RESUMO
The Runt-related transcription factor (Runx) family has been suggested to play roles in stem cell regulation, tissue development, and oncogenesis in various tissues/organs. In this study, we investigated the possible functions of Runx1 and Runx3 in keratinocyte differentiation. Both Runx1 and Runx3 proteins were detected in primary cultures of mouse keratinocytes. Proteins were localized in the nuclei of undifferentiated keratinocytes but translocated to the cytoplasm of differentiated cells. The siRNA-mediated inhibition of Runx1 and Runx3 expression increased expression of keratin 1 and keratin 10, which are early differentiation markers of keratinocytes. In contrast, overexpression of Runx1 and Runx3 suppressed keratin 1 and keratin 10 expression. Endogenous Runx1 and Runx3 proteins were associated with the promoter sequences of keratin 1 and keratin 10 genes in undifferentiated but not differentiated keratinocytes. In mouse skin, the inhibition of Runx1 and Runx3 expression by keratinocyte-specific gene targeting increased the ratios of keratin 1- and keratin 10-positive cells in the basal layer of the epidermis. On the other hand, inhibition of Runx1 and Runx3 expression did not alter the proliferation capacity of cultured or epidermal keratinocytes. These results suggest that Runx1 and Runx3 likely function to directly inhibit differentiation-induced expression of keratin 1 and keratin 10 genes but are not involved in the regulation of keratinocyte proliferation.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Queratina-10 , Queratina-1 , Animais , Diferenciação Celular , Queratina-1/genética , Queratina-10/genética , Queratinócitos/metabolismo , Queratinas/genética , CamundongosRESUMO
PTPRT has been known to regulate synaptic formation and dendritic arborization of hippocampal neurons. PTPRT-/- null and PTPRT-D401A mutant mice displayed enhanced depression-like behaviors compared with wild-type mice. Transient knockdown of PTPRT in the dentate gyrus enhanced the depression-like behaviors of wild-type mice, whereas rescued expression of PTPRT ameliorated the behaviors of PTPRT-null mice. Chronic stress exposure reduced expression of PTPRT in the hippocampus of mice. In PTPRT-deficient mice the expression of GluR2 (also known as GRIA2) was attenuated as a consequence of dysregulated tyrosine phosphorylation, and the long-term potentiation at perforant-dentate gyrus synapses was augmented. The inhibitory synaptic transmission of the dentate gyrus and hippocampal GABA concentration were reduced in PTPRT-deficient mice. In addition, the hippocampal expression of GABA transporter GAT3 (also known as SLC6A11) was decreased, and its tyrosine phosphorylation was increased in PTPRT-deficient mice. PTPRT-deficient mice displayed reduced numbers and neurite length of newborn granule cells in the dentate gyrus and had attenuated neurogenic ability of embryonic hippocampal neural stem cells. In conclusion, our findings show that the physiological roles of PTPRT in hippocampal neurogenesis, as well as synaptic functions, are involved in the pathogenesis of depressive disorder.
