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1.
Proc Natl Acad Sci U S A ; 117(32): 19544-19555, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32747566

RESUMO

Corresponding attributes of neural development and function suggest arthropod and vertebrate brains may have an evolutionarily conserved organization. However, the underlying mechanisms have remained elusive. Here, we identify a gene regulatory and character identity network defining the deutocerebral-tritocerebral boundary (DTB) in Drosophila This network comprises genes homologous to those directing midbrain-hindbrain boundary (MHB) formation in vertebrates and their closest chordate relatives. Genetic tracing reveals that the embryonic DTB gives rise to adult midbrain circuits that in flies control auditory and vestibular information processing and motor coordination, as do MHB-derived circuits in vertebrates. DTB-specific gene expression and function are directed by cis-regulatory elements of developmental control genes that include homologs of mammalian Zinc finger of the cerebellum and Purkinje cell protein 4 Drosophila DTB-specific cis-regulatory elements correspond to regulatory sequences of human ENGRAILED-2, PAX-2, and DACHSHUND-1 that direct MHB-specific expression in the embryonic mouse brain. We show that cis-regulatory elements and the gene networks they regulate direct the formation and function of midbrain circuits for balance and motor coordination in insects and mammals. Regulatory mechanisms mediating the genetic specification of cephalic neural circuits in arthropods correspond to those in chordates, thereby implying their origin before the divergence of deuterostomes and ecdysozoans.


Assuntos
Evolução Molecular , Redes Reguladoras de Genes , Mesencéfalo/fisiologia , Animais , Comportamento Animal , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Drosophila , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Sequências Reguladoras de Ácido Nucleico , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Rombencéfalo/fisiologia , Transdução de Sinais
2.
Anat Sci Int ; 95(4): 523-539, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32476103

RESUMO

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease caused by the destruction of pancreatic ß-cells. Human dental pulp stem cells represent a promising source for cell-based therapies, owing to their easy, minimally invasive surgical access, and high proliferative capacity. It was reported that human dental pulp stem cells can differentiate into a pancreatic cell lineage in vitro; however, few studies have investigated their effects on diabetes. Our study aimed to investigate the therapeutic potential of intravenous and intrapancreatic transplantation of human dental pulp stem cells in a rat model of streptozotocin-induced type 1 diabetes. Forty Sprague Dawley male rats were randomly categorized into four groups: control, diabetic (STZ), intravenous treatment group (IV), and intrapancreatic treatment group (IP). Human dental pulp stem cells (1 × 106 cells) or vehicle were injected into the pancreas or tail vein 7 days after streptozotocin injection. Fasting blood glucose levels were monitored weekly. Glucose tolerance test, rat and human serum insulin and C-peptide, pancreas histology, and caspase-3, vascular endothelial growth factor, and Ki67 expression in pancreatic tissues were assessed 28 days post-transplantation. We found that both IV and IP transplantation of human dental pulp stem cells reduced blood glucose and increased levels of rat and human serum insulin and C-peptide. The cells engrafted and survived in the streptozotocin-injured pancreas. Islet-like clusters and scattered human dental pulp stem cells expressing insulin were observed in the pancreas of diabetic rats with some difference in the distribution pattern between the two injection routes. RT-PCR analyses revealed the expression of the human-specific pancreatic ß-cell genes neurogenin 3 (NGN3), paired box 4 (PAX4), glucose transporter 2 (GLUT2), and insulin in the pancreatic tissues of both the IP and IV groups. In addition, the transplanted cells downregulated the expression of caspase-3 and upregulated the expression of vascular endothelial growth factor and Ki67, suggesting that the injected cells exerted pro-angiogenetic and antiapoptotic effects, and promoted endogenous ß-cell replication. Our study is the first to show that human dental pulp stem cells can migrate and survive within streptozotocin-injured pancreas, and induce antidiabetic effects through the differentiation and replacement of lost ß-cells and paracrine-mediated pancreatic regeneration. Thus, human dental pulp stem cells may have therapeutic potential to treat patients with long term T1DM.


