ABSTRACT
BACKGROUND: In vivo ectopic gene expression is a common approach for prostate research through the use of transgenes in germline transgenic mice. For some other organs, somatic transgenesis with the Sleeping Beauty transposon system has allowed in vivo ectopic gene expression with higher throughput and lower cost than germline transgenic approaches. METHODS: Mouse e16 urogenital sinuses (UGSs) were co-injected with plasmids expressing the Sleeping Beauty transposase and plasmids with control or activated BRAF expressing transposons. Following electroporation, the transduced UGSs were grown as allografts in mouse hosts for 8 weeks, and the resulting allografts were evaluated for several endpoints. RESULTS: Transposon-transduced UGS allografts developed into prostatic tissue with normal tissue structure and cellular differentiation. Integration of transposon vectors into the genomes of transduced allografts was confirmed using linker-mediated PCR, sequencing, and in situ PCR. Transduction of UGS allografts with transposons expressing activated BRAF resulted in ectopic BRAF expression that was detectable at both the mRNA and protein levels. Prostatic ducts over-expressing activated BRAF also had ectopic activation of the ERK1/2 mitogen activated kinases and increased epithelial cell proliferation. CONCLUSIONS: The Sleeping Beauty transposon system can be used to achieve somatic transgenesis of prostatic allografts. This new method for achieving ectopic gene expression in the prostate will complement other existing approaches such as ectopic gene expression in cell lines and in germline transgenic mice. Advantages of this new approach include preservation of stromal-epithelial interactions not possible with cell lines, and higher throughput and lower cost than traditional germline transgenic approaches.
Subject(s)
Gene Transfer Techniques , Prostate/metabolism , Prostatic Neoplasms/genetics , Transposases/genetics , Allografts , Animals , Genetic Vectors , Male , Mice , Mice, Transgenic , Prostatic Neoplasms/metabolism , Transposases/metabolismABSTRACT
Fetal prostate development from urogenital sinus (UGS) epithelium requires androgen receptor (AR) activation in UGS mesenchyme (UGM). Despite growing awareness of sexually dimorphic gene expression in the UGS, we are still limited in our knowledge of androgen-responsive genes in UGM that initiate prostate ductal development. We found that WNT inhibitory factor 1 (Wif1) mRNA is more abundant in male vs. female mouse UGM in which its expression temporally and spatially overlaps androgen-responsive steroid 5α-reductase 2 (Srd5a2). Wif1 mRNA is also present in prostatic buds during their elongation and branching morphogenesis. Androgens are necessary and sufficient for Wif1 expression in mouse UGS explant mesenchyme, and testicular androgens remain necessary for normal Wif1 expression in adult mouse prostate stroma. WIF1 contributes functionally to prostatic bud formation. In the presence of androgens, exogenous WIF1 protein increases prostatic bud number and UGS basal epithelial cell proliferation without noticeably altering the pattern of WNT/ß-catenin-responsive Axin2 or lymphoid enhancer binding factor 1 (Lef1) mRNA. Wif1 mutant male UGSs exhibit increased (Sfrp)2 and (Sfrp)3 expression and form the same number of prostatic buds as the wild-type control males. Collectively our results reveal Wif1 as one of the few known androgen-responsive genes in the fetal mouse UGM and support the hypothesis that androgen-dependent Wif1 expression is linked to the mechanism of androgen-induced prostatic bud formation.
Subject(s)
Androgens/metabolism , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation , Intercellular Signaling Peptides and Proteins/metabolism , Prostate/metabolism , 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/metabolism , Adaptor Proteins, Signal Transducing , Animals , Female , In Situ Hybridization , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , RNA, Messenger/metabolism , Sex Factors , Testosterone/metabolism , Time Factors , Urethra/metabolismABSTRACT
BACKGROUND: Prostate morphogenesis initiates in the urogenital sinus (UGS) with epithelial bud development. Sulfatase-1 (SULF1) inhibits bud development by reducing extracellular heparan sulfate (HS) 6-O sulfation and impairing FGF10 signaling by means of the ERK1/2 mitogen activated kinases. RESULTS: We characterized the expression patterns of HS 6-O sulfation modifying enzymes in the developing prostate by in situ hybridization and showed that Sulf1 and Hs6st1 had overlapping but distinct expression domains. Notably, Hs6st1 was present while Sulf1 was excluded from the tips of elongating epithelial buds. This predicted relatively high HS 6-O sulfation at the tips of elongating epithelial buds that was confirmed by immunohistochemistry. The pattern of Sulf1 expression in the peri-mesenchymal epithelium matched predicted locations of bone morphogenetic protein (BMP) signaling. Exogenous BMP4 and BMP7 induced Sulf1 expression in the UGS, decreased epithelial HS 6-O sulfation, and reduced ERK1/2 activation in response to FGF10. CONCLUSIONS: These data suggest that BMPs limit FGF10 action in the developing prostate at least in part by inducing Sulf1.
