RESUMO
Male penis is required to become erect during copulation. In the upper (dorsal) part of penis, the erectile tissue termed corpus cavernosum (CC) plays fundamental roles for erection by regulating the inner blood flow. When blood flows into the CC, the microvascular complex termed sinusoidal space is reported to expand during erection. A novel in vitro explant system to analyze the dynamic erectile responses during contraction/relaxation is established. The current data show regulatory contraction/relaxation processes induced by phenylephrine (PE) and nitric oxide (NO) donor mimicking dynamic erectile responses by in vitro CC explants. Two-photon excitation microscopy (TPEM) observation shows the synchronous movement of sinusoidal space and the entire CC. By taking advantages of the CC explant system, tadalafil (Cialis) was shown to increase sinusoidal relaxation. Histopathological changes have been generally reported associating with erection in several pathological conditions. Various stressed statuses have been suggested to occur in the erectile responses by previous studies. The current CC explant model enables to analyze such conditions through directly manipulating CC in the repeated contraction/relaxation processes. Expression of oxidative stress marker and contraction-related genes, Hypoxia-inducible factor 1-alpha (Hif1a), glutathione peroxidase 1 (Gpx1), Ras homolog family member A (RhoA), and Rho-associated protein kinase (Rock), was significantly increased in such repeated contraction/relaxation. Altogether, it is suggested that the system is valuable for analyzing structural changes and physiological responses to several regulators in the field of penile medicine.
Assuntos
Ereção Peniana/fisiologia , Pênis/citologia , Animais , Células Cultivadas , Disfunção Erétil/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microscopia/métodos , Modelos Biológicos , Técnicas de Cultura de Órgãos , Pênis/fisiologia , Pênis/ultraestruturaRESUMO
Phosphatidylserine (PS), an anionic phospholipid enriched in the inner leaflet of the plasma membrane, is exposed to the outer leaflet during apoptosis. PS exposure was recently shown to be induced during tumor necrosis factor-induced necroptosis. We herein demonstrated that interferon (IFN)-γ induced necroptosis in Caspase-8-knockout mouse-derived embryonic fibroblasts (C8KO MEFs), as well as in WT MEFs co-treated with the pan-caspase inhibitor, z-VAD-fmk. PS exposure and necroptosis were significant after 6- and 24-h treatments with IFN-γ, respectively. To elucidate the molecular mechanisms underlying IFN-γ-induced PS exposure, we generated C8KO MEF-derived cell lines without the expression of RIPK3 (receptor-interacting protein kinase 3), an essential molecule in tumor necrosis factor-induced necroptosis, and IFN-γ-induced PS exposure and necrotic cell death were shown to be specifically inhibited by the loss of RIPK3 expression. Furthermore, the down-regulated expression of MLKL (mixed lineage kinase domain-like protein), a key molecule for inducing membrane rupture downstream of RIPK3 in necroptosis, abolished IFN-γ-induced PS exposure in C8KO MEFs. In human colorectal adenocarcinoma-derived HT29 cells, PS exposure and necroptosis were similarly induced by treatment with IFN-γ in the presence of Smac mimetics and z-VAD-fmk. The removal of IFN-γ from PS-exposing MEFs after a 6-h treatment completely inhibited necroptotic cell death but not the subsequent increase in the number of PS-exposing cells. Therefore, PS exposure mediated by RIPK3-activated MLKL oligomers was induced by a treatment with IFN-γ for a significant interval of time before the induction of necroptosis by membrane rupture.
