RESUMO
How maternal Ezh1 and Ezh2 function in H3K27 methylation in vivo in pre-implantation embryos and during embryonic development is not clear. Here, we have deleted Ezh1 and Ezh2 alone or simultaneously from mouse oocytes. H3K27me3 was absent in oocytes without Ezh2 alone, while both H3K27me2 and H3K27me3 were absent in Ezh1/Ezh2 (Ezh1/2) double knockout (KO) oocytes. The effects of Ezh1/2 maternal KO were inherited in zygotes and early embryos, in which restoration of H3K27me3 and H3K27me2 was delayed by the loss of Ezh2 alone or of both Ezh1 and Ezh2. However, the ablation of both Ezh1 and Ezh2, but not Ezh1 or Ezh2 alone, led to significantly decreased litter size due to growth retardation post-implantation. Maternal Ezh1/2 deficiency caused compromised H3K27me3 and pluripotent epiblast cells in late blastocysts, followed by defective embryonic development. By using RNA-seq, we examined crucial developmental genes in maternal Ezh1/2 KO embryos and identified 80 putatively imprinted genes. Maternal Ezh1/2-H3K27 methylation is inherited in offspring embryos and has a critical effect on fetal and placental development. Thus, this work sheds light on maternal epigenetic modifications during embryonic development.
Assuntos
Histonas , Complexo Repressor Polycomb 2 , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Camadas Germinativas/metabolismo , Camundongos , Oócitos/metabolismo , Placenta/metabolismo , Complexo Repressor Polycomb 2/metabolismo , GravidezRESUMO
Approximately 75% of failed pregnancies are considered to be due to embryo implantation failure or defects. Nevertheless, the explicit signaling mechanisms governing this process have not yet been elucidated. Here, we found that conditional deletion of the Shp2 gene in mouse uterine stromal cells deferred embryo implantation and inhibited the decidualization of stromal cells, which led to embryonic developmental delay and to the death of numerous embryos mid-gestation, ultimately reducing female fertility. The absence of Shp2 in stromal cells increased the proliferation of endometrial epithelial cells, thereby disturbing endometrial epithelial remodeling. However, Shp2 deletion impaired the proliferation and polyploidization of stromal cells, which are distinct characteristics of decidualization. In human endometrial stromal cells (hESCs), Shp2 expression gradually increased during the decidualization process. Knockout of Shp2 blocked the decidual differentiation of hESCs, while Shp2 overexpression had the opposite effect. Shp2 knockout inhibited the proliferation of hESCs during decidualization. Whole gene expression profiling analysis of hESCs during the decidualization process showed that Shp2 deficiency disrupted many signaling transduction pathways and gene expression. Analyses of hESCs and mouse uterine tissues confirmed that the signaling pathways extracellular regulated protein kinases (ERK), protein kinase B (AKT), signal transducer and activator of transcription 3 (STAT3) and their downstream transcription factors CCAAT/enhancer binding protein ß (C/EBPß) and Forkhead box transcription factor O1 (FOXO-1) were involved in the Shp2 regulation of decidualization. In summary, these results demonstrate that Shp2 plays a crucial role in stromal decidualization by mediating and coordinating multiple signaling pathways in uterine stromal cells. Our discovery possibly provides a novel key regulator of embryo implantation and novel therapeutic target for pregnancy failure.
