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1.
Immunogenetics ; 71(7): 489-499, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31297569

RESUMO

Epigenetic modifications have been shown to be important for immune cell differentiation by regulating gene transcription. However, the role and mechanism of histone methylation in the development and differentiation of iNKT cells in rheumatoid arthritis (RA) mice have yet to be deciphered. The DBA/1 mouse RA model was established by using a modified GPI mixed peptide. We demonstrated that total peripheral blood, thymus, and spleen iNKT cells in RA mice decreased significantly, while iNKT1 in the thymus and spleen was increased significantly. PLZF protein and PLZF mRNA levels were significantly decreased in thymus DP T cells, while T-bet protein and mRNA were significantly increased in thymus iNKT cells. We found a marked accumulation in H3K27me3 around the promoter regions of the signature gene Zbtb16 in RA mice thymus DP T cells, and an accumulation of H3K4me3 around the promoters of the Tbx21 gene in iNKT cells. The expression levels of UTX in the thymus of RA mice were significantly reduced. The changes in the above indicators were particularly significant in the progressive phase of inflammation (11 days after modeling) and the peak phase of inflammation (14 days after modeling) in RA mice. Developmental and differentiation defects of iNKT cells in RA mice were associated with abnormal methylation levels (H3K27me3 and H3K4me3) in the promoters of key genes Zbtb16 (encoding PLZF) and Tbx21 (encoding T-bet). Decreased UTX of thymus histone demethylase levels resulted in the accumulation of H3K27me3 modification.


Assuntos
Artrite Reumatoide/patologia , Lisina/metabolismo , Células T Matadoras Naturais/patologia , Regiões Promotoras Genéticas , Timo/fisiologia , Animais , Artrite Experimental/patologia , Diferenciação Celular , Epigênese Genética , Regulação da Expressão Gênica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Metilação , Camundongos Endogâmicos DBA , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Baço/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
2.
Nat Commun ; 10(1): 2278, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123254

RESUMO

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Processamento de RNA/fisiologia , Espermatogênese/genética , Espermatogônias/metabolismo , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , RNA Helicases DEAD-box/genética , Embrião de Mamíferos , Fertilidade/genética , Fibroblastos , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Cultura Primária de Células , Testículo/citologia
3.
Scand J Immunol ; 90(3): e12794, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31141185

RESUMO

Natural killer T (NKT) cells are αß T cell receptor (TCR) expressing innate-like T cells that display natural killer (NK) cell markers. Based on TCR characteristics, they are divided into two groups restricted to the MHC class I-like molecule CD1d. Type I NKT cells, most extensively studied, are identified by a semi-invariant Vα14-Jα18 (mouse, Vα24-Jα18 in humans) TCR reactive to the prototypic ligand α-galactosylceramide presented on CD1d. In contrast, type II NKT cells display diverse TCR reacting to different CD1d-presented ligands. There are no reagents that identify all type II NKT cells, limiting their exploration. Here, we searched for novel type II NKT cells by comparing Jα18-/- MHCII-/- mice that harbour type II but not type I NKT cells, and CD1d-/- MHCII-/- mice, lacking all NKT cells. We identified significantly larger populations of CD4+ and CD4- CD8- (double negative, DN) TCRß+ cells expressing NKG2D or NKG2A/C/E in Jα18-/- MHCII-/- mice compared with CD1d-/- MHCII-/- mice, suggesting that 30%-50% of these cells were type II NKT cells. They expressed CD122, NK1.1, CXCR3 and intermediate/low levels of CD45RB. Further, the CD4+ subset was CD69+ , while the DN cells were CD49b+ and CD62L+ . Both subsets expressed the NKT cell-associated promyelocytic leukaemia zinc finger (PLZF) transcription factor and Tbet, while fewer cells expressed RORγt. NKG2D+ CD4+ and DN populations were producers of IFN-γ, but rarely IL-4 and IL-17. Taken together, we identify a novel subset of primary CD4+ and DN type II NKT cells that expresses NKG2 receptors have typical NKT cell phenotypes and a TH1-like cytokine production.