Assuntos
Depressão , Neurogênese , Animais , Giro Denteado , Hipocampo , Camundongos , Camundongos Knockout , Neurogênese/genética , Neurônios , SinapsesRESUMO
CD4/CD8 T-cell lineage differentiation is a key process in immune system development; however, a defined regulator(s) that converts the signal from T-cell receptor and co-receptor complexes into lineage differentiation remains unclear. Here, we show that Twist2 is a critical factor in CD4/CD8 thymocyte differentiation. Twist2 expression is differentially regulated by T-cell receptor signaling, leading to differentiation into the CD4 or CD8 lineage. Forced Twist2 expression perturbed CD4+ thymocyte differentiation while enhancing CD8+ thymocyte differentiation. Furthermore, Twist2 expression produced mature CD8+ thymocytes in B2m-/- mice, while its deficiency significantly impaired CD8+ cells in MHC class-II-/- and TCR transgenic mice, favoring CD8 T-cell differentiation. During CD8 lineage differentiation, Twist2 interacted with Runx3 to bind to the silencer region of the ThPOK locus, thereby blocking ThPOK expression. These findings indicate that Twist2 is a part of the transcription factor network controlling CD8 lineage differentiation.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteína 1 Relacionada a Twist/genética , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Timo/citologia , Timo/imunologia , Fatores de Transcrição/biossíntese , Proteína 1 Relacionada a Twist/metabolismoRESUMO
A major advantage of image-based phenotypic profiling of compounds is that numerous image features can be sampled and quantitatively evaluated in an unbiased way. However, since this assay is a discovery-oriented screening, it is difficult to determine the optimal experimental setup in advance. In this study, we examined whether variable cellular stimulation affects the efficacy of the image-based profiling of compounds. Seven different epidermal growth factor receptor (EGFR) ligands were used, and the expression of EGFR signaling molecules was monitored at various time points. Significant quantitative differences in image features were detected among the differentially treated samples. Next, 14 different compounds that affect EGFR signaling were profiled. Nearly half of the compounds were classified into distinct clusters, irrespective of differential ligand stimulation. The results suggest that image-based phenotypic profiling is quite robust in its ability to predict compound interaction with its target. Although this method will have to be validated in other experimental systems, the robustness of image-based compound profiling demonstrated in this work provides a valid basis for further study and its extended application.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células A549 , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , LigantesRESUMO
Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal disease due, in large part, to a high rate of second primary tumor (SPT) formation. The 4-nitroquinoline 1-oxide (4-NQO) mouse model of oral carcinogenesis provides a robust system in which to study chemopreventive agents in the context of chemically induced HNSCC tumors. STAT3 is a potent oncogene that is hyperactivated by tyrosine phosphorylation early in HNSCC carcinogenesis and is a rational therapeutic target. We recently reported that loss-of-function of the STAT3 phosphatase PTPRT promotes STAT3 activation in HNSCC tumors and preclinical models and may serve as a predictive biomarker of response to STAT3 inhibitors, including the small-molecule Stattic. We therefore investigated the hypothesis that Ptprt-knockout (KO) mice would be more susceptible to 4-NQO-induced oral carcinogenesis and more sensitive to Stattic-mediated chemoprevention compared with wild-type (WT) mice. Herein, we demonstrate that Ptprt WT and KO mice develop similar spectra of HNSCC disease severity upon 12 weeks of 4-NQO administration, with no apparent effect of Ptprt genotype on carcinogenesis or treatment outcome. Targeting of STAT3 with Stattic resulted in a chemopreventive effect against 4-NQO-induced oral cancer (P = 0.0402). While these results do not support a central role for PTPRT in 4-NQO-induced HNSCC carcinogenesis, further investigation of STAT3 as a chemoprevention target in this cancer is warranted. Cancer Prev Res; 9(8); 657-63. ©2016 AACR.
