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1.
EBioMedicine ; 47: 427-435, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31481324

RESUMO

In recent years molecules involved on the immune synapse became successful targets for therapeutic immune modulation. CD6 has been extensively studied, yet, results regarding CD6 biology have been controversial, in spite of the ubiquitous presence of this molecule on virtually all CD4 T cells. We investigated the outcome of murine and human antibodies targeting CD6 domain 1. We found that CD6-targeting had a major impact on the functional specialization of CD4 cells, both human and murine. Differentiation of CD4 T cells towards a Foxp3+ Treg fate was prevented with increasing doses of anti-CD6, while Th1 polarization was favoured. No impact was observed on Th2 or Th17 specialization. These in vitro results provided an explanation for the dose-dependent outcome of in vivo anti-CD6 administration where the anti-inflammatory action is lost at the highest doses. Our data show that therapeutic targeting of the immune synapse may lead to paradoxical dose-dependent effects due to modification of T cell fate.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Animais , Biomarcadores , Linfócitos T CD4-Positivos/citologia , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular , Proteínas Fetais/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
2.
J Orthop Res ; 37(11): 2389-2400, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31286562

RESUMO

Intervertebral disc (IVD) degeneration is a major contributor to chronic low back pain and is characterized by decreases in cellularity and proteoglycan synthesis, upregulation of matrix degradation, and increases in pro-inflammatory factors with neurovascular invasion. Current treatments fail to target the underlying pathology or promote tissue repair and approaches such as viral transfection raise safety concerns due to mutagenesis and unwarranted immune responses. To avoid such concerns, nonviral transfection is a viable method of gene delivery into the host cell while bypassing the caveats of viral delivery. Brachyury is expressed in the developing notochord and is associated with an immature healthy nucleus pulposus (NP). We hypothesize that Brachyury can reprogram degenerate NP cells to a healthy pro-anabolic phenotype with increased proteoglycan content and decreased expression of catabolic, inflammatory, and neurovascular markers. NP cells obtained from human autopsy and surgical tissues were transfected with plasmids encoding for Brachyury or an empty vector control via bulk electroporation. Post transfection, cells were seeded in three-dimensional agarose constructs cultured over 4 weeks and analyzed for viability, gene expression, and proteoglycan. Results demonstrated successful transfection of both autopsy and surgical NP cells. We observed long-term Brachyury expression, significant increased expression of NP phenotypic markers FOXF1, KRT19, and chondrogenic marker SOX9 with decreases in inflammatory cytokines IL1-ß/IL6, NGF, and MMPs and significant increases in glycosaminoglycan accumulation. These results highlight nonviral transfection with developmental transcription factors, such as Brachyury, as a promising method to reprogram degenerate human disc cells toward a healthy NP phenotype. Clinical significance: This project proposes a novel translational approach for the treatment of intervertebral disc degeneration via direct reprogramming of diseased human patient-derived IVD cells to a healthy phenotype. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2389-2400, 2019.


Assuntos
Proteínas Fetais/genética , Terapia Genética/métodos , Degeneração do Disco Intervertebral/terapia , Núcleo Pulposo/metabolismo , Proteínas com Domínio T/genética , Transfecção/métodos , Adulto , Idoso , Citocinas/metabolismo , Feminino , Proteínas Fetais/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Fator de Crescimento Neural/metabolismo , Cultura Primária de Células , Estudo de Prova de Conceito , Proteínas com Domínio T/metabolismo , Adulto Jovem
3.
Artif Cells Nanomed Biotechnol ; 47(1): 3021-3028, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31334674

RESUMO

Identification of specific cell markers is crucial for recognizing functionally healthy nucleus pulposus (NP) cells. The objective of this study was to investigate the role of CD24 expression in adult human NP cells. Cells were retrieved from NP tissues of 20 patients (aged 17-44) operated on for lumbar disc herniation. Based on CD24 expression, NP cells were separated by sorting and then used to examine phenotypic behavior, the effects of culture conditions and cellular senescence pathway related proteins. CD24 expression was positive in 35.5 ± 3.7% (range 9.1-65.2%) of NP cells. Consistently, normoxic expansion and serial passages in monolayers decreased percentage positivity for CD24 in NP cells. CD24- NP cells showed a markedly decreased GSK-3ß activity and increased mitogen-activated protein kinase phosphorylation accompanying by an increased ß-catenin expression. Higher levels of matrix metalloproteinases, as well as lower levels of ACAN and COL2 in CD24- cells, indicated the breakdown and reduced the formation of key extracellular matrix components. CD24+ NP cells presented a more favorable phenotype while CD24- cells showed a more prominent cellular senescence fate. CD24 in NP cells may be a surrogate marker of healthy cells, in the cell-based therapeutic treatment of degenerative disc disorders.