Assuntos
Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Biomarcadores/metabolismo , Células Matadoras Naturais/imunologia , Células T Matadoras Naturais/imunologia , Animais , Feminino , Galactosilceramidas/imunologia , Galactosilceramidas/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células T Matadoras Naturais/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
4.
Andrologia ; 51(6): e13283, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30957907

RESUMO

The identification system of spermatogonial stem cell (SSC) was established in alpaca using the molecular expression as well as the reactivity pattern to Dolichos biflorus agglutinin (DBA) by flow cytometry. Twenty-four testicles with their epididymis were recovered from adult alpacas at the slaughterhouse of Huancavelica-Perú. Samples were transported to the Laboratory of Reproductive Physiology at Universidad Nacional Mayor de San Marcos. Testes were selected for our study when the progressive motility of epididymal spermatozoa (ESPM) was above 30%. Isolation of SSC was performed with two enzymatic digestions. Finally, sperm viability was evaluated by means of the trypan blue vital stain in spermatogonial round cells. Samples with more than 80% viability were selected. Isolated cells cultured for 2 days were used for identifying the presence of SSCs by the expression of integrin ß1 (116 bp) and PLZF (206 bp) genes. Spermatogonia were classified according to the DBA reactivity. Spermatogonia with a strong positive to DBA (sDBA+ ) were classified as SSC (Mean ± SEM=4.44 ± 0.68%). Spermatogonia in early differentiation stages stained weakly positive with DBA (wDBA+ ) (Mean ± SEM=37.44 ± 3.07%) and differentiated round cells as DBA negative (Mean ± SEM=54.12 ± 3.18%). With the use of molecular and DBA markers, it is possible to identify easily the spermatogonial stem cells in alpaca.


Assuntos
Células-Tronco Germinativas Adultas/fisiologia , Camelídeos Americanos , Separação Celular/veterinária , Citometria de Fluxo/veterinária , Espermatogônias/fisiologia , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular , Separação Celular/métodos , Células Cultivadas , Conservação dos Recursos Naturais , Citometria de Fluxo/métodos , Inseminação Artificial , Integrina beta1/análise , Integrina beta1/metabolismo , Masculino , Lectinas de Plantas/química , Proteína com Dedos de Zinco da Leucemia Promielocítica/análise , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Coloração e Rotulagem/métodos , Coloração e Rotulagem/veterinária , Testículo/citologia , Testículo/metabolismo
5.
J Biomed Sci ; 26(1): 30, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027502

RESUMO

BACKGROUND: Promyelocytic leukemia zinc finger (Plzf), a transcriptional regulator involved in a lot of important biological processes during development, has been implied to maintain neural stem cells and inhibit their differentiation into neurons. However, the effects of Plzf on brain structures and functions are still not clarified. RESULTS: We showed that Plzf expression was detected as early as embryonic day (E) 9.5 in Pax6+ cells in the mouse brain, and was completely disappeared in telencephalon before the initiation of cortical neurogenesis. Loss of Plzf resulted in a smaller cerebral cortex with a decrease in the number of Tbr1+ deep layer neurons due to a decrease of mitotic cell number in the ventricular zone of forebrain at early developmental stage. Microarray, qRT-PCR, and flow cytometry analysis identified dysregulation of Mash1 proneural gene expression. We also observed an impairment of recognition memory in Plzf-deficient mice. CONCLUSIONS: Plzf is expressed at early stages of brain development and involved in the formation of deep layer cortical neurons. Loss of Plzf results in dysregulation of Mash1, microcephaly with reduced numbers of early-born neurons, and impairment of recognition memory.