Assuntos
Decídua/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Útero/citologia , Animais , Linhagem Celular , Proliferação de Células , Implantação do Embrião , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Camundongos , Gravidez , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais , Células Estromais/citologia , Células Estromais/metabolismo , Útero/metabolismoRESUMO
Maternal histone methyltransferase is critical for epigenetic regulation and development of mammalian embryos by regulating histone and DNA modifications. Here, we reported a novel mechanism by revealing the critical effects of maternal Ezh1/2 deletion on mitochondria in MII oocytes and early embryos in mice. We found that Ezh1/2 knockout in mouse MII oocytes impaired the structure of mitochondria and decreased its number, but membrane potential and respiratory function of mitochondrion were increased. The similar effects of Ezh1/2 deletion have been observed in 2-cell and morula embryos, indicating that the effects of maternal Ezh1/2 deficiency on mitochondrion extend to early embryos. However, the loss of maternal Ezh1/2 resulted in a severe defect of morula: the number, membrane potential, respiratory function, and ATP production of mitochondrion dropped significantly. Content of reactive oxygen species was raised in both MII oocytes and early embryos, suggesting maternal Ezh1/2 knockout induced oxidative stress. In addition, maternal Ezh1/2 ablation interfered the autophagy in morula and blastocyst embryos. Finally, maternal Ezh1/2 deletion led to cell apoptosis in blastocyst embryos in mice. By analyzing the gene expression profile, we revealed that maternal Ezh1/2 knockout affected the expression of mitochondrial related genes in MII oocytes and early embryos. The chromatin immunoprecipitation-polymerase chain reaction assay demonstrated that Ezh1/2 directly regulated the expression of genes Fxyd6, Adpgk, Aurkb, Zfp521, Ehd3, Sgms2, Pygl, Slc1a1, and Chst12 by H3K27me3 modification. In conclusion, our study revealed the critical effect of maternal Ezh1/2 on the structure and function of mitochondria in oocytes and early embryos, and suggested a novel mechanism underlying maternal epigenetic regulation on early embryonic development through the modulation of mitochondrial status.
Assuntos
Mitocôndrias , Oócitos , Complexo Repressor Polycomb 2 , Animais , Feminino , Camundongos , Apoptose/genética , Autofagia/genética , Blastocisto/metabolismo , Desenvolvimento Embrionário/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Potencial da Membrana Mitocondrial , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/genética , Mórula/metabolismo , Oócitos/metabolismo , Estresse Oxidativo/genética , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Espécies Reativas de Oxigênio/metabolismo , Histonas/metabolismoRESUMO
The standard isothermal titration calorimetry (ITC) curve, characterized as a typical sigmoid is strictly confined by the so-called c value, which is a ratio of titrand concentration to KD. The proper c value with a range from 5 to 500 is commonly recommended as a standard protocol in routine detection process for acquiring the reliable fitting results in 1:1 binding mode. However, if the c value is less than "1" due to the weak binding or low concentration of analyte, fitting precision gets unstable and susceptible to the data noise. Herein, we first got a deep discussion into the reliability of the fitting procedure for 1:1 binding mode by data simulation, then quantized the effect of several affecting factors on the precision of parameters estimation through mathematical analysis. Finally, we proposed the value of 2~4 times KD for final ligand concentration is optimal for the ITC titration in low c system (c < 1). All the theoretical derivations were further verified by a practical experiment of Magnesium-EDTA binding test.
Assuntos
Calorimetria/estatística & dados numéricos , Ácido Edético/química , Cloreto de Magnésio/química , TermodinâmicaRESUMO