Assuntos
Antígeno CD24/genética , Antígeno CD24/metabolismo , Senescência Celular , Regulação da Expressão Gênica , Núcleo Pulposo/citologia , Fenótipo , Adolescente , Adulto , Feminino , Proteínas Fetais/metabolismo , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Virais/metabolismo , Proteínas com Domínio T/metabolismo , Adulto Jovem
4.
J Neurooncol ; 144(1): 65-77, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31240525

RESUMO

BACKGROUND: Chordoma is a rare refractory neoplasm that arises from the embryological remnants of the notochord, which is incurable using any multimodality therapy. Vascular endothelial growth factor (VEGF) is a potent activator of angiogenesis that is strongly associated with the tumor-immune microenvironment. These factors have not been elucidated for chordomas. METHODS: To evaluate the characteristics of vascular and tumor cells in chordoma, we first analyzed the expression of VEGF receptor (VEGFR) 1, VEGFR2, CD34, and Brachyury in a cell line and 54 tumor tissues. Patients with primary skull base chordomas were divided into the following two groups as per the tumor growth rate: patients with slow progression (SP: < 3 mm/year) and those with rapid progression (RP: ≥ 3 mm/year). Thus, the expressions of VEGF-A, VEGFR 1, and VEGFR2 on tumor cells; tumor infiltrative immune cells, including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs); and immune-checkpoint molecules (PD-1/PD-L1) were analyzed with the clinical courses, especially in a comparison between the two groups. RESULTS: In chordomas, both VEGFR1 and VEGFR2 were strongly expressed not only on vascular endothelial cells, but also on tumor cells. The recurrent cases showed significantly higher VEGFR1 expressions on tumor cells than the primary cases. The expression of VEGF-A was significantly higher in RP than that in SP group. The numbers of CD163+ TAMs and Foxp3+ Tregs were higher in RP than that in SP group. CONCLUSIONS: Expression of VEGFR1 and VEGFR2 on tumor cells and immunosuppressive tumor-microenvironment were related to tumor growth in patients with chordomas.


Assuntos
Biomarcadores Tumorais/metabolismo , Cordoma/patologia , Recidiva Local de Neoplasia/patologia , Neoplasias da Base do Crânio/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adolescente , Adulto , Idoso , Antígenos CD34/metabolismo , Cordoma/metabolismo , Cordoma/cirurgia , Feminino , Proteínas Fetais/metabolismo , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Neoplasias da Base do Crânio/metabolismo , Neoplasias da Base do Crânio/cirurgia , Proteínas com Domínio T/metabolismo , Adulto Jovem
5.
Invest Ophthalmol Vis Sci ; 60(7): 2696-2704, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31242292

RESUMO

Purpose: Cancer stem cells (CSCs) are a subpopulation of cells with the capacity to drive tumor growth. While there is evidence of the existence of CSCs in uveal melanoma (UM), there is no consensus on their defining markers. In this study, we examined putative CSC markers in UM cell lines, primary UM (PUM), and normal choroidal melanocytes (NCM). Methods: Nonadherent sphere assays were used to assess the tumorigenic potential of 15 PUMs, 8 high (M3) and 7 low (D3) metastatic risk. Flow cytometry was used to compare the expression of CSC markers between 10 PUMs and 4 NCMs, as well as in 8 UM cell lines grown under adherent and nonadherent conditions. Based on the data generated and from TCGA analyses, CD166 was investigated in detail, including its effect on cell migration using a tumor transendothelial migration assay. Results: M3 PUM had a greater melanosphere-forming efficiency than D3 PUM. CD166 and Nestin expression was upregulated in PUM compared to NCM by flow cytometry. UM cell lines resistant to anoikis had increased levels of CD271, Nestin, and CD166 compared with adherent cells. TCGA analysis showed that patients with higher CD166 expression had a poorer prognosis: this was supported by a Mel270 CD166high subpopulation that had enhanced migratory capabilities compared with CD166low cells. IHC showed that CD166 is expressed in the cytoplasm and cell membrane of PUM cells. Conclusions: UM contain a population of cells with characteristics of CSCs. In particular, CD166high UM cells appear to represent a subpopulation with enhanced migratory capacity.


