RESUMO
Although men with testosterone deficiency are at increased risk for type 2 diabetes (T2D), previous studies have ignored the role of testosterone and the androgen receptor (AR) in pancreatic ß cells. We show that male mice lacking AR in ß cells (ßARKO) exhibit decreased glucose-stimulated insulin secretion (GSIS), leading to glucose intolerance. The AR agonist dihydrotestosterone (DHT) enhances GSIS in cultured male islets, an effect that is abolished in ßARKO(-/y) islets and human islets treated with an AR antagonist. In ß cells, DHT-activated AR is predominantly extranuclear and enhances GSIS by increasing islet cAMP and activating the protein kinase A. In mouse and human islets, the insulinotropic effect of DHT depends on activation of the glucagon-like peptide-1 (GLP-1) receptor, and accordingly, DHT amplifies the incretin effect of GLP-1. This study identifies AR as a novel receptor that enhances ß cell function, a finding with implications for the prevention of T2D in aging men.
Assuntos
Núcleo Celular/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Receptores Androgênicos/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Intolerância à Glucose/patologia , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos Knockout , Modelos Biológicos , Receptores Androgênicos/deficiência , Transdução de Sinais/efeitos dos fármacos , Testosterona/farmacologiaAssuntos
Receptores Androgênicos/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Animais , Integrases , Masculino , Camundongos , Camundongos Knockout , Receptores Androgênicos/genética , Túbulos Seminíferos/química , Túbulos Seminíferos/metabolismo , Testículo/química , Testículo/metabolismoRESUMO
Androgens have important cardiometabolic actions in males, but their metabolic role in females is unclear. To determine the physiologic androgen receptor (AR)-dependent actions of androgens on atherogenesis in female mice, we generated female AR-knockout (ARKO) mice on an atherosclerosis-prone apolipoprotein E (apoE)-deficient background. After 8 weeks on a high-fat diet, but not on a normal chow diet, atherosclerosis in aorta was increased in ARKO females (+59% vs. control apoE-deficient mice with intact AR gene). They also displayed increased body weight (+18%), body fat percentage (+62%), and hepatic triglyceride levels, reduced insulin sensitivity, and a marked atherogenic dyslipidemia (serum cholesterol, +52%). Differences in atherosclerosis, body weight, and lipid levels between ARKO and control mice were abolished in mice that were ovariectomized before puberty, consistent with a protective action of ovarian androgens mediated via the AR. Furthermore, the AR agonist dihydrotestosterone reduced atherosclerosis (-41%; thoracic aorta), subcutaneous fat mass (-44%), and cholesterol levels (-35%) in ovariectomized mice, reduced hepatocyte lipid accumulation in hepatoma cells in vitro, and regulated mRNA expression of hepatic genes pivotal for lipid homeostasis. In conclusion, we demonstrate that the AR protects against diet-induced atherosclerosis in female mice and propose that this is mediated by modulation of body composition and lipid metabolism.
Assuntos
Aterosclerose/prevenção & controle , Dislipidemias/prevenção & controle , Obesidade/prevenção & controle , Receptores Androgênicos/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/etiologia , Aterosclerose/metabolismo , Colesterol/metabolismo , Dieta/efeitos adversos , Di-Hidrotestosterona/farmacologia , Dislipidemias/etiologia , Dislipidemias/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Feminino , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismo , Orquiectomia , Ovariectomia , Receptores Androgênicos/deficiência , Receptores Androgênicos/genéticaRESUMO
Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by â¼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.