Our previous studies have found that Growth factor receptor-bound protein 2-associated binding protein 2 (Gab2)-a docking protein-governs the development of fatty liver disease. Here, we further demonstrate that Gab2 mediates hepatocarcinogenesis. Compared with a faint expression in para-carcinoma tissue, Gab2 was highly expressed in â¼60-70% of human hepatocellular carcinoma (HCC) specimens. Deletion of Gab2 dramatically suppressed diethylnitrosamine-induced HCC in mice. The oncogenic effects of Gab2 in HepG2 cells were promoted by Gab2 overexpression but were rescued by Gab2 knockdown. Furthermore, Gab2 knockout in HepG2 cells restrained cell proliferation, migration and tumor growth in nude mice. Signaling pathway analysis with protein kinase inhibitors demonstrated that oncogenic regulation by Gab2 in hepatic cells involved multiple signaling molecules, including ERK, Akt, and Janus kinases (Jaks), especially those that mediate inflammatory signaling. IL-6 signaling was increased by Gab2 overexpression and impaired by Gab2 deletion via regulation of Jak2 and signal transducer and activator of transcription 3 phosphorylation and the expression of downstream genes, such as Bcl-2 (B-cell lymphoma 2), c-Myc, MMP7 (matrix metalloproteinase-7), and cyclin D1in vitro and in vivo These data indicate that Gab2 mediates the pathologic progression of HCC by integrating multiple signaling pathways and suggest that Gab2 might be a powerful therapeutic target for HCC.-Cheng, J., Zhong, Y., Chen, S., Sun, Y., Huang, L., Kang, Y., Chen, B., Chen, G., Wang, F., Tian, Y., Liu, W., Feng, G.-S., Lu, Z. Gab2 mediates hepatocellular carcinogenesis by integrating multiple signaling pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Hepatocelular/metabolismo , Dietilnitrosamina/toxicidade , Neoplasias Hepáticas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Movimento Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transdução de Sinais/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Decidualization of endometrial stroma is a key step in embryo implantation and its abnormality often leads to pregnancy failure. Stromal decidualization is a very complex process that is co-regulated by estrogen, progesterone and many local factors. The signaling protein SHP2 encoded by PTPN11 is dynamically expressed in decidualized endometrial stroma and mediates and integrates various signals to govern the decidualization. In the present study, we investigate the mechanism of PTPN11 gene transcription. Estrogen, progesterone and cAMP co-induced decidualization of human endometrial stromal cell in vitro, but only progesterone and cAMP induced SHP2 expression. Using the luciferase reporter, we refined a region from -229 bp to +1 bp in the PTPN11 gene promoter comprising the transcriptional core regions that respond to progesterone and cAMP. Progesterone receptor (PGR) and cAMP-responsive element-binding protein 1 (CREB1) were predicted to be transcription factors in this core region by bioinformatic methods. The direct binding of PGR and CREB1 on the PTPN11 promoter was confirmed by electrophoretic mobility and chromatin immunoprecipitation in vitro. Knockdown of PGR and CREB1 protein significantly inhibited the expression of SHP2 induced by medroxyprogesterone acetate and cAMP. These results demonstrate that transcription factors PGR and CREB1 bind to the PTPN11 promoter to regulate the expression of SHP2 in response to decidual signals. Our results explain the transcriptional expression mechanism of SHP2 during decidualization and promote the understanding of the mechanism of decidualization of stromal cells.
Assuntos
Progesterona , Receptores de Progesterona , Feminino , Humanos , Gravidez , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Decídua/metabolismo , Endométrio/metabolismo , Estrogênios , Progesterona/farmacologia , Progesterona/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Células Estromais/metabolismoRESUMO
The timely onset of female parturition is a critical determinant for pregnancy success. The highly heterogenous maternal decidua has been increasingly recognized as a vital factor in setting the timing of labor. Despite the cell type specific roles in parturition, the role of the uterine epithelium in the decidua remains poorly understood. This study uncovers the critical role of epithelial SHP2 in parturition initiation via COX1 and COX2 derived PGF2α leveraging epithelial specific Shp2 knockout mice, whose disruption contributes to delayed parturition initiation, dystocia and fetal deaths. Additionally, we also show that there are distinct types of epithelium in the decidua approaching parturition at single cell resolution accompanied with profound epithelium reformation via proliferation. Meanwhile, the epithelium maintains the microenvironment by communicating with stromal cells and macrophages. The epithelial microenvironment is maintained by a close interaction among epithelial, stromal and macrophage cells of uterine stromal cells. In brief, this study provides a previously unappreciated role of the epithelium in parturition preparation and sheds lights on the prevention of preterm birth.
Assuntos
Fenômenos Bioquímicos , Trabalho de Parto , Nascimento Prematuro , Animais , Feminino , Humanos , Recém-Nascido , Camundongos , Gravidez , Parto , ÚteroRESUMO