Assuntos
Caspase 8/genética , Interferon gama/farmacologia , Necroptose/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Proteínas Quinases/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Caspase 8/metabolismo , Linhagem Celular , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases/química , Proteínas Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Histone H3 lysine 9 (H3K9) methylation is a hallmark of heterochromatin. H3K9 demethylation is crucial in mouse sex determination; The H3K9 demethylase Jmjd1a deficiency leads to increased H3K9 methylation at the Sry locus in embryonic gonads, thereby compromising Sry expression and causing male-to-female sex reversal. We hypothesized that the H3K9 methylation level at the Sry locus is finely tuned by the balance in activities between the H3K9 demethylase Jmjd1a and an unidentified H3K9 methyltransferase to ensure correct Sry expression. Here we identified the GLP/G9a H3K9 methyltransferase complex as the enzyme catalyzing H3K9 methylation at the Sry locus. Based on this finding, we tried to rescue the sex-reversal phenotype of Jmjd1a-deficient mice by modulating GLP/G9a complex activity. A heterozygous GLP mutation rescued the sex-reversal phenotype of Jmjd1a-deficient mice by restoring Sry expression. The administration of a chemical inhibitor of GLP/G9a enzyme into Jmjd1a-deficient embryos also successfully rescued sex reversal. Our study not only reveals the molecular mechanism underlying the tuning of Sry expression but also provides proof on the principle of therapeutic strategies based on the pharmacological modulation of epigenetic balance.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Desenvolvimento Sexual/genética , Animais , Feminino , Regulação da Expressão Gênica , Loci Gênicos , Gônadas/embriologia , Gônadas/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histona Desmetilases com o Domínio Jumonji/deficiência , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Análise de Sequência de DNA , Proteína da Região Y Determinante do Sexo/genéticaRESUMO
A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general-purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock-in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP-mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1-Cre-transgenic (tg) embryos almost equally as efficiently as from Nr5a1-hCD271-tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh-Cre-tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types.
Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Recombinação Genética , Células de Sertoli/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Mamíferos/embriologia , Feminino , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Imuno-Histoquímica , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA não Traduzido/genética , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Fator Esteroidogênico 1/genética , Fator Esteroidogênico 1/metabolismoRESUMO
Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.
Assuntos
Apoptose , Caspase 8/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Peptídeo Hidrolases/química , Proteínas de Ligação a RNA/química , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Animais , Autofagia , Caspase 8/metabolismo , Morte Celular , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Humanos , Linfoma de Células T/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Necrose , Espécies Reativas de Oxigênio , Linfócitos T/metabolismoRESUMO
JmjC domain-containing proteins are a class of enzymes responsible for histone demethylation. Previous studies revealed that the JmjC domain-containing protein KDM3A possesses intrinsic demethylase activity toward lysine 9 of histone H3 and plays essential roles in spermiogenesis. In contrast, the biological roles of JMJD1C, a KDM3A homolog in mice, are largely unknown. Here we present the crucial role of JMJD1C in male gametogenesis. Jmjd1c-deficient males became infertile due to the progressive reduction of germ cells after 3 mo of age. Importantly, Jmjd1c-deficient testes frequently contained abnormal tubules lacking developmentally immature germ cells. JMJD1C is most abundantly expressed in undifferentiated spermatogonia in mouse testis. The numbers of ZBTB16-positive spermatogonia and apoptotic germ cells in Jmjd1c-deficient testes decreased and increased in an age-dependent manner, respectively. Our studies demonstrated that JMJD1C contributes to the long-term maintenance of the male germ line.
