Abstract
Although much is known about signal transduction downstream of insulin-like growth factor-1 (IGF-1), relatively little is known about the global changes in protein expression induced by this hormone. In this study, the acute effects of IGF-1 on the proteome of murine C2C12 cells were examined. Cells were treated with IGF-1 for up to 24 hours, lysed, and fractionated into cytosolic, nuclear, and insoluble portions. Proteins from the cytosolic fraction were further separated using a new batch ion-exchange chromatography method to reduce sample complexity, followed by two-dimensional (2D) electrophoresis, and identification of selected proteins by mass spectrometry. PDQuest software was utilized to identify and catalogue temporal changes in protein expression during IGF-1 stimulation. In response to IGF-1 stimulation, expression of 23 proteins increased at least three-fold and expression of 17 proteins decreased at least three-fold compared with control un-stimulated C2C12 cells. Changes in expression of selected proteins from each group, including Rho-GDI, cofillin, RAD50, enolase, IκB kinase b (IκBKb) and Hsp70 were confirmed by Western blotting. Additionally, the position of 136 'landmark' proteins whose expression levels and physicochemical properties did not change appreciably or consistently during IGF-1 treatment were mapped and identified. This characterization of large-scale changes in protein expression in response to growth factor stimulation of C2C12 cells will further help to establish a comprehensive understanding of the networks and pathways involved in the action of IGF-1.
Highlights
The growth factor insulin-like growth factor-1 (IGF-1) is one of the major components of the mammalian hypothalamic-pituitary growth axis
Proteome Science 2009, 7:28 http://www.proteomesci.com/content/7/1/28 phocyte activation [5,6,7,8]. These studies have demonstrated that treatment of cells with functional IGF-1 protein or transduction of mouse or rat skeletal muscle cells with vectors encoding IGF-1 induces pleiotrophic effects in many cellular pathways that result in muscle hypertrophy, enhanced muscle contractility and protection from agerelated muscle wasting
We have previously used a microarray-based approach to identify changes in expression of genes sets in C2C12 cells in response to IGF-1 treatment. To determine if these transcriptional effects are reflected in alterations of the encoded proteins, we have examined the effect of IGF-1 on the proteome of C2C12 cells using two-dimensional (2D) electrophoresis and mass spectrometry
Summary
The growth factor IGF-1 is one of the major components of the mammalian hypothalamic-pituitary growth axis. Proteome Science 2009, 7:28 http://www.proteomesci.com/content/7/1/28 phocyte activation [5,6,7,8] These studies have demonstrated that treatment of cells with functional IGF-1 protein or transduction of mouse or rat skeletal muscle cells with vectors encoding IGF-1 induces pleiotrophic effects in many cellular pathways that result in muscle hypertrophy, enhanced muscle contractility and protection from agerelated muscle wasting. We have previously used a microarray-based approach to identify changes in expression of genes sets in C2C12 cells in response to IGF-1 treatment To determine if these transcriptional effects are reflected in alterations of the encoded proteins, we have examined the effect of IGF-1 on the proteome of C2C12 cells using two-dimensional (2D) electrophoresis and mass spectrometry. Among the interesting protein changes were increased expression of RAD50, enolase, IκBKb, and Hsp and decreased levels of cofilin and Rho-GDI
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