The incidence of liver cancer ranks seventh globally, with nearly half of all cases occurring in East Asia, but currently, there are very few drugs to treat it. Our previous studies demonstrated that the signal integration protein Gab2 is a potential drug target for the prevention and therapy of liver cancer. Here, we screened for and identified two miRNAs that target Gab2 to suppress the proliferation and migration of hepatocellular carcinoma (HCC) cells. First, we predicted Gab2-targeting miRNAs through biological websites, and we selected nine miRNAs that were reported in the literature as being abnormally expressed in liver cancer and fatty liver tissue. Then, we measured the expression of these miRNAs in the hepatic epithelial cell line HL-7702 and the HCC cell line HepG2. The expression levels of miR-9, miR-181a, miR-181c, miR-34a, and miR-134 were high in HL-7702 cells but low in HepG2 cells, and their expression patterns were the opposite of Gab2 in these cells. Furthermore, we transfected miR-9, miR-34a, miR-181a, and miR-181c mimics into HepG2 cells and found that only miR-9 and miR-181a reduced the level of Gab2 proteins. miR-9 also reduced the Gab2 mRNA level, but miR-181a did not affect the Gab2 mRNA levels. Using a miRNA-Gab2 3'UTR binding reporter, we confirmed that miR-9 and miR-181a bind to the Gab2 3'UTR region. Finally, we introduced miR-9 and miR-181a mimics into HepG2 cells and found that cell proliferation and migration were significantly inhibited. In conclusion, we identified two novel miRNAs targeting Gab2 and provided potential drug targets for the prevention and treatment of liver cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Humanos , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Proliferação de Células/genética , Células Hep G2 , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/metabolismoRESUMO
In mammals, the growth and maturation of oocytes within growing follicles largely depends on ovarian granulosa cells (GCs) in response to gonadotropin stimulation. Many signals have been shown to regulate GC proliferation and apoptosis. However, whether the tyrosine phosphatase SHP2 is involved remains unclear. In this study, we identified the crucial roles of SHP2 in modulating GC proliferation and apoptosis. The production of both mature oocytes and pups was increased in mice with Shp2 specifically deleted in ovarian GCs via Fshr-Cre. Shp2 deletion simultaneously promoted GC proliferation and inhibited GC apoptosis. Furthermore, Shp2 deficiency promoted, while Shp2 overexpression inhibited, the proliferation of cultured primary mouse ovarian GCs and the human ovarian granulosa-like tumor cell line KGN in vitro. Shp2 deficiency promoted follicule-stimulating hormone (FSH)-activated phosphorylation of AKT in vivo. SHP2 deficiency reversed the inhibitory effect of hydrogen peroxide (H2O2) on AKT activation in KGN cells. H2O2 treatment promoted the interaction between SHP2 and the p85 subunit of PI3K in KGN cells. Therefore, SHP2 in GCs may act as a negative modulator to balance follicular development by suppressing PI3K/AKT signaling. The novel function of SHP2 in modulating proliferation and apoptosis of GCs provides a potential therapeutic target for the clinical treatment of follicle developmental dysfunction.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Feminino , Camundongos , Humanos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Peróxido de Hidrogênio/metabolismo , Células da Granulosa/metabolismo , Tirosina/metabolismo , Tirosina/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/farmacologia , MamíferosRESUMO
BACKGROUND: The zona pellucida (ZP) of human oocytes plays essential protective roles in sperm-egg interactions during fertilisation and embryo development. ZP4-null female rabbits exhibit a thin and irregular ZP, which severely impairs embryo development and fertility. However, the effects of ZP4 defect on human female reproduction remain unknown. METHODS AND RESULTS: We performed whole-exome sequencing in 26 female patients with abnormal (thin and irregular) ZP and identified heterozygous variants in ZP4 (OMIM: 613514) from 3 patients (approximately 11%). No ZP4 variant was found in the 30 control women with proven fertility. We constructed ZP4-mutated plasmids and found that the variants reduced the secretion of ZP4 in vitro. Lower suction pressure facilitated egg retrieval, and intracytoplasmic sperm injection (ICSI) was a desirable treatment for ZP4-mutated patients with abnormal ZP. CONCLUSIONS: We identified ZP4 as a novel gene for human abnormal ZP and found that lower suction pressure and ICSI are efficient treatment strategies.