Assuntos
Antígenos CD/genética , Biomarcadores Tumorais/genética , Moléculas de Adesão Celular Neuronais/genética , Proteínas Fetais/genética , Melanoma/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Uveais/patologia , Anoikis/fisiologia , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Ensaios de Migração Celular , Movimento Celular/fisiologia , Corioide/metabolismo , Proteínas Fetais/metabolismo , Citometria de Fluxo , Dosagem de Genes , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Melanócitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Reação em Cadeia da Polimerase Multiplex , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina/genética , Nestina/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo
6.
Elife ; 82019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31063128

RESUMO

Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric enzyme consisting of a chaperonin-binding domain fused to the Cα subunit of protein kinase A. Biochemical analyses of clinical samples reveal that a unique property of this fusion enzyme is the ability to recruit heat shock protein 70 (Hsp70). This cellular chaperonin is frequently up-regulated in cancers. Gene-editing of mouse hepatocytes generated disease-relevant AML12DNAJ-PKAc cell lines. Further analyses indicate that the proto-oncogene A-kinase anchoring protein-Lbc is up-regulated in FLC and functions to cluster DNAJ-PKAc/Hsp70 sub-complexes with a RAF-MEK-ERK kinase module. Drug screening reveals Hsp70 and MEK inhibitor combinations that selectively block proliferation of AML12DNAJ-PKAc cells. Phosphoproteomic profiling demonstrates that DNAJ-PKAc biases the signaling landscape toward ERK activation and engages downstream kinase cascades. Thus, the oncogenic action of DNAJ-PKAc involves an acquired scaffolding function that permits recruitment of Hsp70 and mobilization of local ERK signaling.


Assuntos
Carcinoma Hepatocelular/fisiopatologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Fetais/metabolismo , Neoplasias Hepáticas/fisiopatologia , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas Fetais/genética , Proteínas de Choque Térmico HSP70/metabolismo , Hepatócitos/patologia , Humanos , Camundongos , Modelos Teóricos , Chaperonas Moleculares/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais
7.
Biochim Biophys Acta Mol Cell Res ; 1866(8): 1338-1352, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30905597

RESUMO

Galectin-8 (Gal-8), a 'tandem-repeat'-type galectin, has been described as a modulator of cellular functions including adhesion, spreading, growth arrest, apoptosis, pathogen recognition, autophagy, and immunomodulation. We have previously shown that activated leukocyte cell adhesion molecule (ALCAM), also known as CD166, serves as a receptor for endogenous Gal-8. ALCAM is a member of the immunoglobulin superfamily involved in cell-cell adhesion through homophilic (ALCAM-ALCAM) and heterophilic (i.e. ALCAM-CD6) interactions in different tissues. Here we investigated the physiologic relevance of ALCAM-Gal-8 association and glycosylation-dependent mechanisms governing these interactions. We found that silencing of ALCAM in MDA-MB-231 triple negative breast cancer cells decreases cell adhesion and migration onto Gal-8-coated surfaces in a glycan-dependent fashion. Remarkably, either Gal-8 or ALCAM silencing also disrupted cell-cell adhesion, and led to reduced tumor growth in a murine model of triple negative breast cancer. Moreover, structural characterization of endogenous ALCAM N-glycosylation showed abundant permissive structures for Gal-8 binding. Importantly, we also found that cell sialylation controls Gal-8-mediated cell adhesion. Altogether, these findings demonstrate a central role of either ALCAM or Gal-8 (or both) in controlling triple negative breast cancer.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Fetais/metabolismo , Galectinas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antígenos CD/genética , Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular Tumoral , Feminino , Proteínas Fetais/genética , Galectinas/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas de Neoplasias/genética , Neoplasias de Mama Triplo Negativas/genética
8.
Mol Biol Cell ; 30(11): 1298-1313, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30893012