Assuntos
Células-Tronco Adultas/metabolismo , Envelhecimento , Apoptose , Regulação Enzimológica da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Espermatogênese , Espermatogônias/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/patologia , Processamento Alternativo , Androstenodiona/sangue , Animais , Animais Recém-Nascidos , Infertilidade Masculina/sangue , Infertilidade Masculina/enzimologia , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/deficiência , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Mutantes/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Testículo/patologia , Testosterona/sangueRESUMO
Among caspase family members, Caspase-8 is unique, with associated critical activities to induce and suppress death receptor-mediated apoptosis and necroptosis, respectively. Caspase-8 inhibits necroptosis by suppressing the function of receptor-interacting protein kinase 1 (RIPK1 or RIP1) and RIPK3 to activate mixed lineage kinase domain-like (MLKL). Disruption of Caspase-8 expression causes embryonic lethality in mice, which is rescued by depletion of either Ripk3 or Mlkl, indicating that the embryonic lethality is caused by activation of necroptosis. Here, we show that knockdown of Caspase-8 expression in embryoid bodies derived from ES cells markedly enhances retinoic acid (RA)-induced cell differentiation and necroptosis, both of which are dependent on Ripk1 and Ripk3; however, the enhancement of RA-induced cell differentiation is independent of Mlkl and necrosome formation. RA treatment obviously enhanced the expression of RA-specific target genes having the retinoic acid response element (RARE) in their promoter regions to induce cell differentiation, and induced marked expression of RIPK1, RIPK3, and MLKL to stimulate necroptosis. Caspase-8 knockdown induced RIPK1 and RIPK3 to translocate into the nucleus and to form a complex with RA receptor (RAR), and RAR interacting with RIPK1 and RIPK3 showed much stronger binding activity to RARE than RAR without RIPK1 or RIPK3. In Caspase-8-deficient as well as Caspase-8- and Mlkl-deficient mouse embryos, the expression of RA-specific target genes was obviously enhanced. Thus, Caspase-8, RIPK1, and RIPK3 regulate RA-induced cell differentiation and necroptosis both in vitro and in vivo.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Tretinoína/farmacologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Embrião de Mamíferos/metabolismo , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos C57BL , Proteínas Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The mammalian sex-determining gene Sry induces male development. Since its discovery 30 years ago, Sry has been believed to be a single-exon gene. Here, we identified a cryptic second exon of mouse Sry and a corresponding two-exon type Sry (Sry-T) transcript. XY mice lacking Sry-T were sex-reversed, and ectopic expression of Sry-T in XX mice induced male development. Sry-T messenger RNA is expressed similarly to that of canonical single-exon type Sry (Sry-S), but SRY-T protein is expressed predominantly because of the absence of a degron in the C terminus of SRY-S. Sry exon2 appears to have evolved recently in mice through acquisition of a retrotransposon-derived coding sequence to replace the degron. Our findings suggest that in nature, SRY-T, not SRY-S, is the bona fide testis-determining factor.
Assuntos
Éxons , Genes Essenciais , Processos de Determinação Sexual , Proteína da Região Y Determinante do Sexo/genética , Animais , Sistemas CRISPR-Cas , Edição de Genes , Loci Gênicos , Masculino , Camundongos , Camundongos Transgênicos , RNA não Traduzido/genética , Deleção de Sequência , Transcrição GênicaRESUMO
Histone H3 lysine 9 (H3K9) methylation is dynamically regulated by methyltransferases and demethylases. In spermatogenesis, prospermatogonia differentiate into differentiating or undifferentiated spermatogonia after birth. However, the epigenetic regulation of prospermatogonia to spermatogonia transition is largely unknown. We found that perinatal prospermatogonia have extremely low levels of di-methylated H3K9 (H3K9me2) and that H3K9 demethylases, JMJD1A and JMJD1B, catalyze H3K9me2 demethylation in perinatal prospermatogonia. Depletion of JMJD1A and JMJD1B in the embryonic germline resulted in complete loss of male germ cells after puberty, indicating that H3K9me2 demethylation is essential for male germline maintenance. JMJD1A/JMJD1B-depleted germ cells were unable to differentiate into functional spermatogonia. JMJD1 isozymes contributed to activation of several spermatogonial stem cell maintenance genes through H3K9 demethylation during the prospermatogonia to spermatogonia transition, which we propose is key for spermatogonia development. In summary, JMJD1A/JMJD1B-mediated H3K9me2 demethylation promotes prospermatogonia to differentiate into functional spermatogonia by establishing proper gene expression profiles.