Assuntos
Infertilidade Feminina/genética , Glicoproteínas da Zona Pelúcida/genética , Desenvolvimento Embrionário , Feminino , Fertilidade , Expressão Gênica , Humanos , Infertilidade Feminina/patologia , Mutação , Sequenciamento do Exoma , Zona Pelúcida/patologia , Glicoproteínas da Zona Pelúcida/metabolismoRESUMO
Tyrosine phosphatase Src-homology phosphotyrosyl phosphatase 2 (Shp2) was identified as a potential molecular target for therapeutic treatment of diabetes and obesity. However, there is still no systematic research on the enhancers for the Shp2 enzyme. The present study established a novel powerful model for the high-throughput screening of Shp2 enhancers and successfully identified a new specific Shp2 enhancer, oleanolic acid, from Chinese herbs.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Medicamentos de Ervas Chinesas/química , Forsythia/química , Hipoglicemiantes/uso terapêutico , Obesidade/tratamento farmacológico , Ácido Oleanólico/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus/enzimologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/enzimologia , Humanos , Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/farmacologia , Camundongos , Obesidade/enzimologia , Ácido Oleanólico/isolamento & purificação , Ácido Oleanólico/farmacologiaRESUMO
Src homology-2 domain-containing protein tyrosine phosphatase (Shp2), a novel proto-oncogenic protein, is an important target in cancer therapy research. Approximately 2000 plant extracts were screened to find its natural specific inhibitors, with the ethyl acetate (EtOAc) active extract of the root of Angelica dahurica showing considerable inhibitory effects (IC(50)=21.6 mg/L). Bioguided isolation of EtOAc extract led to 13 compounds, including 10 fatty acids and derivatives. All these compounds were isolated from the plant for the first time. The inhibitory effects of these compounds on the enzyme activities of Shp2, VH1-related human protein (VHR), and hematopoietic protein tyrosine phosphatase (HePTP) were investigated. 8Z,11Z-Feptadecadienoic acid (4), 14Z,17Z-tricosadienoic acid (5), caffeic acid (9), and 2-hydroxy-3-[(1-oxododecyl) oxy]propyl-ß-d-glucopyranoside (11) showed considerable selective inhibition of Shp2 activity. After treatment of HepG2 cells with the compounds, only compound 5, a polyunsaturated fatty acid, strongly induced poly (ADP-ribose) polymerase (PARP) cleavage in a dose- and time-dependent manner and increased the activities of caspase-3, caspase-8, and caspase-9 at 100 µM. Compound 5 also inhibited colony formation of HepG2 cells in a dose-dependent manner. Thus, this study reported fatty acids as specific Shp2 inhibitors and provided the molecular basis of one active compound as novel potential anticancer drug.
Assuntos
Angelica/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Ácidos Graxos/isolamento & purificação , Células Hep G2 , Humanos , Neoplasias/tratamento farmacológico , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
Obesity is caused by a long-term imbalance between energy intake and consumption and is regulated by multiple signals. This study investigated the effect of signaling scaffolding protein Gab2 on obesity and its relevant regulation mechanism. Gab2 knockout (KO) and wild-type (WT) mice were fed with a standard diet (SD) or high-fat diet (HFD) for 12 weeks. The results showed that the a high-fat diet-induced Gab2 expression in adipose tissues, but deletion of Gab2 attenuated weight gain and improved glucose tolerance in mice fed with a high-fat diet. White adipose tissue and systemic inflammations were reduced in HFD-fed Gab2 deficiency mice. Gab2 deficiency increased the expression of Ucp1 and other thermogenic genes in brown adipose tissue. Furthermore, the regulation of Gab2 on the mature differentiation and function of adipocytes was investigated in vitro using primary or immortalized brown preadipocytes. The expression of brown fat-selective genes was found to be elevated in differentiated adipocytes without Gab2. The mechanism of Gab2 regulating Ucp1 expression in brown adipocytes involved with its downstream PI3K (p85)-Akt-FoxO1 signaling pathway. Our research suggests that deletion of Gab2 suppresses diet-induced obesity by multiple pathways and Gab2 may be a novel therapeutic target for the treatment of obesity and associated complications.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Metabolismo Energético , Obesidade/prevenção & controle , Paniculite/prevenção & controle , Proteínas Adaptadoras de Transdução de Sinal/genética , Tecido Adiposo Marrom/fisiopatologia , Tecido Adiposo Branco/fisiopatologia , Adiposidade , Animais , Glicemia/metabolismo , Linhagem Celular , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Proteína Forkhead Box O1/metabolismo , Resistência à Insulina , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Obesidade/fisiopatologia , Paniculite/genética , Paniculite/metabolismo , Paniculite/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteína Desacopladora 1/metabolismo , Aumento de PesoRESUMO
Spermatocyte meiosis is the cornerstone of mammalian production. Thousands of long noncoding RNAs (lncRNAs) have been reported to be functional in various cellular processes, but the function of lncRNAs in meiosis remains largely unknown. Here, we profiled lncRNAs in spermatocytes at stage I of meiosis and identified a testis-specific lncRNA, Rbakdn, as a vital regulator of meiosis. Rbakdn is dynamically expressed during meiosis I, and Rbakdn knockdown inhibits meiosis in vitro. Furthermore, Rbakdn knockdown in testes in mice by intratesticular injection disturbs meiosis, reduces testicular volume, and increases apoptosis of spermatocytes, resulting in vacuolation of the seminiferous tubules. Rbakdn can bind to Ptbp2, an RNA-binding protein that is important in the regulation of the alternative splicing of many genes in spermatogenesis. Rbakdn knockdown leads to a decrease in Ptbp2 through the ubiquitination degradation pathway, indicating that Rbakdn maintains the stability of Ptbp2. In conclusion, our study identified an lncRNA, Rbakdn, with a crucial role in meiosis.
