ABSTRACT
The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of both IGF1 and IGF2. In recent years, evidence has accumulated showing that, in addition to its classical cell-surface distribution, IGF1R translocates to cell nucleus via an apparently SUMO-1-dependent mechanism. While the role of IGF1R in nucleus has not yet been settled, available information suggests that the nuclear receptor displays activities usually linked to transcription factors, including DNA binding and transcription regulation. To gain insight into the biological pathways associated with nuclear IGF1R action we conducted a mass spectrometry-based proteomic analysis aimed at identifying interactors of IGF1R in nucleus of both benign and malignant breast cells. The nucleolar NOM1 molecule belongs to a family of proteins that contain the middle domain of eukaryotic initiation factor 4G (MIF4G) and/or interaction module (MA3), and functions in translation, cell growth and proliferation. Using a combination of co-immunoprecipitation and silencing assays we provide evidence of a complex, bi-directional interplay between nuclear IGF1R and nucleolar protein NOM1. Inhibition of nuclear IGF1R translocation by dansylcadaverine reduced NOM1 levels in nuclei of MCF7 cells. On the other hand, IGF1R overexpression enhanced NOM1 levels in the nuclear fraction. Of interest, NOM1 silencing led to a major increase in IGF1R biosynthesis. In summary, results are consistent with a physiologically-relevant interplay between the nuclear IGF1 signaling pathway and nucleolar protein NOM1.
Subject(s)
Insulin-Like Growth Factor I/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Somatomedin/metabolism , Signal Transduction , Cell Line , Cell Nucleus , Cell Proliferation , Gene Silencing , Humans , Insulin-Like Growth Factor I/genetics , MCF-7 Cells , Nuclear Proteins/antagonists & inhibitors , Phosphorylation , Protein Binding , Proteomics , RNA, Small Interfering , RNA-Binding Proteins/antagonists & inhibitors , Receptor, IGF Type 1 , Receptors, Somatomedin/geneticsABSTRACT
The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF1 and IGF2. The IGF1R is involved in both physiological and pathological activities and is usually overexpressed in most types of cancer. In addition to its classical mechanism of action, recent evidence has shown a nuclear presence of IGF1R, associated with novel genomic/transcriptional types of activities. The present study was aimed at evaluating the hypothesis that nuclear IGF1R localization is not restricted to cancer cells and might constitute a novel physiologically relevant regulatory mechanism. Our data shows that nuclear translocation takes place in a wide array of cells, including normal diploid fibroblasts. In addition, we provide evidence for a synergistic effect of a nuclear translocation blocker along with selective IGF1R inhibitors in terms of decreasing cell proliferation. Given the important role of the IGF1R in mitogenesis, the present results may be of translational relevance in cancer research. In conclusion, results are consistent with the concept that nuclear IGF1R fulfills important physiological and pathological roles.
Subject(s)
Cell Proliferation/physiology , Receptors, Somatomedin/physiology , Active Transport, Cell Nucleus/drug effects , Cadaverine/analogs & derivatives , Cadaverine/pharmacology , Cell Line , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/physiology , Cell Nucleus/physiology , Cell Proliferation/drug effects , Cell Transformation, Neoplastic , Cells, Cultured , Fibroblasts/metabolism , Gene Knockdown Techniques , Humans , MCF-7 Cells , Microscopy, Confocal , Receptor, IGF Type 1 , Receptors, Somatomedin/antagonists & inhibitors , Receptors, Somatomedin/genetics , Signal TransductionABSTRACT
BACKGROUND: We have previously shown that both insulin and IGF1 lead to increased proliferation of keratinocytes. However, whereas insulin supports keratinocytes differentiation, IGF1 inhibits this process. The aim of the present study was to examine the proliferative and differentiative effects of insulin analogues (glargine, detemir, lispro and aspart) in primary keratinocytes in comparison with insulin and IGF1. METHODS: Primary keratinocytes cultures were produced from newborn BALB/c mice skin. Proliferation rates were assessed by [(3)H]-thymidine incorporation and XTT assays and differentiation was evaluated by Western blots analysis. Insulin receptor and IGF1 receptor phosphorylation was assessed by immunoprecipitation assays. RESULTS: Treatment with glargine or detemir resulted in an insulin-like effect on the differentiation process whereas lispro and aspart treatment led to an IGF1-like effect. In addition, treatment of keratinocytes with aspart led to a rapid phosphorylation of the IGF1 receptor. CONCLUSIONS: Our study provides evidence that insulin analogues elicit atypical actions in the skin.
