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 membrane products of these genes operate. Previously, we showed that inhibition of Notch function causes the loss of the insulin-dependent differentiating ability of preadipocyte 3T3-L1 cells. These effects are associated with the concomitant down regulation of the expression of the transcription factors that control the differentiation program. Since differentiation of 3T3-L1 cells is dependent upon ras activation, and transfection with oncogenic ras leads to insulin-independent adipocyte differentiation, we studied whether the inhibitory effect of Notch on differentiation could be bypassed by oncogenic ras. The results obtained indicate that oncogenic ras is not able to trigger differentiation of antisense Notch 3T3-L1 cells and, instead, causes their transformation in a way that correlates with the decrease in the levels of Notch expression. These results suggest that Notch may be involved in the interpretation of extracellular signals to direct cells towards growth or differentiation. The dlk gene was originally identified and cloned by Dr. Laborda. We continued our studies on the role of dlk on adipocyte differentiation on the 3T3-L1 and Balb/c 3T3 cell differentiation models. We found that transfection with antisense dlk decreased dlk protein levels and enhanced adipocyte differentiation in response to insulin. This enhanced response was associated with an increased activation of insulin signaling effectors, such as MAP kinases. These results suggests the hypothesis that dlk may function as a molecular checkpoint to allow extracellular signals to proceed to the interior of the cell. Recently, it has been reported that dlk may play a role on hematopoiesis. In collaboration with Dr. Steven R. Bauer, we tested this hypothesis in an in vitro model of PreB cell differentiation. In vitro PreB cultures require both soluble IL-7 and stromal cell interactions to promote preB cell growth and prevent preB cell maturation and death. We found that Balb/c 3T3 cells can function as stromal cells in the presence of IL-7. When antisense dlk Balb/c 3T3 were used as stromal cells, however, preB cells grew in a manner inversely correlated with the expression levels of stromal dlk, in the absence of IL-7. We also found that preB cells and mature B cells express dlk. These results demonstrate that dlk plays a role in regulating differentiation signals to both the stromal cells and the lymphocyte precursors and are compatible with the previous hypothesis on a role of dlk in the regulation of extracellular signals leading to cell growth or differentiation.