RESUMO
The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.
Assuntos
Meiose/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Espermatócitos/metabolismo , Espermatogênese/genética , Espermatogônias/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Infertilidade Masculina , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética , Reação em Cadeia da Polimerase em Tempo Real , Espermatócitos/citologia , Espermatogônias/citologiaRESUMO
Fatty liver disease is a serious health problem worldwide and is the most common cause for chronic liver disease and metabolic disorders. The major challenge in the prevention and intervention of this disease is the incomplete understanding of the underlying mechanism and thus lack of potent therapeutic targets due to multifaceted and interdependent disease factors. In this study, we investigated the role of a signaling adaptor protein, GRB2-associated-binding protein 2 (Gab2), in fatty liver using an animal disease model. Gab2 expression in hepatocytes responded to various disease factor stimulations, and Gab2 knockout mice exhibited resistance to fat-induced obesity, fat- or alcohol-stimulated hepatic steatosis, as well as methionine and choline deficiency-induced steatohepatitis. Concordantly, the forced expression or knockdown of Gab2 enhanced or diminished oleic acid (OA)- or ethanol-induced lipid production in hepatocytes in vitro, respectively. During lipid accumulation in hepatocytes, both fat and alcohol induced the recruitment of PI3K or Socs3 by Gab2 and the activation of their downstream signaling proteins AKT, ERK, and Stat3. Therefore, Gab2 may be a disease-associated protein that is induced by pathogenic factors to amplify and coordinate multifactor-induced signals to govern disease development in the liver. Our research provides a novel potential target for the prevention and intervention of fatty liver disease.
Assuntos
Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Deleção de Genes , Terapia de Alvo Molecular , Fosfoproteínas/metabolismo , Substâncias Protetoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Modelos Animais de Doenças , Etanol , Proteína Adaptadora GRB2/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lipídeos/biossíntese , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ácido Oleico , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de SinaisRESUMO
The male's ability to reproduce is completely dependent on Sertoli cells. However, the mechanisms governing the functional integrity of Sertoli cells have remained largely unexplored. Here, we demonstrate that deletion of Shp2 in Sertoli cells results in infertility in mice. In Shp2 knockout mice (SCSKO), a normal population of Sertoli cells was observed, but the blood-testis barrier (BTB) was not formed. Shp2 ablation initiated the untimely and excessive differentiation of spermatogonial stem cells (SSCs) by disturbing the expression of paracrine factors. As a consequence, the process of spermatogenesis was disrupted, and the germ cells were depleted. Furthermore, Shp2 deletion impaired the cell junctions of the primary Sertoli cells and failed to support the clonal formation of SSCs co-cultured with SCSKO Sertoli cells. As expected, Shp2 restoration largely restores the cell junctions of the primary Sertoli cells and the clonal formation of SSCs. To identify the underlying mechanism, we further demonstrated that the absence of Shp2 suppressed Erk phosphorylation, and thus, the expression of follicle-stimulating hormone (FSH)- and testosterone-induced target genes. These results collectively suggest that Shp2 is a critical signaling protein that is required to maintain Sertoli cell function and could serve as a novel target for male infertility therapies.