Assuntos
4-Nitroquinolina-1-Óxido/toxicidade , Carcinoma de Células Escamosas/prevenção & controle , Óxidos S-Cíclicos/uso terapêutico , Neoplasias de Cabeça e Pescoço/prevenção & controle , Neoplasias Bucais/induzido quimicamente , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Biomarcadores/metabolismo , Carcinógenos/toxicidade , Carcinoma de Células Escamosas/induzido quimicamente , Quimioprevenção , Óxidos S-Cíclicos/farmacologia , Neoplasias de Cabeça e Pescoço/induzido quimicamente , Camundongos , Camundongos Knockout , Neoplasias Bucais/prevenção & controle , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Fator de Transcrição STAT3/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fatores de TempoRESUMO
Background. Members of the Runx gene family encode transcription factors that bind to DNA in a sequence-specific manner. Among the three Runx proteins, Runx2 comprises 607 amino acid (aa) residues, is expressed in bone, and plays crucial roles in osteoblast differentiation and bone development. We examined whether the Runx2 gene is also expressed in testes. Methods. Murine testes from 1-, 2-, 3-, 4-, and 10-week-old male mice of the C57BL/6J strain and W∕W (v) strain were used throughout the study. Northern Blot Analyses were performed using extracts form the murine testes. Sequencing of cDNA clones and 5'-rapid amplification of cDNA ends were performed to determine the full length of the transcripts, which revealed that the testicular Runx2 comprises 106 aa residues coding novel protein. Generating an antiserum using the amino-terminal 15 aa of Runx2 (Met(1) to Gly(15)) as an antigen, immunoblot analyses were performed to detect the predicted polypeptide of 106 aa residues with the initiating Met(1). With the affinity-purified anti-Runx2 antibody, immunohistochemical analyses were performed to elucidate the localization of the protein. Furthermore, bioinformatic analyses were performed to predict the function of the protein. Results. A Runx2 transcript was detected in testes and was specifically expressed in germ cells. Determination of the transcript structure indicated that the testicular Runx2 is a splice isoform. The predicted testicular Runx2 polypeptide is composed of only 106 aa residues, lacks a Runt domain, and appears to be a basic protein with a predominantly alpha-helical conformation. Immunoblot analyses with an anti-Runx2 antibody revealed that Met(1) in the deduced open reading frame of Runx2 is used as the initiation codon to express an 11 kDa protein. Furthermore, immunohistochemical analyses revealed that the Runx2 polypeptide was located in the nuclei, and was detected in spermatocytes at the stages of late pachytene, diplotene and second meiotic cells as well as in round spermatids. Bioinformatic analyses suggested that the testicular Runx2 is a histone-like protein. Discussion. A variant of Runx2 that differs from the bone isoform in its splicing is expressed in pachytene spermatocytes and round spermatids in testes, and encodes a histone-like, nuclear protein of 106 aa residues. Considering its nuclear localization and differentiation stage-dependent expression, Runx2 may function as a chromatin-remodeling factor during spermatogenesis. We thus conclude that a single Runx2 gene can encode two different types of nuclear proteins, a previously defined transcription factor in bone and cartilage and a short testicular variant that lacks a Runt domain.
RESUMO
The Runx1 transcription factor cooperates with or antagonizes other transcription factors and plays essential roles in the differentiation and function of T lymphocytes. Previous works showed that Runx1 is expressed in peripheral CD4(+) T cells which level declines after T cell receptor (TCR) activation, and artificial deletion of Runx1 causes autoimmune lung disease in mice. The present study addresses the mechanisms by which Runx1 contributes to the maintenance of peripheral CD4(+) T cell quiescence. Microarray and quantitative RT-PCR analyses were employed to compare the transcriptome of Runx1 -/- CD4(+) T cells to those of unstimulated and TCR-stimulated Runx1 +/- cells. The results identified genes whose expression was modulated similarly by Runx1 deletion and TCR activation. Among them, genes encoding cytokines, chemokines, and Jak/STAT signaling molecules were substantially induced. In Runx1-deleted T cells, simultaneous increases in Il-17A and Rorγc, a known master gene in TH17 differentiation, were observed. In addition, we observed that the loss of Runx1 reduced the transcription of genes encoding quiescence-associated transcription factors, including Foxp1, Foxo1, and Klf2. Interestingly, we identified consensus Runx1 binding sites at the promoter regions of Foxp1, Foxo1, and Klf2 genes, which can be enriched by chromatin immunoprecipitation assay with an anti-Runx1 antibody. Therefore, we suggest that Runx1 may activate, directly or indirectly, the expression of quiescence-associated molecules and thereby contribute to the maintenance of quiescence in CD4(+) T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Tolerância Periférica/genética , Proteínas Repressoras/imunologia , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/citologia , Subunidade alfa 2 de Fator de Ligação ao Core/deficiência , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interleucina-17/genética , Interleucina-17/imunologia , Janus Quinases/genética , Janus Quinases/imunologia , Fatores de Transcrição Kruppel-Like/genética , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Transdução de Sinais , Baço/citologia , Baço/imunologiaRESUMO
Arf GTPase-activating proteins (Arf GAP) play important roles in the formation of the membrane vesicles that traffic between subcellular membranous organelles. The small Arf GTPase-activating protein (SMAP) subfamily of Arf GAPs has two members, SMAP1 and SMAP2, in mammals. The present study investigated whether these two proteins may have an overlapping function in addition to their previously reported distinct functions. Results showed that the presence of either SMAP1 or SMAP2 was sufficient for endocytosis of the transferrin receptor, and that transferrin incorporation was impaired only by the absence of both SMAP1 and SMAP2. This suggests the involvement of both SMAP1 and SMAP2 in transferrin endocytosis. Results also demonstrated a physical association between SMAP1 and SMAP2, which might serve as a basis for a functional interaction, and identified the intramolecular domains responsible for this association.