Assuntos
Polpa Dentária/citologia , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Pâncreas/fisiologia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caspase 3/metabolismo , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Modelos Animais de Doenças , Transportador de Glucose Tipo 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração , Estreptozocina
3.
Cancer Res ; 80(4): 832-842, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31888889

RESUMO

The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS. SIGNIFICANCE: These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aurora Quinase A/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/tratamento farmacológico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Aurora Quinase A/antagonistas & inibidores , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/metabolismo , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Development ; 147(1)2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31806659

RESUMO

The GATA and PAX-SIX-EYA-DACH transcriptional networks (PSEDNs) are essential for proper development across taxa. Here, we demonstrate novel PSEDN roles in vivo in Drosophila hematopoiesis and in human erythropoiesis in vitro Using Drosophila genetics, we show that PSEDN members function with GATA to block lamellocyte differentiation and maintain the prohemocyte pool. Overexpression of human SIX1 stimulated erythroid differentiation of human erythroleukemia TF1 cells and primary hematopoietic stem-progenitor cells. Conversely, SIX1 knockout impaired erythropoiesis in both cell types. SIX1 stimulation of erythropoiesis required GATA1, as SIX1 overexpression failed to drive erythroid phenotypes and gene expression patterns in GATA1 knockout cells. SIX1 can associate with GATA1 and stimulate GATA1-mediated gene transcription, suggesting that SIX1-GATA1 physical interactions contribute to the observed functional interactions. In addition, both fly and human SIX proteins regulated GATA protein levels. Collectively, our findings demonstrate that SIX proteins enhance GATA function at multiple levels, and reveal evolutionarily conserved cooperation between the GATA and PSEDN networks that may regulate developmental processes beyond hematopoiesis.


Assuntos
Proteínas de Drosophila/metabolismo , Eritropoese/genética , Redes Reguladoras de Genes , Hematopoese/genética , Animais , Linhagem Celular Tumoral , Drosophila , Fatores de Transcrição GATA/metabolismo , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição Box Pareados/metabolismo
5.
Dev Cell ; 52(2): 223-235.e5, 2020 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-31866202

RESUMO

Cell polarity is a key feature in the development of multicellular organisms. For instance, asymmetrically localized plasma-membrane-integral PIN-FORMED (PIN) proteins direct transcellular fluxes of the phytohormone auxin that govern plant development. Fine-tuned auxin flux is important for root protophloem sieve element differentiation and requires the interacting plasma-membrane-associated BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX) proteins. We observed "donut-like" polar PIN localization in developing sieve elements that depends on complementary, "muffin-like" polar localization of BRX and PAX. Plasma membrane association and polarity of PAX, and indirectly BRX, largely depends on phosphatidylinositol-4,5-bisphosphate. Consistently, mutants in phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) display protophloem differentiation defects similar to brx mutants. The same PIP5Ks are in complex with BRX and display "muffin-like" polar localization. Our data suggest that the BRX-PAX module recruits PIP5Ks to reinforce PAX polarity and thereby the polarity of all three proteins, which is required to maintain a local PIN minimum.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Polaridade Celular , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Mutação , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento
6.
Int J Mol Sci ; 20(24)2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817798

RESUMO

Gestational diabetes mellitus (GDM), a metabolic disease that develops with the increase in insulin resistance during late pregnancy, is currently one of the most common complications affecting pregnancy. The polygenic nature of GDM, together with the interplay between different genetic variants with nutritional and environmental factors has hindered the full understanding of the etiology of this disease. However, an important genetic overlap has been found with type 2 diabetes mellitus (T2DM) and, as in the case of T2DM, most of the identified loci are associated with ß-cell function. Early detection of GDM and adequate interventions to control the maternal glycemia are necessary to avoid the adverse outcomes for both the mother and the offspring. The in utero exposure to the diabetic milieu predispose these children for future diseases, among them T2DM, originating a vicious circle implicated in the increased prevalence of both GDM and T2DM. The involvement of inflammatory processes in the development of GDM highlights the importance of pancreatic ß-cell factors able to favor the adaptation processes required during gestation, concomitantly with the protection of the islets from an inflammatory milieu. In this regard, two members of the Pax family of transcription factors, PAX4 and PAX8, together with the chromatin remodeler factor HMG20A, have gained great relevance due to their involvement in ß-cell mass adaptation together with their anti-inflammatory properties. Mutations in these factors have been associated with GDM, highlighting these as novel candidates for genetic screening analysis in the identification of women at risk of developing GDM.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Diabetes Gestacional/metabolismo , Diabetes Gestacional/fisiopatologia , Ilhotas Pancreáticas/fisiologia , Glicemia/metabolismo , Feminino , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Fator de Transcrição PAX8/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Gravidez
8.
Genes (Basel) ; 10(10)2019 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614829

