TUMOR NECROSIS FACTOR AND IRON METABOLISM

肿瘤坏死因子和铁代谢

• 批准号: 2142261
• 负责人: FRANK M. TORTI
• 金额: $ 13.34万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2142261
• 关键词: cell type; cytotoxicity; ferritin; fusion gene; gel mobility shift assay; genetic regulation; interleukin 1; intracellular transport; iron metabolism; nucleic acid methylation; oxidative stress; protein structure function; transcription factor; transfection; tumor necrosis factor alpha

We have observed that the cytokines, tumor necrosis factor alpha (TNF, cachectin) and interleukin-1 alpha (IL-1), induce the synthesis of the H subunit of ferritin, a major intracellular iron binding protein. The overall goals of our experiments are to elucidate the mechanism of the response of ferritin to these cytokines, and to assess the implications of altered ferritin composition on intracellular iron balance and selected iron-dependent pathways. Specifically, our first aim is to determine whether the increase in synthesis of H subunit-rich ferritins which occurs as a consequence of TNF or IL- 1 treatment modulates intracellular iron levels. This will be assessed by measuring the effects of TNF and IL-1 treatment on the iron content of ferritin and on chelatable iron pools. Ferritin H expression vectors will be used to distinguish effects on iron metabolism directly attributable to the increase in ferritin H from other effects of cytokine treatment. Our second aim is to determine the consequences of altered ferritin subunit composition on specific cellular functions. For these experiments we will use cells transfected with ferritin H expression vectors to measure the effect of altered ferritin subunit composition on the response to oxidant stress and the cytotoxic response to TNF. Our third aim is to explore the molecular mechanisms underlying the response of the ferritin H gene to TNF . Using chimeric genes, gel retardation assays and methylation interference, we will assess the role of specific transcription factors in mediating the response of the ferritin H gene to TNF and IL-1. Our fourth aim is to explore our very recent observation that treatment of myoblasts with iron induces the synthesis of a 23 kDa protein with properties thus far consistent with those predicted for a secreted ferritin: induction by iron, precipitation with anti-ferritin antibodies, glycosylation, and correct molecular mass. We propose to definitively assess whether this protein is a component of secreted ferritin, in what cell types it is synthesized, and what factors (including cytokines) influence its synthesis. This 23 kDa protein will be isolated and purified and its relationship to known ferritin subunits assessed. Taken together, these experiments will be used to clarify the effects exerted on intracellular iron metabolism by the cytokines TNF and IL-1.

我们观察到细胞因子，肿瘤坏死因子α（TNF， Cachectin）和白介素-1α（IL-1），诱导H的合成 铁蛋白的亚基，一种主要的细胞内铁结合蛋白。 这 我们实验的总体目标是阐明 铁蛋白对这些细胞因子的反应，并评估含义 在细胞内铁平衡上改变铁蛋白组成的改变和 选定的铁依赖性途径。 具体来说，我们的第一个目标是 确定富含h亚基的铁蛋白的合成是否增加 由于TNF或IL-1治疗调节而发生的结果 细胞内铁水平。 这将通过测量 TNF和IL-1处理对铁蛋白的铁含量以及对 可螯合的铁池。 铁蛋白H表达载体将用于 区分对铁代谢的影响直接归因于 细胞因子治疗的其他作用增加铁蛋白H。 我们的 第二个目的是确定改变铁蛋白亚基的后果 特定细胞功能的组成。 对于这些实验，我们 将使用用铁蛋白H表达向量转染的细胞测量 铁蛋白亚基组成改变的影响对 氧化应激和对TNF的细胞毒性反应。 我们的第三个目标是 探索铁蛋白反应背后的分子机制 h基因至TNF。使用嵌合基因，凝胶延迟测定和 甲基化干扰，我们将评估特定的作用 转录因子介导铁蛋白H基因对 TNF和IL-1。 我们的第四个目标是探索我们最近的观察 用铁对成肌细胞的治疗诱导了23 kDa的合成 到目前为止，具有特性的蛋白质与预测的蛋白质一致 分泌铁蛋白：铁诱导，抗铁蛋白的降水 抗体，糖基化和正确的分子质量。 我们建议 明确评估该蛋白是否是分泌的组成部分 铁蛋白，合成的细胞类型以及哪些因素 （包括细胞因子）影响其合成。 这个23 kDa蛋白将 被隔离和纯化及其与已知铁蛋白亚基的关系 评估。 综上所述，这些实验将用于澄清 细胞因子TNF和细胞内铁代谢产生的影响 IL-1。