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首页> 外文学位 >The lactoferrin receptor mediates lactoferrin endocytosis and is involved in the effects of lactoferrin on intestinal proliferation and characterization of the human lactoferrin receptor gene promoter.
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The lactoferrin receptor mediates lactoferrin endocytosis and is involved in the effects of lactoferrin on intestinal proliferation and characterization of the human lactoferrin receptor gene promoter.

机译：乳铁蛋白受体介导乳铁蛋白的内吞作用，并参与乳铁蛋白对人乳铁蛋白受体基因启动子的肠道增殖和特征的影响。

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摘要

Lactoferrin (Lf) is a major iron-binding protein in breast milk. Multiple functions of Lf, such as positive regulation of intestinal proliferation, appear dependent upon the iron-saturation of Lf and are postulated to be mediated through Lf internalization by a Lf receptor (LfR). However, mechanisms by which LfR mediates Lf internalization and stimulation of proliferation in enterocytes are unknown.;We now demonstrate that a LfR mediates Lf endocytosis in a human enterocyte model (Caco-2 cells). LfR was detected at the plasma membrane by cell surface biotinylation; both apo-Lf and holo-Lf uptake were significantly inhibited in cells transfected with LfR siRNA. Hypertonic and cytochalasin D treatments indicate that LfR regulates Lf endocytosis via clathrin-mediated endocytosis rather than macropinocytosis. The role of clathrin in LfR mediated Lf endocytosis was confirmed by clathrin siRNA and co-immunoprecipitation of LfR with clathrin adaptor AP2.;We next hypothesized that Lf stimulates proliferation of intestinal epithelial cells by activating ERK and PI3K signaling cascades and that LfR is involved. Mouse crypt cells were isolated from 7-10 days old pups. Confocal microscopy showed that LfR is expressed and localized at the plasma membrane of crypt cells, specifically binding both iron-free Lf (apo-Lf) and iron-saturated Lf (holo-Lf). Our results showed that apo-Lf but not holo-Lf initiated ERK1/2 signaling by ERK1/2 phosphorylation and both apo- and holo-Lf were capable of activating the PI3K/Akt signaling pathway. Function-blocking antibody against LfR and LfR siRNA inhibited activation of the ERK1/2 signaling pathway but not the PI3K/Akt signaling pathway by apo-Lf, suggesting that LfR is involved in initiation of the ERK1/2 but not the PI3K signaling pathway. Thus, apo-Lf and holo-Lf stimulate intestinal proliferation but through various signaling pathways.;The important role of LfR in Lf endocytosis and signaling transduction focused our interest in transcription regulation of LfR. Therefore, the promoter region of the LfR gene was cloned and characterized. Neither a TATA box nor a CCAAT box is found at the typical positions. The transcription start site was identified as 298 bp upstream of the translation start codon (+1) by 5' RLM-RACE. The transfection assays demonstrated a fragment of -299/+63 sufficient for eliciting maximal promoter activity in transiently transfected Caco-2 cells, in which two Sp1 binding sites play critical roles in transcriptional regulation of the LfR gene.
机译：乳铁蛋白（Lf）是母乳中的主要铁结合蛋白。 Lf的多种功能，例如肠道增生的阳性调控，似乎取决于Lf的铁饱和度，并被认为是通过Lf受体（LfR）通过Lf内在化介导的。然而，LfR介导Lf内在化和刺激肠上皮细胞增殖的机制尚不清楚。；我们现在证明LfR在人肠上皮细胞模型（Caco-2细胞）中介导Lf内吞。通过细胞表面生物素化在质膜上检测到LfR；在用LfR siRNA转染的细胞中，载脂蛋白Lf和全脂Lf的摄取均被显着抑制。高渗和细胞松弛素D处理表明LfR通过网格蛋白介导的内吞作用而不是巨胞饮作用来调节Lf内吞作用。网格蛋白siRNA以及LfR与网格蛋白衔接子AP2的共免疫沉淀证实了网格蛋白在LfR介导的Lf内吞作用中的作用;我们接下来假设Lf通过激活ERK和PI3K信号级联反应刺激肠上皮细胞增殖，并且涉及到LfR。从7-10天大的幼崽中分离出小鼠隐窝细胞。共聚焦显微镜显示LfR表达并定位在隐窝细胞的质膜上，特异性结合无铁Lf（apo-Lf）和铁饱和Lf（holo-Lf）。我们的结果表明，apo-Lf而非holo-Lf通过ERK1 / 2磷酸化引发ERK1 / 2信号传导，apo-和holo-Lf均能够激活PI3K / Akt信号传导途径。针对LfR和LfR siRNA的功能阻断抗体通过apo-Lf抑制ERK1 / 2信号通路的激活，但不抑制PI3K / Akt信号通路的激活，这表明LfR参与了ERK1 / 2的启动，但不参与PI3K信号通路。因此，载脂蛋白Lf和完整Lf刺激肠增殖，但通过各种信号途径。； LfR在Lf内吞作用和信号转导中的重要作用使我们对LfR的转录调控感兴趣。因此，克隆并鉴定了LfR基因的启动子区。在典型位置都找不到TATA盒或CCAAT盒。转录起始位点被5'RLM-RACE鉴定为翻译起始密码子（+1）上游298bp。转染分析表明，-299 / + 63片段足以在瞬时转染的Caco-2细胞中引发最大的启动子活性，其中两个Sp1结合位点在LfR基因的转录调控中起关键作用。

著录项

  • 作者

    Jiang, Rulan.;

  • 作者单位

    University of California, Davis.;

  • 授予单位 University of California, Davis.;
  • 学科 Biology Molecular.;Agriculture Animal Culture and Nutrition.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 141 p.
  • 总页数 141
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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