ABSTRACT
We report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants.
Subject(s)
12E7 Antigen/metabolism , Growth Differentiation Factor 6/metabolism , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology , Signal Transduction , src-Family Kinases/metabolism , Animals , CSK Tyrosine-Protein Kinase/metabolism , Cell Proliferation , Down-Regulation , Gene Expression Regulation, Neoplastic , Growth Differentiation Factor 6/chemistry , Humans , Klippel-Feil Syndrome/genetics , Mice, SCID , Mutation/genetics , Oncogene Proteins, Fusion/metabolism , Protein Domains , Proteome/metabolism , Proteomics , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS/metabolism , Transcription, GeneticABSTRACT
BACKGROUND: During visual system development, multiple signalling pathways cooperate to specify axial polarity within the retina and optic tectum. This information is required for the topographic mapping of retinal ganglion cell axons on the tectum. Meis1 is a TALE-class homeodomain transcription factor known to specify anterior-posterior identity in the hindbrain, but its role in visual system patterning has not been investigated. RESULTS: meis1 is expressed in both the presumptive retina and tectum. An analysis of retinal patterning reveals that Meis1 is required to correctly specify both dorsal-ventral and nasal-temporal identity in the zebrafish retina. Meis1-knockdown results in a loss of smad1 expression and an upregulation in follistatin expression, thereby causing lower levels of Bmp signalling and a partial ventralization of the retina. Additionally, Meis1-deficient embryos exhibit ectopic Fgf signalling in the developing retina and a corresponding loss of temporal identity. Meis1 also positively regulates ephrin gene expression in the tectum. Consistent with these patterning phenotypes, a knockdown of Meis1 ultimately results in retinotectal mapping defects. CONCLUSIONS: In this work we describe a novel role for Meis1 in regulating Bmp signalling and in specifying temporal identity in the retina. By patterning both the retina and tectum, Meis1 plays an important role in establishing the retinotectal map and organizing the visual system.
Subject(s)
Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Retina/embryology , Superior Colliculi/embryology , Amino Acids , Animals , Animals, Genetically Modified , Apolipoprotein A-II/genetics , Apolipoprotein A-II/metabolism , Body Patterning/genetics , Carbocyanines , Embryo, Nonmammalian , Gene Expression Profiling/methods , Growth Differentiation Factor 6/chemistry , Growth Differentiation Factor 6/genetics , Homeodomain Proteins/chemistry , Mutation/genetics , Myeloid Ecotropic Viral Integration Site 1 Protein , Neoplasm Proteins/chemistry , Oligodeoxyribonucleotides, Antisense/pharmacology , RNA, Messenger/pharmacology , Retina/cytology , Retina/drug effects , Retinal Ganglion Cells/physiology , Signal Transduction/genetics , Smad5 Protein/chemistry , Smad5 Protein/genetics , Superior Colliculi/cytology , Superior Colliculi/drug effects , Zebrafish , Zebrafish Proteins/chemistry , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletal development; however, because of their widespread expression and functional redundancy, less progress has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and demonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spectrum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formation and altered expression of somite markers noggin1 and noggin2. Gdf6(+/-) mice exhibited variable ocular phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident between patients and animal models included pleiotropic effects, variable expressivity and incomplete penetrance. These data establish the important role of this determinant in ocular and vertebral development, demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMP function and its contributions to human disease.
