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, (3) the molecular targets of induction, and (4) alterations in inductive signaling during tumorigenesis. 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. From microarray analysis, we have observed high levels of interleukin-1 expression by our ureteric bud cell line and gro-alpha expression in mesenchyme. Il-1 can stimulate gro-alpha in the mesenchyme, which in turn, binds to the chemokine receptors CXCR1 and 2. A block in signaling through this receptor dramatically inhibits development of the metanephros, suggesting that it plays a critical role in the growth and maintenance of metanephric tissues and that it may also function in a similar capacity during tumorigenesis. 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 patterns of Stat phosphorylation, which mediates LIF signaling. We have established that these altered patterns exist in primary human tumor tissues from nephroblastomas but not in adjacent normal tissues. Furthermore, abrogation of altered Stat signaling inhibits tumorigenesis as measured by nephroblastoma cell growth in soft agar, suggesting that the altered patterns contribute to the neoplastic process. 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 both physically and functionally with members of the COUP family of transcription factors. 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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