RESUMO

Development requires the careful orchestration of several biological events in order to create any structure and, eventually, to build an entire organism. On the other hand, the fate transformation of terminally differentiated cells is a consequence of erroneous development, and ultimately leads to cancer. In this review, we elaborate how development and cancer share several biological processes, including molecular controls. Transcription factors (TF) are at the helm of both these processes, among many others, and are evolutionarily conserved, ranging from yeast to humans. Here, we discuss four families of TFs that play a pivotal role and have been studied extensively in both embryonic development and cancer-high mobility group box (HMG), GATA, paired box (PAX) and basic helix-loop-helix (bHLH) in the context of their role in development, cancer, and their conservation across several species. Finally, we review TFs as possible therapeutic targets for cancer and reflect on the importance of natural resistance against cancer in certain organisms, yielding knowledge regarding TF function and cancer biology.


Assuntos
Desenvolvimento Embrionário , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular/genética , Movimento Celular/imunologia , Desenvolvimento Embrionário/genética , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Fatores de Transcrição GATA/química , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Proteínas HMGB/química , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fatores de Transcrição Box Pareados/química , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética
9.
Cell Rep ; 29(4): 961-973.e4, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31644916

RESUMO

Taste drives appropriate food preference and intake. In Drosophila, taste neurons are housed in both external and internal organs, but the latter have been relatively underexplored. Here, we report that Poxn mutants with a minimal taste system of pharyngeal neurons can avoid many aversive tastants, including bitter compounds, acid, and salt, suggesting that pharyngeal taste is sufficient for rejecting intake of aversive compounds. Optogenetic activation of selected pharyngeal bitter neurons during feeding events elicits changes in feeding parameters that can suppress intake. Functional dissection experiments indicate that multiple classes of pharyngeal neurons are involved in achieving behavioral avoidance, by virtue of being inhibited or activated by aversive tastants. Tracing second-order pharyngeal circuits reveals two main relay centers for processing pharyngeal taste inputs. Together, our results suggest that the pharynx can control the ingestion of harmful compounds by integrating taste input from different classes of pharyngeal neurons.


Assuntos
Aprendizagem da Esquiva , Células Quimiorreceptoras/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Paladar , Animais , Agentes Aversivos/farmacologia , Células Quimiorreceptoras/efeitos dos fármacos , Células Quimiorreceptoras/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Preferências Alimentares , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição Box Pareados/genética , Faringe/citologia , Percepção Gustatória
10.
Genes (Basel) ; 10(9)2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480361

RESUMO

Rhabdomyosarcoma is subclassified by the presence or absence of a recurrent chromosome translocation that fuses the FOXO1 and PAX3 or PAX7 genes. The fusion protein (FOXO1-PAX3/7) retains both binding domains and becomes a novel and potent transcriptional regulator in rhabdomyosarcoma subtypes. Many studies have characterized and integrated genomic, transcriptomic, and epigenomic differences among rhabdomyosarcoma subtypes that contain the FOXO1-PAX3/7 gene fusion and those that do not; however, few investigations have investigated how gene co-expression networks are altered by FOXO1-PAX3/7. Although transcriptional data offer insight into one level of functional regulation, gene co-expression networks have the potential to identify biological interactions and pathways that underpin oncogenesis and tumorigenicity. Thus, we examined gene co-expression networks for rhabdomyosarcoma that were FOXO1-PAX3 positive, FOXO1-PAX7 positive, or fusion negative. Gene co-expression networks were mined using local maximum Quasi-Clique Merger (lmQCM) and analyzed for co-expression differences among rhabdomyosarcoma subtypes. This analysis observed 41 co-expression modules that were shared between fusion negative and positive samples, of which 17/41 showed significant up- or down-regulation in respect to fusion status. Fusion positive and negative rhabdomyosarcoma showed differing modularity of co-expression networks with fusion negative (n = 109) having significantly more individual modules than fusion positive (n = 53). Subsequent analysis of gene co-expression networks for PAX3 and PAX7 type fusions observed 17/53 were differentially expressed between the two subtypes. Gene list enrichment analysis found that gene ontology terms were poorly matched with biological processes and molecular function for most co-expression modules identified in this study; however, co-expressed modules were frequently localized to cytobands on chromosomes 8 and 11. Overall, we observed substantial restructuring of co-expression networks relative to fusion status and fusion type in rhabdomyosarcoma and identified previously overlooked genes and pathways that may be targeted in this pernicious disease.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma/genética , Redes Reguladoras de Genes , Humanos , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rabdomiossarcoma/classificação , Transcriptoma
11.
Diagn Pathol ; 14(1): 103, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31493794