Assuntos
Expressão Gênica/fisiologia , Neurogênese/genética , Neurônios/fisiologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Animais , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/fisiologia , Camundongos , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo
6.
Theriogenology ; 130: 120-124, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30884332

RESUMO

Microminipigs are one of the smallest miniature pigs characterized as sexually precocious; the males achieve sexual maturity at around 3-4.5 months of age. However, the physiology of this sexual precocity is still unclear. To understand sexual precocity in male microminipigs, we analyzed their testes at five developmental stages: neonatal (<7 days), 30-day-old, 45-day-old, 80-day-old, and adult (>24 months) stages. We used 4 pigs in each of the stages. To analyze testicular development histologically, the seminiferous tubule diameter (SD) was measured, and the presence or absence of the seminiferous lumen was confirmed. Changes in the expression of pluripotency markers, DBA, UCHL1, ZBTB16, and vimentin, were evaluated immunohistologically. For the analyses, cells positive for DBA, UCHL1, and ZBTB16 per 150 round seminiferous tubules in cross sections from each testis were counted to evaluate the total number of positive cells. The number of positive cells per 100 Sertoli cells (DBA+/Sertoli, UCHL1+/Sertoli, and ZBTB16+/Sertoli) was calculated to compare the five developmental stages. Histologically, SDs became larger with piglet growth, and precocity was confirmed; seminiferous lumens were observed from the 30-day-old stage. Immunohistologically, the number of DBA+/Sertoli, which indicates the number of gonocytes, decreased rapidly to an undetectable level by the 45-day-old stage. In the same period, the number of UCHL1+/Sertoli, which indicates total SSCs, increased significantly, suggesting that the proliferation of SSCs was accelerated before 30 days of age. Consequently, our study clarified that differentiation of SSCs in microminipigs started during the fetal period, the differentiation of gonocytes and proliferation of SSCs was then accelerated before 30 days of age, and the early phase of spermatogenesis was finally completed at around 45 days after birth. Consequently, sexual precocity in male microminipigs was characterized by a shorter duration of the early phase of spermatogenesis.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Maturidade Sexual/fisiologia , Suínos/fisiologia , Animais , Biomarcadores , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Espermatogênese/fisiologia , Porco Miniatura , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Vimentina/genética , Vimentina/metabolismo
7.
Oncol Rep ; 41(2): 1007-1018, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30431129

RESUMO

Promyelocytic leukemia zinc finger (PLZF) plays important roles in tumorigenic and developmental processes of various types of cancers. However, the expression of PLZF in gastric cancer (GC) has not been reported. The aim of the present study was to investigate the expression level and potential status of PLZF in GC as well as its possible mechanism. In the present study, we found that PLZF was downregulated in the majority of GC cell lines and tumor tissues and that alteration of PLZF expression was closely correlated with a malignant phenotype, epithelial­mesenchymal transformation and overall survival. Evaluation of in vitro proliferation, colony information, migration and invasion indicated that PLZF gene transduction induced a less malignant phenotype, which was also confirmed through in vivo studies performed in athymic nude mice. Furthermore, we assessed the expression levels of the lncRNA ANRIL in GC and found that it was negatively associated with the level of PLZF and that ANRIL indirectly methylated PLZF to suppress its expression via binding with polycomb repressive complex 2. When GC cells were treated with the methylation inhibitor 5­Aza­2'­deoxycytidine, the expression of PLZF increased, which further confirmed that PLZF was methylated. These results indicated that constitutive ANRIL activation was a possible cause of the lack of PLZF expression in GC cells. Coupled deregulation of PLZF and ANRIL may account for most of the alterations described in GC, and PLZF may become a potential target of GC therapy.


