Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
PLoS One. 2012;7(8):e42483. doi: 10.1371/journal.pone.0042483. Epub 2012 Aug 3.
Type I insulin-like growth factor receptor (IGF-1R) and insulin receptor (INSR) are highly homologous molecules, which can heterodimerize to form an IGF-1R/INSR hybrid (Hybrid-R). The presence and biological significance of the Hybrid-R in human corneal epithelium has not yet been established. In addition, while nuclear localization of IGF-1R was recently reported in cancer cells and human corneal epithelial cells, the function and profile of nuclear IGF-1R is unknown. In this study, we characterized the nuclear localization and function of the Hybrid-R and the role of IGF-1/IGF-1R and Hybrid-R signaling in the human corneal epithelium.
METHODOLOGY/PRINCIPLE FINDINGS: IGF-1-mediated signaling and cell growth were examined in a human telomerized corneal epithelial (hTCEpi) cell line using co-immunoprecipitation, immunoblotting and cell proliferation assays. The presence of Hybrid-R in hTCEpi and primary cultured human corneal epithelial cells was confirmed by immunofluorescence and reciprocal immunoprecipitation of whole cell lysates. We found that IGF-1 stimulated Akt and promoted cell growth through IGF-1R activation, which was independent of the Hybrid-R. The presence of Hybrid-R, but not IGF-1R/IGF-1R, was detected in nuclear extracts. Knockdown of INSR by small interfering RNA resulted in depletion of the INSR/INSR and preferential formation of Hybrid-R. Chromatin-immunoprecipitation sequencing assay with anti-IGF-1R or anti-INSR was subsequently performed to identify potential genomic targets responsible for critical homeostatic regulatory pathways.
CONCLUSION/SIGNIFICANCE: In contrast to previous reports on nuclear localized IGF-1R, this is the first report identifying the nuclear localization of Hybrid-R in an epithelial cell line. The identification of a nuclear Hybrid-R and novel genomic targets suggests that IGF-1R traffics to the nucleus as an IGF-1R/INSR heterotetrameric complex to regulate corneal epithelial homeostatic pathways. The development of novel therapeutic strategies designed to target the IGF-1/IGF-1R pathway must take into account the modulatory roles IGF-1R/INSR play in the epithelial cell nucleus.
Ⅰ型胰岛素样生长因子受体（IGF-1R）和胰岛素受体（INSR）是高度同源的分子，它们可以异二聚化形成 IGF-1R/INSR 杂合体（Hybrid-R）。Hybrid-R 在人角膜上皮中的存在及其生物学意义尚未确定。此外，尽管最近有报道称 IGF-1R 在癌细胞和人角膜上皮细胞中存在核定位，但核 IGF-1R 的功能和特征尚不清楚。在这项研究中，我们对 Hybrid-R 的核定位和功能以及 IGF-1/IGF-1R 和 Hybrid-R 信号通路在人角膜上皮中的作用进行了表征。
方法/主要发现：使用免疫共沉淀、免疫印迹和细胞增殖测定法，在人端粒化角膜上皮（hTCEpi）细胞系中检查 IGF-1 介导的信号转导和细胞生长。通过免疫荧光和全细胞裂解物的相互免疫沉淀证实了 hTCEpi 和原代培养的人角膜上皮细胞中 Hybrid-R 的存在。我们发现，IGF-1 通过 IGF-1R 激活刺激 Akt 并促进细胞生长，这与 Hybrid-R 无关。Hybrid-R 存在于核提取物中，但 IGF-1R/IGF-1R 不存在。通过小干扰 RNA 敲低 INSR 导致 INSR/INSR 的耗尽和 Hybrid-R 的优先形成。随后进行了用抗 IGF-1R 或抗 INSR 的染色质免疫沉淀测序分析，以鉴定负责关键稳态调节途径的潜在基因组靶标。
结论/意义：与之前关于核定位 IGF-1R 的报告相反，这是首次在细胞系中鉴定出核定位的 Hybrid-R。核 Hybrid-R 和新型基因组靶标的鉴定表明，IGF-1R 作为 IGF-1R/INSR 异四聚体复合物转运到核内，以调节角膜上皮稳态途径。设计用于靶向 IGF-1/IGF-1R 通路的新型治疗策略必须考虑 IGF-1R/INSR 在上皮细胞核中发挥的调节作用。