RESUMO

BACKGROUND: The most frequent histological types of rhabdomyosarcoma (RMS) in children are embryonal (ERMS) and alveolar (ARMS) tumours. The majority of ARMS are characterized by the presence of PAX3/7-FOXO1 gene fusion and have a worse prognosis than fusion gene-negative ARMS. However, identification of PAX3/7-FOXO1 fusion status is challenging when using formalin-fixed, paraffin-embedded (FFPE) material. Microarray analyses revealed that high expression of several genes is associated with PAX3/7-FOXO1 fusion status. Therefore, we investigated if immunohistochemical approach may detect surrogate marker genes as indicators of fusion gene-positive RMS. METHODS: Forty five RMS patients were included in the analysis and immunohistochemistry was applied to FFPE tissues collected at diagnosis. Protein expression of OLIG2, a novel marker in RMS, was investigated using antibody EP112 (Cell Marque). In addition already known two markers were also analyzed: TFAP2B using rabbit anti-TFAP2ß antibody (Santa Cruz Biotechnology) and ALK using anti-ALK antibody clone D5F3 #3633 (Cell Signalling). Fluorescence in situ hybridization (FISH) was performed on FFPE sections with FOXO1/PAX3 and/or FOXO1/PAX7 probes (Dual Colour Single Fusion Probe, Zytovision). RESULTS: Our analysis revealed that all three immunohistochemical markers are associated with the presence of PAX3/7-FOXO1 fusion: TFAP2B (p < 0.00001), OLIG2 (p = 0.0001) and ALK (p = 0.0007). Four ARMS had negative PAX3/7-FOXO1 status and none of them displayed positive reaction with the analysed markers. Positive reaction with OLIG2 (6 tumours) was always associated with the presence of PAX3/7-FOXO1 rearrangement. Two additional OLIG2 positive cases showed inconclusive FISH results, but were positive for TFAP2B and ALK, what suggests that these tumours expressed fusion positive signature. CONCLUSION: Our results indicate that TFAP2B, ALK and a novel marker OLIG2 may serve as surrogate markers for PAX3/7-FOXO1 status what is especially beneficial in cases where poor quality tumour tissue is not suitable for reliable genetic analyses or shows inconclusive result.


Assuntos
Imuno-Histoquímica , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Rabdomiossarcoma/diagnóstico , Rabdomiossarcoma/metabolismo , Adolescente , Biomarcadores/análise , Criança , Pré-Escolar , Feminino , Proteína Forkhead Box O1/metabolismo , Fatores de Transcrição Forkhead/genética , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Lactente , Masculino , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX3/metabolismo , Fatores de Transcrição Box Pareados/metabolismo
12.
Physiol Rep ; 7(17): e14200, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31496052

RESUMO

Massage is a widely accepted manual therapy used to modulate the inflammatory response of muscle and restore function, but prolonged compression of muscle potentially causes overt injury and damage to muscle fibers. Therefore, a balance exists between the positive effects of massage and the induction of mechanical damage and injury. In addition, skeletal muscle of aged individuals displays increased stiffness, and therefore, the response to massage is likely different compared with young. We hypothesized that the aged skeletal muscle exhibits increased sarcolemmal permeability when subjected to massage compared with young skeletal muscle. Male Brown Norway/F344 rats, 10 and 30 months of age, were each divided into control, non-massaged (n = 8) and massaged (n = 8) groups. The right gastrocnemius muscle received one bout of cyclic compressive loading for 30 min at 4.5 N as a massage-mimetic. Muscles were dissected and frozen 24 h after massage. Alterations in sarcolemma permeability were quantified by measuring the level of intracellular IgG within the muscle fibers. Immunohistochemistry was performed to determine IgG inside fibers and Pax7+ cell number as an indicator of stem cell abundance. Average IgG intensity was not different between control and massaged animals at either age. However, a significant shift to the right of the density histogram indicated that massaged animals had more fibers with higher IgG intensity than control at 10 months. In addition, Pax7+ cell number was significantly elevated in massaged muscles compared with control at both ages. One bout of massage did not induce overt muscle injury, but facilitated membrane permeability, which was associated with an increase in satellite cell number. Data suggest that the load applied here, which was previously shown to induce immunomodulatory changes, does not induce overt muscle injury in young and old muscles but may result in muscle remodeling. Funded by NIH grant AG042699 and AT009268.