RESUMO

Fibroblasts transformed by the proto-oncogene Src form individual invadopodia that can spontaneously self-organize into large matrix-degrading superstructures called rosettes. However, the mechanisms by which the invadopodia can spatiotemporally reorganize their architecture is not well understood. Here, we show that Hic-5, a close relative of the scaffold protein paxillin, is essential for the formation and organization of rosettes in active Src-transfected NIH3T3 fibroblasts and cancer-associated fibroblasts. Live cell imaging, combined with domain-mapping analysis of Hic-5, identified critical motifs as well as phosphorylation sites that are required for the formation and dynamics of rosettes. Using pharmacological inhibition and mutant expression, we show that FAK kinase activity, along with its proximity to and potential interaction with the LD2,3 motifs of Hic-5, is necessary for rosette formation. Invadopodia dynamics and their coalescence into rosettes were also dependent on Rac1, formin, and myosin II activity. Superresolution microscopy revealed the presence of formin FHOD1 and INF2-mediated unbranched radial F-actin fibers emanating from invadopodia and rosettes, which may facilitate rosette formation. Collectively, our data highlight a novel role for Hic-5 in orchestrating the organization of invadopodia into higher-order rosettes, which may promote the localized matrix degradation necessary for tumor cell invasion.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas com Domínio LIM/metabolismo , Podossomos/metabolismo , Processamento de Proteína Pós-Traducional , Quinases da Família src/genética , Actinas/metabolismo , Actinas/fisiologia , Animais , Linhagem Celular Transformada , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Fetais/metabolismo , Proteínas Fetais/fisiologia , Fibroblastos/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , /fisiologia , Proteínas com Domínio LIM/fisiologia , Camundongos , Miosina Tipo II/metabolismo , Miosina Tipo II/fisiologia , Células NIH 3T3 , Neuropeptídeos/metabolismo , Neuropeptídeos/fisiologia , Fosforilação , Podossomos/fisiologia , Formação de Roseta , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/fisiologia
9.
Cancer Sci ; 110(5): 1644-1652, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30784169

RESUMO

Cancer tissues contain small populations of highly tumorigenic cells termed cancer stem cells (CSCs). Immortalized cell lines containing CSCs are valuable and powerful experimental tools for research into the characteristics of these stem cells. We previously reported that the hepatocellular carcinoma cell line Li-7 includes abundant CD13+ CD166- CSCs; however, the number of these cells decreases after long-term culture as a result of differentiation to non-CSC populations. To ensure consistent and reproducible results in experiments using Li-7 cells, it is important that the CSC population is maintained stably regardless of culture duration and passage. In the present study, we found that a commercially available culture medium for maintenance of embryonic stem cells and induced pluripotent stem cells, mTeSR1, effectively prevented spontaneous differentiation by CD13+ CD166- cells to CD13- CD166+ cells and therefore maintained the CSC population in Li-7 cell cultures. CD13+ CD166- CSCs maintained using this culture medium retained high tumorigenicity after transplantation into mice; they also showed the ability to differentiate in vitro into non-CSC populations in RPMI-1640 with 10% FBS medium. We analyzed gene expression profiles of CSC and non-CSC populations in Li-7 cultures using an RNA sequencing method. Genes such as FGFR, NOTCH1, and JAG1, that are associated with tumorigenicity and stemness, were upregulated in the CSC population. Our results suggest that CSCs can be maintained in immortalized cancer cell lines cultured over an extended period using a medium developed for culture of embryonic/induced pluripotent stem cells.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Técnicas de Cultura de Células/métodos , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/transplante , Animais , Antígenos CD/metabolismo , Antígenos CD13/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultura/farmacologia , Proteínas Fetais/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína Jagged-1/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Receptor Notch1/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Análise de Sequência de RNA , Regulação para Cima
10.
Dev Biol ; 448(2): 119-135, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30661645