Assuntos
Células-Tronco Adultas/fisiologia , Androgênios/fisiologia , Desenvolvimento Fetal/fisiologia , Células Intersticiais do Testículo/fisiologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Callithrix , Linhagem da Célula/fisiologia , Dibutilftalato/toxicidade , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/fisiologia , Humanos , Técnicas In Vitro , Células Intersticiais do Testículo/efeitos dos fármacos , Hormônio Luteinizante/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Animais , Gravidez , Ratos , Ratos Transgênicos , Ratos Wistar , Receptores Androgênicos/deficiência , Receptores Androgênicos/genética , Receptores Androgênicos/fisiologia , Regeneração , Testículo/embriologia , Testículo/fisiologia , Testosterona/deficiência , Testosterona/fisiologiaRESUMO
An understanding of the molecular mechanisms by which androgens drive spermatogenesis has been thwarted by the fact that few consistent androgen receptor (AR) target genes have been identified. Here, we addressed this issue using next-generation sequencing coupled with the RiboTag approach, which purifies translated mRNAs expressed in cells that express cyclic recombinase (CRE). Using RiboTag mice expressing CRE in Sertoli cells (SCs), we identified genes expressed specifically in SCs in both prepubertal and adult mice. Unexpectedly, this analysis revealed that the SC-specific gene program is already largely defined at the initiation of spermatogenesis despite the subsequent dramatic maturational changes known to occur in SCs. To identify AR-regulated genes, we generated triple-mutant mice in which the SCs express the RiboTag but lack ARs. RNA sequencing analysis revealed hundreds of SC-expressed AR-regulated genes that had previously gone unnoticed, including suppressed genes involved in ovarian development. Comparison of the SC-enriched dataset with that from the whole testes allowed us to classify genes in terms of their degree of expression in SCs. This revealed that a greater fraction of AR-up-regulated genes than AR-down-regulated genes were expressed predominantly in SCs. Our results also revealed that AR signaling in SCs causes a large number of genes not detectably expressed in SCs to undergo altered expression, thereby providing genome-wide evidence for wide-scale communication between SCs and other cells. Taken together, our results identified novel classes of genes expressed in a hormone-dependent manner in different testicular cell subsets and highlight a new approach to analyze cell type-specific gene regulation.
Assuntos
Regulação da Expressão Gênica , Técnicas Genéticas , Genoma/genética , Biossíntese de Proteínas/genética , Receptores Androgênicos/metabolismo , Células de Sertoli/metabolismo , Androgênios/farmacologia , Animais , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Células de Sertoli/efeitos dos fármacos , Maturidade Sexual/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The androgen receptor (AR) recognizes two types of DNA elements that are dimers of 5'-AGAACA-3'-like hexamers, either organized as inverted or direct repeats. We developed a mouse model [(specificity affecting AR knock-in (SPARKI)] in which the AR DNA-binding domain was mutated such that it lost binding to direct repeats but not to inverted elements. The impaired fertility of the male SPARKI mice correlates with the reduced motility of the spermatozoa, a characteristic that is developed during transit through the epididymis. Comparative transcriptome analyses revealed that the expression of 39 genes is changed in SPARKI epididymis. Remarkably, the expression of the steroid 5α-reductase type II (Srd5α2) gene, which metabolizes testosterone into the more potent dihydrotestosterone, is reduced 4-fold in SPARKI vs. wild type. The comparison of the SPARKI phenotype with that of Srd5α2-knockout mice shows, however, that the reduced Srd5α2 expression cannot explain all defects of the SPARKI epididymis. Moreover, we describe three new selective androgen response elements (AREs), which control the androgen responsiveness of the Srd5α2 gene. We conclude that the SPARKI model can be considered a knockout model for AR functioning via selective AREs and that this has a dramatic effect on sperm maturation in the epididymis.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Androgênios/farmacologia , Epididimo/metabolismo , Receptores Androgênicos/metabolismo , Elementos de Resposta/fisiologia , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Orquiectomia , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/genética , Elementos de Resposta/genéticaRESUMO
Androgens play a critical role in the development of the male reproductive system, including the positioning of the gonads. It is not clear, however, which developmental processes are influenced by androgens and what are the target tissues and cells mediating androgen signaling during testicular descent. Using a Cre-loxP approach, we have produced male mice (GU-ARKO) with conditional inactivation of the androgen receptor (Ar) gene in the gubernacular ligament connecting the epididymis to the caudal abdominal wall. The GU-ARKO males had normal testosterone levels but developed cryptorchidism with the testes located in a suprascrotal position. Although initially subfertile, the GU-ARKO males became sterile with age. We have shown that during development, the mutant gubernaculum failed to undergo eversion, a process giving rise to the processus vaginalis, a peritoneal outpouching inside the scrotum. As a result, the cremasteric sac did not form properly, and the testes remained in the low abdominal position. Abnormal development of the cremaster muscles in the GU-ARKO males suggested the participation of androgens in myogenic differentiation; however, males with conditional AR inactivation in the striated or smooth muscle cells had a normal testicular descent. Gene expression analysis showed that AR deficiency in GU-ARKO males led to the misexpression of genes involved in muscle differentiation, cell signaling, and extracellular space remodeling. We therefore conclude that AR signaling in gubernacular cells is required for gubernaculum eversion and outgrowth. The GU-ARKO mice provide a valuable model of isolated cryptorchidism, one of the most common birth defects in newborn boys.