Assuntos
Endocitose/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Receptores da Transferrina/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Ligação ProteicaRESUMO
Ligand-stimulated receptor tyrosine kinases (RTKs) are phosphorylated/ubiquitinated, endocytosed and transported to the lysosomes via endosomes/multivesicular bodies, resulting in the attenuation of signal transmission. If this physiological mechanism of RTK signal downregulation is perturbed, signal transduction persists and may contribute to cellular transformation. This article presents several such examples. In some cases, endocytosis is impaired, and the activated RTK remains on the plasma membrane. In other cases, the activated RTK is endocytosed into endosomes/multivesicular bodies, but not subsequently sorted to the lysosomes for degradation. The latter cases indicate that even endocytosed RTKs can transmit signals. Transport of RTKs is accomplished via the formation and movement of membrane vesicles. Blockage or delay of endocytosis/trafficking can be caused by genetic alterations in the RTK itself or by mutations in CBL, Arf GAPs, or other components involved in internalization and vesicle transport. A survey of the literature indicates that, in some cases, even RTKs synthesized de novo can initiate signaling at the endoplasmic reticulum/Golgi before reaching the plasma membrane. The spectrum of molecules targeted by the signal is likely to be different between cell surface- and endoplasmic reticulum/Golgi-localized RTKs.
RESUMO
Obesity is a risk factor for many human diseases. However, the underlying molecular causes of obesity are not well understood. Here, we report that protein tyrosine phosphatase receptor T (PTPRT) knockout mice are resistant to high-fat diet-induced obesity. Those mice avoid many deleterious side effects of high-fat diet-induced obesity, displaying improved peripheral insulin sensitivity, lower blood glucose and insulin levels. Compared to wild type littermates, PTPRT knockout mice show reduced food intake. Consistently, STAT3 phosphorylation is up-regulated in the hypothalamus of PTPRT knockout mice. These studies implicate PTPRT-modulated STAT3 signaling in the regulation of high-fat diet-induced obesity.
Assuntos
Resistência à Insulina/genética , Obesidade/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Fator de Transcrição STAT3/biossíntese , Animais , Glicemia , Dieta Hiperlipídica , Gorduras na Dieta , Humanos , Hipotálamo/metabolismo , Insulina/sangue , Camundongos , Camundongos Knockout , Obesidade/sangue , Obesidade/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genéticaRESUMO
Runx1 transcription factor is a key player in the development and function of T cells. Runx1 transcripts consist of two closely related isoforms (proximal and distal Runx1) whose expressions are regulated by different promoters. Which Runx1 isoform is expressed appears to be tightly regulated. The regulatory mechanism for differential transcription is, however, not fully understood. In this study, we investigated the regulation of the proximal Runx1 promoter in T cells. We showed that proximal Runx1 was expressed at a low level in naïve T cells from C57BL/6 mice, but its expression was remarkably induced upon T-cell activation. In the promoter of proximal Runx1, a highly conserved region was identified which spans from -412 to the transcription start site and harbors a NFAT binding site. In a luciferase reporter assay, this region was found to be responsive to T-cell activation through Lck and calcineurin pathways. Mutagenesis studies and chromatin immunoprecipitation assay indicated that the NFAT site was essential for NFAT binding and transactivation of the proximal Runx1 promoter. Furthermore, TCR signaling-induced expression of proximal Runx1 was blocked by treatment of cells with cyclosporin A. Together, these results demonstrate that the calcineurin-NFAT pathway regulates proximal Runx1 transcription upon TCR stimulation.
