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1.
Biochim Biophys Acta Mol Cell Res ; 1866(9): 1450-1462, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31212003

RESUMO

During Freund's adjuvant induced inflammation rat mesenteric mesothelial cells transdifferentiate into mesenchymal cell. They express macrophage markers, inflammatory cytokines (TGF-ß, TNFα, IL-6), and specific receptors. When primary mesenteric cultures were treated with GM-CSF and/or TGF-ß (in vitro), similar phenotypic and biological changes were induced. It seemed likely that GM-CSF receptor-ligand complex should be internalized to initiate mesothelial-macrophage transition. To follow the intracellular route of GM-CSF receptor ß, we co-localized this receptor with various endocytic markers (Cav-1, EEA1, Rab7, and Rab11a), and carried out detailed immunocytochemical, statistical and biochemical analyses. Since STAT5 is one of the downstream element of GM-CSF signaling, we followed the expression and phosphorylation level of this transcription factor. Our results showed that in mesenteric mesothelial cells GM-CSF receptor ß is internalized by caveolae, delivered into early endosomes where the signaling events occur, STAT5A is phosphorylated by JAK2, and then translocated into the nucleus. When dynamin-dependent endocytosis of GM-CSFR ß is inhibited by dynasore, phosphorylation of STAT5A is not occurred, confirming, that the internalization of receptor ß is indispensable for signal transduction. At the early time of inflammation a significant receptor recycling can be found to the plasma membrane. Later (day 8) the receptor is delivered into late endosomes, indicating that its degradation has already started, and the regeneration of mesothelial cells can start. All of these data strongly support that the internalization of GM-CSF receptor ß is required and essential for signal transduction.


Assuntos
Transdiferenciação Celular/fisiologia , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Endocitose/fisiologia , Macrófagos/metabolismo , Transdução de Sinais , Animais , Cavéolas/efeitos dos fármacos , Cavéolas/metabolismo , Subunidade beta Comum dos Receptores de Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Hidrazonas/farmacologia , Inflamação/metabolismo , Janus Quinase 2/metabolismo , Macrófagos/citologia , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT5/metabolismo , Fator de Crescimento Transformador beta/metabolismo
2.
J Diabetes Res ; 2019: 2583047, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31001561

RESUMO

Recent studies showed that alpha cells, especially immature cells and proalpha cells, might be the precursors of beta cells. Exposure to glucagon-like peptide 1 (GLP1) can ameliorate hyperglycemia in diabetic mice and restore the beta cell mass. In the present study, we adopted single high-dose (60 mg/kg, i.p.) streptozotocin (STZ) to model diabetes mellitus (DM) and randomly assigned short-tail (SD) rats to a normal group, a diabetic group, GLP1 groups (50 µg/kg, 100 µg/kg, and 200 µg/kg), a GLP1 (200 µg/kg) with exendin (9-39) group, and a GLP1 with LY294002 group. We found that the pancreatic insulin-glucagon-positive cell populations increased according to the increase in GLP1 exposure. By contrast, no insulin-amylase-positive cell populations or insulin/pan-cytokeratin cells were observed in the pancreatic sections. The GLP1 receptor antagonist exendin (9-39) and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) family inhibitor LY294002 not only suppressed protein kinase B (Akt), pancreatic and duodenal homeobox 1 (Pdx1), forkhead box O 1 (FoxO1), and mast cell function-associated antigen A (MafA) mRNA expression but also increased MAFB expression. We concluded that treatment with GLP1 might result in beta cell neogenesis by promoting the transdifferentiation of alpha cells but not by pancreatic acinar cells, ductal cells, or the self-replication of beta cells. The regulation on the GLP1 receptor and its downstream transcription factor PI3K/AKT/FOXO1 pathway, which causes increased pancreatic and duodenal homeobox 1 (Pdx1) and MafA mRNA expression but causes decreased MAFB expression, may be the mechanism involved in this process.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Animais , Transdiferenciação Celular/fisiologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína Forkhead Box O1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fator de Transcrição MafB/metabolismo , Masculino , Morfolinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
3.
Int J Mol Sci ; 20(7)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987365