Assuntos
Células Germinativas/citologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Espermatogônias/citologia , Animais , Biocatálise , Cromossomos de Mamíferos/genética , Desmetilação , Perfilação da Expressão Gênica , Isoenzimas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Modelos Biológicos , Transcrição GênicaRESUMO
Orbital decompression (OD) for Graves' orbitopathy usually includes removal of orbital medial wall. While OD occasionally cause or worsen chronic sinusitis because of the obstructed sinus drainage by prolapsed orbital fat, to date few reports have been published on the management or prevention of sinusitis associated with OD. Here, we present two patients (three sides) with newly developed and one patient (one side) with worsening obstructive frontal sinusitis following OD. These three patients had OD including the removal of the superior attachment of uncinated process to lamina papyracea. Endoscopic modified Lothrop procedure (EMLP) was useful to relieve symptoms and keep an enough access to frontal sinuses for all cases. We also performed EMLP for another two patients (four sides) with pre-existing sinusitis before OD. Worsening of sinusitis could be avoided by EMLP before OD. EMLP was useful approach for both treatment and prevention of sinusitis related to OD. The superior attachment site of uncinated process and the pattern of frontal sinus drainage might predict the occurrence of obstructive frontal sinusitis following OD.
Assuntos
Descompressão Cirúrgica/efeitos adversos , Endoscopia/métodos , Sinusite Frontal/etiologia , Oftalmopatia de Graves/cirurgia , Complicações Pós-Operatórias , Adulto , Descompressão Cirúrgica/métodos , Feminino , Seio Frontal/diagnóstico por imagem , Sinusite Frontal/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: We conducted a 3-year prospective study on olfaction of patients with Parkinson's disease (PD) in order to examine the severity and frequency of smell disorder in PD using odor identification test, Open Essence (OE) and to verify the validity of olfactory tests as a predictor of cognitive symptom onset of PD. PATIENTS AND METHODS: We conducted a prospective study by performing an annual examination over a 3-year period. For 56 cases diagnosed with PD by the Department of Neurology at our hospital, OE and Jet Stream Olfactometry (JSO) were performed to assess the olfactory function, and Mini-Mental-State Examination (MMSE) was conducted to measure cognitive impairment. RESULTS: At the beginning, 56 cases were examined, of which 42 remained to be followed up for 3 years. Based on the results of baseline, we found a correlation between OE and the average cognitive thresholds of JSO, but did not find any correlation between OE and MMSE. OE (median 4.0â4.0) and the average cognitive thresholds of JSO (median 2.2â1.6)ãdecreased after 3 years, and MMSE (median 29â29) also declined, but not significantly. At the 3rd year, 6 cases with MMSE score of 23 or less were identified as suspected dementia and 36 cases with more than 24 points were defined as an invariant group. In order to distinguish these two groups, OE scores of baselines were evaluated with a combination of 12 odors. Sensitivity 1.0 and specificity 0.722 were obtained and the sensitivity+specificity value (1.722) was the highest when the number of correct answers was 4 or less using an odor combination of lumber, menthol, Japanese orange, gas for household use, Hinoki cypress and condensed milk. CONCLUSION: When the number of correct answers of 6 odors (lumber, menthol, Japanese orange, gas for household use, Hinoki cypress and condensed milk) is 4 or less in patients with PD, there is a possibility that MMSE declines in 3 years.
Assuntos
Demência/diagnóstico , Transtornos do Olfato/etiologia , Doença de Parkinson/complicações , Idoso , Idoso de 80 Anos ou mais , Demência/etiologia , Feminino , Humanos , Masculino , Testes de Estado Mental e Demência , Pessoa de Meia-Idade , Doença de Parkinson/fisiopatologia , Doença de Parkinson/psicologia , Estudos Prospectivos , Sensibilidade e Especificidade , Olfato/fisiologiaRESUMO
SRY is the master regulator of male sex determination in eutherian mammals. In mice, Sry expression is transcriptionally and epigenetically controlled in a developmental stage-specific manner. The Sry promoter undergoes demethylation in embryonic gonadal somatic cells at the sex-determining period. However, its molecular mechanism and in vivo significance remain unclear. Here, we report that the Sry promoter is actively demethylated during gonadal development, and TET2 plays a fundamental role in Sry demethylation. Tet2-deficient mice showed absence of 5-hydroxymethylcytosine in the Sry promoter. Furthermore, Tet2 deficiency diminished Sry expression, indicating that TET2-mediated DNA demethylation regulates Sry expression positively. We previously showed that the deficiency of the H3K9 demethylase Jmjd1a compromises Sry expression and induces male-to-female sex reversal. Tet2 deficiency enhanced the sex reversal phenotype of Jmjd1a-deficient mice. Thus, TET2-mediated active DNA demethylation and JMJD1A-mediated H3K9 demethylation contribute synergistically to sex determination.
