ABSTRACT
The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.
Subject(s)
Fibroblasts/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Receptor, Notch1/metabolism , Signal Transduction , 3T3 Cells , 3T3-L1 Cells , Adaptor Proteins, Signal Transducing , Adipocytes/cytology , Adipocytes/metabolism , Animals , Binding, Competitive , Blotting, Western , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cells, Cultured , Embryo, Mammalian/cytology , Fibroblasts/cytology , HEK293 Cells , Humans , Immunoprecipitation , Intercellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Protein Binding , Receptor, Notch1/genetics , Serrate-Jagged Proteins , Two-Hybrid System TechniquesABSTRACT
The Dlk1 gene appears to function as a regulator of adipogenesis. Adult Dlk1-deficient mice are obese, but adipose tissue still develops in transgenic mice overexpressing an Fc-dlk1 fusion protein, and neither type of genetically modified mice displays serious abnormalities. It was therefore possible that one yet unidentified gene might either compensate or antagonize for the absence or for overexpression, respectively, of Dlk1 in those animals. In database searches, we found a novel gene, EGFL9, encoding for a protein whose structural features are virtually identical to those of dlk1, suggesting it may function in a similar way. As dlk1 does, the protein encoded by EGFL9/Dlk2 affects adipogenesis of 3T3-L1 preadipocytes and mesenchymal C3H10T1/2 cells; however, it does so in an opposite way to that of dlk1. In addition, expression levels of both genes appear to be inversely correlated in both cell lines. Moreover, enforced changes in the expression of one gene affect the expression levels of the other. Our data suggest that adipogenesis may be modulated by the coordinated expression of Dlk1 and EGFL9/Dlk2.