Assuntos
Criptorquidismo/metabolismo , Receptores Androgênicos/genética , Testículo/metabolismo , Actinas/metabolismo , Animais , Criptorquidismo/genética , Criptorquidismo/patologia , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Liso/metabolismo , Músculo Liso/patologia , Tamanho do Órgão , Receptores Androgênicos/metabolismo , Transdução de Sinais , Contagem de Espermatozoides , Testículo/crescimento & desenvolvimento , Testículo/patologia , Testosterona/genética , Testosterona/metabolismoRESUMO
This review aims to evaluate the contribution of individual cell-selective knockout models to our current understanding of androgen action. Cre/loxP technology has allowed the generation of cell-selective knockout models targeting the androgen receptor (AR) in distinct putative target cells in a wide variety of organs and tissues including: testis, ovary, accessory sex tissues, muscle, bone, fat, liver, skin and myeloid tissue. In some androgen-regulated processes such as spermatogenesis and folliculogenesis this approach has lead to the identification of a key cellular mediator of androgen action (Sertoli and granulosa cells, respectively). In many target tissues, however, the final response to androgens appears to be more complex. Here, cell-selective knockout technology offers a platform upon which we can begin to unravel the more complex interplay and signaling pathways of androgens. A prototypic example is the analysis of mesenchymal-epithelial interactions in many accessory sex glands. Furthermore, for some actions of testosterone, in which part of the effect is mediated by the active metabolite 17ß-estradiol, conditional knockout technology offers a novel strategy to study the relative contribution of AR and estrogen receptor-mediated signaling. The latter approach has already resulted in a better understanding of androgen action in brain and bone. Finally, cell-selective knockout technology has generated valuable models to search for AR-controlled molecular mediators of androgen action, a strategy that has successfully been applied to the study of androgen action in the testis and in the epididymis. Although some conditional knockout models have provided clear answers to physiologic questions, it should be noted that others have pointed to unexpected complexities or technical limitations confounding interpretation of the results.
Assuntos
Especificidade de Órgãos , Receptores Androgênicos/metabolismo , Animais , Camundongos , Camundongos Knockout , Receptores Androgênicos/fisiologia , Transdução de SinaisRESUMO
Our previous analysis of Sertoli cell androgen receptor (AR) knockout (SCARKO) mice revealed that several cytoskeletal components are a potential target of androgen action. Here, we found that one of these components, the beta-tubulin isotype Tubb3, is differentially regulated in testes from SCARKO mice (relative to littermate controls) from Postnatal Day 10 to adulthood. The Tubb3 gene is unique in this respect, as at Day 10, no other beta-tubulin genes are significantly regulated by AR. We further characterized androgen regulation of Tubb3 in vivo and in vitro and demonstrated that it is a conserved feature in both mice and rats. To investigate whether androgens directly regulate Tubb3 expression, we screened for androgen response elements (AREs) in the Tubb3 gene. In silico analysis revealed the presence of four ARE motifs in Tubb3 intron 1, two of which bind to AR in vitro. Mutation of one of these (ARE1) strongly reduced androgen-dependent reporter gene expression. These results, coupled with the finding that the AR binds to the Tubb3 ARE region in vivo, suggest that Tubb3 is a direct target of AR. Our data strengthen the contention that androgens exert their effects on spermatogenesis, in part, through modulation of the Sertoli cell cytoskeleton. Androgen regulation of beta-tubulin has also been described in neurons, fortifying the already known similarity in microtubule organization in Sertoli cell processes and neurons, the only other cell type in which Tubb3 is known to be expressed.
