RESUMEN
The prostate gland develops from the urogenital sinus in response to circulating androgens. Androgens initiate and stimulate branching morphogenesis in the urogenital sinus via unknown mediators. Heparan sulfate proteoglycans are important extracellular molecules that sequester many growth factors in the extracellular matrix and facilitate signaling by some growth factors as part of ternary complexes that include growth factors, receptors, and heparan sulfate chains. Several enzymes modify the chemical structure of heparan sulfate to further regulate its activity. An examination of these enzymes for sexually dimorphic expression in the urogenital sinus identified Sulfatase 1 (Sulf1) as an enzyme that was down-regulated in the male urogenital sinus coincident with the initiation of prostatic morphogenesis. Down-regulation of Sulf1 was accompanied by an increase in the most highly sulfated forms of heparan sulfate, and a similar increase was observed in female urogenital sinuses treated with testosterone. Inhibiting de novo sulfation of heparan sulfate blocked prostatic morphogenesis, supporting the importance of heparan sulfate modification for prostate development. To functionally test the specific role of Sulf1 during prostate development, Sulf1 was ectopically expressed in the urogenital sinus. It partially inhibited testosterone-stimulated ductal morphogenesis, and it reduced the activation of fibroblast growth factor receptors as well as the ERK1 and ERK2 MAPKs. These data identify sulfatase 1 as an inhibitor of prostatic branching morphogenesis and growth factor signaling that is down-regulated as part of the normal response to androgen action in the male urogenital sinus.
Asunto(s)
Próstata/embriología , Sulfotransferasas/metabolismo , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Femenino , Proteoglicanos de Heparán Sulfato/metabolismo , Inmunohistoquímica , Masculino , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Fosforilación , Próstata/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfotransferasas/genética , Testosterona/farmacologíaAsunto(s)
Antineoplásicos/uso terapéutico , Investigación Biomédica/tendencias , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Humanos , Inmunoterapia , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/metabolismoRESUMEN
Retroviral and transposon-based mutagenesis screens in mice have been useful for identifying candidate cancer genes for some tumor types. However, many of the organs that exhibit the highest cancer rates in humans, including the prostate, have not previously been amenable to these approaches. This study shows for the first time that the Sleeping Beauty transposon system can be used to identify candidate prostate cancer genes in mice. Somatic mobilization of a mutagenic transposon resulted in focal epithelial proliferation and hyperplasia in the prostate. Efficient methods were established to identify transposon insertion sites in these lesions, and analysis of transposon insertions identified candidate prostate cancer genes at common insertion sites, including Pde4d. PDE4D was also overexpressed in human prostate cancer patient samples and cell lines, and changes in PDE4D mRNA isoform expression were observed in human prostate cancers. Furthermore, knockdown of PDE4D reduced the growth and migration of prostate cancer cells in vitro, and knockdown of PDE4D reduced the growth and proliferation rate of prostate cancer xenografts in vivo. These data indicate that PDE4D functions as a proliferation promoting factor in prostate cancer, and the Sleeping Beauty transposon system is a useful tool for identifying candidate prostate cancer genes.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Neoplasias de la Próstata/genética , Transposasas/genética , Animales , División Celular , ADN Complementario/genética , ADN de Neoplasias/genética , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Antígeno Ki-67/metabolismo , Masculino , Ratones , Ratones Desnudos , Mutagénesis , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Isoformas de Proteínas/genética , ARN Ligasa (ATP)/metabolismo , ARN Neoplásico/genética , Trasplante HeterólogoRESUMEN
The urogenital sinus (UGS) is specified as prostate in mice around embryonic day 15.5 as indicated by expression of the transcription factor Nkx3.1. Shortly thereafter, growth of epithelial buds into the UGS mesenchyme initiates prostatic morphogenesis. A comparison of male and female UGSs in vivo demonstrated sexually dimorphic expression of branching morphogenesis regulatory genes coincident with epithelial budding including Bmp7, Gli1, Gli2, Fgf10, Ptch1, and Shh. A comparison of UGSs grown with or without testosterone in serum-free organ cultures showed that some, but not all sexually dimorphic differences observed during prostate bud induction, were effectively modeled in vitro. Organ cultures were then used to investigate the role of fibroblast growth factor receptor (FGFR) signaling during prostatic induction. Blocking FGFR activation with PD173074 showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the UGS is dependent on FGFR signaling. Furthermore, inhibiting either FGFR activation with PD173074 or ERK1/2 activation with UO126 blocked all morphogenesis, proliferation, and gene expression changes induced by androgens in the UGS. These data reveal a previously unknown role for ERK1/2 during prostate bud induction. They also show that signaling by FGFRs through ERK1/2 is required for androgen-induced budding morphogenesis, proliferation, and gene expression during prostate bud induction.