RESUMO

In a multitude of organisms, transcription factors of the basic helix-loop-helix (bHLH) family control the expression of genes required for organ development and tissue differentiation. The functions of different bHLH transcription factors in the specification of nervous system and paraxial mesoderm have been widely investigated in various model systems. Conversely, the knowledge of the role of these regulators in the development of the axial mesoderm, the embryonic territory that gives rise to the notochord, and the identities of their target genes, remain still fragmentary. Here we investigated the transcriptional regulation and target genes of Bhlh-tun1, a bHLH transcription factor expressed in the developing Ciona notochord as well as in additional embryonic territories that contribute to the formation of both larval and adult structures. We describe its possible role in notochord formation, its relationship with the key notochord transcription factor Brachyury, and suggest molecular mechanisms through which Bhlh-tun1 controls the spatial and temporal expression of its effectors.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciona/embriologia , Ciona/genética , Redes Reguladoras de Genes , Notocorda/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Padronização Corporal/genética , Embrião não Mamífero/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Notocorda/embriologia , Reprodutibilidade dos Testes , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Regulação para Cima/genética
11.
Nat Med ; 25(2): 292-300, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30664779

RESUMO

Chordoma is a primary bone cancer with no approved therapy1. The identification of therapeutic targets in this disease has been challenging due to the infrequent occurrence of clinically actionable somatic mutations in chordoma tumors2,3. Here we describe the discovery of therapeutically targetable chordoma dependencies via genome-scale CRISPR-Cas9 screening and focused small-molecule sensitivity profiling. These systematic approaches reveal that the developmental transcription factor T (brachyury; TBXT) is the top selectively essential gene in chordoma, and that transcriptional cyclin-dependent kinase (CDK) inhibitors targeting CDK7/12/13 and CDK9 potently suppress chordoma cell proliferation. In other cancer types, transcriptional CDK inhibitors have been observed to downregulate highly expressed, enhancer-associated oncogenic transcription factors4,5. In chordoma, we find that T is associated with a 1.5-Mb region containing 'super-enhancers' and is the most highly expressed super-enhancer-associated transcription factor. Notably, transcriptional CDK inhibition leads to preferential and concentration-dependent downregulation of cellular brachyury protein levels in all models tested. In vivo, CDK7/12/13-inhibitor treatment substantially reduces tumor growth. Together, these data demonstrate small-molecule targeting of brachyury transcription factor addiction in chordoma, identify a mechanism of T gene regulation that underlies this therapeutic strategy, and provide a blueprint for applying systematic genetic and chemical screening approaches to discover vulnerabilities in genomically quiet cancers.


Assuntos
Cordoma/metabolismo , Proteínas Fetais/metabolismo , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Proliferação de Células/efeitos dos fármacos , Cordoma/genética , Cordoma/patologia , Quinases Ciclina-Dependentes/metabolismo , Regulação para Baixo/efeitos dos fármacos , Genes Essenciais , Humanos , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia
12.
J Crohns Colitis ; 13(4): 510-524, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30395204

RESUMO

BACKGROUND AND AIMS: CD6 is a crucial regulator of T cell activation and is implicated in the pathogenesis of multiple autoimmune diseases. ALCAM is the first identified endogenous ligand of CD6. We sought to investigate potential roles of CD6 in regulating intestinal mucosal inflammation in inflammatory bowel disease [IBD]. METHODS: We analysed the expression of CD6 and ALCAM in the inflamed mucosa of IBD patients using qRT-PCR and immunohistochemistry. Phenotypic properties of CD6low/- and CD6highCD4+ T cells were determined by flow cytometry, qRT-PCR, and ELISA. ALCAM Fc chimeric protein was used to evaluate the role of CD6-ALCAM engagement in regulating IBD CD4+ T cell activation and differentiation. RESULTS: Expression of CD6 and its ligand ALCAM was markedly increased in the inflamed mucosa of IBD patients compared with that in normal controls, and was significantly correlated with disease activity indices of IBD patients. Interestingly, CD6highCD4+ T cells of IBD patients exhibited significantly higher pathogenicity compared with CD6low/-CD4+ T cells, characterized by enhanced T cell activation and preferential Th1 and Th17 cell phenotypes, but a markedly decreased proportion of nTreg [CD25highFoxp3+, CD25highCD127low] cells. Importantly, inclusion of ALCAM Fc chimeric protein significantly facilitated IBD CD4+ T cell, especially CD6highCD4+ T cell, differentiation into Th1/Th17 cells compared with hIgG1 Fc-treated controls. CONCLUSIONS: These data indicate that overexpression of CD6 and ALCAM in the inflamed mucosa of IBD patients accelerates intestinal mucosal immune responses via promoting CD4+ T cell proliferation and differentiation into Th1/Th17 cells. Thus, CD6 may serve as a novel therapeutic target for treatment of IBD.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Fetais/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/imunologia , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Linfócitos T CD4-Positivos/fisiologia , Estudos de Casos e Controles , Moléculas de Adesão Celular Neuronais/genética , Diferenciação Celular , Proteínas Fetais/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/metabolismo , Ativação Linfocitária , Índice de Gravidade de Doença , Células Th1/imunologia , Células Th17/imunologia
13.
Dev Biol ; 445(1): 1-7, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30389344