Assuntos
Desmetilação do DNA , Proteínas de Ligação a DNA/metabolismo , Gônadas , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proteína da Região Y Determinante do Sexo/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Dioxigenases , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/embriologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Processos de Determinação Sexual , Fator Esteroidogênico 1/metabolismoRESUMO
OBJECTIVES: Parkinson disease (PD) is frequently associated with olfactory disorder at early stage, which is caused by deposition of Lewy bodies emerging from the olfactory bulb to higher olfactory centers. Early detection of olfactory disorder in the patients with PD may lead to the early diagnosis and treatment for this refractory disease. METHODS: Visual analog scale (VAS), Jet Stream Olfactometry, and Japanese smell identification test, Open Essence (OE), were carried out on 39 patients with PD. Thirty-one patients with postviral olfactory disorder (PVOD), which was caused by the olfactory mucosal dysfunction, were also enrolled in this study as control. RESULTS: There were no significant differences in detection thresholds (2.2 vs. 1.4, P=0.13), recognition thresholds (3.9 vs. 3.5, P=0.39) and OE (4.8 vs. 4.2, P=0.47) between PVOD and PD, while VAS scores of PVOD and PD were significantly different (2.0 and 6.2, P<0.01). In OE, significant differences were observed in the accuracy rates of menthol (68% vs. 44%, P=0.04) and Indian ink (42% vs. 15%, P=0.01) between PVOD and PD. Of particular interest, patients with PVOD tended to select "no detectable," while patients with PD tended to select wrong alternative other than "no smell detected." CONCLUSION: Discrepancy between VAS and OE, and high selected rates of wrong alternative other than "undetectable" in OE might be significant signs of olfactory dysfunction associated with PD.
RESUMO
Transforming growth factor beta (TGF-beta) has been implicated in the maintenance of homeostasis in various organs, including the gastric epithelium. In particular, TGF-beta-induced signaling was shown to be required for the differentiation-associated physiological apoptosis of gastric epithelial cells, but its mechanism has not been well understood. In this study, the molecular mechanism of TGF-beta-induced apoptosis was analyzed in a human gastric epithelial cell line, SNU16, as an in vitro model. Expression of Smad7 and Bcl-X(L), but not viral FLIP, was shown to prevent TGF-beta-induced apoptosis, indicating an exclusive requirement of the activation of Smad signaling pathway and mitochondrial dysfunction followed by activation of caspase-9. In addition, treatment with TGF-beta induced binding of Bim, a proapoptotic Bcl-2 homology domain 3 (BH3)-only protein, to Bcl-X(L), which is dependent on the activation of Smad, and reduction in the expression of Bim by RNA interference decreased the sensitivity to TGF-beta-induced apoptosis. Moreover, we found abnormalities in the gastric epithelium of both Bim and caspase-9 knockout mice; these abnormalities were associated with a defect of physiological apoptosis in gastric epithelial cells. These results indicate for the first time that TGF-beta is involved in the physiological loss of gastric epithelial cells by activating apoptosis mediated by Smad, Bim, and caspase-9.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Caspases/metabolismo , Epitélio/metabolismo , Mucosa Gástrica/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Caspase 9 , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Cruzamentos Genéticos , Cicloeximida/farmacologia , Feminino , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Ligação Proteica , Conformação Proteica , Interferência de RNA , Sensibilidade e Especificidade , Transdução de Sinais , Proteína Smad7/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta1 , Proteína bcl-X/metabolismoRESUMO
Regulation of gene expression without changing the DNA sequence is governed by epigenetic mechanisms. Epigenetic regulation is important for changing chromatin structure in response to environmental cues as well as maintaining chromatin structure after cell division. The epigenetic machinery can reversibly change chromatin function, allowing a wide variety of biological processes in multicellular organisms to be controlled. Epigenetic regulation ensures spatial and temporal accuracy of the expression of developmentally regulated genes. So far, few studies have focused on the relationship between epigenetic regulation and mammalian sex development, despite this being an interesting area of research. Sex development consists of three sequential stages: the undifferentiated stage, gonadal differentiation into testes or ovaries, and differentiation of internal and external genitalia. Some genetic studies have revealed that epigenetic regulation is required for proper gonadal differentiation in mice. Particularly, the epigenetic machinery plays an integral part in sex determination, which is the first step of gonadal differentiation. Mammalian sex determination is triggered by activation of the mammalian sex-determining gene, Sry, in a spatially and temporally accurate manner. Several studies have demonstrated that expression of Sry is controlled not only by specific transcription factors but also by the epigenetic machinery. Here, we focus on the epigenetic regulation of Sry expression.
