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1.
J Environ Pathol Toxicol Oncol ; 38(3): 271-283, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31679313

RESUMO

Certain mechanical stimuli-particularly low-magnitude, high-frequency vibration-could induce bone marrow stem cell osteogenic differentiation and promote bone formation via Wnt signaling pathway, although the molecular mechanism is still unclear. In this study, we found that miR-335-5p is significantly upregulated after low-magnitude, high-frequency vibration, which suppresses the expression of the Wnt signaling inhibitor Dickkopf-related protein 1. Inhibition of miR-335-5p greatly reduced the osteogenic differentiation. Furthermore, the increase of miR-335-5p level was also confirmed in vivo after LMHF vibration in rabbit. Our study elucidates the prominent role of miRNAs that links the LMHF vibration and osteogenic differentiation.


Assuntos
Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Osteogênese/fisiologia , Vibração/efeitos adversos , Animais , Diferenciação Celular/fisiologia , Masculino , MicroRNAs/metabolismo , Coelhos
2.
Isr Med Assoc J ; 21(7): 471-474, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31507123

RESUMO

BACKGROUND: Microvascular damage, clinically expressed by Raynaud's phenomenon, is generally the first symptom of the disease and the injured vascular cells, both endothelial and perivascular, may transdifferentiate to myofibroblasts, thus leading to collagen deposition in the tissue and consequent fibrosis. Systemic sclerosis (SSc, scleroderma) is complex disease characterized by autoimmunity, vasculopathy, and fibrosis. It has been shown that microvascular damage may be the first symptom of SSc. Injured endothelial cells and pericytes may transdifferentiate into myofibroblasts, the cells responsible for fibrosis and collagen deposition in the tissue. Based on these factors, the process of myofibroblast generation may link two pivotal events of SSc: microvascular damage and fibrosis. Understanding the development, differentiation, and function of myofibroblasts is therefore crucial to individuate early pathogenetic events and develop new therapeutic target for SSc, a condition in which no disease-modifying agents are available. The aim of this review was to discuss the possible origins of myofibroblasts in SSc, highlighting the process of endothelial mesenchymal transition and pericytes to myofibroblast transition and to show how these events may contribute to pathogenesis of the disease.


Assuntos
Miofibroblastos/citologia , Doença de Raynaud/fisiopatologia , Escleroderma Sistêmico/fisiopatologia , Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Transição Epitelial-Mesenquimal/fisiologia , Fibrose/patologia , Humanos , Pericitos/citologia
3.
Braz J Med Biol Res ; 52(9): e8551, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482977

RESUMO

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Fibroblastos/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Western Blotting , Fibroblastos/metabolismo , Imunofluorescência , Camundongos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
4.
Nat Biotechnol ; 37(10): 1198-1208, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501559

RESUMO

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington's disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening.


Assuntos
Ectoderma/fisiologia , Células-Tronco Embrionárias/fisiologia , Doença de Huntington , Neurulação/fisiologia , Telencéfalo/crescimento & desenvolvimento , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Neurogênese , Telencéfalo/fisiologia
5.
Life Sci ; 232: 116625, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276691

