Morphogenesis in the differentiating metanephros is regulated by reciprocal interactions between ureteric bud epithelia and the metanephric mesenchyme. The ureteric bud invades the overlying metanephric mesenchyme and induces conversion of the mesenchyme into stromal and epithelial elements, which form the nephron. Concurrently, the mesenchyme stimulates the ureteric bud to grow, branch, and eventually form the collecting duct system of the adult kidney. The Differentiation and Neoplasia Section has focused on the elucidation of mechanisms of inductive signaling in metanephric development, seeking (1) the ligands responsible for nephronic differentiation, (2) other non-inductive regulatory factors of nephrogenesis, and (3) the molecular targets of induction. 1) Previously, we reported the establishment of a cell line from the renal inductor, ureteric bud, and have now identified three families of secreted growth factors/cytokines that cooperate to induce epithelial conversion, tubule formation, and glomerulogenesis. These include the fibroblast growth factors (notably FGF2 and 9), gp130-binding proteins (leukemia inhibitory factor; LIF), and transforming growth factor-beta (Tgf-beta2, activin A and B, and gdf11). Individually, these factors induce differentiation, but at a rate significantly slower than in vivo, while in combination, they cooperate in accelerating tubule formation to a rate consistent with the in vivo process. In efforts to understand the basis for this cooperation and to define the signaling pathways responsible for induction, we have begun a systematic assessment of potential pathways that may be involved. As part of this effort, we have generated an inducible immortalized cell line of metanephric mesenchyme that can, under inductive conditions, differentiate to form epithelial-like structures consistent with tubule formation. We have characterized this line extensively now and found that it responds to the three families of inductive factors and expresses markers appropriate to nephronic epithelialization. This line is now being applied to the elucidation of signaling events that mediate or regulate differentiation of this progenitor. In addition to the direct effects of the aforementioned secreted cytokines, there is ample evidence from our lab to indicate that other secreted proteins can positively or negatively regulate nephronic development. For example, we have described a clear inhibitory effect of secreted Frizzled-related protein-1 (sFRP-1) on tubulogenesis of metanephric mesenchyme and Wnt signaling and have provided evidence that another family member, sFRP-2 can counter this effect. Now we have identified an additional member, sFRP-4, which is expressed in patterns similar to that of sFRP-2, but that can actually stimulate signaling through the Wnt pathway, which is critical to tubule formation. As an extension to these studies of normal development, we have also now initiated investigations into the status of inductive signaling pathways in renal tumors. Thus far, we have evaluated several cell lines derived from nephroblastomas and renal cell carcinomas. Remarkably, we are finding a consistent alteration in signaling through the JAK/Stat pathway, which mediates LIF signaling. We are currently assessing the status of this pathway in primary tumors. Finally, we previously observed the down regulation of a transcriptional coactivating factor with epithelial conversion, i.e., CITED1. We have now found that the protein interacts with members of the COUP family of transcription factors and that this interaction is necessary for CITED1 to translocate to the nucleus. Furthermore, COUP apparently mediates an interaction between CITED1 and Smad4 to regulate signaling through this pathway. Since we have also observed an ability of CITED1 to inhibit Wnt/Tcf activation, it may represent one of the first examples of a factor capable of simultaneously regulating two critical pathways in nephrogenesis and is consistent with our hypothesis that CITED1 may function as a gatekeeper for metanephric blastemal cell differentiation.
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