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Title: Regulation of Fgf10 gene expression in the prostate
Author: Tomlinson, Darren Charles
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Fibroblast growth factor 10 (FGF10) is a mesenchymal paracrine-acting factor that stimulates epithelial growth and is involved in the development of several branched organs, including the lungs, lachrymal glands and prostate. During branching morphogenesis in the lung, FGF10 is expressed in discrete areas of mesenchyme juxtaposed to branching epithelial tips. It has been proposed that paracrine factors produced in the epithelium and by differentiated stroma, such as sonic hedgehog (SHH) and transforming growth factor beta 1 (TGFbetal) regulate the discrete expression pattern of FGF10. In the prostate, FGF10 expression is also confined to the mesenchyme that surrounds growing epithelial buds. In the prostate it has been proposed that FGF10 is involved in prostatic induction and epithelial branching morphogenesis. However, little is known about how FgflO is regulated in the prostate, and the aim of this thesis was to investigate some of these regulatory mechanisms. This was done by developing a primary mesenchymal cell system in which to study FgflO regulation; investigating how TGFbetal and testosterone affect FgflO transcript expression in prostate cells and organs, and analysing the FgflO promoter. In addition the effects of TGFbetal on prostate growth were assessed to determine if TGFbetal might have opposing effects to that of FGF10. A primary stromal cell system, derived from the Ventral Mesenchymal Pad (VMP) was established and characterised. The VMP is a condensed area of mesenchyme found in both males and females that is required for prostatic induction in males, and is known to express FgflO. After the first passage in vitro, primary VMP cells (VMPC) became larger and their growth rate slowed, suggesting that primary VMPC senesced after being plated out. VMPC maintained expression of FgflO, Tgfbetal, 2, and 3 transcripts at levels similar to those in the VMP in vivo. VMPC also expressed androgen receptor but did not show androgen responsive growth in vitro. It was concluded that primary VMPC were a good cellular system in which to study the regulation of FgflO gene expression, and were used on their first passage. It has been shown that TGFbetal and testosterone regulate FgflO transcript expression in cells in culture. In studies presented here treatment of both cells and organs showed no direct regulation of FgflO by testosterone. However, treatment of primary VMPC with TGFbetal reduced FgflO expression fourteen-fold after 3 hours, but levels returned to control levels after 48 hours. A seven-hour TGFbetal treatment of VMP organ rudiments grown in vitro also decreased FgflO transcript levels by three-fold, similar to the decrease in expression after seven hours in primary VMPC. Furthermore, regulation of FgflO transcripts by TGFbetal was found to be specific for cells of the VMP and was not observed in urethral stroma. We next sought to extend our study into ventral prostate (VP) to determine if TGFbetal could regulate FgflO transcript levels in the prostate. TGFbetal only decreased FgflO transcript levels in the VP by approximately 1.5-fold, in contrast to VMP and VMPC that showed over a 3-fold repression. The reasons for the lower response of FgflO to TGFbetal in VPs were not determined but may be due to epithelium inhibiting TGFbetal repression of FgflO. This implies that factors present in the epithelium regulate the temporal and spatial expression of FgflO in the prostate, similar to observations in the lung. To further analyse FgflO regulation, 6 kb of mouse genomic sequence 5' to the translation start, which was thought to contain the FgflO promoter, was characterised. A transcription start site for FgflO was mapped 704 nts 5' to the translation start site, by RNase protection assay. Comparisons of the mouse and human genomic sequences 5' to the FgflO gene revealed several regions of high homology, suggestive of sites that control FgflO gene expression. Deletion analysis of the FgflO promoter identified a conserved element that mediated the majority of FgflO promoter activity above basal core promoter activity, as well as mediating promoter downregulation by TGFbetal. This element was located between nucleotides -50/-198, and contained a consensus Spl binding site. The promoter study provided further evidence that suggests TGFbetal regulates FgflO gene expression in the prostate, as well as identifying a potential mechanism. Next the effect of TGFbetal on VP development was characterised. The addition of TGFbetal to VPs inhibited 86% of testosterone-induced growth in vitro and significantly increased numbers of epithelial-branched tips. After a six day TGFbetal treatment in the presence of testosterone, TGFbetal decreased proliferation of epithelial (67%) and stromal (70%) cells in the proximal to urethra region of the VP, but increased proliferation of epithelial (89%) and stromal (40%) cells in the distal to urethra region of the VP. This suggested that TGFbetal has different effects on proliferation depending on the location of the cells within the prostate and perhaps the level of cellular differentiation. Previously it has been demonstrated that testosterone and TGFbetas regulate expression of Tgfbeta transcripts in adult prostates. To determine if the same effect was observed during development, VPs and VMPs were treated with testosterone and TGFbetal and Tgfbeta transcript levels were analysed by RPA. Testosterone and TGFbetal regulated Tgfbetal, Tgfbeta2, and Tgfbeta3 transcript levels in VPs and female VMPs cultured for three days, but the same effects were not observed over a six-day culture. As a previous study has shown that testosterone and TGFbetal did not affect Tgfbeta transcript levels in isolated prostatic stromal or epithelial cells it can be suggested that interactions between stroma and epithelium may be involved in the regulation of Tgfbeta transcript levels in prostatic rudiments. Overall, I have provided an insight into the regulation of FgflO and identified a possible mechanism involved in branching morphogenesis in the prostate. However, these data suggests that complex regulatory pathways, involving interactions between TGFbeta, testosterone and FGF10 are involved in regulating prostate morphogenesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available