RESUMO

The somatic embryogenesis (SE) process of plants, as one of the typical responses to abiotic stresses with hormone, occurs through the dynamic expression of different proteins that constitute a complex regulatory network in biological activities and promotes plant totipotency. Plant SE includes two critical stages: primary embryogenic calli redifferentiation and somatic embryos development initiation, which leads to totipotency. The isobaric labels tandem mass tags (TMT) large-scale and quantitative proteomics technique was used to identify the dynamic protein expression changes in nonembryogenic calli (NEC), primary embryogenic calli (PEC) and globular embryos (GEs) of cotton. A total of 9369 proteins (6730 quantified) were identified; 805, 295 and 1242 differentially accumulated proteins (DAPs) were identified in PEC versus NEC, GEs versus PEC and GEs versus NEC, respectively. Eight hundred and five differentially abundant proteins were identified, 309 of which were upregulated and 496 down regulated in PEC compared with NEC. Of the 295 DAPs identified between GEs and PEC, 174 and 121 proteins were up- and down regulated, respectively. Of 1242 differentially abundant proteins, 584 and 658 proteins were up- and down regulated, respectively, in GEs versus NEC. We have also complemented the authenticity and accuracy of the proteomic analysis. Systematic analysis indicated that peroxidase, photosynthesis, environment stresses response processes, nitrogen metabolism, phytohormone response/signal transduction, transcription/posttranscription and modification were involved in somatic embryogenesis. The results generated in this study demonstrate a proteomic molecular basis and provide a valuable foundation for further investigation of the roles of DAPs in the process of SE transdifferentiation during cotton totipotency.


Assuntos
Transdiferenciação Celular/fisiologia , Gossypium/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Transdiferenciação Celular/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Gossypium/embriologia , Gossypium/genética , Proteínas de Plantas/genética , Proteômica
4.
Arterioscler Thromb Vasc Biol ; 39(4): 765-773, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30816803

RESUMO

Objective- To determine the role of a cytokine-like protein DKK3 (dikkopf-3) in directly transdifferentiating fibroblasts into endothelial cells (ECs) and the underlying mechanisms. Approach and Results- DKK3 overexpression in human fibroblasts under defined conditions for 4 days led to a notable change in cell morphology and progenitor gene expression. It was revealed that these cells went through mesenchymal-to-epithelial transition and subsequently expressed KDR (kinase insert domain receptor) at high levels. Further culture in EC defined media led to differentiation of these progenitors into functional ECs capable of angiogenesis both in vitro and in vivo, which was regulated by the VEGF (vascular endothelial growth factor)/miR (microRNA)-125a-5p/Stat3 (signal transducer and activator of transcription factor 3) axis. More importantly, fibroblast-derived ECs showed the ability to form a patent endothelium-like monolayer in tissue-engineered vascular grafts ex vivo. Conclusions- These data demonstrate that DKK3 is capable of directly differentiating human fibroblasts to functional ECs under defined media and provides a novel potential strategy for endothelial regeneration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Transdiferenciação Celular/fisiologia , Células Endoteliais/citologia , Fibroblastos/efeitos dos fármacos , Animais , Reatores Biológicos , Células Cultivadas , Meios de Cultura , Transição Epitelial-Mesenquimal/fisiologia , Fibroblastos/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/fisiologia , Neovascularização Fisiológica , Proteínas Recombinantes/biossíntese , Fator de Transcrição STAT3/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
5.
Mol Biol Rep ; 46(3): 2749-2759, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30835040

