Our research focuses on the role of the epidermal growth factor-like homeotic genes, dlk and Notch, in mammalian cell differentiation and the signal transduction pathways by which the products of these genes operate. The dlk gene was originally identified and cloned by Dr. Laborda. By using 3T3-L1 fibroblasts, an insulin and ras-dependent in vitro adipocyte differentiation model, we found that dlk mRNA was down regulated during the induction of differentiation, reaching minimum levels 16 hours after induction. Ectopic expression of dlk in these cells inhibited their ability to differentiate. The cells cannot be rescued with increased amounts of insulin. During differentiation, dlk protein levels are down regulated more slowly, with minimum levels reached more than four days after induction. Disappearance of the dlk from the membrane is coincident with the appearance of the adipocyte phenotype. Additional experiments indicate that functional dlk is not released to the external media, suggesting that dlk intervene in cell to cell signaling. Inhibition of Notch expression by transfection with an antisense Notch construct under the control of a constitutive promoter causes the loss of the differentiating ability of 3T3-L1 cells, accompanied by morphological changes. Differentiation can also be blocked by addition of a recombinat Notch protein encompasing the ligand-binding region of Notch, or by addition of an antiserum againts the same region. We have found that the morphological changes and the effect on differentiation are associated to the concomitant down regulation of the expression of fatty acid activated receptor transcription factors that control the differentiation program, and for that reason are called the master regulators of the adipocyte differentiation. In these cells, Notch expression and dlk down regulation are, therefore, needed to maintain their commitment to differentiation.
