dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats.
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.
Baladrón
Victoriano
V
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.
Ruiz-Hidalgo
María José
MJ
Nueda
María Luisa
ML
Díaz-Guerra
María José M
MJ
García-Ramírez
José Javier
JJ
Bonvini
Ezio
E
Gubina
Elena
E
Laborda
Jorge
J
D041721:3T3-L1 Cells; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002460:Cell Line; D016373:DNA, Antisense / Q000235:genetics; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D000072056:Transcription Factor HES-1; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D051792:Basic Helix-Loop-Helix Proteins; D016373:DNA, Antisense; D004815:Epidermal Growth Factor; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases; D008565:Membrane Proteins; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000072056:Transcription Factor HES-1; D014157:Transcription Factors; C489734:Hes1 protein, mouse; C497761:NOTCH1 protein, human; C497762:Notch1 protein, mouse; C064045:delta protein