 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

La compréhension des interactions intra- et inter-moléculaires à l’échelle atomique représente un enjeu scientifique important. A l’heure actuelle, les techniques de RMN ont déjà prouvé leur efficacité pour l’analyse de ces interactions in vitro, dans les solutions tampons. Toutefois, il a également été montré que la plupart des biomolécules ont une structure et une dynamique différentes in vivo, à l’intérieur des cellules, de celle in vitro. Il est donc crucial d’analyser les biomolécules dans leur milieu naturel, la cellule. Récemment, les progrès dans le domaine de la RMN dans les cellules ont permis de mieux comprendre la dynamique et les interactions des biomolécules présentes dans le milieu cellulaire complexe. Cependant, la biomolécule étudiée étant présente en faibles concentrations, elle possède un faible signal sur le spectre RMN, qu’il est difficile de suivre. De plus, du fait de la forte viscosité du milieu cellulaire, la relaxation rapide de l’aimantation transverse se traduit par un élargissement des raies spectrales. L’utilisation des états de spin à longs temps de vie et de la Polarisation Dynamique Nucléaire suivie par la dissolution de l’échantillon (dissolution-DNP) pourraient permettre de pallier aux problèmes d’élargissement de raies et de sensibilité. L’objectif de ce travail de thèse a été d’explorer les bénéfices des ces avancées récentes de la RMN pour l’étude des petites molécules, peptides et protéines à l’intérieur des cellules. Pour la protéine c-Src, qui appartient à la classe des protéines intrinsèquement désordonnées (IDP), la dynamique de l’ensemble des conformations de l’extrémité N-terminale a été suivie utilisant des états de spin à longs temps de vie LLS. Le signal du noyau de carbone-13 de la molécule de pyruvate a été augmenté utilisant la Polarisation Dynamique Nucléaire (DNP) afin de mieux l’observer dans le milieu cellulaire. Un peptide représentatif pour la partie active d’une autre protéine, IκBα, a été introduit dans des cellules HepG2 par l’électroporation. Les observations faites lors des ces expériences sont discutées dans la perspective de faciliter les études RMN des biomolécules à l’intérieur des cellules. / Most NMR studies are carried out in vitro, but the structure and dynamics of some biomolecules inside cells differ from those in vitro. It thus becomes interesting to analyze biomolecules such as proteins in their natural environment: the cell. Recent progress of in cell NMR allowed to better understand the behaviour of proteins: their dynamics and their interactions with other biomolecules in the cell. But the low concentration of proteins leads to low signal intensity. Moreover, the viscosity of the environment induces faster transverse relaxation, resulting in line broadening for proteins signals. The use of the Long-Lived States and Coherences (LLS and LLC, respectively) as well as dissolution Dynamic Nuclear Polarization (dissolution-DNP) can improve NMR observations in cells. LLS were used to understand and characterize the structure of the N-terminal domain of c-Src, which is intrinsically disordered. To follow the phosphorylation of proteins, a first preliminary study of a 21-aa peptides derived from IKBα electroporated into HepG2 cell lines was carried out.

The transcription factor nuclear factor κB (NF-κB) regulates multiple immediate-early gene expressions involved in immune and inflammatory responses and cellular defenses. Ischemia-reperfusion induces many immediate- early gene expressions, but little is known about the NF-κB activation in myocardium during ischemia and reperfusion. This study demonstrated that ischemia alone rapidly induced NF-κB activation in the myocardium of isolated working rat hearts. Electrophoretic mobility shift assay showed that NF-κB binding activity significantly increased in the nucleus after 5 min of ischemia and remained elevated for up to 30 min. Western blot analysis suggested that the levels of inhibitory IκBα protein in the cytoplasm became markedly decreased at 4, 5, 7.5, and 10 min of ischemia but were gradually restored following 10 min of ischemia. Reduction of IκBα protein in the cytoplasm by ischemia resulted in NF-κB translocation to the nucleus. Northern blot hybridization showed that IκBα mRNA levels were not significantly elevated during myocardial ischemia. Pyrrolidine dithiocarbamate, an antioxidant, significantly inhibited the loss of IκBα protein from the cytoplasm and prevented NF-κB binding activity in the nucleus. Reperfusion following short periods of ischemia augmented NF-κB binding activity in the nucleus induced by ischemia. The results suggest that early activation of NF-κB induced by ischemia in the myocardium could be a signal mechanism for controlling and regulating immediate-early gene expression during ischemia-reperfusion.

Background: Human mast cells are capable of a wide variety of inflammatory responses and play a vital role in the pathogenesis of inflammatory diseases such as allergy, asthma, and atherosclerosis. We have reported that cigarette smoke extract (CSE) significantly increased IL-6 and IL-8 production in IL-1β-activated human mast cell line (HMC-1). Baicalein (BAI) has anti-inflammatory properties and inhibits IL-1β- and TNF-α-induced inflammatory cytokine production from HMC-1. The goal of the present study was to examine the effect of BAI on IL-6 and IL-8 production from CSE-treated and IL-1β-activated HMC-1.Methods: Main-stream (Ms) and Side-stream (Ss) cigarette smoke were collected onto fiber filters and extracted in RPMI-1640 medium. Two ml of HMC-1 at 1 × 10 6 cells/mL were cultured with CSE in the presence or absence of IL-1β (10 ng/mL) for 24 hrs. A group of HMC-1 cells stimulated with both IL-1β (10 ng/ml) and CSE was also treated with BAI. The expression of IL-6 and IL-8 was assessed by ELISA and RT-PCR. NF-κB activation was measured by electrophoretic mobility shift assay (EMSA) and IκBα degradation by Western blot.Results: Both Ms and Ss CSE significantly increased IL-6 and IL-8 production (p < 0.001) in IL-1β-activated HMC-1. CSE increased NF-κB activation and decreased cytoplasmic IκBα proteins in IL-1β-activated HMC-1. BAI (1.8 to 30 μM) significantly inhibited production of IL-6 and IL-8 in a dose-dependent manner in IL-1β-activated HMC-1 with the optimal inhibition concentration at 30 μM, which also significantly inhibited the enhancing effect of CSE on IL-6 and IL-8 production in IL-1β-activated HMC-1. BAI inhibited NF-κB activation and increased cytoplasmic IκBα proteins in CSE-treated and IL-1β-activated HMC-1.Conclusions: Our results showed that CSE significantly increased inflammatory cytokines IL-6 and IL-8 production in IL-1β-activated HMC-1. It may partially explain why cigarette smoke contributes to lung and cardiovascular diseases. BAI inhibited the production of inflammatory cytokines through inhibition of NF-κB activation and IκBα phosphorylation and degradation. This inhibitory effect of BAI on the expression of inflammatory cytokines induced by CSE suggests its usefulness in the development of novel anti-inflammatory therapies.