Assuntos
Permeabilidade da Membrana Celular , Massagem , Músculo Esquelético/metabolismo , Sarcolema/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Imunoglobulina G/metabolismo , Masculino , Músculo Esquelético/crescimento & desenvolvimento , Fatores de Transcrição Box Pareados/metabolismo , Ratos , Ratos Endogâmicos F344
13.
Med Sci Monit ; 25: 6980-6989, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31527569

RESUMO

BACKGROUND The pathogenesis of idiopathic congenital clubfoot (CCF) is unknown. Although some familial patients have Pitx1 mutations, and the Pitx1+/- genotype causes a clubfoot-like phenotype in mice, the mechanism of Pitx1-induced CCF is unknown. MATERIAL AND METHODS We used tibialis anterior tendon samples to detect the expression of Pitx1 in idiopathic and neurogenic clubfoot patients. After obtaining Sprague-Dawley (SD) rat Achilles tendon cells, the expression of Pitx1 was knocked down by SiRNA. After 48 h of culture, mass spectrometry was used to quantitatively analyze proteins. Then, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to assess the downstream pathway of PITX1. The relationship between Pitx1 and the promoter region of deacetylase 1 (Sirtuin-1 and Sirt1) was examined by luciferase and ChIP assays. RESULTS We found that Pitx1 expression in the tendon samples of idiopathic CCF patients was downregulated. Mass spectrometry analysis revealed that the inhibition of Pitx1 induced the downregulation of Sirt1 expression in tendon cells. Luciferase and ChIP assays confirmed that Pitx1 binds to the promoter region of SIRT1 and promotes Sirt1 gene transcription. Further results showed that, after the inhibition of Pitx1 in tendon cells, CRABP2 acetylation increased, the nuclear import of CRABP2 was enhanced, and the expression of RARß2 increased. After the inhibition of Pitx1, RARß2 expression was further increased by RA treatment in tendon cells. In the presence of retinoic acid, the expression of Pitx1 was inhibited in tendon cells. CONCLUSIONS Pitx1 binds to the promoter region of SIRT1 and promotes the transcription of SIRT1. Positive feedback occurs between RA signaling and Pitx1.


Assuntos
Pé Torto Equinovaro/metabolismo , Pé Torto Equinovaro/patologia , Fatores de Transcrição Box Pareados/metabolismo , Transdução de Sinais , Tendões/patologia , Tretinoína/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico , Criança , Pré-Escolar , Retroalimentação Fisiológica , Humanos , Masculino , Regiões Promotoras Genéticas/genética , Ratos Sprague-Dawley , Sirtuína 1/genética , Sirtuína 1/metabolismo
14.
PLoS One ; 14(8): e0217605, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31404068

RESUMO

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex.


Assuntos
Regulação Enzimológica da Expressão Gênica , Melanoma/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , RNA Mensageiro/metabolismo , Telomerase/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética , Humanos , Melanoma/genética , Fatores de Transcrição Box Pareados/genética , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , Telomerase/genética , Fatores de Transcrição/genética , Transcrição Genética , Células Tumorais Cultivadas
15.
Diabetes Res Clin Pract ; 155: 107792, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31325538

RESUMO

Gene regulatory factors that govern the expression of heritable information come in an array of flavors, chiefly with transcription factors, the proteins which bind to regions of specific genes and modulate gene transcription, subsequently altering cellular function. PAX transcription factors are sequence-specific DNA-binding proteins exerting its regulatory activity in many tissues. Notably, three members of the PAX family namely PAX2, PAX4 and PAX6 have emerged as crucial players at multiple steps of pancreatic development and differentiation and also play a pivotal role in the regulation of pancreatic islet hormones synthesis and secretion. Providing a comprehensive outline of these transcription factors and their primordial and divergent roles in the pancreas is far-reaching in contemporary diabetes research. Accordingly, this review furnishes an outline of the role of pancreatic specific PAX regulators in the development of the pancreas and its associated disorders.