Assuntos
Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Metilação de DNA/genética , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , RNA Longo não Codificante/genética , Estômago/patologia , Estômago/cirurgia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Análise de Sobrevida , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Int J Biochem Cell Biol ; 105: 104-114, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30393202

RESUMO

Jak-Stat pathway is the first pathway identified to stimulate spermatogonial stem cells (SSCs) self-renewal and maintenance activity. Recent studies have showed that stat3 a crucial gene implicated in this pathway can regulate self-renewal in male germline stem cell. In our previous study, we demonstrated that miR-19b-3p induces cell proliferation and reduces heterochromatin through Plzf which also regulates the balance between cell self-renewal and differentiation. Because miRNA can target several genes and to understand more about Plzf, a crucial transcription factor of SSCs, we performed microarray and found that miR-19b-3p integrate Jak-Stat through Plzf to regulate cell self-renewal. Our results demonstrated that miR-19b-3p induces Jak-Stat when Plzf is downregulated; overexpression of Plzf reversed the trend and shown an existence of feedback (-/+) between Plzf and GHR. The cell self-renewal markers CD49f, GFRα1, Oct4 and cKIT analyzed in the both groups miR-19b-3p and Plzf-overexpressing compared to their respective control confirm miR-19b-3p regulates cell pluripotency and self-renewal in goat male germline stem cells through Plzf. Together our finding revels that miR-19b-3p control Jak-Stat signaling through Plzf.


Assuntos
Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Cabras/genética , Cabras/metabolismo , MicroRNAs/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Espermatogônias/citologia , Espermatogônias/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Técnicas de Silenciamento de Genes , Cabras/crescimento & desenvolvimento , Janus Quinases/metabolismo , Masculino , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transfecção
9.
Nat Commun ; 9(1): 3749, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218105

RESUMO

While CD69 may regulate thymocyte egress by inhibiting S1P1 expression, CD69 expression is not thought to be required for normal thymocyte development. Here we show that CD69 is in fact specifically required for the differentiation of mature NKT2 cells, which do not themselves express CD69. Mechanistically, CD69 expression is required on CD24+ PLZFhi innate precursors for their retention in the thymus and completion of their differentiation into mature NKT2 cells. By contrast, CD69-deficient CD24+ PLZFhi innate precursors express S1P1 and prematurely exit the thymus, while S1P1 inhibitor treatment of CD69-deficient mice retains CD24+ PLZFhi innate precursors in the thymus and restores NKT2 cell differentiation. Thus, CD69 prevents S1P1 expression on CD24+ PLZFhi innate precursor cells from aborting NKT2 differentiation in the thymus. This study reveals the importance of CD69 to prolong the thymic residency time of developing immature precursors for proper differentiation of a T cell subset.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Lectinas Tipo C/genética , Linfopoese/genética , Células T Matadoras Naturais/citologia , Receptores de Lisoesfingolipídeo/genética , Subpopulações de Linfócitos T/citologia , Timócitos/citologia , Animais , Antígeno CD24/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Subpopulações de Linfócitos T/metabolismo , Timócitos/metabolismo
10.
Exp Cell Res ; 373(1-2): 71-79, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30266657

RESUMO

During spermatogenesis, a group of undifferentiated spermatogonia undergoes an essential transition to a differentiating stage, which involves gain of Kit receptor. In the current study, we showed that a small non-coding RNA, miRNA-26b could induce transition from Kit- to Kit+ and inhibit proliferation of spermatogonia. A key transcriptional factor for undifferentiated spermatogonia, Plzf, was proven as a direct target of miR-26b. When undifferentiated spermatogonia were treated with Retinoic acid (RA), miR-26b was increased, further promoting RA-induced differentiation of spermatogonia. In addition, miR-26b could repress 5-hydroxymethylcytosine (5hmC) via repression of Tet3 in spermatogonia. These findings demonstrate that miR-26b might play a role in promoting the transition from Kit- to Kit+ SSCs.


