ABSTRACT
Activated forms of the platelet derived growth factor receptor alpha (PDGFRα) have been described in various tumors, including FIP1L1-PDGFRα in patients with myeloproliferative diseases associated with hypereosinophilia and the PDGFRα(D842V) mutant in gastrointestinal stromal tumors and inflammatory fibroid polyps. To gain a better insight into the signal transduction mechanisms of PDGFRα oncogenes, we mutated twelve potentially phosphorylated tyrosine residues of FIP1L1-PDGFRα and identified three mutations that affected cell proliferation. In particular, mutation of tyrosine 720 in FIP1L1-PDGFRα or PDGFRα(D842V) inhibited cell growth and blocked ERK signaling in Ba/F3 cells. This mutation also decreased myeloproliferation in transplanted mice and the proliferation of human CD34(+) hematopoietic progenitors transduced with FIP1L1-PDGFRα. We showed that the non-receptor protein tyrosine phosphatase SHP2 bound directly to tyrosine 720 of FIP1L1-PDGFRα. SHP2 knock-down decreased proliferation of Ba/F3 cells transformed with FIP1L1-PDGFRα and PDGFRα(D842V) and affected ERK signaling, but not STAT5 phosphorylation. Remarkably, SHP2 was not essential for cell proliferation and ERK phosphorylation induced by the wild-type PDGF receptor in response to ligand stimulation, suggesting a shift in the function of SHP2 downstream of oncogenic receptors. In conclusion, our results indicate that SHP2 is required for cell transformation and ERK activation by mutant PDGF receptors.
Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , mRNA Cleavage and Polyadenylation Factors/genetics , mRNA Cleavage and Polyadenylation Factors/metabolism , Animals , Cell Line , Cell Proliferation , Cells, Cultured , Humans , MAP Kinase Signaling System , Mice , Mutation , Phosphorylation , Protein Binding , STAT5 Transcription Factor/metabolism , Signal TransductionABSTRACT
FOXO (Forkhead box O) transcription factors induce cell growth arrest and apoptosis, which can be prevented by FOXO phosphorylation by AKT in response to growth factors such as platelet-derived growth factors (PDGF) and insulin-like growth factor I (IGF-I). In addition to this well characterized post-translational modification, we showed that FOXO1, FOXO3, and FOXO4 were also regulated at the transcriptional level. PDGF, fibroblast growth factors (FGF), and IGF-I repressed the expression of FOXO genes in human fibroblasts. This process was sensitive to phosphatidylinositol 3-kinase inhibition by LY294002. FOXO1-specific shRNA decreased FOXO1 mRNA expression and enhanced fibroblast proliferation, mimicking the effects of growth factors. Conversely, ectopic FOXO3 activation blocked the proliferation of fibroblasts and induced the expression of FOXO1, FOXO4, and p27-KIP1. Using luciferase reporter assays and chromatin immunoprecipitations, we identified a conserved FOXO-binding site in the promoter of the FOXO1 gene, which was required for regulation by PDGF, and mediated the up-regulation of FOXO1 by itself and by FOXO3. Altogether, our results suggest that the expression of FOXO1 and FOXO4 genes is stimulated by FOXO3 and possibly by other FOXO factors in a positive feedback loop, which is disrupted by growth factors.
Subject(s)
Forkhead Transcription Factors , Insulin-Like Growth Factor I/metabolism , Platelet-Derived Growth Factor/metabolism , Transcription Factors , Transcription, Genetic , Active Transport, Cell Nucleus/physiology , Animals , Cell Cycle Proteins , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/physiology , Forkhead Box Protein O1 , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Humans , Phosphatidylinositol 3-Kinases/metabolism , Promoter Regions, Genetic , RNA Interference , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
Sterol-regulatory element binding proteins (SREBPs) control the expression of genes involved in fatty acid and cholesterol biosynthesis. Using microarrays, we observed that mature SREBP-1 also induced the expression of genes unrelated to lipid metabolism, such as heme oxygenase 1 (HMOX1), plasma glutathione peroxidase, the phosphatidylinositol-3 kinase regulatory subunit p55 gamma, synaptic vesicle glycoprotein 2A, and COTE1. The expression of these genes was repressed upon addition of sterols, which block endogenous SREBP cleavage, and was induced by the statin drug mevinolin. Stimulation of fibroblasts with platelet-derived growth factor, which activates SREBP-1, had a similar effect. Fasted mice that were refed with a high-carbohydrate diet presented an increased expression of HMOX1 and p55 gamma in the liver. Overall, the transcriptional signature of SREBP-1 in fibroblasts stimulated by growth factors was very similar to that described in liver cells. We analyzed the HMOX1 promoter and found one SREBP binding site of the E-box type, which was required for regulation by SREBP-1a and SREBP-1c but was insensitive to SREBP-2. In conclusion, our data suggest that SREBP-1 regulates the expression of stress response and signaling genes, which could contribute to the metabolic response to insulin and growth factors in various tissues.