Assuntos
Fatores de Transcrição Box Pareados/metabolismo , Pâncreas/patologia , Pancreatopatias/patologia , Animais , Humanos , Pâncreas/metabolismo , Pancreatopatias/metabolismo
16.
Am J Physiol Cell Physiol ; 317(3): C492-C501, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31216190

RESUMO

The transcription factor aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1) is an essential regulator of the circadian clock, which controls the 24-h cycle of physiological processes such as nutrient absorption. To examine the role of BMAL1 in small intestinal glucose absorption, we used differentiated human colon adenocarcinoma cells (Caco-2 cells). Here, we show that BMAL1 regulates glucose uptake in differentiated Caco-2 cells and that this process is dependent on the glucose transporter sodium-glucose cotransporter 1 (SGLT1). Mechanistic studies show that BMAL1 regulates glucose uptake by controlling the transcription of SGLT1 involving paired-homeodomain transcription factor 4 (PAX4), a transcriptional repressor. This is supported by the observation that clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated endonuclease Cas9 (Cas9) knockdown of PAX4 increases SGLT1 and glucose uptake. Chromatin immunoprecipitation (ChIP) and ChIP-quantitative PCR assays show that the knockdown or overexpression of BMAL1 decreases or increases the binding of PAX4 to the hepatocyte nuclear factor 1-α binding site of the SGLT1 promoter, respectively. These findings identify BMAL1 as a critical mediator of small intestine carbohydrate absorption and SGLT1.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Diferenciação Celular/fisiologia , Glucose/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição ARNTL/genética , Células CACO-2 , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição Box Pareados/genética
17.
Sci Rep ; 9(1): 9195, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235851

RESUMO

DNA methylation alteration, such as global hypomethylation and localized hypermethylation, within the promoters of tumor suppressor genes, is an important risk factor in cervical cancer. The potential use of DNA methylation detection, in cervical cancer screening or triage of mildly abnormal cytology, has recently been demonstrated. In particular, PAX1 DNA methylation testing was approved as an adjunct to cytology, in Taiwan, and is now undergoing registration trials in China. However, the function of PAX1 in cancer biology remains largely unknown. Here, we show that PAX1 inhibits malignant phenotypes upon oncogenic stress. Specifically, PAX1 expression inhibited the phosphorylation of multiple kinases, after challenges with oncogenic growth factors such as EGF and IL-6. Analogously, PAX1 activated a panel of phosphatases, including DUSP1, 5, and 6, and inhibited EGF/MAPK signaling. PAX1 also interacted with SET1B, increasing histone H3K4 methylation and DNA demethylation of numerous phosphatase-encoding genes. Furthermore, hypermethylated PAX1 associated with poor prognosis in cervical cancer. Taken together, this study reveals, for the first time, the functional relevance of PAX1 in cancer biology, and further supports the prospect of targeting multifold oncogenic kinase cascades, which jointly contribute to multiresistance, via epigenetic reactivation of PAX1.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Neoplasias do Colo do Útero/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Metilação de DNA , Fosfatases de Especificidade Dupla/metabolismo , Epigênese Genética , Feminino , Células HeLa , Humanos , Fosfotransferases/metabolismo , Neoplasias do Colo do Útero/diagnóstico
18.
Brain Struct Funct ; 224(7): 2325-2341, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31203451

