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.

Olfactomedin 4 下调中性粒细胞对革兰氏阳性和革兰氏阴性细菌的杀伤我们最近证明 OLFM4 负向调节宿主针对幽门螺杆菌的先天免疫。为了扩展我们之前的研究，我们通过研究 OLFM4-/- 小鼠中性粒细胞的杀菌活性来确定 OLFM4 是否可能调节针对广泛细菌感染的先天免疫。在这项研究中，我们证明了 OLFM4 是一种新型的中性粒细胞颗粒蛋白。 OLFM4-/- 小鼠的中性粒细胞在体外增强了对金黄色葡萄球菌和大肠杆菌的细胞内杀伤力。 OLFM4-/- 小鼠体内细菌清除能力增强，并且在腹膜内注射金黄色葡萄球菌或大肠杆菌时对败血症具有更强的抵抗力。使用酵母二次杂交测定，发现 OLFM4 与组织蛋白酶 C 相互作用，组织蛋白酶 C 是一种半胱氨酸蛋白酶，在细菌清除和炎症反应中发挥重要作用。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。 OLFM4-/-小鼠的中性粒细胞中的组织蛋白酶C活性显着高于野生型同窝小鼠的中性粒细胞。丝氨酸蛋白酶、中性粒细胞弹性蛋白酶、组织蛋白酶 G 和蛋白酶 3（其加工和成熟需要组织蛋白酶 C 活性）的活性在 OLFM4-/- 中性粒细胞中也较高。这些结果表明，OLFM4 通过限制组织蛋白酶 C 活性及其下游颗粒相关丝氨酸蛋白酶，是中性粒细胞针对多种细菌的杀菌活性的重要负调节剂。 Olfactomedin 4 是 Wnt/β-catenin 通路的分泌型拮抗剂 OLFM4是一种分泌型糖蛋白，但其对细胞内信号通路的影响尚未被研究。先前的研究表明，olfactomedin 家族的成员 olfactomedin 1 和 myocillin 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 通路。为了解决这个问题，我们用纯化的 OLFM4 蛋白处理 293T 细胞。在总细胞裂解物中观察到β-连环蛋白水平的下调，并且在包括细胞膜、细胞质和细胞核在内的亚细胞部分中观察到更明显。 β-catenin 的降解依赖于 GSK3 介导的磷酸化并经过蛋白酶体介导的降解。 293T 细胞中 OLFM4 的过度表达也降低了 β-连环蛋白水平。我们发现外源添加OLFM4蛋白以及OLFM4的过表达可以拮抗Wnt3a诱导的293T细胞的细胞质和细胞核中β-catenin的上调。初步研究表明 OLFM4 可能与 293T 细胞中的 Frizzled 受体相互作用。我们已经确定 OLFM4 是一种细胞外 Wnt 拮抗剂，可能在与其表面受体相互作用的水平上抑制 Wnt 信号传导。 OLFM4 可能被证明是干扰涉及 Wnt 及其受体的 Wnt 通路的有效治疗剂。