RESUMO

MESP1 is a key transcription factor in development of early cardiovascular tissue and it is required for induction of the cardiomyocyte (CM) gene expression program, but its role in vascular development is unclear. Here, we used inducible CRISPRi knock-down of MESP1 to analyze the molecular processes of the early differentiation stages of human induced pluripotent stem cells into mesoderm and subsequently vascular progenitor cells. We found that expression of the mesodermal marker, BRACHYURY (encoded by T) was unaffected in MESP1 knock-down cells as compared to wild type cells suggesting timely movement through the primitive streak whereas another mesodermal marker MIXL1 was slightly, but significantly decreased. In contrast, the expression of the vascular cell surface marker KDR was decreased and CD31 and CD34 expression were substantially reduced in MESP1 knock-down cells supporting inhibition or delay of vascular specification. In addition, mRNA microarray data revealed several other altered gene expressions including the EMT regulating transcription factors SNAI1 and TWIST1, which were both significantly decreased indicating that MESP1 knock-down cells are less likely to undergo EMT during vascular progenitor differentiation. Our study demonstrates that while leaving primitive streak markers unaffected, MESP1 expression is required for timely vascular progenitor specification. Thus, MESP1 expression is essential for the molecular features of early CM, EC and VSMC lineage specification.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Linha Primitiva/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/fisiologia , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Sequências Hélice-Alça-Hélice/fisiologia , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mesoderma/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Linha Primitiva/citologia , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo
14.
Oncogene ; 38(3): 421-444, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30120411

RESUMO

Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RARγ) are commonly reduced in prostate cancer (PCa). Therefore, we sought to establish the cellular and gene regulatory consequences of reduced RARγ expression, and determine RARγ regulatory mechanisms. RARG shRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RARγ levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RARγ cistrome, which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RARγ to regulate androgen signaling, RARγ knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RARγ downregulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RARγ expression and function. Capture of the miR-96 targetome by biotin-miR-96 identified that RARγ and a number of RARγ interacting co-factors including TACC1 were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels of RARG and TACC1 and upper quartile miR-96, compared to the reverse, identified a gene network including several RARγ target genes (e.g., SOX15) that significantly associated with worse disease-free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p = 0.015). In summary, miR-96 targets a RARγ network to govern AR signaling, PCa progression and disease outcome.


Assuntos
Adenocarcinoma/patologia , Androgênios , MicroRNAs/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/patologia , RNA Neoplásico/fisiologia , Receptores do Ácido Retinoico/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Animais , Linhagem Celular Tumoral , Progressão da Doença , Elementos Facilitadores Genéticos , Proteínas Fetais/metabolismo , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/mortalidade , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais
15.
Environ Sci Pollut Res Int ; 26(5): 4801-4820, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30565106

