Our research focuses on the role of the epidermal growth factor-like homeotic gene, dlk, in mammalian cell differentiation, the signal transduction pathways by which the membrane product of this gene operates and its role in the interpretation of extracellular signals leading to growth or differentiation. The dlk gene was originally identified and cloned by Dr. Laborda. A soluble and a membrane form of Dlk have been indentified. The role of dlk on differentiation is being studied by using the 3T3-L1 and Balb/c 3T3 adipocyte differentiation models. We have found that downregulation of dlk is required for adipocyte differentiation. Transfection with antisense dlk decreased Dlk protein levels and enhanced adipocyte differentiation in response to insulin. In preliminary experiments, the enhanced differentiation response of the antisense dlk-transfected cells was associated with an increased activation of insulin signaling effectors (e.g., MAP kinases). Selection of cells with undetectable membrane Dlk expression by cell sorting, however, showed that they were impaired in their ability to differen- tiate. This indicate a complex function for Dlk in adipocyte differentiation and suggests an opposite role of soluble and membrane-associated Dlk proteins. We will continue to investigate this phenomenon with different dlk-transfected cell lines in the presence or absence of soluble Dlk peptide ligands. In collaboration with Dr. S.R. Bauer we have studied the role of Dlk in an in vitro model of pre-B cell differentiation. Pre-B cell cultures require both soluble IL-7 and stromal cell interactions to promote pre-B cell growth and prevent terminal differentiation and death. We have found that, when antisense dlk Balb/c 3T3 were used as stromal cells, pre-B cells grew in the absence of exogenous IL-7 and in a manner that inversely correlated with the expression levels of stromal Dlk. We extended these observations to other stromal cell lines, such as the S10 cell line and ruled out the production of IL-7 or other growth factors by stromal or pre-B cells under these conditions. The results of our studies are consistent with the hypothesis that Dlk functions as a molecular checkpoint for extracellular signals to proceed to the cell interior. Future studies will focus on the production of dlk knockout cell lines to further explore its function in cell growth and differentiation in vitro, as well as in the generation of a transgenic dlk-knockout mouse. We have also initiated the characterization of the murine and human dlk promoter regions and successfully subcloned a 2.0 kb fragment of the mouse promoter region that appears to contain the complete promoter, thus allowing us to study the transcriptional regulation crucial for the differentiation process.