RESUMO

AIMS: The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is critical for cartilage regeneration. Tissues constructed from BMSCs through cartilage tissue engineering still exhibit some histological, morphological, and biomechanical differences from normal cartilage tissues. Cyclic tensile strain (CTS) can increase chondrogenic gene expression and reduce hypertrophic gene expression in chondrocytes. miR-365 has been identified as a mechanoresponsive microRNA and is an important regulator of both chondrocyte hypertrophy and differentiation. Therefore, we hypothesized that CTS may promote the chondrogenesis of BMSCs by upregulating the expression of miR-365. METHODS: BMSCs were subjected to CTS to investigate the effects and mechanism on chondrogenesis. An Agilent miRNA microarray was used to profile miRNAs in the CTS-treated BMSCs and 3D-cultured control BMSCs. miR-365 was shown to interact with HDAC4 mRNA through a luciferase reporter assay. An animal cartilage defect model was constructed and different groups of BMSCs were implanted to investigate their in vivo effect. KEY FINDINGS: CTS promoted BMSC chondrogenesis. miR-365 was significantly upregulated in CTS-treated cells and played an important role in CTS-mediated chondrogenesis. Luciferase assays showed that HDAC4 is a direct target of miR-365. An in vivo study showed that CTS treatment and miR-365 overexpression could promote cartilage regeneration from BMSCs. SIGNIFICANCE: CTS can promote the expression of miR-365, a crucial mechanosensitive microRNA involved in the chondrogenesis of BMSCs, which directly inhibits the expression of HDAC4, in turn, enhancing the chondrogenesis of BMSCs.


Assuntos
Condrogênese/genética , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/genética , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Cartilagem/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Condrócitos/metabolismo , Condrogênese/fisiologia , MicroRNAs/metabolismo , Ratos , Transdução de Sinais , Resistência à Tração/fisiologia , Engenharia Tecidual
6.
Life Sci ; 232: 116620, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31291594

RESUMO

AIMS: Cell-based biological pacemakers aim to overcome limitations and side effects of electronic pacemaker devices. We here developed and tested different approaches to achieve nodal-type differentiation using human adipose- and bone marrow-derived mesenchymal stem cells (haMSC, hbMSC). MAIN METHODS: haMSC and hbMSC were differentiated using customized protocols. Quantitative RT-PCR was applied for transcriptional pacemaker-gene profiling. Protein membrane expression was analyzed by immunocytochemistry. Pacemaker current (If) was studied in haMSC with and without lentiviral HCN4-transduction using patch clamp recordings. Functional characteristics were evaluated by co-culturing with neonatal rat ventricular myocytes (NRVM). KEY FINDINGS: Culture media-based differentiation for two weeks generated cells with abundant transcription of ion channel genes (Cav1.2, NCX1), transcription factors (TBX3, TBX18, SHOX2) and connexins (Cx31.9 and Cx45) characteristic for cardiac pacemaker tissue, but lack adequate HCN transcription. haMSC-derived cells revealed transcript levels, which were closer related to sinoatrial nodal cells than hbMSC-derived cells. To substitute for the lack of If, we performed lentiviral HCN4-transduction of haMSC resulting in stable If. Co-culturing with NRVM demonstrated that differentiated haMSC expressing HCN4 showed earlier onset of spontaneous contractions and higher beating regularity, synchrony and rate compared to co-cultures with non-HCN4-transduced haMSC or HCN4-transduced, non-differentiated haMSC. Confocal imaging indicated increased membrane expression of cardiac gap junctional proteins in differentiated haMSC. SIGNIFICANCE: By differentiation haMSC, rather than hbMSC attain properties favorable for cardiac pacemaking. In combination with lentiviral HCN4-transduction, a cellular phenotype was generated that sustainably controls and stabilizes rate in co-culture with NRVM.


Assuntos
Relógios Biológicos/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/metabolismo , Canais de Potássio/metabolismo , Tecido Adiposo/fisiologia , Animais , Células da Medula Óssea/fisiologia , Diferenciação Celular/fisiologia , Técnicas de Cocultura , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células Musculares/metabolismo , Proteínas Musculares/fisiologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Ratos , Nó Sinoatrial
7.
Nat Commun ; 10(1): 3028, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292434