RESUMO

Tumor microenvironment (TME) crucially functions in tumor initiation and progression. Stroma-tumor interactions and cellular transdifferentiation are the prerequisite for tumor formation. Transforming growth factor-ß (TGF-ß), a major cytokine secreted by tumor-associated fibroblasts (TAFs) and cancer cells, is a crucial player involving cell transdifferentiation. Therefore, we hypothesized that these TAFs and cancer cells also affect normal colon epithelium. In our study, we found for the first time that colon cancer cells HCT116 and TAF-like CCD-18Co cells induced epithelial-mesenchymal transition (EMT)-like transdifferentiation in colon epithelial cells HCoEpiCs, with enhanced migratio. Dysfunction of TGF-ß/Smads signal was also observed in the EMT-transformed HCoEpiCs. We wondered whether these phenomena were regulated by TGF-ß/Smads signaling pathway. A TGFß receptor kinase I (TßRI) inhibitor LY364947 was used. We found that the EMT induced by the HCT116- and CCD-18Co-derived CM was suppressed by the LY364947. Besides, different expression profiles for the components of TGF-ß/Smads pathway were found in the EMT-like HCoEpiCs, but high expression of p-Smad2/3 and Smad4 was the common feature. Our observations suggest that the mechanisms of phenotypic transition of colon epithelial cells are cellular environment-dependent, which maybe a basis of potential therapy targeting TME.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Transdiferenciação Celular/fisiologia , Neoplasias do Colo/metabolismo , Linhagem Celular Tumoral , Transdiferenciação Celular/efeitos dos fármacos , Colo/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Células HCT116 , Humanos , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Microambiente Tumoral/efeitos dos fármacos
6.
Cell Mol Life Sci ; 76(9): 1681-1695, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30721319

RESUMO

Despite significant developments in medical and surgical strategies, cardiac diseases remain the leading causes of morbidity and mortality worldwide. Numerous studies involving preclinical and clinical trials have confirmed that stem cell transplantation can help improve cardiac function and regenerate damaged cardiac tissue, and stem cells isolated from bone marrow, heart tissue, adipose tissue and umbilical cord are the primary candidates for transplantation. During the past decade, menstrual blood-derived endometrial stem cells (MenSCs) have gradually become a promising alternative for stem cell-based therapy due to their comprehensive advantages, which include their ability to be periodically and non-invasively collected, their abundant source material, their ability to be regularly donated, their superior proliferative capacity and their ability to be used for autologous transplantation. MenSCs have shown positive therapeutic potential for the treatment of various diseases. Therefore, aside from a brief introduction of the biological characteristics of MenSCs, this review focuses on the progress being made in evaluating the functional improvement of damaged cardiac tissue after MenSC transplantation through preclinical and clinical studies. Based on published reports, we conclude that the paracrine effect, transdifferentiation and immunomodulation by MenSC promote both regeneration of damaged myocardium and improvement of cardiac function.


Assuntos
Reabilitação Cardíaca/métodos , Doenças Cardiovasculares/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Endométrio/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Adulto , Idoso , Transdiferenciação Celular/fisiologia , Feminino , Humanos , Masculino , Menstruação/sangue , Adulto Jovem
7.
Invest Ophthalmol Vis Sci ; 60(1): 113-122, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30629726

RESUMO

Purpose: To develop a culture regime for the in vitro human lens capsular bag model that better reflects clinical events following cataract surgery and to use this refined model to evaluate the putative impact of IOLs on PCO formation. Methods: Capsulorhexis and lens extraction were performed on human donor eyes to generate capsular bags attached to the ciliary body by the zonules. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining in 6 mL serum-free EMEM for 28 days or in a graded culture system (days 1-3, 5% human serum and 10 ng/mL TGFß2; days 4-7, 2% human serum and 1 ng/mL TGFß2; days 8-14, 1% human serum and 0.1 ng/mL TGFß2; days 15-28, serum-free EMEM), which better mimics clinical changes. Preparations were monitored with phase-contrast and modified-dark-field microscopy. Cell coverage and light scatter were quantified using image analysis software. The transdifferentiation marker, α-SMA and matrix component, fibronectin were assessed by immunocytochemistry. To assess IOLs in the model, Alcon Acrysof or Hoya Vivinex IOLs were implanted in match-paired capsular bags. Results: Match-paired experiments showed that graded culture enhanced growth, facilitated matrix contraction, increased transdifferentiation, and promoted matrix deposition relative to serum-free culture. The graded culture protocol was applied to match-paired bags implanted with a Hoya Vivinex or an Alcon Acrysof IOL. The Vivinex demonstrated a lag in growth across the posterior capsule. However, by day 28, coverage was similar, but light-scatter was greater with Acrysof implanted. Cell growth on the Acrysof IOL anterior surface was significantly greater than Vivinex. Conclusions: The graded culture human capsular bag model serves as an excellent system to evaluate and develop intraocular lenses. The Hoya Vivinex IOL showed an overall better level of performance against postsurgical wound healing and PCO than the Alcon Acrysof using this model.