Assuntos
Androgênios/metabolismo , Células de Sertoli/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Receptores Androgênicos/deficiência , Receptores Androgênicos/genética , Células de Sertoli/citologia , Espermatogênese/fisiologia , Tubulina (Proteína)/genéticaRESUMO
The steroid hormone signaling axis is thought to play a central role in initiation and progression of many hormonally regulated epithelial tumors. It is unclear whether all cancer-initiating signals depend on an intact hormone receptor signaling machinery. To ascertain whether cell autonomous androgen receptor (AR) is essential for initiation of prostate intraepithelial neoplasia (PIN), the response of AR-null prostate epithelia to paracrine and cell autonomous oncogenic signals was assessed in vivo by using the prostate regeneration model system. Epithelial-specific loss of AR blocked paracrine FGF10-induced PIN, whereas the add back of exogenous AR restored this response. In contrast, PIN initiated by cell-autonomous, chronic-activated AKT developed independent of epithelial AR signaling. Our findings demonstrate a selective role for AR in the initiation of PIN, dependent on the signaling pathways driving tumor formation. Insights into the role of hormone receptor signaling in the initiation of epithelial tumors may help define this axis as a target for chemoprevention of carcinomas.
Assuntos
Neoplasias Hormônio-Dependentes/etiologia , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias da Próstata/etiologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Animais , Sequência de Bases , Carcinógenos/metabolismo , Primers do DNA/genética , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Comunicação Parácrina , Neoplasia Prostática Intraepitelial/etiologia , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Receptores Androgênicos/deficiência , Receptores Androgênicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The epithelial lining of the epididymal duct expresses the androgen receptor (Ar) along its entire length and undergoes rapid and profound degeneration when androgenic support is withdrawn. However, experiments involving orchidectomy with systemic testosterone replacement, and testicular efferent duct ligation, have indicated that structural and functional integrity of the initial segment cannot be maintained by circulating androgen alone, leaving the role of androgen in this epididymal zone unclear. We addressed this question in a mouse model with intact testicular output and selective Ar inactivation in the proximal epididymis by creating double-transgenic males carrying a conditional Ar(loxP) allele and expressing Cre recombinase under the promoter of Rnase10, a gene specifically expressed in proximal epididymis. At 20-25 d of life, on the onset of Rnase10 expression, Ar became selectively inactivated in the principal cells of proximal epididymis, resulting in epithelial hypoplasia and hypotrophy. Upon the subsequent onset of spermiation, epididymal obstruction ensued, with the consequent development of spermatic granulomata, back pressure-induced atrophy of the seminiferous epithelium, orchitis, and fibrosis of the testicular parenchyma. Consistent with these findings, the mice were infertile. When the effect of Ar knockout on gene expression in the proximal epididymis was compared with that of efferent duct ligation and orchidectomy, we identified genes specifically regulated by androgen, testicular efferent fluid, and both. Our findings demonstrate that the development and function of the epididymal initial segment is critically dependent on direct androgen regulation. The phenotype of the produced knockout mouse provides a novel model for obstructive azoospermia.
Assuntos
Azoospermia/etiologia , Epididimo/metabolismo , Receptores Androgênicos/metabolismo , Animais , Azoospermia/genética , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A mouse model with a Sertoli cell (SC)-selective ablation of the androgen receptor (AR)-the SCARKO mouse-demonstrated that the effects of androgens on spermatogenesis depend on the presence of an active AR in SC. This model has been extremely valuable in the study of the effects of androgens on the initiation of spermatogenesis. However, due to the early (prenatal) inactivation of the AR SCARKO mice develop a complete block in meiosis, making it impossible to study the effects of androgens on postmeiotic steps of germ cell development. It would therefore be of interest to develop a model in which the AR can be ablated at any chosen time point. Here we used a mouse line ubiquitously expressing a tamoxifen (TM)-inducible Cre recombinase to develop an inducible AR knockout model (iARKO). It is shown that treatment with TM (3 mg/day) for five consecutive days efficiently inactivates the AR in testicular tissue and decreases the expression of known AR-target genes in SC (Rhox5, Spinlw1) without markedly affecting testicular cell composition one day after the final injection. TM treatment did, however, decrease serum gonadotropin levels and the expression of several Leydig cell genes (StAR, Cyp17a1, Insl3), resulting in decreased testosterone levels. Nevertheless, the intratesticular testosterone concentration still far exceeds the estimated concentrations required to saturate the AR. It may be concluded that the study of androgen-responsive postmeiotic genes in SC may be feasible using a TM-inducible AR knockout model provided that appropriate controls are included correcting for off-target effects of TM.
RESUMO
The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.