RESUMO

Cerebellar neuronal progenitors undergo a series of divisions before irreversibly exiting the cell cycle and differentiating into neurons. Dysfunction of this process underlies many neurological diseases including ataxia and the most common pediatric brain tumor, medulloblastoma. To better define the pathways controlling the most abundant neuronal cells in the mammalian cerebellum, cerebellar granule cell progenitors (GCPs), we performed RNA-sequencing of GCPs exiting the cell cycle. Time-series modeling of GCP cell cycle exit identified downregulation of activity of the epigenetic reader protein Brd4. Brd4 binding to the Gli1 locus is controlled by Casein Kinase 1δ (CK1 δ)-dependent phosphorylation during GCP proliferation, and decreases during GCP cell cycle exit. Importantly, conditional deletion of Brd4 in vivo in the developing cerebellum induces cerebellar morphological deficits and ataxia. These studies define an essential role for Brd4 in cerebellar granule cell neurogenesis and are critical for designing clinical trials utilizing Brd4 inhibitors in neurological indications.


Assuntos
Ataxia Cerebelar/genética , Córtex Cerebelar/crescimento & desenvolvimento , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Caseína Quinase Idelta , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Ataxia Cerebelar/patologia , Córtex Cerebelar/citologia , Córtex Cerebelar/patologia , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Proteínas Nucleares/genética , Fosforilação/fisiologia , Cultura Primária de Células , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
8.
Life Sci ; 232: 116676, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31340165

RESUMO

Bone is one of the most dynamic organs in the body that continuously undergoes remodeling through bone formation and resorption. A cascade of molecules and pathways results in the osteoblast differentiation that is attributed to osteogenesis, or bone formation. The process of osteogenesis is achieved through participation of the Wnt pathway, FGFs, BMPs/TGF-ß, and transcription factors such as Runx2 and Osx. The activity and function of the master transcription factor, Runx2, is of utmost significance as it can induce the function of osteoblast differentiation markers. A number of microRNAs [miRNAs] have been recently identified in the regulation of Runx2 expression/activity, thus affecting the process of osteogenesis. miRNAs that target Runx2 corepressors favor osteogenesis, while miRNAs that target Runx2 coactivators inhibit osteogenesis. In this review, we focus on the regulation of Runx2 by miRNAs in osteoblast differentiation and their potential for treating bone and bone-related diseases.


Assuntos
Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , MicroRNAs/fisiologia , Osteoblastos/citologia , Animais , Humanos
9.
J Surg Oncol ; 120(3): 382-388, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31206726

RESUMO

BACKGROUND AND OBJECTIVES: Well-differentiated liposarcomas (WDL) are often partly composed of sclerotic tissue, however, the amount varies widely between tumors, and its prognostic significance is unknown. We hypothesized that tumors with more sclerosis would behave more aggressively. METHODS: Primary retroperitoneal WDL from 29 patients resected at our institution with follow-up were histologically evaluated by soft tissue pathologists blinded to outcome. Tumors with ≥ 10% sclerosis were designated "sclerotic" while tumors with < 10% sclerosis were designated as "minimally sclerotic". Cellular and dedifferentiated tumors were excluded. Clinical parameters and radiologic assessments on computed tomography (CT) were recorded. RESULTS: Histological evaluation identified 13 minimally sclerotic WDL and 16 sclerotic WDL. Median follow-up was 9 years (range, 3-20). Median recurrence-free survival (RFS) and median overall survival (OS) were 6.16 and 13.9 years, respectively. Compared with patients with sclerotic WDL, those with minimally sclerotic WDL had superior RFS (HR = 0.17 [95% CI, 0.06-0.53], P = .002) and OS (log-rank test, P = .002). Sclerotic WDL exhibited higher Houndsfield Units than minimally sclerotic WDL (26 vs 1, P = .040). CONCLUSIONS: Minimally sclerotic WDL were associated with more favorable outcome compared with sclerotic tumors. Assessment of sclerosis in WDL is likely a useful prognostic marker.