Assuntos
MicroRNAs/fisiologia , Espermatogênese , Espermatogônias/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Apoptose , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos , MicroRNAs/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-kit/análise , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Tretinoína/farmacologia
11.
Cell Physiol Biochem ; 48(6): 2528-2538, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30121655

RESUMO

BACKGROUND/AIMS: Our study aims to characterize functions of ZBTB16 gene in the process of intramuscular fat (IMF) deposition and metabolism of bovine, thereby providing insights into mechanisms for the use of ZBTB16 in fat management. METHODS: Primary preadipocytes derived from bovine IMF tissue were isolated and used as the in vitro cell model. An adenovirus Ad-ZBTB16 was transfected into bovine preadipocytes to overexpress the ZBTB16 gene. By using real-time quantitative PCR (RT-qPCR), western blotting, Oil Red-O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity assay, and cell counting kit-8 (CCK-8) test, adipogenic and proliferative signals in adipocytes were monitored to investigate effects of ZBTB16 on adipogenesis of bovine preadipocytes. RESULTS: After transfection, mRNA and protein levels of ZBTB16 gene were significantly increased. Enhanced ZBTB16 significantly promoted preadipocyte differentiation, as evidenced by accelerated lipid accumulation, enhanced GPDH activity, consistently increased mRNA expressions of adipogenic key transcription factors PPARγ, C/EBPα, FABP4, and ADIPOQ, and markedly increased protein expressions of PPARγ and FABP4. No difference was observed concerning proliferation of preadipocytes after treatment with Ad-ZBTB16. Furthermore, relative mRNA levels of brown adipocyte selective genes (PRDM16, UCP1, Cidea, Cox8b, and PGC-1α) and beige adipocyte selective genes (CD137, TMEM26, and Tbx1) as well as UCP1 protein expression were significantly increased by Ad-ZBTB16. Meanwhile, Ad-ZBTB16 treatment remarkably induced mitochondrial biogenesis and increased relative mitochondrial DNA (mtDNA) copy number in bovine adipocytes. CONCLUSION: These results suggest that ZBTB16 overexpression can promote white adipogenesis and induce brown-like adipocyte formation for bovine white intramuscular preadipocytes.


Assuntos
Adipogenia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Adenoviridae/genética , Adipócitos/citologia , Adipócitos/metabolismo , Adiponectina/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Bovinos , Proliferação de Células , Células Cultivadas , DNA Mitocondrial/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Metabolismo dos Lipídeos , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Regulação para Cima
12.
Nat Commun ; 9(1): 2819, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30026551

RESUMO

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismo , Transativadores/genética , Animais , Diferenciação Celular , Linhagem da Célula/genética , Efeito Fundador , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/metabolismo , Integrases/genética , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Análise de Célula Única , Espermatogônias/citologia , Células-Tronco/citologia , Testículo/citologia , Testículo/crescimento & desenvolvimento , Transativadores/metabolismo
13.
J Transl Med ; 16(1): 188, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29976201

RESUMO

BACKGROUND: Abnormal microRNAs (miRNAs) were reported to be involved in the mechanism of Graves' disease (GD). Dysregulated miRNAs may be overlapping in different cells and can be secreted to circulation. We chose miRNAs which were previously reported to be differentially expressed in peripheral blood mononuclear cells (PBMCs) in patients with GD with different disease stage, detected the expression of those miRNAs in serum, corroborated the findings in thyroid tissue, and validated the target gene in vitro to investigate the possible role of circulating miRNAs in GD. METHODS: A total of 54 individuals with untreated GD, 12 individuals with GD in remission and 14 disease-free controls were enrolled. The expression of miR-142-3p, miR-154-3p, miR-431-3p, miR-590-5p, and let-7b was detected in the serum. Ten thyroid tissue samples from patients with GD and six disease-free thyroid samples were used for further validation. The potential target genes were identified and validated in vitro. RESULTS: miR-142-3p, miR-154-3p, miR-431-3p, miR-590-5p, and let-7b were present in serum and two of them (miR-142-3p and let-7b) were significantly increased in serum of patients with untreated GD (for serum miR-142-3p, P = 0.033, for serum let-7b, P = 0.026) and gradually decreased to normal levels in patients with GD in remission. Correlation analysis showed that let-7b level was strongly correlated with TRAb level (r = 0.305, P = 0.001). let-7b directly inhibited promyelocytic leukemia zinc finger (PLZF) expression and increased the expression of TSHR in thyroid cells in vitro. Furthermore, let-7b levels in GD thyroid tissue were found to be inversely correlated with PLZF levels (r = - 0.849, P = 0.033). Decreased PLZF and increased TSHR was validated in thyroid tissue in patients with GD. CONCLUSIONS: The present study confirmed that a portion of miRNAs in PBMCs were also presented and differentially expressed in serum and thyroid tissue. Upregulated in all these three compartments, let-7b may be used as a disease biomarker and therapeutic targets in patients with GD. Circulating let-7b had a strong correlation with disease severity and let-7b may participate in the production of TRAb via targeting PLZF in patients with GD.