RESUMO

In mammals, the development of the olfactory bulb (OB) relies in part on the expression of transcription factors involved in the specifications/differentiation of glutamatergic cells. In a previous study from our group, a high molecular similarity was reported between mammals and cartilaginous fishes regarding the neurogenic mechanisms underlying the development of glutamatergic cells in the telencephalon. However, information about the transcriptional program operating in the development of the glutamatergic system (mainly represented by mitral cells) in the OB is lacking in the catshark Scyliorhinus canicula, a cartilaginous fish. Using immunohistochemistry and in situ hybridization techniques, we have found that, previously to the appearance of the olfactory primordium (OP), proliferating cells expressing Pax6 with molecular hallmarks of progenitor radial glia were located in the ventrolateral pallial ventricular zone. Later in development, when the OP is recognizable, a stream of Pax6-positive cells were observed between the ventricular zone and the OP, where transcription factors involved in mitral cell development in mammals (ScTbr2, ScNeuroD, Tbr1) are expressed. Later in development, these transcription factors became expressed in a layered-like structure where ScVglut1, a marker of mitral cells, is also present. Our data suggest that the transcriptional program related with the specification/differentiation of glutamatergic cells in the telencephalon has been conserved throughout the evolution of vertebrates. These results, in combination with previous studies concerning GABAergic neurogenesis in sharks, have evidenced that the OB of mammals and sharks shares similarities in the timing and molecular programs of development.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Bulbo Olfatório/metabolismo , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Tubarões/metabolismo , Telencéfalo/metabolismo
19.
Dev Biol ; 454(2): 128-144, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31247188

RESUMO

The tetrapod limb is a stunning example of evolutionary diversity, with dramatic variation not only among distantly related species, but also between the serially homologous forelimbs (FLs) and hindlimbs (HLs) within species. Despite this variation, highly conserved genetic and developmental programs underlie limb development and identity in all tetrapods, raising the question of how limb diversification is generated from a conserved toolkit. In some breeds of domestic pigeon, shifts in the expression of two conserved limb identity transcription factors, PITX1 and TBX5, are associated with the formation of feathered HLs with partial FL identity. To determine how modulation of PITX1 and TBX5 expression affects downstream gene expression, we compared the transcriptomes of embryonic limb buds from pigeons with scaled and feathered HLs. We identified a set of differentially expressed genes enriched for genes encoding transcription factors, extracellular matrix proteins, and components of developmental signaling pathways with important roles in limb development. A subset of the genes that distinguish scaled and feathered HLs are also differentially expressed between FL and scaled HL buds in pigeons, pinpointing a set of gene expression changes downstream of PITX1 and TBX5 in the partial transformation from HL to FL identity. We extended our analyses by comparing pigeon limb bud transcriptomes to chicken, anole lizard, and mammalian datasets to identify deeply conserved PITX1- and TBX5-responsive components of the limb identity program. Our analyses reveal a suite of predominantly low-level gene expression changes that are conserved across amniotes to regulate the identity of morphologically distinct limbs.


Assuntos
Padronização Corporal/genética , Pé/embriologia , Membro Posterior/embriologia , Animais , Columbidae/genética , Extremidades/embriologia , Plumas , Pé/fisiologia , Membro Anterior/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/metabolismo , Botões de Extremidades/metabolismo , Morfogênese/genética , Organogênese/genética , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Transdução de Sinais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
20.
Sci Rep ; 9(1): 4605, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30872687

RESUMO

A previously identified enhancer 10 kb upstream of the Aggrecan (Acan) gene (UE) can drive cartilage specific reporter expression in vivo. Here, we report that the paralogous transcription factors PAX1 and PAX9 differentially drive UE, depending on the presence or absence of SOX9-driven transactivation. In the developing vertebral column, PAX1/9 expression was inversely correlated with Acan expression. Moreover, PAX1/9 was co-expressed with SOX9/5/6 in the intervertebral mesenchyme and the inner annulus fibrosus (AF), and with SOX9 in the outer AF. Significant Acan upregulation was observed during chondrification of Pax1-silenced AF cells, while, Acan was significantly downregulated by persistent expression of Pax1 in cartilage. Deletion of UE using CRISPR/Cas9 resulted in ~30% and ~40% reduction of Acan expression in cartilage and the AF, respectively. In the UE, PAX1/9 acts as weak transactivators through a PAX1/9-binding site partially overlapped with a SOX9-binding site. In the presence of SOX9, which otherwise drives robust Acan expression along with SOX5/6, PAX1/9 competes with SOX9 for occupancy of the binding site, resulting in reduced transactivation of Acan. Coimmunoprecipitation revealed the physical interaction of Pax1 with SOX9. Thus, transactivation of the UE is differentially regulated by concerted action of PAX1/9, SOX9, and SOX5/6 in a context-dependent manner.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição SOX9/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Biomarcadores , Imunofluorescência , Inativação Gênica , Camundongos , Camundongos Transgênicos , Fatores de Transcrição Box Pareados/química , Fatores de Transcrição Box Pareados/genética , Fenótipo
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