Assuntos
Opacificação da Cápsula/etiologia , Cápsula do Cristalino/fisiologia , Implante de Lente Intraocular/efeitos adversos , Lentes Intraoculares , Modelos Biológicos , Cápsula Posterior do Cristalino/patologia , Actinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Opacificação da Cápsula/patologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Transdiferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Fibronectinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Luz , Masculino , Pessoa de Meia-Idade , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Técnicas de Cultura de Órgãos , Espalhamento de Radiação , Doadores de Tecidos
8.
Exp Neurol ; 319: 112813, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30171864

RESUMO

Glial cells which are indispensable for the central nervous system development and functioning, are proven to be vulnerable to a harmful influence of pathological cues and tissue misbalance. However, they are also highly sensitive to both in vitro and in vivo modulation of their commitment, differentiation, activity and even the fate-switch by different types of bioactive molecules. Since glial cells (comprising macroglia and microglia) are an abundant and heterogeneous population of neural cells, which are almost uniformly distributed in the brain and the spinal cord parenchyma, they all create a natural endogenous reservoir of cells for potential neurogenerative processes required to be initiated in response to pathophysiological cues present in the local tissue microenvironment. The past decade of intensive investigation on a spontaneous and enforced conversion of glial fate into either alternative glial (for instance from oligodendrocytes to astrocytes) or neuronal phenotypes, has considerably extended our appreciation of glial involvement in restoring the nervous tissue cytoarchitecture and its proper functions. The most effective modulators of reprogramming processes have been identified and tested in a series of pre-clinical experiments. A list of bioactive compounds which are potent in guiding in vivo cell fate conversion and driving cell differentiation includes a selection of transcription factors, microRNAs, small molecules, exosomes, morphogens and trophic factors, which are helpful in boosting the enforced neuro-or gliogenesis and promoting the subsequent cell maturation into desired phenotypes. Herein, an issue of their utility for a directed glial differentiation and transdifferentiation is discussed in the context of elaborating future therapeutic options aimed at restoring the diseased nervous tissue.


Assuntos
Diferenciação Celular/fisiologia , Transdiferenciação Celular/fisiologia , Regeneração Nervosa/fisiologia , Neuroglia/fisiologia , Animais , Humanos , Tecido Nervoso/citologia , Tecido Nervoso/crescimento & desenvolvimento , Traumatismos dos Nervos Periféricos/terapia
9.
Curr Stem Cell Res Ther ; 14(1): 57-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30227822

RESUMO

Low back pain (LBP) is one of the world's most common musculoskeletal diseases and is frequently associated with intervertebral disc degeneration (IDD). While the main cause of IDD is commonly attributed to a reduced number of nucleus pulposus (NP) cells, current treatment strategies (both surgical and more conservative) fail to replenish NP cells or reverse the pathology. Cell replacement therapies are an attractive alternative for treating IDD. However, injecting intervertebral disc (IVD) cells, chondrocytes, or mesenchymal stem cells into various animal models of IDD indicate that transplanted cells generally fail to survive and engraft into the avascular IVD niche. Whereas pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), hold great potential for revolutionizing regenerative medicine, current protocols for differentiating these cells into NP-like cells are inadequate. Nucleus pulposus progenitor cells (NPPCs), which are derived from the embryonic notochord, can not only survive within the harsh hypoxic environment of the IVD, but they also efficiently differentiate into NP-like cells. Here we provide an overview of the latest progress in repairing degenerated IVDs using PSCs and NPPCs. We also discuss the molecular pathways by which PSCs differentiate into NPPCs in vitro and in vivo and propose a new, in vivo IDD therapy.