Assuntos
Doença de Graves/sangue , Doença de Graves/genética , MicroRNAs/metabolismo , Receptores da Tireotropina/imunologia , Glândula Tireoide/metabolismo , Adulto , Sequência de Bases , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo
14.
Syst Biol Reprod Med ; 64(4): 225-239, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29911897

RESUMO

Per- and polyfluoroalkyl substances (PFASs) represent a highly ubiquitous group of synthetic chemicals used in products ranging from water and oil repellents and lubricants to firefighting foam. These substances can enter and accumulate in multiple tissue matrices in up to 100% of people assessed. Though animal models strongly identify these compounds as male reproductive toxicants, with exposed rodents experiencing declines in sperm count, alterations in hormones, and DNA damage in spermatids, among other adverse outcomes, human studies report conflicting conclusions as to the reproductive toxicity of these chemicals. Using an innovative, human stem-cell-based model of spermatogenesis, we assessed the effects of the PFASs perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and a mixture of PFOS, PFOA, and PFNA for their impacts on human spermatogenesis in vitro under conditions relevant to the general and occupationally exposed populations. Here, we show that PFOS, PFOA, PFNA, and a mixture of PFOS, PFOA, and PFNA do not decrease in vitro germ cell viability, consistent with reports from human studies. These compounds do not affect mitochondrial membrane potential or increase reactive oxygen species generation, and they do not decrease cell viability of spermatogonia, primary spermatocytes, secondary spermatocytes, or spermatids in vitro under the conditions examined. However, exposure to PFOS, PFOA, and PFNA reduces expression of markers for spermatogonia and primary spermatocytes. While not having direct effects on germ cell viability, these effects suggest the potential for long-term impacts on male fertility through the exhaustion of the spermatogonial stem cell pool and abnormalities in primary spermatocytes. ABBREVIATIONS: CDC: Centers for Disease Control; DMSO: dimethyl sulfoxide; GHR: growth hormone receptor; hESCs: human embryonic stem cells; PFASs: per- and polyfluoroalkyl substances; PFCs: perfluorinated compounds; PFNA: perfluorononanoic acid; PFOS: perfluorooctanesulfonic acid; PFOA: perfluorooctanoic acid; PLZF: promyelocytic leukemia zinc finger; ROS: reactive oxygen species; HILI: RNA-mediated gene silencing 2; SSC: spermatogonial stem cell.