Assuntos
Calcineurina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Fatores de Transcrição NFATC/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Ativação Transcricional , Animais , Sítios de Ligação , Sequência Conservada , Ciclosporina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mutação , Fatores de Transcrição NFATC/genética , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The c-ABL non-receptor tyrosine kinase and the p53 tumor suppressor protein are pivotal modulators of cellular responses to DNA damage. However, a comprehensive understanding of the role of c-ABL kinase in p53-dependent transcription of p21(CIP1/WAF1) and ensuing cell fate decision is still obscure. Here, we demonstrate that c-ABL tyrosine kinase regulates p53-dependent induction of p21. As a result, it modulates cell fate decision by p53 in response to DNA damage differently according to the extent of DNA damage. When human cancer cells were treated with DNA damaging agent, adriamycin (0.08 µg/ml), p21 was induced following p53 induction. Owing largely to p21, a substantial fraction of cells treated with adriamycin were blocked at the G2 phase of the cell cycle and most cells eventually became senescent. When these cells were simultaneously treated with a c-ABL kinase inhibitor, STI571, or a c-ABL-specific siRNA along with adriamycin, the p53-dependent p21 induction was dramatically diminished, even though p53 is substantially induced. Accordingly, G2-arrest, and cellular senescence largely dependent on p21 were substantially abrogated. On the contrary, when cells were treated with a relatively high dose of adriamycin (0.4 µg/ml) cells became apoptotic, and the simultaneous presence of a c-ABL kinase inhibitor STI571 augmented the extent of apoptosis. We speculate this is due to abrogation of p53-dependent p21 induction, which leads to elimination of anti-apoptotic function of p21. In summary, c-ABL appears to promote senescence or inhibit apoptosis, depending on the extent of DNA damage. These findings suggest that the combined use of ABL kinase inhibitor and DNA damaging drug in chemotherapy against tumors retaining wild type p53 should be carefully designed.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/toxicidade , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células HeLa , Humanos , Mesilato de Imatinib , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/genética , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.
Assuntos
Acrossomo/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Espermatogênese , Rede trans-Golgi/metabolismo , Animais , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Modelos Animais de Doenças , Feminino , Marcação de Genes , Humanos , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Proteínas Monoméricas de Montagem de Clatrina/genética , Espermátides/metabolismo , Sintaxina 1/metabolismoRESUMO
The RUNX family genes encode transcription factors that are involved in development and human diseases. RUNX1 is one of the most frequently mutated genes in human hematological malignancies and is a critical factor for the generation and maintenance of hematopoietic stem cells. Another Runx family gene, Runx3, is known to be expressed in hematopoietic cells. However, its involvement in hematopoiesis remains unclear. Here we show the hematopoietic phenotypes in Runx3 conditional knockout (KO) mice (Runx3(fl/fl);Mx1-Cre(+)): whereas young Runx3 KO mice did not exhibit any significant hematopoietic defects, aged Runx3 KO mice developed a myeloproliferative disorder characterized by myeloid-dominant leukocytosis, splenomegaly, and an increase of hematopoietic stem/progenitor cells (HSPCs). Notably, Runx3-deficient cells showed hypersensitivity to granulocyte-colony stimulating factor, suggesting enhanced proliferative and mobilization capability of Runx3-deficient HSPCs when stimulated. These results suggest that, besides Runx1, Runx3 also plays a role in hematopoiesis.
