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

• 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
• 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

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.

嗅觉调节素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作用 我们最近证实，OLFM4负性调节宿主对幽门螺杆菌的天然免疫。为了扩展我们之前的研究，我们通过研究OLFM4-/-鼠中性粒细胞的杀菌活性来确定OLFM4是否可能调节对广泛细菌感染的先天免疫。在本研究中，我们证明了OLFM4是一种新的中性粒细胞颗粒蛋白。来自OLFM4-/-小鼠的中性粒细胞在体外增加了对金黄色葡萄球菌和大肠杆菌的细胞内杀伤力。OLFM4-/-小鼠增强了体内细菌的清除，并在腹腔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。使用酵母双杂交试验，OLFM4被发现与组织蛋白酶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，在体外和体内都能抑制组织蛋白酶C的活性。OLFM4-/-小鼠中性粒细胞的组织蛋白酶C活性显著高于野生型小鼠的中性粒细胞。在OLFM4-/-中性粒细胞中，丝氨酸蛋白酶、中性粒细胞弹性蛋白酶、组织蛋白酶G和蛋白酶3的活性也较高，这些酶的加工和成熟需要组织蛋白酶C的活性。这些结果表明，OLFM4通过限制组织蛋白酶C及其下游颗粒相关丝氨酸蛋白酶的活性，是中性粒细胞对多种细菌杀菌活性的重要负调控因子。 Ofactomedin 4是Wnt/-catenin途径的分泌型拮抗剂 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。以往的研究表明，olfactomedin家族成员、olfactomedin 1和myoclin是Wnt信号通路的调节者。在这里，我们研究了OLFM4是否能调节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连环素蛋白水平的下调，在包括细胞膜、细胞质和细胞核在内的亚细胞部分中更为明显。-连环蛋白的降解依赖于GSK3介导的磷酸化，并经历蛋白酶体介导的降解。在293T细胞中过表达OLFM4也降低了-连环蛋白水平。我们发现外源添加OLFM4蛋白和过表达OLFM4可以拮抗Wnt3a诱导的293T细胞胞浆和胞核中连环蛋白的上调。初步研究表明，OLFM4可能与293T细胞中的Frizzled型受体相互作用。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信号转导。Olfm4可能是一种有效的治疗药物，可以干扰涉及Wnts及其受体的Wnt途径。