ABSTRACT
pVHL, the protein product of the von Hippel-Lindau (VHL) tumor suppressor gene, is a ubiquitin ligase that targets hypoxia-inducible factor α (HIF-α) for proteasomal degradation. Although HIF-α activation is necessary for VHL disease pathogenesis, constitutive activation of HIF-α alone did not induce renal clear cell carcinomas and pheochromocytomas in mice, suggesting the involvement of an HIF-α-independent pathway in VHL pathogenesis. Here, we show that the transcription factor B-Myb is a pVHL substrate that is degraded via the ubiquitin-proteasome pathway and that vascular endothelial growth factor (VEGF)- and/or platelet-derived growth factor (PDGF)-dependent tyrosine 15 phosphorylation of B-Myb prevents its degradation. Mice injected with B-Myb knockdown 786-O cells developed dramatically larger tumors than those bearing control cell tumors. Microarray screening of B-Myb-regulated genes showed that the expression of HIF-α-dependent genes was not affected by B-Myb knockdown, indicating that B-Myb prevents HIF-α-dependent tumorigenesis through an HIF-α-independent pathway. These data indicate that the regulation of B-Myb by pVHL plays a critical role in VHL disease.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Tyrosine/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , von Hippel-Lindau Disease/pathology , Animals , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Neoplasm Transplantation , Phosphorylation , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Signal Transduction , Ubiquitin/metabolism , von Hippel-Lindau Disease/genetics , von Hippel-Lindau Disease/metabolismABSTRACT
Fibroblast growth factors (FGFs) expressed in the apical ectodermal ridge (AER) and FGF10 expressed in the underlying mesoderm are essential for limb bud outgrowth. Their expression is maintained through a positive feedback loop. We identified the cis-regulatory element and trans-acting factors involved in the AER-FGF-dependent transactivation of Fgf10. Etv1 and Ewsr1 stimulated transcription from the Fgf10 promoter in the sub-AER mesenchyme of mouse and chick limb buds in a conserved AGAAAR cluster-dependent manner. We found that both Etv1 and Ewsr1 were necessary for Fgf10 expression and elongation of the limb bud. In addition, Etv1 and AER-FGF synergistically stimulated Fgf10 promoter activity in an Ewsr1-dependent manner. We also found that Etv1 and Ewsr1 bound to the segment of DNA containing the AGAAAR cluster in vivo and in vitro. Moreover, Etv1 directly bound to the AGAAAR sequence in vitro. Our results suggest that Etv1 and Ewsr1 transactivate Fgf10 directly and cooperatively in response to AER-FGFs.