Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia
Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia
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DOI：
10.1371/journal.pone.0071586
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发表时间：
2013-08-05
期刊：
影响因子：
3.7
通讯作者：
Thevenod, Frank
中科院分区：
文献类型：
--
作者：
Langelueddecke, Christian;Roussa, Eleni;Thevenod, Frank
脂钙蛋白2//NGAL/24p3受体（NGAL- r /24p3- r）在啮齿动物肾远端表达，介导蛋白内吞作用。小肠中蛋白质和多肽的根尖内吞作用和胞吞作用机制尚不清楚。本研究采用免疫荧光、共聚焦激光扫描显微镜、免疫组织化学和免疫印迹技术，研究了啮齿动物24p3-R （r24p3-R）和人NGAL-R （hNGAL-R）在肠道段的表达和定位。通过免疫荧光显微镜、RT-PCR和质膜富集囊泡（PM）免疫印迹技术，研究了瞬时转染r24p3-R的人Caco-2 BBE细胞和CHO细胞中r24p3-R/hNGAL-R的表达。为了测定其功能，通过测量在塑料或Transwell插入物上单层生长的cco -2 BBE细胞中荧光标记配体的内化，测定了植物螯合素（PC3）、金属硫蛋白（MT）和转铁蛋白（Tf）的内吞作用/胞吞作用。用微尺度热泳法（MST）测定了Alexa 488-PC3与结肠样Caco-2 BBE PM的结合亲和力。r24p3-R/hNGAL-R在各肠段均有表达，但在回肠和结肠中表达量最高。结肠样而非十二指肠样Caco-2 BBE细胞在其表面表达hNGAL-R。结肠样Caco-2 BBE细胞或r24p3-R转染的CHO细胞内化荧光标记的PC3或MT，在亚微摩尔浓度下达到半最大饱和度。Caco-2 BBE细胞对PC3和MT （0.7 μ M）的摄取被hNGAL （500 pM）部分阻断，MST测定Alexa 488-PC3与pM囊泡结合的EC50为18.6 +/- 12.2 nM。Transwell实验显示，荧光PC3/MT/Tf （0.7 μ M）在0.5-2 h内迅速被根尖吸收，并向基底侧输送。500 pM hNGAL显著阻断了配体的顶端摄取。hNGAL-R依赖性摄取在MT中更为突出，但与PC3和Tf相比，胞吞效率降低。因此，r24p3-R/hNGAL-R可能是一种高亲和力的多配体受体，用于下肠的根尖内化和完整蛋白/肽的胞吞。
The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC3), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC3 to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC3 or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC3 and MT (0.7 mu M) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC50 of 18.6 +/- 12.2 nM was determined for binding of Alexa 488-PC3 to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC3/MT/Tf (0.7 mu M). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC3 and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.