Assuntos
Diferenciação Celular/fisiologia , Citoesqueleto/metabolismo , Receptores Androgênicos/fisiologia , Células de Sertoli/metabolismo , Animais , Caderinas/genética , Diferenciação Celular/genética , Claudina-3 , Claudinas , Feminino , Expressão Gênica , Imuno-Histoquímica , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/genética , Receptores Androgênicos/deficiência , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Sertoli/citologia , Células de Sertoli/ultraestrutura , Espermatogênese/genética , Espermatogênese/fisiologia , Testículo/metabolismo , Testículo/ultraestrutura , Fatores de Tempo , Vimentina/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
Aberrant activation of fatty acid synthesis is a key feature of many advanced human cancers. Unlike in classical lipogenic tissues, this process has been implicated in membrane production required for rapid cell proliferation. Here, to gain further insight into the consequences of tumor-associated fatty acid synthesis, we have mimicked the lipogenic phenotype of cancer cells in Xenopus embryos by microinjection of RNA encoding the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c). Dramatic morphologic changes were observed that could be linked to alterations in Wnt and Hedgehog signaling, and ultimately to a distortion of the primary cilium. This is a sophisticated microtubular sensory organelle that is expressed on the surface of nearly every cell type and that is lost in many cancers. SREBP1c-induced loss of the primary cilium could be confirmed in mammalian Madin-Darby canine kidney (MDCK) cells and was mediated by changes in the supply of fatty acids. Conversely, inhibition of fatty acid synthesis in highly lipogenic human prostate cancer cells restored the formation of the primary cilium. Lipid-induced ciliary loss was associated with mislocalization of apical proteins, distortion of cell polarization, and aberrant epithelial tissue development as revealed in three-dimensional cultures of MDCK cells and in the developing mouse prostate. These data imply that tumor-associated lipogenesis, in addition to rendering cells more autonomous in terms of lipid supply, disturbs cilium formation and contributes to impaired environmental sensing, aberrant signaling, and distortion of polarized tissue architecture, which are all hallmarks of cancer.
Assuntos
Cílios/metabolismo , Embrião não Mamífero/metabolismo , Ácidos Graxos/biossíntese , RNA/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Linhagem Celular Tumoral , Cílios/genética , Embrião não Mamífero/embriologia , Feminino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Lipogênese/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Microinjeções , Microscopia Confocal , Microscopia Eletrônica de Varredura , Próstata/crescimento & desenvolvimento , Próstata/metabolismo , RNA/administração & dosagem , RNA/genética , Interferência de RNA , Transdução de Sinais/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
The atheroprotective effect of testosterone is thought to require aromatization of testosterone to estradiol, but no study has adequately addressed the role of the androgen receptor (AR), the major pathway for the physiological effects of testosterone. We used AR knockout (ARKO) mice on apolipoprotein E-deficient background to study the role of the AR in testosterone atheroprotection in male mice. Because ARKO mice are testosterone deficient, we sham operated or orchiectomized (Orx) the mice before puberty, and Orx mice were supplemented with placebo or a physiological testosterone dose. From 8 to 16 wk of age, the mice consumed a high-fat diet. In the aortic root, ARKO mice showed increased atherosclerotic lesion area (+80%, P < 0.05). Compared with placebo, testosterone reduced lesion area both in Orx wild-type (WT) mice (by 50%, P < 0.001) and ARKO mice (by 24%, P < 0.05). However, lesion area was larger in testosterone-supplemented ARKO compared with testosterone-supplemented WT mice (+57%, P < 0.05). In WT mice, testosterone reduced the presence of a necrotic core in the plaque (80% among placebo-treated vs. 12% among testosterone-treated mice; P < 0.05), whereas there was no significant effect in ARKO mice (P = 0.20). In conclusion, ARKO mice on apolipoprotein E-deficient background display accelerated atherosclerosis. Testosterone treatment reduced atherosclerosis in both WT and ARKO mice. However, the effect on lesion area and complexity was more pronounced in WT than in ARKO mice, and lesion area was larger in ARKO mice even after testosterone supplementation. These results are consistent with an AR-dependent as well as an AR-independent component of testosterone atheroprotection in male mice.