Assuntos
Lipossarcoma/patologia , Neoplasias Retroperitoneais/patologia , Esclerose/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/fisiologia , Procedimentos Cirúrgicos de Citorredução , Intervalo Livre de Doença , Feminino , Humanos , Lipossarcoma/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Retroperitoneais/cirurgia , Esclerose/cirurgia , Adulto Jovem
10.
Cell Prolif ; 52(4): e12637, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31168899

RESUMO

OBJECTIVES: Chondrocyte proliferation and differentiation are crucial for endochondral ossification, but their regulatory mechanism remains unclear. The present study aimed to determine the physiological function of TGFß1 signalling in the proliferation and differentiation of antler chondrocytes and explore its relationship with Notch, Shh signalling and Foxa. MATERIALS AND METHODS: Immunofluorescence, Western blot, MTS assay, flow cytometry, RNA interference and real-time PCR were used to analyse the function and regulatory mechanisms of TGFß1 signalling in antler chondrocyte proliferation and differentiation. RESULTS: TGFß1, TGFBR1 and TGFBR2 were highly expressed in antler cartilage. TGFß1 promoted chondrocyte proliferation, increased the proportion of S-phase cells and induced the expression of hypertrophic chondrocyte markers Col X, Runx2 and Alpl. However, this induction was weakened by TGFß receptor inhibitor SB431542 and Smad3 inhibitor SIS3. Simultaneously, TGFß1 activated Notch and Shh signalling whose blockage attenuated the above effects of rTGFß1, whereas addition of rShh rescued the defects in chondrocyte proliferation and differentiation elicited by SB431542 and SIS3. Further analysis revealed that inhibition of Notch signalling impeded TGFß1 activation of the Shh pathway. Knockdown of Foxa1, Foxa2 and Foxa3 abrogated the effects of TGFß1 on chondrocyte differentiation. Notch and Shh signalling mediated the regulation of Foxa transcription factors by TGFß1. CONCLUSIONS: TGFß1 signalling could induce the proliferation and differentiation of antler chondrocytes through Notch-Shh-Foxa pathway.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Chifres de Veado , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Dioxóis/farmacologia , Proteínas Hedgehog/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Isoquinolinas/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Receptores Notch/metabolismo , Fase S/efeitos dos fármacos , Fase S/fisiologia , Transdução de Sinais/efeitos dos fármacos
11.
Cancer Sci ; 110(8): 2667-2675, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31175699

RESUMO

Multicellular structures, such as tumor buddings and poorly differentiated clusters (PDC), exist at the invasive front of colorectal cancers (CRC). Although it has been reported that CRC with PDC showed frequent lymph node metastases with a worse prognosis, the molecular markers of PDC that are responsible for prognosis have not been identified. We here noticed for the first time that Ezrin, a regulator of the actin cytoskeleton, is expressed in the corner cells of PDC. We then aimed to verify whether heterogeneous Ezrin expression in PDC predicts the prognosis of CRC patients. We immunohistochemically analyzed Ezrin expression in PDC of 184 patients with completely resected stages I-III CRC. We established the Ezrin corner score (ECS), which quantifies the tendency of Ezrin-positive cells to accumulate at the corners of PDC. On the basis of ECS values, 2 indices, the mean ECS and the number of PDC with high ECS, were obtained. Both indices were significantly higher in CRC with lymphatic invasion, higher PDC grade, and presence of micropapillary (MP) PDC. The mean ECS-high group showed shorter recurrence-free survival than the mean ECS-low group but without significance. The other index, the number of ECS-high PDC, was significantly associated with recurrence-free survival. These results suggest that Ezrin is involved in PDC progression and lymphatic invasion, and that ECS may be a marker for aggressive PDC.


