dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats |
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Authors: | Baladrón Victoriano Ruiz-Hidalgo María José Nueda María Luisa Díaz-Guerra María José M García-Ramírez José Javier Bonvini Ezio Gubina Elena Laborda Jorge |
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Affiliation: | Biochemistry and Molecular Biology Branch, Department of Inorganic Chemistry, Organic Chemistry and Biochemistry, Medical School/RCBR (Regional Center for Biomedical Research), University of Castilla-La Mancha, Campus of Albacete, Avda. Almansa s/n, Spain. |
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Abstract: | The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis. |
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Keywords: | 3T3-L1 Balb/c 14 dlk Notch1 CSL/RBP-Jk/CBF-1 Luciferase Yeast two-hybrid system |
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