Assuntos
Aorta/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Receptores Androgênicos/metabolismo , Testosterona/metabolismo , Animais , Aorta/efeitos dos fármacos , Doenças da Aorta/genética , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/prevenção & controle , Pressão Sanguínea/fisiologia , Citocinas/sangue , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , Orquiectomia , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Testosterona/farmacologiaRESUMO
Activation of de novo lipogenesis in cancer cells is increasingly recognized as a hallmark of aggressive cancers and has been implicated in the production of membranes for rapid cell proliferation. In the current report, we provide evidence that this activation has a more profound role. Using a mass spectrometry-based phospholipid analysis approach, we show that clinical tumor tissues that display the lipogenic phenotype show an increase in the degree of lipid saturation compared with nonlipogenic tumors. Reversal of the lipogenic switch in cancer cells by treatment with the lipogenesis inhibitor soraphen A or by targeting lipogenic enzymes with small interfering RNA leads to a marked decrease in saturated and mono-unsaturated phospholipid species and increases the relative degree of polyunsaturation. Because polyunsaturated acyl chains are more susceptible to peroxidation, inhibition of lipogenesis increases the levels of peroxidation end products and renders cells more susceptible to oxidative stress-induced cell death. As saturated lipids pack more densely, modulation of lipogenesis also alters lateral and transversal membrane dynamics as revealed by diffusion of membrane-targeted green fluorescent protein and by the uptake and response to doxorubicin. These data show that shifting lipid acquisition from lipid uptake toward de novo lipogenesis dramatically changes membrane properties and protects cells from both endogenous and exogenous insults. These findings provide important new insights into the role of de novo lipogenesis in cancer cells, and they provide a rationale for the use of lipogenesis inhibitors as antineoplastic agents and as chemotherapeutic sensitizers.
Assuntos
Radicais Livres/farmacologia , Lipogênese/fisiologia , Lipídeos de Membrana/metabolismo , Neoplasias/metabolismo , Antibióticos Antineoplásicos/metabolismo , Divisão Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Colesterol/metabolismo , Doxorrubicina/metabolismo , Células HCT116/efeitos dos fármacos , Células HCT116/metabolismo , Humanos , Immunoblotting , Peroxidação de Lipídeos , Masculino , Neoplasias/patologia , Fosfolipídeos/metabolismo , Próstata/metabolismo , Próstata/patologia , Prostatectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Espectrometria de Massas por Ionização por Electrospray , Transfecção , Triglicerídeos/metabolismoRESUMO
Transgenic mouse models have contributed considerably to our understanding of the cellular and molecular mechanisms by which androgens control spermatogenesis. Cell-selective ablation of the androgen receptor (AR) in Sertoli cells (SC) results in a complete block in meiosis and unambiguously identifies the SC as the main cellular mediator of the effects of androgens on spermatogenesis. This conclusion is corroborated by similar knockouts in other potential testicular target cells. Mutations resulting in diminished expression of the AR or in alleles with increased length of the CAG repeat mimick specific human forms of disturbed fertility that are not accompanied by defects in male sexual development. Transcriptional profiling studies in mice with cell-selective and general knockouts of the AR, searching for androgen-regulated genes relevant to the control of spermatogenesis, have identified many candidate target genes. However, with the exception of Rhox5, the identified subsets of genes show little overlap. Genes related to tubular restructuring, cell junction dynamics, the cytoskeleton, solute transportation and vitamin A metabolism are prominently present. Further research will be needed to decide which of these genes are physiologically relevant and to identify genes that can be used as diagnostic tools or targets to modulate the effects of androgens in spermatogenesis.
Assuntos
Androgênios/fisiologia , Espermatogênese/fisiologia , Testículo/fisiologia , Androgênios/genética , Animais , Masculino , Camundongos , Camundongos Transgênicos , Receptores Androgênicos/fisiologia , Células de Sertoli/citologia , Células de Sertoli/fisiologia , Espermatogênese/genética , Testículo/citologiaRESUMO
Rhox5, the founding member of the reproductive homeobox on the X chromosome (Rhox) gene cluster, encodes a homeodomain-containing transcription factor that is selectively expressed in Sertoli cells, where it promotes the survival of male germ cells. To identify Rhox5-regulated genes, we generated 15P-1 Sertoli cell clones expressing physiological levels of Rhox5 from a stably transfected expression vector. Microarray analysis identified many genes altered in expression in response to Rhox5, including those encoding proteins controlling cell cycle regulation, apoptosis, metabolism, and cell-cell interactions. Fifteen of these Rhox5-regulated genes were chosen for further analysis. Analysis of Rhox5-null male mice indicated that at least nine of these are Rhox5-regulated in the testes in vivo. Many of them have distinct postnatal expression patterns and are regulated by Rhox5 at different postnatal time points. Most of them are expressed in Sertoli cells, indicating that they are candidates to be directly regulated by Rhox5. Transfection analysis with expression vectors encoding different mouse and human Rhox family members revealed that the regulatory response of a subset of these Rhox5-regulated genes is both conserved and redundant. Given that Rhox5 depends on androgen receptor (AR) for expression in Sertoli cells, we examined whether some Rhox5-regulated genes are also regulated by AR. We provide several lines of evidence that this is the case, leading us to propose that RHOX5 serves as a key intermediate transcription factor that directs some of the actions of AR in the testes.