Assuntos
Diferenciação Celular/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas do Citoesqueleto/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Prognóstico
12.
Med Oncol ; 36(8): 66, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31183633

RESUMO

Nuclear receptor subfamily 4, group A, member 3 (NR4A3) is a member of the NR4A subgroup of orphan nuclear receptors, implicated in the regulation of diverse biological functions, including metabolism, angiogenesis, inflammation, cell proliferation, and apoptosis. Although many reports have suggested the involvement of NR4A3 in the development and/or progression of tumors, its role varies among tumor types. Previously, we reported that DNA hypomethylation at NR4A3 exon 3 is associated with lower survival rate of neuroblastoma (NB) patients. As hypomethylation of this region results in reduced expression of NR4A3, our observations suggested that NR4A3 functions as a tumor suppressor in NB. However, the exact mechanisms underlying its functions have not been clarified. In the present study, we analyzed public databases and showed that reduced NR4A3 expression was associated with shorter survival period of NB in two out of three datasets. An in vitro study revealed that forced expression of NR4A3 in human NB-derived cell line NB1 resulted in elongation of neurites along with overexpression of GAP43, one of the differentiation markers of NB. On the other hand, siRNA-mediated knockdown of NR4A3 suppressed the expression level of GAP43. Interestingly, the forced expression of NR4A3 induced only the GAP43 but not the other molecules involved in NB cell differentiation, such as MYCN, TRKA, and PHOX2B. These results indicated that NR4A3 directly activates the expression of GAP43 and induces differentiated phenotypes of NB cells, without affecting the upstream signals regulating GAP43 expression and NB differentiation.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Neuroblastoma/metabolismo , Receptores de Esteroides/biossíntese , Receptores dos Hormônios Tireóideos/biossíntese , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Progressão da Doença , Proteína GAP-43/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Neuritos/metabolismo , Neuritos/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Regulação para Cima
13.
Yakugaku Zasshi ; 139(6): 853-859, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31155525

RESUMO

In most mammalian species, adult neurogenesis appears to occur only in the olfactory bulb and hippocampal dentate gyrus, where neural stem/progenitor cells exist to create new neurons. The discovery of multi-potential neural stem/progenitor cells (NPCs) in the adult brain has precipitated a novel therapeutic strategy for harnessing these endogenous cells to aid in recovery from neurodegenerative disorders. During neurodegeneration, a plethora of endogenous factors, including cytokines, chemokines, neurotransmitters, blood-derived factors, and reactive oxygen species, are released by the activation of resident microglia, astrocytes, and infiltrating peripheral macrophages. It is interesting that these endogenous factors affect the proliferation, migration, differentiation, and survival of newly generated cells involved in the incorporation of newly generated neurons into the brain's circuitry. The unique profile of these endogenous factors can vary the degree of neuroregeneration after neurodegeneration. We show that adult neurogenesis-activating signals are regulated by endogenous factors produced during neurodegeneration.


Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Terapia de Alvo Molecular , Células-Tronco Multipotentes/fisiologia , Células-Tronco Neurais/fisiologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/terapia , Neurogênese/genética , Neurogênese/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Animais , Encéfalo/citologia , Quimiocinas/fisiologia , Citocinas/fisiologia , Humanos , Camundongos , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Neurotransmissores/fisiologia , Espécies Reativas de Oxigênio
15.
Mol Med Rep ; 19(6): 4719-4726, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059010

RESUMO

Leukemia inhibitory factor (LIF) modulates various biological processes. Although previous studies have described the effects of LIF on adipocyte differentiation, the role of LIF receptor (LIFR) on adipocyte differentiation remains unclear. Using reverse transcription­quantitative PCR (RT­qPCR), LIFR expression was demonstrated to increase during adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs), indicating that LIFR may be involved in this process. To further evaluate the association between LIFR and adipogenic differentiation, lentivirus­mediated LIFR knockdown was performed in hMSCs. Cells were divided into two groups: Negative control group and LIFR­knockdown group. During the adipogenic differentiation process, intracellular lipid accumulation was assessed with Oil Red O staining at various time points (days 3, 6 and 9). Additionally, the mRNA and protein expression levels of LIF, LIFR and three molecular indicators of adipogenesis, peroxisome proliferator­activated receptor Î³ (PPARγ), CCAAT enhancer binding protein α (C/EBPα) and fatty acid binding protein 4 (FABP4/aP2), were assessed by RT­qPCR and western blotting. The culture supernatant was collected to evaluate the concentration of LIF using ELISA. The present results suggested that LIFR expression progressively increased during adipogenic differentiation of hMSCs. Conversely, LIFR knockdown significantly suppressed this process. Additionally, PPARγ, C/EBPα and aP2 were inhibited following LIFR knockdown. In contrast with LIFR, the expression levels of LIF were significantly decreased after the initiation of adipogenic differentiation. Therefore, the expression levels of LIF and LIFR exhibited opposite trends. Collectively, the present results suggested that LIFR promoted adipogenic differentiation, whereas LIF may negatively regulate this process.


