Olfactomedin 4 down-regulates neutrophil killing of Gram-positive and Gram-negat

Olfactomedin 4 下调中性粒细胞对革兰氏阳性菌和革兰氏阴性菌的杀伤

基本信息

批准号：
8344821
负责人：
GRIFFIN RODGERS
金额：
$ 83.2万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8344821
关键词：
Address Amino Acid Sequence Apoptosis Attenuated Bacteria Biological Assay CSF3 gene Cathepsin C Cathepsin G Cell Nucleus Cell membrane Cells Cysteine Protease Cytoplasm Cytoplasmic Granules Down-Regulation Epithelium Escherichia coli Family Genes Glycoproteins Gram-Negative Bacteria Granulocyte Colony-Stimulating Factor Helicobacter pylori Hematopoietic Human In Vitro Infection Inflammation Inflammatory Bowel Diseases Inflammatory Response Injection of therapeutic agent Interferons Laboratories Leukocyte Elastase Link Malignant Neoplasms Mediating Mucous Membrane Mus Myelogenous Natural Immunity Noelin-1 Pathway interactions Peptide Sequence Determination Peritoneal Phosphorylation Process Protein Overexpression Proteinase 3 Proteins Regulation Reporting Resistance Role Sepsis Serine Protease Signal Pathway Signal Transduction Staphylococcus aureus Stream Subcellular Fractions Surface Therapeutic Agents Translations Tretinoin Up-Regulation Yeasts alpha-latrotoxin receptor bactericide extracellular gastrointestinal infection in vitro activity in vivo killings member multicatalytic endopeptidase complex neutrophil novel olfactomedin overexpression precursor cell receptor

项目摘要

Olfactomedin 4 down-regulates neutrophil killing of Gram-positive and Gram-negative bacteria We recently demonstrated that OLFM4 negatively regulates host innate immunity against Helicobacter pylori. To extend our previous study, we determined whether OLFM4 might regulate innate immunity against broad bacteria infections by investigating the bactericidal activity in neutrophils from OLFM4-/- mice. In this study, we have demonstrated that OLFM4 is a novel neutrophil granule protein. Neutrophils from OLFM4-/- mice have increased intracellular killing of Staphlocococus aureus and Escherichia coli in vitro. The OLFM4-/- mice have enhanced in vivo bacteria clearance and are more resistant to sepsis when challenged with intra-peritoneal injection of S. aureus or E. coli. Using a yeast two hybridization assay, OLFM4 is found to interact with cathepsin C, a cysteine protease that pays an important role in the bacterial clearance and inflammatory response. We have demonstrated that OLFM4 is a direct substrate of cathepsin C and inhibits cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4-/- mice is significantly higher than that in neutrophils from wild-type littermate mice. The activities of serine proteinases, neutrophil elastase, cathepsin G and proteinase 3, whose processing and maturity require cathepsin C activity, are also higher in OLFM4-/- neutrophils. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity against broad range of bacteria via restricting cathepsin C activity and its down stream granule-associated serine proteases. Olfactomedin 4 is a secreted antagonist of Wnt/β-catenin pathway OLFM4 is a secreted glycoprotein, but its effect on intracellular signal pathway has not been investigated. Previous studies have shown that members of olfactomedin family, olfactomedin 1 and myocillin are modulators of Wnt signaling pathways. Here we investigated whether OLFM4 can regulate Wnt pathway. To address this question, we treated 293T cells with purified OLFM4 protein. Downregulation of β-catenin protein levels were observed in total cell lysate and more obviously in subcellular fractions including cell membrane, cytoplasm and nucleus. The degradation of β-catenin was dependent on GSK3-mediated phosphorylation and go through proteasome mediated degradation. Overexpression of OLFM4 in 293T cells also decreased the β-catenin level. We have found exogenously added OLFM4 protein and overexpression of OLFM4 can antagonize Wnt3a induced upregulation of β-catenin in the cytoplasm and nucleus of 293T cells. Primary study demonstrated that OLFM4 might interact with Frizzled receptor in 293T cells. We have identified OLFM4 as an extracellular Wnt antagonist that inhibits Wnt signaling probably at the level of interaction with its surface receptors. OLFM4 may prove to be an effective therapeutic agent to interfere with Wnt pathway involving Wnts and their receptors.

嗅觉素4下调革兰氏阳性和革兰氏阴性细菌的中性粒细胞杀死我们最近证明OLFM4负责调节宿主对幽门螺杆菌的先天免疫力。为了扩展我们先前的研究，我们确定了OLFM4是否可以通过研究OLFM4 - / - 小鼠中性粒细胞的杀菌活性来调节对广泛细菌感染的先天免疫。在这项研究中，我们已经证明OLFM4是一种新型的中性粒细胞颗粒蛋白。来自OLFM4 - / - 小鼠的嗜中性粒细胞在体外的细胞内杀死金黄色葡萄球菌和大肠杆菌的细胞内杀死增加。 OLFM4 - / - 小鼠在用腹膜内注射金黄色葡萄球菌或大肠杆菌的挑战时，具有增强的体内细菌清除率，并且对败血症具有更大的抵抗力。使用酵母两个杂交测定法，发现OLFM4与组织蛋白酶C相互作用，这是一种半胱氨酸蛋白酶，在细菌清除和炎症反应中起着重要作用。我们已经证明OLFM4是组织蛋白酶C的直接底物，并在体外和体内抑制组织蛋白酶C活性。来自OLFM4 - / - 小鼠的嗜中性粒细胞中的组织蛋白酶C活性明显高于野生型同窝小鼠的中性粒细胞中的中性粒细胞。在OLFM4 - / - 中性粒细胞中，丝氨酸蛋白酶，中性粒细胞弹性酶，组织蛋白酶G和蛋白酶3的活性也更高。这些结果表明，通过限制组织蛋白酶C活性及其下水道相关的丝氨酸蛋白酶，OLFM4是嗜中性粒细胞活性对广泛细菌的重要负调节剂。嗅觉素4是Wnt/β-catenin途径的分泌拮抗剂 OLFM4是一种分泌的糖蛋白，但尚未研究其对细胞内信号途径的影响。先前的研究表明，橄榄素家族，嗅觉素1和肌林的成员是Wnt信号通路的调节剂。在这里，我们研究了OLFM4是否可以调节WNT途径。为了解决这个问题，我们用纯化的OLFM4蛋白处理了293T细胞。在总细胞裂解物中观察到β-catenin蛋白水平的下调，在包括细胞膜，细胞质和细胞核在内的亚细胞部分中更明显。 β-catenin的降解取决于GSK3介导的磷酸化，并通过蛋白酶体介导的降解。 OLFM4在293T细胞中的过表达也降低了β-catenin水平。我们发现外源添加的OLFM4蛋白和OLFM4的过表达可以拮抗Wnt3a在293T细胞的细胞质和细胞核中诱导的β-catenin上调。主要研究表明，OLFM4可能与293T细胞中的毛躁受体相互作用。我们已经将OLFM4鉴定为细胞外Wnt拮抗剂，它可能在与其表面受体的相互作用水平上抑制Wnt信号。 OLFM4可能被证明是一种有效的治疗剂，以干扰涉及WNT及其受体的Wnt途径。