RESUMO
Inflammation is critical for host defense, but without appropriate control, it can cause chronic disease or even provoke fatal responses. Here we identify a mechanism that limits the inflammatory response. Probing the responses of macrophages to the key sensory Toll-like receptors, we identify that the Broad-complex, Tramtrack and Bric-a-brac/poxvirus and zinc finger (BTB/POZ), transcriptional regulator promyelocytic leukemia zinc finger (PLZF) limits the expression of inflammatory gene products. In accord with this finding, PLZF-deficient animals express higher levels of potent inflammatory cytokines and mount exaggerated inflammatory responses to infectious stimuli. Temporal quantitation of inflammatory gene transcripts shows increased gene induction in the absence of PLZF. Genome-wide analysis of histone modifications distinguish that PLZF establishes basal activity states of early response genes to maintain immune homeostasis and limit damaging inflammation. We show that PLZF stabilizes a corepressor complex that encompasses histone deacetylase activity to control chromatin. Together with our previous demonstration that PLZF promotes the antiviral response, these results suggest a strategy that could realize one of the major goals of immune therapy to retain immune resistance to pathogens while curbing damaging inflammation.
Assuntos
Cromatina/metabolismo , Inflamação/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Transdução de Sinais , Animais , Infecções Bacterianas/metabolismo , Imunoprecipitação da Cromatina , Transferência Ressonante de Energia de Fluorescência , Histona Desacetilases/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína com Dedos de Zinco da Leucemia Promielocítica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
To identify mechanisms of anabolic androgen action in muscle, we generated male and female genomic androgen receptor (AR) knockout (ARKO) mice, and characterized muscle mass, contractile function, and gene expression. Muscle mass is decreased in ARKO males, but normal in ARKO females. The levator ani muscle, which fails to develop in normal females, is also absent in ARKO males. Force production is decreased from fast-twitch ARKO male muscle, and slow-twitch muscle has increased fatigue resistance. Microarray analysis shows up-regulation of genes encoding slow-twitch muscle contractile proteins. Real-time PCR confirms that expression of genes encoding polyamine biosynthetic enzymes, ornithine decarboxylase (Odc1), and S-adenosylmethionine decarboxylase (Amd1), is reduced in ARKO muscle, suggesting androgens act through regulation of polyamine biosynthesis. Altered expression of regulators of myoblast progression from proliferation to terminal differentiation suggests androgens also promote muscle growth by maintaining myoblasts in the proliferate state and delaying differentiation (increased Cdkn1c and Igf2, decreased Itg1bp3). A similar pattern of gene expression is observed in orchidectomized male mice, during androgen withdrawal-dependent muscle atrophy. In conclusion, androgens are not required for peak muscle mass in females. In males, androgens act through the AR to regulate multiple gene pathways that control muscle mass, strength, and fatigue resistance.
Assuntos
Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/fisiopatologia , Receptores Androgênicos/deficiência , Androgênios/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Expressão Gênica , Redes Reguladoras de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/patologia , Mioblastos Esqueléticos/patologia , Mioblastos Esqueléticos/fisiologia , Orquiectomia , Tamanho do Órgão , Receptores Androgênicos/genética , Receptores Androgênicos/fisiologia , Caracteres Sexuais , Testículo/fisiologiaRESUMO
The ability to rapidly and unequivocally distinguish heterozygous from homozygous transgenic mice is an integral part of any breeding strategy. Here we describe a quick and simple protocol for determining the zygosity of transgenic mice at multiple loci in a single reaction. This involved the development of a multiplex ligation-dependent probe amplification (MLPA) probe mix to simultaneously measure common transgenic alleles such as Cre recombinase (Cre), neomycin (Neo), beta-galactosidase (LacZ) and enhanced green fluorescent protein (eGFP), as well as loci specific to the X and Y chromosome to allow sexing. Each reaction required as little as 100 ng of genomic DNA isolated from a tail biopsy using a simple procedure. Normalization against autosomal control loci resulted in 100% call accuracy, with no ambiguous results. This probe mix can be easily implemented in any laboratory with access to a PCR machine and a DNA sequencer, and can be rapidly adapted to genotype any additional loci of interest.
