EphB signalling in rat prostate development
Ashley, George Russell
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In male mammals, the formation of the prostate gland is driven by androgens and involves cell-cell signalling between mesenchymal and epithelial cells. Gene profiling studies of prostate mesenchyme, using serial analysis of gene expression (SAGE), identified many transcripts that encode potential regulators of prostate development. The studies identified transcripts expressed in the ventral mesenchymal pad (VMP), a subset of the prostate mesenchyme known to express key growth factors and to regulate prostate organ development. These candidate mRNAs were used in a whole mount in-situ hybridisation (WISH) screen to identify those showing mesenchyme specific expression. The transcripts selected for WISH analysis were placed in three groups. The first group of transcripts were identified as enriched in the VMP based upon statistical analysis of their SAGE tag count. The second group of transcripts shared a SAGE tag count that was not statistically significant, and were a control for the first group. The third group encompassed transcripts that encoded either secretory or transmembrane proteins that were likely mediators of cell-cell communication. From 194 candidates, 30 were analysed by WISH and 13 were identified as mesenchymal. The tyrosine kinase receptor, EphB3, was selected from the WISH analysis and its role in prostate development was examined.EphB signalling has been characterised as a chemotactic guidance cue in neuronal development and has also been implicated in organogenesis of the kidney, lung and colon. The Eph tyrosine kinase family is the largest of its type and is divided into two classes of receptor, Eph A and EphB. The EphB family has five receptors (EphB 1-4, B6) and three ligands (EphrinBl-3) in mammals. The EphrinB ligands are transmembrane proteins.PCR analysis was used to examine the expression of the EphB and EphrinB transcripts in the developing rat prostate. The PCR analysis showed that mRNAs for the EphB2 and EphB3 receptors, and the EphrinB 1 and EphrinB2 ligands, were highly expressed in the rat prostate compared with the other EphB and EphrinB family members. The PCR analysis did not establish whether EphB receptors or EphrinB ligands were expressed in epithelia, mesenchyme or both. The EphB2 and EphB3 receptors, and the EphrinB 1 and EphrinB2 ligands, were further characterised by WISH, quantitative real-time PCR and immunohistochemical analysis during prostate development. At both the mRNA and protein levels, EphB3 and EphrinB 1 were expressed in a restricted area of the prostate mesenchyme, in close association with the developing epithelial buds. The EphB3 and EphrinB 1 transcripts were detected by the SAGE analysis, suggesting that they were expressed in the mesenchyme. The EphB2 and EphrinB2 transcripts were not detected by the SAGE analysis, suggesting that they were expressed in the epithelium. The EphB2 receptor and EphrinB2 ligand were predominantly expressed in the developing epithelial buds, as shown by immunohistochemical analysis. The SAGE analysis of VMP mesenchyme identified EphB3 and EphrinB 1 but not EphB2 and EphrinB2. This was consistent with their expression in mesenchyme or epithelium respectively.The addition of EphB2-Fc and EphB3-Fc to in vitro organ cultures of neonatal prostates, acting as a ligand trap, decreased prostate growth. The addition of EphrinB 1-Fc and EphrinB2-Fc ligands increased prostate organ size. The addition of EphrinB 1-Fc and EphrinB2-Fc produced a significant increase in the mesenchymal and epithelial cell proliferation rates. This increase in cell proliferation in response to EphrinB 1-Fc and EphrinB2-Fc was consistent with the observed increase in prostate organ size. The addition of EphB2-Fc and EphB3-Fc produced no significant increase in the mesenchymal and epithelial cell proliferation rates. This lack of a significant increase in cell proliferation in response to EphB2-Fc and EphB3-Fc was consistent with the observed decrease in prostate organ size. These findings suggest a role for EphB signalling in the regulation of prostate growth.The addition of either EphB-Fc or EphrinB-Fc proteins to in vitro organ cultures resulted in a decrease epithelial branching morphogenesis. Larger epithelial buds were observed in organs treated with EphrinB 1-Fc and EphrinB2-Fc, when compared to control organs. No visible change in the size of the epithelial buds was observed in response to EphB-Fc9 treatment. Furthermore, p63 and Smooth Muscle Actin immunohistochemical analysis of EphrinBl-Fc and EphrinB2-Fc treated organs showed larger epithelial buds, and proliferation analysis showed greater epithelial cell proliferation in EphrinB-Fc treated organs. The increased size of each epithelial bud may be caused by the decreased epithelial branching and the increased epithelial proliferation rate, in response to the addition of EphrinB-Fc proteins. These findings suggest a role for EphB-EphrinB signalling in the regulation of prostate epithelial branching.Collectively, we report the first reported functional link between EphB signalling and prostate development. EphB-EphrinB signalling may act as a novel juxtacrine or autocrine signal within the mesenchyme or as a novel paracrine signalling mechanism during prostate organogenesis.