Assuntos
Ácidos Alcanossulfônicos/toxicidade , Caprilatos/toxicidade , Fluorcarbonetos/toxicidade , Espermatogênese/efeitos dos fármacos , Proteínas Argonauta/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias , Humanos , Masculino , Mitocôndrias/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espermatócitos/efeitos dos fármacos , Espermatócitos/metabolismo , Espermatogônias/efeitos dos fármacos , Espermatogônias/metabolismo
15.
Stem Cells Dev ; 27(9): 624-636, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29649409

RESUMO

Continuous spermatogenesis from puberty to old age in males relies on spermatogonial stem cells (SSCs) that possess the property of self-renewal and differentiation. The delicate balance between self-renewal and differentiation is of great importance. In mice, SSCs exist as a subpopulation of undifferentiated spermatogonia. SSCs are controlled by intrinsic molecular pathways that can be activated by extrinsic signals. Our results here first show that the expression of forkhead box C2 (FOXC2) is restricted to GFRα1-positive spermatogonia in the testis. Whole-mount immunofluorescence results reveal that FOXC2 is expressed predominately in As and Apr spermatogonia. Reduction of Foxc2 gene expression by shRNA lentivirus treatment significantly impairs the maintenance of SSCs in vitro. Furthermore, knock-down of Foxc2 decreases SSC colonization to only 10.42% compared to the control by transplantation. Reverse transcription and real-time quantitative PCR gene analyses following knock-down of Foxc2 indicate that Foxc2 may act as a suppressor for SSC differentiation. Extrinsic stimuli treatments show that glial cell line-derived neurotrophic factor and retinoic acid act in opposite ways to regulate FOXC2 expression and subsequent SSC property. These results suggest that FOXC2 is a critical intrinsic regulator of SSC self-renewal and differentiation.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Espermatogônias/citologia , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Regulação para Baixo/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogônias/efeitos dos fármacos , Espermatogônias/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Testículo/metabolismo , Tretinoína/farmacologia
16.
Elife ; 72018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29664401

RESUMO

We report that Histone Deacetylase 7 (HDAC7) controls the thymic effector programming of Natural Killer T (NKT) cells, and that interference with this function contributes to tissue-specific autoimmunity. Gain of HDAC7 function in thymocytes blocks both negative selection and NKT development, and diverts Vα14/Jα18 TCR transgenic thymocytes into a Tconv-like lineage. Conversely, HDAC7 deletion promotes thymocyte apoptosis and causes expansion of innate-effector cells. Investigating the mechanisms involved, we found that HDAC7 binds PLZF and modulates PLZF-dependent transcription. Moreover, HDAC7 and many of its transcriptional targets are human risk loci for IBD and PSC, autoimmune diseases that strikingly resemble the disease we observe in HDAC7 gain-of-function in mice. Importantly, reconstitution of iNKT cells in these mice mitigated their disease, suggesting that the combined defects in negative selection and iNKT cells due to altered HDAC7 function can cause tissue-restricted autoimmunity, a finding that may explain the association between HDAC7 and hepatobiliary autoimmunity.


Assuntos
Autoimunidade , Histona Desacetilases/metabolismo , Células T Matadoras Naturais/imunologia , Animais , Animais Geneticamente Modificados , Deleção de Genes , Expressão Gênica , Humanos , Camundongos , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo
17.
Eur J Immunol ; 48(8): 1329-1335, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29677387

RESUMO

Innate lymphocytes are selectively enriched in the liver where they have important roles in liver immunology. Murine studies have shown that type I NKT cells can promote liver inflammation, whereas type II NKT cells have an anti-inflammatory role. In humans, type II NKT cells were found to accumulate in the gut during inflammation and IL13Rα2 was proposed as a marker for these cells. In the human liver, less is known about type I and II NKT cells. Here, we studied the phenotype and function of human liver T cells expressing IL13Rα2. We found that IL13Rα2 was expressed by around 1% of liver-resident memory T cells but not on circulating T cells. In support of their innate-like T-cell character, the IL13Rα2+ T cells had higher expression of promyelocytic leukaemia zinc finger (PLZF) compared to IL13Rα2- T cells and possessed the capacity to produce IL-22. However, only a minority of human liver sulfatide-reactive type II NKT cells expressed IL13Rα2. Collectively, these findings suggest that IL13Rα2 identifies tissue-resident intrahepatic T cells with innate characteristics and the capacity to produce IL-22.