Assuntos
Camundongos Transgênicos/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Genótipo , Camundongos , Reação em Cadeia da Polimerase , TransgenesRESUMO
We previously generated a conditional floxed mouse line to study androgen action, in which exon 3 of the androgen receptor (AR) gene is flanked by loxP sites, with the neomycin resistance gene present in intron 3. Deletion of exon 3 in global AR knockout mice causes androgen insensitivity syndrome, characterized by genotypic males lacking normal masculinization. We now report that male mice carrying the floxed allele (AR(lox)) have the reverse phenotype, termed hyperandrogenization. AR(lox) mice have increased mass of androgen-dependent tissues, including kidney, (P < 0.001), seminal vesicle (P < 0.001), levator ani muscle (P = 0.001), and heart (P < 0.05). Serum testosterone is not significantly different. Testis mass is normal, histology shows normal spermatogenesis, and AR(lox) males are fertile. AR(lox) males also have normal AR mRNA levels in kidney, brain, levator ani, liver, and testis. This study reaffirms the need to investigate the potential phenotypic effects of floxed alleles in the absence of cre in tissue-specific knockout studies. In addition, this androgen hypersensitivity model may be useful to further investigate the effects of subtle perturbations of androgen action in a range of androgen-responsive systems in the male.
Assuntos
Hiperandrogenismo/genética , Perda de Heterozigosidade/fisiologia , Receptores Androgênicos/genética , Animais , Peso Corporal/genética , Cruzamentos Genéticos , Feminino , Regulação da Expressão Gênica/fisiologia , Coração/anatomia & histologia , Integrases/genética , Integrases/metabolismo , Rim/anatomia & histologia , Fígado/anatomia & histologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Testículo/anatomia & histologia , Testículo/citologia , Testosterona/sangueRESUMO
Spermatogenesis requires androgen but, paradoxically, oestradiol (E2) treatment stimulates spermatogenic development in gonadotrophin- and androgen-deficient hypogonadal (hpg) mice. The mechanisms of E2-induced spermatogenesis were investigated by determining intratesticular E2 levels and testis cell populations in E2-treated hpg male mice, and E2 spermatogenic actions were determined in androgen receptor-knockout (ARKO) mice. Despite increased serum E2 concentrations (150-300 pmol L(-1)), intratesticular E2 concentrations declined fivefold (P < 0.001) in E2-treated v. untreated hpg male mice. Serum FSH reached 40% of normal and total testicular numbers of known FSH-responsive Sertoli, spermatogonia and meiotic spermatocyte populations were significantly (P < 0.001) elevated 1.7-, 4- and 13-fold, respectively. However, E2 administration also increased androgen-dependent pachytene spermatocytes and post-meiotic spermatids to levels comparable with testosterone-treated hpg testes. Selective investigation of androgen receptor involvement used E2-treated ARKO mice, which were found to exhibit increased (1.6-fold; P < 0.05) intratesticular E2 concentrations and suppression of the elevated serum gonadotrophins, although FSH remained twofold higher than normal. However, testis size and total Sertoli, spermatogonia and spermatocyte numbers were not increased in E2-treated ARKO male mice. Therefore, E2-stimulated murine spermatogenic development occurs with markedly suppressed and not elevated intratesticular E2 levels and displays an absolute requirement for functional androgen receptors. We propose that this paradoxical E2 spermatogenic response is explained by predominantly extratesticular E2 actions, increasing FSH to combine with residual androgen activity in hpg testes to stimulate pre- to post-meiotic development.
Assuntos
Estradiol/farmacologia , Receptores Androgênicos/fisiologia , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Estradiol/metabolismo , Hormônio Foliculoestimulante/sangue , Hipogonadismo/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão/efeitos dos fármacos , Receptores Androgênicos/genética , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacos , Transdução de Sinais/fisiologia , Espermatogênese/fisiologia , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Testículo/citologia , Testículo/metabolismo , Testosterona/metabolismoRESUMO
UNLABELLED: The mechanism of androgen action on bone was studied in male mice with the AR deleted in mature osteoblasts. These mice had decreased trabecular bone volume associated with a decrease in trabecular number, suggesting that androgens may act directly on osteoblasts to maintain trabecular bone. INTRODUCTION: Androgens modulate bone cell activity and are important for the maintenance of bone mass. However, the mechanisms by which they exert these actions on bone remain poorly defined. The aim of this study was to investigate the role of androgens acting through the classical androgen receptor (AR) signaling pathways (i.e., DNA-binding dependent pathways) in osteoblasts using male mice in which exon 3 of the AR gene was deleted specifically in mature osteoblasts. MATERIALS AND METHODS: Mice with a floxed exon 3 of the AR gene were bred with Col 2.3-cre transgenic mice, in which Cre recombinase is expressed in mineralizing osteoblasts. The skeletal phenotype of mutant mice was assessed by histomorphometry and quantitative microCT at 6, 12, and 32 weeks of age (n=8 per group). Wildtype, hemizygous exon 3 floxed and hemizygous Col 2.3-cre male littermates were used as controls. Data were analyzed by one-way ANOVA and Tukey's posthoc test. RESULTS: microCT analysis of the fifth lumbar vertebral body showed that these mice had reduced trabecular bone volume (p<0.05) at 32 weeks of age compared with controls. This was associated with a decrease in trabecular number (p<0.01) at 12 and 32 weeks of age, suggesting increased bone resorption. These effects were accompanied by a reduction in connectivity density (p<0.01) and an increase in trabecular separation (p<0.01). A similar pattern of trabecular bone loss was observed in the distal femoral metaphysis at 32 weeks of age. CONCLUSIONS: These findings show that inactivation of the DNA binding-dependent functions of the AR, specifically in mature osteoblasts in male mice, results in increased bone resorption and decreased structural integrity of the bone, leading to a reduction in trabecular bone volume at 32 weeks of age. These data provide evidence of a role for androgens in the maintenance of trabecular bone volume directly through DNA binding-dependent actions of the AR in mature osteoblasts.