Assuntos
Infertilidade Masculina/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Células de Sertoli/enzimologia , Animais , Diferenciação Celular , Hormônio Foliculoestimulante/fisiologia , Expressão Gênica/fisiologia , Masculino , Camundongos , Células de Sertoli/citologia , Testosterona/fisiologiaRESUMO
The extranuclear estrogen receptor pathway opens up novel perspectives in many physiological and pathological processes, especially in breast carcinogenesis. However, its function and mechanisms are not fully understood. Herein we present data identifying Shp2, a SH2-containing tyrosine phosphatase, as a critical component of extranuclear ER pathway in breast cancer. The research checked that the effect of Shp2 on the tumor formation and growth in animal model and investigated the regulation of Shp2 on the bio-effect and signaling transduction of estrogen in breast cancer cell lines. The results showed that Shp2 was highly expressed in more than 60% of total 151 breast cancer cases. The inhibition of Shp2 activity by PHPS1 (a Shp2 inhibitor) delayed the development of dimethylbenz(a)anthracene (DMBA)-induced tumors in the rat mammary gland and also blocked tumor formation in MMTV-pyvt transgenic mice. Estradiol (E2) stimulated protein expression and phosphorylation of Shp2, and induced Shp2 binding to ERα and IGF-1R around the membrane to facilitate the phosphorylation of Erk and Akt in breast cancer cells MCF7. Shp2 was also involved in several biological effects of the extranuclear ER-initiated pathway in breast cancer cells. Specific inhibitors (phps1, phps4 and NSC87877) or small interference RNAs (siRNA) of Shp2 remarkably suppressed E2-induced gene transcription (Cyclin D1 and trefoil factor 1 (TFF1)), rapid DNA synthesis and late effects on cell growth. These results introduced a new mechanism for Shp2 oncogenic action and shed new light on extranuclear ER-initiated action in breast tumorigenesis by identifying a novel associated protein, Shp2, for extranuclear ER pathway, which might benefit the therapy of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Estrogênios/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Benzenossulfonatos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Humanos , Hidrazonas/farmacologia , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Ratos , Ratos Sprague-DawleyRESUMO
Identification of novel cancer genes for molecular therapy and diagnosis is a current focus of breast cancer research. Although a few small gene sets were identified as prognosis classifiers, more powerful models are still needed for the definition of effective gene sets for the diagnosis and treatment guidance in breast cancer. In the present study, we have developed a novel statistical approach for systematic analysis of intrinsic correlations of gene expression between development and tumorigenesis in mammary gland. Based on this analysis, we constructed a predictive model for prognosis in breast cancer that may be useful for therapy decisions. We first defined developmentally associated genes from a mouse mammary gland epithelial gene expression database. Then, we found that the cancer modulated genes were enriched in this developmentally associated genes list. Furthermore, the developmentally associated genes had a specific expression profile, which associated with the molecular characteristics and histological grade of the tumor. These result suggested that the processes of mammary gland development and tumorigenesis share gene regulatory mechanisms. Then, the list of regulatory genes both on the developmental and tumorigenesis process was defined an 835-member prognosis classifier, which showed an exciting ability to predict clinical outcome of three groups of breast cancer patients (the predictive accuracy 64â¼72%) with a robust prognosis prediction (hazard ratio 3.3â¼3.8, higher than that of other clinical risk factors (around 2.0-2.8)). In conclusion, our results identified the conserved molecular mechanisms between mammary gland development and neoplasia, and provided a unique potential model for mining unknown cancer genes and predicting the clinical status of breast tumors. These findings also suggested that developmental roles of genes may be important criteria for selecting genes for prognosis prediction in breast cancer.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Modelos Estatísticos , Algoritmos , Neoplasias da Mama/mortalidade , Análise por Conglomerados , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Humanos , Internet , PrognósticoRESUMO
Lim only protein (LMO) 4 acts as a transcriptional adapter and modulates mammary gland morphogenesis as well as breast oncogenesis in transgenic mice. Yet, the molecular and cellular mechanisms of these effects remain to be fully elucidated. Engrailed LMO4 fusion protein is a powerful dominant repressor of LMO4 activated transcription that was successfully used to discover the role of LMO4 as a transcriptional activator in mammary gland development in our previous studies using mouse models. In this manuscript, we investigated the cellular effects of LMO4 in human normal mammary epithelial cells (HMECs) and breast cancer cell lines using the Engrailed-LMO4 fusion protein. HMEC cell growth was inhibited by the expression of the Engrailed-LMO4 fusion protein. The decrease in cell number was due to both decreased cell proliferation and enhanced apoptosis, suggesting that LMO4 promotes proliferation and survival of normal mammary epithelial cells. The expression of the Engrailed-LMO4 fusion protein also suppressed cell growth, and induced apoptosis in two breast cancer cell lines, MDA-MB-231 and T47D, suggesting that LMO4 contributes to oncogenesis by similar mechanisms of enhanced cell survival and proliferation. Taken together, our data indicate that LMO4 has similar cellular effects in normal mammary epithelial cells and breast cancer cells, and also provide direct evidence for the idea that normal development and carcinogenesis share conserved molecular mechanisms.