Clusterin Promotes Corneal Epithelial Cell Growth through Upregulation of Hepatocyte Growth Factor by Mesenchymal Cells In Vitro
Clusterin Promotes Corneal Epithelial Cell Growth through Upregulation of Hepatocyte Growth Factor by Mesenchymal Cells In Vitro
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DOI：
10.1167/iovs.10-6348
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发表时间：
2011-05-01
影响因子：
4.4
通讯作者：
Tsubota, Kazuo
中科院分区：
文献类型：
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作者：
Okada, Naoko;Kawakita, Tetsuya;Tsubota, Kazuo
目的.虽然角膜表达高水平的簇蛋白（CLU），但CLU在角膜中的作用知之甚少。本研究旨在探讨CLU在角膜上皮稳态中的可能作用。通过转染编码全长CLU的载体（Clu-3 T3）在3 T3细胞中过表达CLU。在与Clu-3 T3和mock-3 T3共培养的小鼠角膜细胞系（TKE 2）和人原代角膜/角膜缘上皮细胞中比较集落形成功效（CFE）。为了确定饲养层细胞是否具有接触效应，还进行了没有饲养层-上皮接触的共培养。用抗CLU中和抗体检测分泌型CLU对与Clu-3 T3细胞共培养的TKE 2细胞的影响。RT-PCR和Western blot分析与角膜缘干/祖细胞维持和生长相关的生长因子的表达。与Clu-3 T3饲养层共培养的TKE 2细胞显示出更高的CFE，并且比与mock-3 T3饲养层共培养的TKE 2细胞的集落大小更大。在不与Clu-3 T3细胞直接接触的情况下观察到TKE 2的CFE增加，这被CLU中和抗体处理显著阻断。Clu-3 T3细胞比mock-3 T3细胞表达更高水平的HGF，其被抗HGF中和抗体显著抑制。结论：HGF对Clu-3 T3细胞集落形成和细胞增殖的促进作用部分是由HGF诱导的。白藜芦醇通过诱导饲养细胞产生HGF间接增强角膜/角膜缘上皮细胞的CFE，表明其在上皮-间充质相互作用中的作用。（Invest Ophthalmol维斯科学。2011;52：2905-2910）DOI：10.1167/iovs.10-6348
PURPOSE. Although the cornea expresses high levels of clusterin (CLU), the role of CLU in the cornea is poorly understood. This study was performed to investigate the possible role of CLU in corneal epithelial homeostasis.METHODS. CLU was overexpressed in 3T3 cells by transfection of a vector encoding full-length CLU (Clu-3T3). Colony-forming efficacy (CFE) was compared in mouse corneal cell line (TKE2) and human primary corneal/limbal epithelial cells that were cocultured with Clu-3T3 and mock-3T3. To determine whether feeder cells have a contact effect, cocultures without feeder-epithelium contact were also performed. Neutralizing antibody against CLU was used to assess the effects of secretory CLU in TKE2 cells cocultured with Clu-3T3 cells. The expression of growth factors associated with limbal stem/progenitor cell maintenance and growth were analyzed by RT-PCR and Western blot analysis.RESULTS. TKE2 cells cocultured with Clu-3T3 feeders showed higher CFE and were larger in colony size than TKE2 cells cocultured with mock-3T3 feeders. Increased CFE of TKE2 was observed without direct contact with Clu-3T3 cells, which was significantly blocked by treatment with CLU neutralizing antibody. Clu-3T3 cells expressed higher levels of HGF than mock-3T3 cells, which were significantly suppressed with anti-HGF neutralizing antibodies. Collectively, the promotion of colony-forming and cell proliferation by Clu-3T3 cells was partially mediated by the induction of HGF.CONCLUSIONS. Clusterin indirectly enhances the CFE of corneal/limbal epithelial cells by inducing the production of HGF by feeder cells, suggesting a role in epithelial-mesenchymal interaction. (Invest Ophthalmol Vis Sci. 2011;52:2905-2910) DOI:10.1167/iovs.10-6348