Assuntos
Sequência de Bases , Reabsorção Óssea/metabolismo , Éxons , Osteoblastos/metabolismo , Receptores Androgênicos/metabolismo , Deleção de Sequência , Envelhecimento , Animais , Reabsorção Óssea/genética , Cruzamento , Éxons/genética , Vértebras Lombares/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Fenótipo , Receptores Androgênicos/genéticaRESUMO
UNLABELLED: A PTH gene has been isolated from the fish Fugu rubripes. The encoded protein of 80 amino acid has the lowest homology with any of the PTH family members. Fugu PTH(1-34) had 5-fold lower potency than human PTH(1-34) in a mammalian cell system. INTRODUCTION: Parathyroid hormone (PTH) is the major hypercalcemic hormone in higher vertebrates. Fish lack parathyroid glands, but there have numerous attempts to identify and isolate PTH from fish. MATERIALS AND METHODS: Polymerase chain reaction (PCR) was performed with primers based on preliminary data from the Joint Genome Institute database. PCR amplification was performed on genomic DNA isolated from Fugu rubripes. PCR products were purified and DNA was sequenced. All sequence was confirmed from more than one independently amplified PCR product. Multiple sequence alignments were carried out, and the percentage of identities and similarities were calculated. An unrooted phylogenetic tree, using all the known PTH and PTH-related protein (PTHrP) amino acid sequences, was determined. Synthetic peptides were tested in a biological assay that measured cyclic adenosine 3',5'-monophosphate formation in UMR106.1 cells. Rabbit polyclonal antisera specific for N-terminal human PTHrP and one rabbit polyclonal antiserum specific for N terminus hPTH were used to test the cross-reactivity with fPTH(1-34) in immunoblots.
Assuntos
Hormônio Paratireóideo/análise , Hormônio Paratireóideo/genética , Takifugu/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Molecular , Humanos , Dados de Sequência Molecular , Hormônio Paratireóideo/química , Hormônio Paratireóideo/imunologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments. RESULTS: This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape. CONCLUSION: The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines.
Assuntos
Carcinoma Embrionário/genética , Epigênese Genética , Perfilação da Expressão Gênica , Neoplasias Embrionárias de Células Germinativas/genética , Seminoma/genética , Carcinoma Embrionário/patologia , Linhagem Celular Tumoral , Metilação de DNA , Humanos , Neoplasias Embrionárias de Células Germinativas/patologia , Seminoma/patologiaRESUMO
Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.
Assuntos
Variações do Número de Cópias de DNA/genética , Disgenesia Gonadal 46 XY/genética , Algoritmos , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Aberrações Cromossômicas , Deleção Cromossômica , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 8/genética , Feminino , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica , Rearranjo Gênico/genética , Gônadas/embriologia , Gônadas/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição SOX9/genéticaRESUMO
We used our genomic androgen receptor (AR) knockout (ARKO) mouse model, in which the AR is unable to bind DNA to: 1) document gender differences between males and females; 2) identify the genomic (DNA-binding-dependent) AR-mediated actions in males; 3) determine the contribution of genomic AR-mediated actions to these gender differences; and 4) identify physiological genomic AR-mediated actions in females. At 9 weeks of age, control males had higher body, heart and kidney mass, lower spleen mass, and longer and larger bones compared to control females. Compared to control males, ARKO males had lower body and kidney mass, higher splenic mass, and reductions in cortical and trabecular bone. Deletion of the AR in ARKO males abolished the gender differences in heart and cortical bone. Compared with control females, ARKO females had normal body weight, but 14% lower heart mass and heart weight/body weight ratio. Relative kidney mass was also reduced, and relative spleen mass was increased. ARKO females had a significant reduction in cortical bone growth and changes in trabecular architecture, although with no net change in trabecular bone volume. In conclusion, we have shown that androgens acting via the genomic AR signaling pathway mediate, at least in part, the gender differences in body mass, heart, kidney, spleen, and bone, and play a physiological role in the regulation of cardiac, kidney and splenic size, cortical bone growth, and trabecular bone architecture in females.