Assuntos
Epigênese Genética , Mamíferos/genética , Processos de Determinação Sexual/genética , Animais , Metilação de DNA/genética , Histonas/metabolismo , Humanos , Modelos BiológicosRESUMO
Histone H3 lysine 9 (H3K9) methylation is unevenly distributed in mammalian chromosomes. However, the molecular mechanism controlling the uneven distribution and its biological significance remain to be elucidated. Here, we show that JMJD1A and JMJD1B preferentially target H3K9 demethylation of gene-dense regions of chromosomes, thereby establishing an H3K9 hypomethylation state in euchromatin. JMJD1A/JMJD1B-deficient embryos died soon after implantation accompanying epiblast cell death. Furthermore, combined loss of JMJD1A and JMJD1B caused perturbed expression of metabolic genes and rapid cell death in embryonic stem cells (ESCs). These results indicate that JMJD1A/JMJD1B-meditated H3K9 demethylation has critical roles for early embryogenesis and ESC maintenance. Finally, genetic rescue experiments clarified that H3K9 overmethylation by G9A was the cause of the cell death and perturbed gene expression of JMJD1A/JMJD1B-depleted ESCs. We summarized that JMJD1A and JMJD1B, in combination, ensure early embryogenesis and ESC viability by establishing the correct H3K9 methylated epigenome.
Assuntos
Desmetilação , Desenvolvimento Embrionário , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/deficiência , Lisina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Cromossomos de Mamíferos/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Metilação , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Mutação/genéticaRESUMO
Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1+ subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche.
Assuntos
Células Germinativas/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Nicho de Células-Tronco , Animais , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Masculino , Camundongos , Fenótipo , Espermatogônias/citologia , Tretinoína/farmacologiaRESUMO
Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Processamento de Proteína Pós-Traducional , Processos de Determinação Sexual/genética , Animais , Epididimo/anormalidades , Feminino , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Metilação , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Ovário/anormalidades , Ovário/enzimologia , Testículo/anormalidades , Testículo/enzimologia , Útero/anormalidadesRESUMO
The Y-linked gene Sry regulates mammalian sex determination in bipotential embryonic gonads. Here, we report that the transcription factors Six1 and Six4 are required for male gonadal differentiation. Loss of Six1 and Six4 together, but neither alone, resulted in a male-to-female sex-reversal phenotype in XY mutant gonads accompanied by a failure in Sry activation. Decreased gonadal precursor cell formation at the onset of Sry expression and a gonadal size reduction in both sexes were also found in mutant embryos. Forced Sry transgene expression in XY mutant gonads rescued testicular development but not the initial disruption to precursor growth. Furthermore, we identified two downstream targets of Six1/Six4 in gonadal development, Fog2 (Zfpm2) and Nr5a1 (Ad4BP/Sf1). These two distinct Six1/Six4-regulated pathways are considered to be crucial for gonadal development. The regulation of Fog2 induces Sry expression in male sex determination, and the regulation of Nr5a1 in gonadal precursor formation determines gonadal size.