RESUMO

Deca-bromodiphenyl ether (BDE-209) regulates various aspects of spermatogenesis and male fertility through its effect on estrogen receptor α (ERα), but the underlying mechanism remains unclear. Because molecular mechanisms such as remodeling of the blood-testis barrier (BTB) play crucial roles in spermatogenesis, we investigated the disruptive effects of ERα agonists on the BTB in spermatogenesis. In this study, 0, 300, and 500 mg/kg/day of BDE-209 were administered to pregnant adult mice by oral gavage from gestation day 7 to postnatal day 21. SerW3 cells were treated with methylpiperidino pyrazole (MPP) for 30 min before being treated with 50 µg/mL of BDE-209. BDE-209 increases ERα in time- and dose-dependent manners and decreases formin 1 and BTB-associated protein in F1 male mice. Furthermore, BDE-209 impairs the structure and function of the BTB. Activation of ERα signaling could disrupt the BTB, leading to spermatogenesis dysfunction. The results identified the role of ERα in BTB disruption during spermatogenesis and suggested that BTB disruption occurs because of exposure to BDE-209, which could potentially affect spermatogenesis. In conclusion, Sertoli cells seem to be the primary target of BDE-209 in the perinatal period, and this period constitutes a critical window of susceptibility to BDE-209. Also, the SerW3 cell model may not be a particularly useful cell model for studying the function of the cytoskeleton.


Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Éteres Difenil Halogenados/toxicidade , Células de Sertoli/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Animais , Barreira Hematotesticular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Proteínas Fetais/metabolismo , Éteres Difenil Halogenados/administração & dosagem , Éteres Difenil Halogenados/farmacocinética , Masculino , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Gravidez , Transdução de Sinais/efeitos dos fármacos , Espermatogênese/fisiologia , Testículo/efeitos dos fármacos
16.
FASEB J ; 33(4): 4688-4702, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30592646

RESUMO

Folate deficiency in early development leads to disturbance in multiple processes, including neurogenesis during which fibroblast growth factor (FGF) pathway is one of the crucial pathways. Whether folic acid (FA) directly affects FGF pathways to influence neurodevelopment and the possible mechanism remains unclear. In this study, we presented evidence that in human FA-insufficient encephalocele, the FGF pathway was interfered. Furthermore, in Brachyury knockout mice devoid of such T-box transcription factors regulating embryonic neuromesodermal bipotency and a key component of FGF pathway, change in expression of Brachyury downstream targets, activator Fgf8 and suppressor dual specificity phosphatase 6 was detected, along with the reduction in expression of other key FGF pathway genes. By using a FA-deficient cell model, we further demonstrated that decrease in Brachyury expression was through alteration in hypermethylation at the Brachyury promoter region under FA deficiency conditions, and suppression of Brachyury promoted the inactivation of the FGF pathway. Correspondingly, FA supplementation partially reverses the effects seen in FA-deficient embryoid bodies. Lastly, in mice with maternal folate-deficient diets, aberrant FGF pathway activity was found in fetal brain dysplasia. Taken together, our findings highlight the effect of FA on FGF pathways during neurogenesis, and the mechanism may be due to the low expression of Brachyury gene via hypermethylation under FA-insufficient conditions.-Chang, S., Lu, X., Wang, S., Wang, Z., Huo, J., Huang, J., Shangguan, S., Li, S., Zou, J., Bao, Y., Guo, J., Wang, F., Niu, B., Zhang, T., Qiu, Z., Wu, J., Wang, L. The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects.


Assuntos
Proteínas Fetais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Deficiência de Ácido Fólico/metabolismo , Ácido Fólico/uso terapêutico , Defeitos do Tubo Neural/tratamento farmacológico , Defeitos do Tubo Neural/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Imunoprecipitação da Cromatina , Encefalocele/metabolismo , Feminino , Deficiência de Ácido Fólico/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Sulfitos/farmacologia
17.
J Biol Chem ; 294(6): 2098-2108, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30563838

RESUMO

The endoplasmic reticulum (ER) represents the entry point into the secretory pathway where nascent proteins encounter a specialized environment for their folding and maturation. Inherent to these processes is a dedicated quality-control system that detects proteins that fail to mature properly and targets them for cytosolic degradation. An imbalance in protein folding and degradation can result in the accumulation of unfolded proteins in the ER, resulting in the activation of a signaling cascade that restores proper homeostasis in this organelle. The ER heat shock protein 70 (Hsp70) family member BiP is an ATP-dependent chaperone that plays a critical role in these processes. BiP interacts with specific ER-localized DnaJ family members (ERdjs), which stimulate BiP's ATP-dependent substrate interactions, with several ERdjs also binding directly to unfolded protein clients. Recent structural and biochemical studies have provided detailed insights into the allosteric regulation of client binding by BiP and have enhanced our understanding of how specific ERdjs enable BiP to perform its many functions in the ER. In this review, we discuss how BiP's functional cycle and interactions with ERdjs enable it to regulate protein homeostasis in the ER and ensure protein quality control.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fetais/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Resposta a Proteínas não Dobradas , Animais , Retículo Endoplasmático/genética , Proteínas Fetais/genética , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética
18.
Int J Mol Sci ; 19(11)2018 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-30453543

