Through mouse gene targeting studies, we have identified Fgf8, a factor expressed by metanephric mesenchyme (MM), to be essential for nephrogenesis through its upstream effects on expression of the secreted patterning molecule Wnt4 and homeodomain transcription factor Lim1. We have also discovered an epistatic relationship between Fgf8 and Wnt signaling in nephron formation. Moreover, we have now also established a role for Fgf8 in the development of male sex accessory tissues. During development of the mesonephros, the rostral aspect of the Wolffian duct (WD) (or cranial tubules) is absent in mutant animals, resulting in the loss of the head and body of the epididymis, efferent ductules, and much of the vas deferens. In the female, the epoophoron is missing but development of the mature female reproductive tract, including the ovaries and oviducts, is normal, since the epoophoron does not contribute to these tissues. Surprisingly, the Mullerian duct appears unaffected despite its putative growth requirement for interaction with the WD, suggesting that this relationship is mediated by caudal elements of the WD. These findings demonstrate for the first time the critical role of Fgf8 signaling in formation of the male reproductive tract tissues. Moreover, they suggest that formation of the WD is a discontinuous process. Since the loss of Lim1 expression in the WD also results in a similar phenotype in the male reproductive tract, Lim1 expression in the WD may occur downstream of Fgf8 signaling as it does in nephronic differentiation and may indicate a common mechanism for Fgf8 activity. In addition to the critical requirement for Fgf signaling during nephronic differentiation, Wnt signaling appears to play a complementary role in this process as demonstrated in studies of our conditional knockout of Fgf8. Since Wnt4 and Wnt9 are essential for the epithelial conversion of MM, but Wnt-mediated transcriptional activation is not evident using a transgenic reporter, we have assessed the role of the canonical Wnt mediator beta-catenin in cells from MM. A GSK-3beta inhibitor BIO proved to be a potent inducer of MM progenitor survival, proliferation, and tubule formation. We have found that BIO stabilizes beta-catenin and activates Wnt-dependent signaling in cultured cells from MM. It also induces adhesion complex formation necessary for mesenchymal- epithelial transition (MET). A small molecule called Wnt agonist, which activates TCF-dependent signaling independent of beta-catenin stabilization, fails to induce tubule formation. Furthermore, interference with adhesion complex formation using neutralizing antibodies blocks MET. These findings are consistent with MET being a TCF-independent process. In order to better understand the role of canonical Wnt signaling (beta-catenin/TCF-dependent transcriptional activation) in MET, we have developed a model using the transformed cell line HEK293, which was derived from MM. These cells rapidly form adhesion complexes when exposed to BIO. This process is not inhibited when cells are transfected with dominant-negative TCF constructs or beta-catenin that is incapable of interaction with TCF; however, MET is disrupted in cells expressing a dominant-negative cadherin construct, suggesting that MET is beta-catenin-dependent, but TCF-independent and therefore not the result of canonical signaling. Since Wilms tumor cells show significant activity through canonical signaling, i.e., beta-catenin-mediated TCF-dependent transcription, these studies suggest that if we block TCF activity and promote instead beta-catenin-dependent cell adhesion complex formation, we may be able to re-regulate tumor cells and inhibit tumor formation. The ultimate goal of the DNS is to define inappropriate signaling in tumorigenesis based upon mechanisms required for the differentiation of normal tissue progenitors during development and tissue renewal. In evaluations of one implicated pathway, i.e., STAT signaling, we delineated the role of STAT1 serine 727 phosphorylation in Wilms tumor pathogenesis and identified at least three genes that function downstream of STAT1 signaling, and these are involved in either blocking apoptosis or stimulating proliferation in Wilms tumor cells. These include the BCL-2 family member MCL-1, the stress-related protein HSP-27, and transcriptional repressor and proliferation promoting factor CDP or CUX-1. Targeting of any of these factors with RNAi inhibits tumor cell growth and for MCL-1 and HSP27 induces apoptosis, suggesting that they are required to sustain the tumorigenic phenotype. We are now evaluating the role of STAT1 signaling in the differentiation of normal MM from which Wilms tumor is derived. S727 phosphorylation of STAT1 is demonstrable in embryonic metanephroi, beginning prior to induction at E13.5 in rat and is lost with tissue maturation. Furthermore, induction of STAT1 activation (both Y701 and S727) with gamma-interferon treatment stimulates cell proliferation and inhibits apoptosis in primary MM but also blocks tubule formation. In a collaboration with the Structural Biophysics Laboratory, we have helped in the characterization and development of a penetratin-fusion peptide designed to interact specifically with the N-terminal portion of STAT1, preventing oligomerization. This peptide shows a potent and selective toxicity for tumor cells which express STAT1 and interferes with STAT1 transcriptional activation. When applied to MMs treated with gamma-interferon, this inhibitor blocks STAT1 activity and induces tubule formation. These studies provide compelling evidence that STAT1 signaling in normal renal progenitors inhibits differentiation in opposition to our observed induction of differentiation with STAT3 activation following treatment of MM with leukemia inhibitory factor. Thus, in this particular embryonic progenitor population, STAT1 is oncogenic, which contrasts with its putative role as a tumor suppressor in other tissues. In collaboration with Dr. Mark de Caestecker, Vanderbilt University Medical School, we have continued studies on transcriptional co-activator CITED1, which is highly expressed in condensed cap cell MM that overlays the ureteric bud. We previously proposed a role for CITED1 as a gatekeeper in its differentiation to the epithelia of the nephron. Now we have characterized the expression of this factor in Wilms tumor and have determined that it is an important marker for blastemal populations in these tumors and that its expression and nuclear localization correlates with the more aggressive tumors. We also demonstrate that ectopic expression of CITED1 in Wilms tumor cells stimulates proliferation and tumor growth in xenografts while a dominant-negative form inhibits proliferation in and tumor growth by these cells. These findings indicate the CITED1 functions in tumorigenesis. Finally, we reported the surprising finding that ELR+-CXC chemokine, CXCL-7, and its receptors CXCR1 and 2 play a fundamental role in nephrogenesis by allowing competence to respond to a variety of signaling ligands through induction of receptor expression as well as inducing vasculogenesis in metanephric blastemal cells. Furthermore, we established that Wilms tumors express these receptors and that Wilms tumor cells depend upon signaling through these receptors for survival. This may provide a possible target for therapeutic purposes
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