Assuntos
Memória Imunológica/imunologia , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Interleucinas/metabolismo , Fígado/imunologia , Células T Matadoras Naturais/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Biomarcadores/metabolismo , Humanos , Fígado/citologia
18.
Mol Brain ; 11(1): 19, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29631635

RESUMO

Alzheimer's disease (AD) is characterized by neurotoxicity mediated by the accumulation of beta amyloid (Aß) oligomers, causing neuronal loss and progressive cognitive decline. Genetic deletion or chronic pharmacological inhibition of mGluR5 by the negative allosteric modulator CTEP, rescues cognitive function and reduces Aß aggregation in both APPswe/PS1ΔE9 and 3xTg-AD mouse models of AD. In late onset neurodegenerative diseases, such as AD, defects arise at different stages of the autophagy pathway. Here, we show that mGluR5 cell surface expression is elevated in APPswe/PS1ΔE9 and 3xTg-AD mice. This is accompanied by reduced autophagy (accumulation of p62) as the consequence of increased ZBTB16 expression and reduced ULK1 activity, as we have previously observed in Huntington's disease (HD). The chronic (12 week) inhibition of mGluR5 with CTEP in APPswe/PS1ΔE9 and 3xTg-AD mice prevents the observed increase in mGluR5 surface expression. In addition, mGluR5 inactivation facilitates the loss of ZBTB16 expression and ULK1 activation as a consequence of ULK-Ser757 dephosphorylation, which promotes the loss of expression of the autophagy marker p62. Moreover, the genetic ablation of mGluR5 in APPswe/PS1ΔE9 mice activated autophagy via similar mechanisms to pharmacological blockade. This study provides further evidence that mGluR5 overactivation contributes to inhibition of autophagy and can result in impaired clearance of neurotoxic aggregates in multiple neurodegenerative diseases. Thus, it provides additional support for the potential of mGluR5 inhibition as a general therapeutic strategy for neurodegenerative diseases such as AD and HD.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Autofagia , Inativação Gênica , Receptor de Glutamato Metabotrópico 5/metabolismo , Transdução de Sinais , Animais , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Modelos Animais de Doenças , Feminino , Inativação Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Camundongos Endogâmicos C57BL , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Piridinas/farmacologia
19.
J Exp Med ; 215(4): 1079-1090, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29490936

RESUMO

Appropriate regulation of IL-17 production in the host can mean the difference between effective control of pathogens and uncontrolled inflammation that causes tissue damage. Investigation of conventional CD4+ T cells (Th17 cells) has yielded invaluable insights into IL-17 function and its regulation. More recently, we and others reported production of IL-17 from innate αß+ T cell populations, which was shown to occur primarily via IL-23R signaling through the transcription factor STAT-3. In our current study, we identify promyelocytic leukemia zinc finger (PLZF)-expressing iNKT, CD4-/CD8+, and CD4-/CD8- (DN) αß+T cells, which produce IL-17 in response to TCR and IL-1 receptor ligation independently of STAT-3 signaling. Notably, this noncanonical pathway of IL-17 production may be important in mucosal defense and is by itself sufficient to control pathogenic Staphylococcus aureus infection at the ocular surface.


Assuntos
Infecções Oculares/imunologia , Infecções Oculares/patologia , Imunidade Inata , Interleucina-17/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Memória Imunológica , Interleucinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membrana Mucosa/imunologia , Membrana Mucosa/microbiologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Fosforilação , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Transdução de Sinais , Staphylococcus aureus/fisiologia , Linfócitos T/metabolismo , Células Th17/metabolismo , Timo/metabolismo
20.
Int J Mol Sci ; 19(3)2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29518903

RESUMO

Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints. We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunity.


Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Receptores Imunológicos/genética , Animais , Biomarcadores , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Imunofenotipagem , Contagem de Linfócitos , Camundongos , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Receptores Imunológicos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
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