RESUMO

Recent studies suggest that epithelial⁻mesenchymal transition (EMT) correlates with cancer metastasis. In addition, there is growing evidence of the association of EMT with cancer stem cells (CSCs). Recently, we showed that the T-box transcription factor BRACHYURY could be a strong regulator of EMT and the CSC phenotype, which were effectively suppressed by a BRACHYURY knockdown in an adenoid cystic carcinoma cell line. In this study, we further tested whether BRACHYURY is a regulator of cancer stemness by means of forced expression of BRACHYURY in oral cancer cell lines. BRACHYURY, SOX2, or both were stably transfected into oral carcinoma cell lines. We analysed these transfectants with respect to self-renewal phenotypes using a sphere-formation assay, and we assessed the expression levels of EMT markers and stem cell markers using real-time reverse transcription-polymerase chain reaction (RT-PCR). Cell migration and invasiveness in vitro were evaluated using a wound healing assay and a tumour cell dissemination assay, respectively. Forced expression of BRACHYURY or SOX2 slightly increased expression of EMT and stem cell markers and the self-renewal phenotype. The expression levels, however, were much lower compared to those of cancer stem cell-like cells. Forced co-expression of BRACHYURY and SOX2 strongly upregulated EMT and stem cell markers and the self-renewal phenotype. Cell migration and invasiveness in vitro were also remarkably enhanced. These synergistic effects increased expression levels of FIBRONECTIN, SNAIL, SLUG, ZEB1, and TGF-ß2. In particular, the effects on FIBRONECTIN and TGF-ß2 were significant. We found that BRACHYURY and SOX2 synergistically promote cancer stemness in oral cancer cells. This finding points to the importance of gene or protein networks associated with BRACHYURY and SOX2 in the development and maintenance of the CSC phenotype.


Assuntos
Autorrenovação Celular , Proteínas Fetais/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Fatores de Transcrição SOXB1/metabolismo , Proteínas com Domínio T/metabolismo , Transfecção , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Fenótipo
19.
Cell Rep ; 25(7): 1756-1771, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30428346

RESUMO

The pluripotent state of embryonic stem cells (ESCs) is defined by its transcriptome and epigenome. The chromatin reader Brd4 determines ESC identity. Although Brd4 regulation in gene transcription has been well described, its contribution to the chromatin landscape is less known. Here, we show that Brd4's bromodomains partner with the histone acetyltransferase P300, increasing its enzymatic activities. Augmenting histone acetylation by Brd4-P300 interaction recruits the chromatin remodeler Brg1 altering chromatin structure. This pathway is important for maintaining the expression and chromatin patterns of pluripotency-associated genes, such as Oct4, Nanog, and the X chromosome regulatory long noncoding RNAs Tsix and Xite. Furthermore, we show that the Brd4-P300 interaction regulates the de novo formation of chromatin marks during ESC differentiation, as exemplified by controlling the master regulators of mesoderm formation. Collectively, we delineate the function of Brd4 in organizing the chromatin structure that contributes to gene transcriptional regulation and cell fate determination.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Acetilação , Animais , Diferenciação Celular/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Proteínas Fetais/metabolismo , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Sialoglicoproteínas/metabolismo , Proteínas com Domínio T/metabolismo , Transcrição Genética
20.
Proc Natl Acad Sci U S A ; 115(45): 11537-11542, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30348801

RESUMO

During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Membrana Basal/metabolismo , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mecanotransdução Celular , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Animais , Membrana Basal/citologia , Fenômenos Biomecânicos , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Movimento Celular , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Laminina/genética , Laminina/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Morfogênese/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimerização
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