Assuntos
Androgênios/farmacologia , Regulação da Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Receptores Androgênicos/fisiologia , Células de Sertoli/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Envelhecimento , Animais , Linhagem Celular , Células Clonais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Receptores Androgênicos/deficiência , Receptores Androgênicos/genética , Elementos de Resposta/genética , Espermatogênese/genética , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
In female mice, estrogen receptor-alpha (ERalpha) mediates the anabolic response of bone to mechanical loading. Whether ERalpha plays a similar role in the male skeleton and to what extent androgens and androgen receptor (AR) affect this response in males remain unaddressed. Therefore, we studied the adaptive response of in vivo ulna loading in AR-ERalpha knockout (KO) mice and corresponding male and female single KO and wild-type (WT) littermates using dynamic histomorphometry and immunohistochemistry. Additionally, cultured bone cells from WT and AR KO mice were subjected to mechanical loading by pulsating fluid flow in the presence or absence of testosterone. In contrast with female mice, ERalpha inactivation in male mice had no effect on the response to loading. Interestingly, loading induced significantly more periosteal bone formation in AR KO (+320%) and AR-ERalpha KO mice (+256%) compared with male WT mice (+114%) and had a stronger inhibitory effect on SOST/sclerostin expression in AR KO versus WT mice. In accordance, the fluid flow-induced nitric oxide production was higher in the absence of testosterone in bone cells from WT but not AR KO mice. In conclusion, AR but not ERalpha activation limits the osteogenic response to loading in male mice possibly via an effect on WNT signaling.
Assuntos
Osso e Ossos/fisiologia , Osteogênese , Receptores Androgênicos/metabolismo , Estresse Mecânico , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/enzimologia , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Feminino , Marcadores Genéticos , Glicoproteínas , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico/biossíntese , Osteogênese/efeitos dos fármacos , Periósteo/efeitos dos fármacos , Periósteo/metabolismo , Receptores Androgênicos/deficiência , Testosterona/farmacologia , Suporte de CargaRESUMO
BACKGROUND: Testicular microliths are calcifications found within the seminiferous tubules. In humans, testicular microlithiasis (TM) has an unknown etiology but may be significantly associated with testicular germ cell tumors. Factors inducing microlith development may also, therefore, act as susceptibility factors for malignant testicular conditions. Studies to identify the mechanisms of microlith development have been hampered by the lack of suitable animal models for TM. METHODS: This was an observational study of the testicular phenotype of different mouse models. The mouse models were: cryptorchid mice, mice lacking androgen receptors (ARs) on the Sertoli cells (SCARKO), mice with a ubiquitous loss of androgen ARs (ARKO), hypogonadal (hpg) mice which lack circulating gonadotrophins, and hpg mice crossed with SCARKO (hpg.SCARKO) and ARKO (hpg.ARKO) mice. RESULTS: Microscopic TM was seen in 94% of hpg.ARKO mice (n=16) and the mean number of microliths per testis was 81+/-54. Occasional small microliths were seen in 36% (n=11) of hpg testes (mean 2+/-0.5 per testis) and 30% (n=10) of hpg.SCARKO testes (mean 8+/-6 per testis). No microliths were seen in cryptorchid, ARKO or SCARKO mice. There was no significant effect of FSH or androgen on TM in hpg.ARKO mice. CONCLUSION: We have identified a mouse model of TM and show that lack of endocrine stimulation is a cause of TM. Importantly, this model will provide a means with which to identify the mechanisms of TM development and the underlying changes in protein and gene expression.