Assuntos
Adipogenia/fisiologia , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Células-Tronco Mesenquimais/fisiologia , Adipócitos/metabolismo , Adipócitos/patologia , Adipogenia/genética , Células da Medula Óssea , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Células-Tronco Mesenquimais/patologia , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/metabolismo
16.
Int J Exp Pathol ; 100(2): 83-93, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31090128

RESUMO

Schwann cells (SCs) critically maintain the plasticity of the peripheral nervous system. Peripheral nerve injuries and infections stimulate SCs in order to retrieve homeostasis in neural tissues. Previous studies indicate that Mycobacterium leprae (ML) regulates the expression of key factors related to SC identity, suggesting that alterations in cell phenotype may be involved in the pathogenesis of neural damage in leprosy. To better understand whether ML restricts the plasticity of peripheral nerves, the present study sought to determine the expression of Krox-20, Sox-10, c-Jun and p75NTR in SC culture and mice sciatic nerves, both infected by ML Thai-53 strain. Primary SC cultures were stimulated with two different multiplicities of infection (MOI 100:1; MOI 50:1) and assessed after 7 and 14 days. Sciatic nerves of nude mice (NU-Foxn1nu ) infected with ML were evaluated after 6 and 9 months. In vitro results demonstrate downregulation of Krox-20 and Sox-10 along with the increase in p75NTR-immunolabelled cells. Concurrently, sciatic nerves of infected mice showed a significant decrease in Krox-20 and increase in p75NTR. Our results corroborate previous findings on the interference of ML in the expression of factors involved in cell maturation, favouring the maintenance of a non-myelinating phenotype in SCs, with possible implications for the repair of adult peripheral nerves.


Assuntos
Regulação para Baixo , Proteína 2 de Resposta de Crescimento Precoce/biossíntese , Hanseníase/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Hanseníase/microbiologia , Hanseníase/patologia , Camundongos Nus , Mycobacterium leprae/isolamento & purificação , Plasticidade Neuronal/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Células de Schwann/microbiologia , Células de Schwann/patologia , Nervo Isquiático/microbiologia , Nervo Isquiático/patologia , Técnicas de Cultura de Tecidos
17.
Int J Mol Sci ; 20(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31085991

RESUMO

It is now widely believed that mammary epithelial cell plasticity, an important physiological process during the stages of mammary gland development, is exploited by the malignant cells for their successful disease progression. Normal mammary epithelial cells are heterogeneous and organized in hierarchical fashion, in which the mammary stem cells (MaSC) lie at the apex with regenerative capacity as well as plasticity. Despite the fact that the majority of studies supported the existence of multipotent MaSCs giving rise to both basal and luminal lineages, others proposed lineage restricted unipotent MaSCs. Consistent with the notion, the latest research has suggested that although normal MaSC subsets mainly stay in a quiescent state, they differ in their reconstituting ability, spatial localization, and molecular and epigenetic signatures in response to physiological stimuli within the respective microenvironment during the stages of mammary gland development. In this review, we will focus on current research on the biology of normal mammary stem cells with an emphasis on properties of cellular plasticity, self-renewal and quiescence, as well as the role of the microenvironment in regulating these processes. This will include a discussion of normal breast stem cell heterogeneity, stem cell markers, and lineage tracing studies.


