Novel Nuclear Localization and Potential Function of Insulin-Like Growth Factor-1 Receptor/Insulin Receptor Hybrid in Corneal Epithelial Cells

Abstract

BackgroundType 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 FindingsIGF-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/SignificanceIn 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.

High level of GHR nuclear translocation in skeletal muscle of a hyperplasic transgenic zebrafish

“…Nuclear internalization of cytokine receptors has been associated with cellular proliferation and, in most cases, to cancer (Aleksic et al 2010, Wang et al 2010, Wang & Hung 2012, Wu et al 2012). According to Conway-Campbell et al (2008), nuclear GHR is involved in transcriptional regulation, exhibiting many proprieties normally associated with transcription factors.…”

Cell-based assays for IGF-I bioactivity measurement: overview, limitations and current trends

“…Recently this assay was also used to investigate the role of GH and IGF-I on testosterone on the androgen-responsive muscle (Serra et al, 2011) and the determination of nuclear localization and protein function of IGF-I/insulin receptor hybrids in corneal epithelial cells (Wu et al, 2012). This convenient assay that requires no wash steps and lends itself to automation seems to be the future with regard to the use of nucleic acid dyes for measurement of cell proliferation.…”

Cornea and Ocular Surface Disease

“…Our laboratory has been the first to identify the presence of the Hybrid-R in the corneal epithelium (Figure 10). 18 Importantly, studies using gene knockdown techniques have demonstrated preferential Hybrid-R formation compared to classical IGF-1R or INSR formation in corneal epithelial cells, underscoring the significance of this receptor in ocular surface biology.…”

“…First, the Hybrid-R is present in the nucleus of corneal epithelial cells. 18 The presence of the Hybrid-R in the nucleus, where it interacts with DNA, means that it functions to regulate gene expression. Using state-of-art chromatin immunoprecipitation-DNA sequencing technology (ChIP-seq), we have been able to identify potential genes from enriched genomic DNA sequences that are targeted by the IGF-1R/INSR Hybrid-R (Table 1).…”

“…18 This is critical at the ocular surface, where both IGF-1 and insulin have been shown to be expressed in the preocular tear fluid. 19,20 In corneal epithelial cells, the Hybrid-R is stimulated by IGF-1 and not insulin (Figure 11).…”