Asunto(s)
Inducción Embrionaria/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Próstata/embriología , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Sistema Urogenital/embriología , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Endogámicos , Técnicas de Cultivo de Órganos , Próstata/enzimología , Caracteres Sexuales , Sistema Urogenital/enzimologíaRESUMEN
The mouse seminal vesicle shape (svs) mutation is a spontaneous recessive mutation that causes branching morphogenesis defects in the prostate gland and seminal vesicles. Unlike many other mutations that reduce prostatic and/or seminal vesicle branching, the svs mutation dramatically reduces branching without reducing organ growth. Using a positional cloning approach, we identified the svs mutant lesion as a 491 bp insertion in the tenth intron of Fgfr2 that results in changes in the pattern of Fgfr2 alternative splicing. An engineered null allele of Fgfr2 failed to complement the svs mutation proving that a partial loss of FGFR2(IIIb) isoforms causes svs phenotypes. Thus, the svs mutation represents a new type of adult viable Fgfr2 allele that can be used to elucidate receptor function during normal development and in the adult. In the developing seminal vesicles, sustained activation of ERK1/2 was associated with branching morphogenesis and this was absent in svs mutant seminal vesicles. This defect appears to be the immediate downstream effect of partial loss of FGFR2(IIIb) because activation of FGFR2(IIIb) by FGF10 rapidly induced ERK1/2 activation, and inhibition of ERK1/2 activation blocked seminal vesicle branching morphogenesis. Partial loss of FGFR2(IIIb) was also associated with down-regulation of several branching morphogenesis regulators including Shh, Ptch1, Gli1, Gli2, Bmp4, and Bmp7. Together with previous studies, these data suggest that peak levels of FGFR2(IIIb) signaling are required to induce branching and sustain ERK1/2 activation, whereas reduced levels support ductal outgrowth in the prostate gland and seminal vesicles.
Asunto(s)
Mutación , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Vesículas Seminales/anomalías , Vesículas Seminales/metabolismo , Alelos , Empalme Alternativo , Animales , Secuencia de Bases , Cruzamientos Genéticos , Cartilla de ADN/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Datos de Secuencia Molecular , Fenotipo , Embarazo , Próstata/anomalías , Próstata/crecimiento & desarrollo , Próstata/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Vesículas Seminales/crecimiento & desarrollo , Transducción de SeñalRESUMEN
Expression of the c-myc gene is frequently dysregulated in malignant tumors and translocations of c-myc into the Ig H chain locus are associated with Burkitt's-type lymphoma. There is indirect evidence that bcl-x, an anti-apoptotic member of the bcl-2 gene family, may also contribute to a variety of B lymphoid tumors. In this study, we show that mice transgenic for both B cell-restricted c-myc and bcl-x(L) developed aggressive, acute leukemias expressing early B lineage and stem cell surface markers. Of interest, the tumor cells proliferated and differentiated down the B cell developmental pathway following in vitro treatment with IL-7. Analysis of sorted leukemic cells from spleen indicated constitutive expression of sterile micro and kappa transcripts in combination with evidence for D-J(H) DNA rearrangements. Several B cell-specific genes were either not expressed or were expressed at low levels in primary tumor cells and were induced following culture with IL-7. IL-7 also increased V-Jkappa and V-DJ(H) rearrangements. These data demonstrate oncogenic synergy between c-myc and bcl-x(L) in a new mouse model for acute lymphoblastic leukemia. Tumors in these animals target an early stage in B cell development characterized by the expression of both B lineage and stem cell genes.