Assuntos
Envelhecimento/fisiologia , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Hematopoese/genética , Transtornos Mieloproliferativos/genética , Envelhecimento/sangue , Envelhecimento/genética , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 3 de Fator de Ligação ao Core/deficiência , Técnicas de Transferência de Genes , Fator Estimulador de Colônias de Granulócitos/farmacologia , Hematopoese/efeitos dos fármacos , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos Mieloproliferativos/patologiaRESUMO
The common marmoset is a new world monkey, which has become a valuable experimental animal for biomedical research. This study developed cDNA libraries for the common marmoset from five different tissues. A total of 290 426 high-quality EST sequences were obtained, where 251 587 sequences (86.5%) had homology (1E(-100)) with the Refseqs of six different primate species, including human and marmoset. In parallel, 270 673 sequences (93.2%) were aligned to the human genome. When 247 090 sequences were assembled into 17 232 contigs, most of the sequences (218 857 or 15 089 contigs) were located in exonic regions, indicating that these genes are expressed in human and marmoset. The other 5578 sequences (or 808 contigs) mapping to the human genome were not located in exonic regions, suggesting that they are not expressed in human. Furthermore, a different set of 118 potential coding sequences were not similar to any Refseqs in any species, and, thus, may represent unknown genes. The cDNA libraries developed in this study are available through RIKEN Bio Resource Center. A Web server for the marmoset cDNAs is available at http://marmoset.nig.ac.jp/index.html, where each marmoset EST sequence has been annotated by reference to the human genome. These new libraries will be a useful genetic resource to facilitate research in the common marmoset.
Assuntos
Callithrix/genética , DNA Complementar/genética , Biblioteca Gênica , Animais , Mapeamento de Sequências Contíguas , DNA Complementar/química , Éxons , Etiquetas de Sequências Expressas , Genoma Humano , Humanos , Fases de Leitura Aberta , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
The formation of clathrin-coated vesicles is essential for intracellular membrane trafficking between subcellular compartments and is triggered by the ARF family of small GTPases. We previously identified SMAP1 as an ARF6 GTPase-activating protein that functions in clathrin-dependent endocytosis. Because abnormalities in clathrin-dependent trafficking are often associated with oncogenesis, we targeted Smap1 in mice to examine its physiological and pathological significance. Smap1-deficent mice exhibited healthy growth, but their erythroblasts showed enhanced transferrin endocytosis. In mast cells cultured in SCF, Smap1 deficiency did not affect the internalization of c-KIT but impaired the sorting of internalized c-KIT from multivesicular bodies to lysosomes, resulting in intracellular accumulation of undegraded c-KIT that was accompanied by enhanced activation of ERK and increased cell growth. Interestingly, approximately 50% of aged Smap1-deficient mice developed anemia associated with morphologically dysplastic cells of erythroid-myeloid lineage, which are hematological abnormalities similar to myelodysplastic syndrome (MDS) in humans. Furthermore, some Smap1-deficient mice developed acute myeloid leukemia (AML) of various subtypes. Collectively, to our knowledge these results provide the first evidence in a mouse model that the deregulation of clathrin-dependent membrane trafficking may be involved in the development of MDS and subsequent AML.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas de Membrana/deficiência , Síndromes Mielodisplásicas/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Cinética , Lisossomos/metabolismo , Mastócitos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Transporte Proteico , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Baço/patologia , Transferrina/metabolismoRESUMO
After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G(1) cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G(1)-phase extension and represses CD8 expression in a G(1) extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G(1) extension and CD8 repression. Importantly, forced G(1) extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G(1) progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G(1)-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem da Célula/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/fisiologia , Fase G1/imunologia , Fatores de Transcrição/fisiologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Células Tumorais CultivadasRESUMO
Mast cells (MCs) are developed from hematopoietic progenitor cells and play an important role in inflammation. Study of the kinetics of development and accumulation of primate MC in vivo is crucial for the control of human inflammatory diseases, as evolution of the immune system is quite rapid and inflammation including MC response is considered to be different between mouse and human. In the present study, we examined the development of MC from hematopoietic progenitors of Callithrix jacchus (common marmoset), an experimental animal of nonhuman primates. Bone marrow cells were fractionated for the expression of CD34 and CD117 by cell sorting. MCs were developed in vitro or by transplanting the cells to NOD/SCID/IL-2γc knockout (NOG) mice. In vitro culture of CD34(+)CD117(+) (double positive, DP) cells with stem cell factor could generate high-affinity Fc epsilon receptor (FcεR)-expressing CD117(+) cells with typical granules. The developed MC released ß-hexosaminidase and produced leukotriene C(4) after the stimulation of FcεRI. Transplantation of DP cells gave rise to a marked expansion of CD34(-)CD45(+)CD117(+)FcεR(+) cells in NOG mice. They expressed transcripts encoding chymase 1 and tryptase ß. Differentiation of CD34(-)CD117(+) cells to MCs was relatively limited compared with the DP cells, similarly to human MCs. These results suggest that this marmoset system provides a good model for human MC development.