Assuntos
Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/fisiologia , Feminino , Humanos
18.
Br Poult Sci ; 60(4): 395-403, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31132872

RESUMO

1. In this study, geese (Anas cygnoides) embryonic pituitary cells were cultured in vitro to determine if glucocorticoids could induce growth hormone (GH) expression and to investigate the molecular mechanisms involved in this process. 2. On embryonic day 15 (e15) and e20 the pituitary cells were treated with corticosterone (CORT), membrane impermeable bovine serum albumin-conjugate corticosterone (CORT-BSA), dexamethasone (DEX), and a glucocorticoid receptor (GR) antagonist (RU486) to detect responsiveness of somatotrophs to glucocorticoids. 3. Treatment with CORT, CORT-BSA, and DEX for as little as 6 h increased the percentage of GH-positive cells (P < 0.01) and increased GH mRNA expression (P < 0.01) in e15 goose pituitary cells compared to the control. CORT significantly increased the level of GH protein secreted from cultured e15 goose embryonic pituitary cells, and CORT-BSA increased GH secretion from e20 goose embryonic pituitary cells. 4. A significant increase was observed in the glucocorticoid receptor in GR transcription levels (P < 0.01) with CORT, CORT-BSA, and DEX treatment. Furthermore, the CORT-stimulated GH mRNA expression was completely negated by pre-treatment with RU486. 5. These findings demonstrate that glucocorticoids can stimulate somatotroph differentiation in vitro, as characterised by enhanced GH protein secretion andmRNA expression in cultured geese embryonic pituitary cells. The membrane GR was involved in pituitary somatotroph differentiation induced by glucocorticoids during the embryonic development of geese.


Assuntos
Diferenciação Celular/fisiologia , Corticosterona/farmacologia , Cortisona/farmacologia , Dexametasona/farmacologia , Gansos/fisiologia , Receptores de Glucocorticoides/metabolismo , Soroalbumina Bovina/farmacologia , Somatotrofos/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Gansos/genética , Antagonistas de Hormônios/farmacologia , Mifepristona/farmacologia , Hipófise/fisiologia , RNA Mensageiro/metabolismo
19.
Nat Commun ; 10(1): 2110, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068593

RESUMO

Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Ribossomos/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral/transplante , Movimento Celular/fisiologia , Nucléolo Celular/metabolismo , Embrião de Galinha , Proteínas Cromossômicas não Histona/metabolismo , DNA Ribossômico/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Ribossômico/metabolismo , Ribossomos/genética
20.
Nat Commun ; 10(1): 1960, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036859

RESUMO

Lin28-dependent oligo-uridylylation of precursor let-7 (pre-let-7) by terminal uridylyltransferase 4/7 (TUT4/7) represses let-7 expression by blocking Dicer processing, and regulates cell differentiation and proliferation. The interaction between the Lin28:pre-let-7 complex and the N-terminal Lin28-interacting module (LIM) of TUT4/7 is required for pre-let-7 oligo-uridylylation by the C-terminal catalytic module (CM) of TUT4/7. Here, we report crystallographic and biochemical analyses of the LIM of human TUT4. The LIM consists of the N-terminal Cys2His2-type zinc finger (ZF) and the non-catalytic nucleotidyltransferase domain (nc-NTD). The ZF of LIM adopts a distinct structural domain, and its structure is homologous to those of double-stranded RNA binding zinc fingers. The interaction between the ZF and pre-let-7 stabilizes the Lin28:pre-let-7:TUT4 ternary complex, and enhances the oligo-uridylylation reaction by the CM. Thus, the ZF in LIM and the zinc-knuckle in the CM, which interacts with the oligo-uridylylated tail, together facilitate Lin28-dependent pre-let-7 oligo-uridylylation.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Humanos , MicroRNAs/genética , Ligação Proteica , RNA Nucleotidiltransferases/genética , Proteínas de Ligação a RNA/genética
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