Assuntos
Biomarcadores/metabolismo , Células da Medula Óssea/imunologia , Callithrix/imunologia , Mastócitos/imunologia , Células-Tronco/imunologia , Animais , Antígenos CD34/metabolismo , Diferenciação Celular , Proliferação de Células , Separação Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Mastócitos/transplante , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Modelos Animais , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de IgE/metabolismoRESUMO
The Runx1 transcription factor is abundantly expressed in naive T cells but rapidly downregulated in activated T cells, suggesting that it plays an important role in a naive stage. In the current study, Runx1(-/-)Bcl2(tg) mice harboring Runx1-deleted CD4(+) T cells developed a fatal autoimmune lung disease. CD4(+) T cells from these mice were spontaneously activated, preferentially homed to the lung, and expressed various cytokines, including IL-17 and IL-21. Among these, the deregulation of IL-21 transcription was likely to be associated with Runx binding sites located in an IL-21 intron. IL-17 produced in Runx1-deleted cells mobilized innate immune responses, such as those promoted by neutrophils and monocytes, whereas IL-21 triggered humoral responses, such as plasma cells. Thus, at an initial stage, peribronchovascular regions in the lung were infiltrated by CD4(+) lymphocytes, whereas at a terminal stage, interstitial regions were massively occupied by immune cells, and alveolar spaces were filled with granular exudates that resembled pulmonary alveolar proteinosis in humans. Mice suffered from respiratory failure, as well as systemic inflammatory responses. Our data indicate that Runx1 plays an essential role in repressing the transcription of cytokine genes in naive CD4(+) T cells and, thereby, maintains cell quiescence.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/deficiência , Pneumopatias/imunologia , Ativação Linfocitária/imunologia , Animais , Doenças Autoimunes/mortalidade , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Citocinas/antagonistas & inibidores , Citocinas/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/mortalidade , Células Jurkat , Pneumopatias/mortalidade , Pneumopatias/patologia , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologiaRESUMO
In this study, we investigated the evolution of vertebrate tissues by examining the potential association among gene expression, duplication, and base substitution patterns. In particular, we compared whole-genome duplication (WGD) with small-scale duplication (SSD), as well as tissue restricted with ubiquitously expressed genes. All patterns were also analysed in the light of gene evolutionary rates. Among those genes characterized by rapid evolution and expressed in a restricted range of tissues, SSD was represented in a larger proportion than WGD. Conversely, genes with ubiquitous expression were associated with slower evolutionary rates and a larger proportion of WGD. The results also show that evolutionary rates were faster in genes expressed in endodermal tissues and slower in ectodermal genes. Accordingly, the proportion of the SSD and WGD genes was highest in the endoderm and ectoderm, respectively. Therefore, quickly evolving SSD genes might have contributed to the faster evolution of endodermal tissues, whereas the comparatively slowly evolving WGD genes might have functioned to maintain the basic characteristics of ectodermal tissues. Mesenchymal tissues occupied an intermediate position in this regard, whereas the patterns observed for haemocytes were unique. Rapid tissue evolution could be related to a specific gene duplication mode (SSD) and faster molecular evolution in response to exposure to the external environment. These findings reveal general patterns underlying the evolution of tissues and their corresponding genes.