Assuntos
Diferenciação Celular , Disco Intervertebral/fisiologia , Núcleo Pulposo/fisiologia , Células-Tronco Pluripotentes/fisiologia , Regeneração , Medicina Regenerativa , Animais , Biomarcadores/metabolismo , Transdiferenciação Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração do Disco Intervertebral/fisiopatologia , Degeneração do Disco Intervertebral/terapia , Dor Lombar/fisiopatologia , Dor Lombar/terapia
10.
Hepatol Int ; 13(1): 25-39, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30302735

RESUMO

The process of wound healing in response to chronic liver injury leads to the development of liver fibrosis. Regardless of etiology, the profound impact of the degree of liver fibrosis on the prognosis of chronic liver diseases has been well demonstrated. While disease-specific therapy, such as treatments for viral hepatitis, has been shown to reverse liver fibrosis and cirrhosis in both clinical trials and real-life practice, subsets of patients do not demonstrate fibrosis regression. Moreover, where disease-specific therapies are not available, the need for antifibrotics exists. Increased understanding into the pathogenesis of liver fibrosis sets the stage to focus on antifibrotic therapies attempting to: (1) Minimize liver injury and inflammation; (2) Inhibit liver fibrogenesis by enhancing or inhibiting target receptor-ligand interactions or intracellular signaling pathways; and (3) Promote fibrosis resolution. While no antifibrotic therapies are currently available, a number are now being evaluated in clinical trials, and their use is becoming closer to reality for select subsets of patients.


Assuntos
Cirrose Hepática/tratamento farmacológico , Imunidade Adaptativa/fisiologia , Adipocinas/fisiologia , Amidas/uso terapêutico , Antagonistas de Receptores de Angiotensina/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Antagonistas de Receptores de Canabinoides/uso terapêutico , Comunicação Celular/fisiologia , Transdiferenciação Celular/fisiologia , Senescência Celular/efeitos dos fármacos , Antagonistas dos Receptores de Endotelina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética/fisiologia , Células Epiteliais/fisiologia , Matriz Extracelular/patologia , Galectinas/antagonistas & inibidores , Células Estreladas do Fígado/fisiologia , Humanos , Imunidade Inata/fisiologia , Integrinas/fisiologia , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , NF-kappa B/fisiologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/fisiologia , Piridinas/uso terapêutico , Receptores CCR/antagonistas & inibidores , Receptores de Canabinoides/fisiologia , Sistema Renina-Angiotensina/fisiologia , Transdução de Sinais/fisiologia , Receptores Toll-Like/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/fisiologia
11.
Exp Eye Res ; 180: 102-109, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30578787

RESUMO

PURPOSE: Transforming growth factor ß1 (TGFß1) is elevated in wounds after injury and promotes the transdifferentiation of quiescent cells in the stroma (keratocytes, to activated fibroblasts and subsequently myofibroblasts-KFM transformation). Coactivators of transcription, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif), are mechanotransducers that intersect with the TGFß pathway via interactions with Smad proteins. Here, we examined the distinct role of YAP and TAZ on TGFß1 induced myofibroblast transformation of primary human corneal fibroblasts (HCFs). METHODS: A knockdown approach was used to silence YAP and TAZ individually in HCFs. Forty-eight hours post siRNA transfection, cells were cultured in the presence or absence of 2 ng/ml TGFß1 for 24h. The cells were subjected to nuclear and cytoplasmic fractionation. The expression of α-smooth muscle actin (αSMA), Smad 2, 3 and 4, CTGF and phospho-Smad2, 3, and 4 were assessed by qPCR and Western blotting. RESULTS: TGFß1 stimulation resulted in the decreased phosphorylation of YAP in the cytosol, and increased levels of phosphorylated TAZ and Smad2/3/4 in the nucleus. Knockdown of TAZ resulted in elevated YAP expression but not vice versa. Additionally, knockdown of TAZ but not YAP resulted in upregulation of αSMA expression in the presence and absence of TGFß1. In the presence of TGFß1 YAP knockdown increased Smad2/3/4 expression and Smad4 phosphorylation, while TAZ knockdown had no effect on Smad2/3/4 expression and phosphorylation. YAP knockdown inhibited CTGF expression while TAZ knockdown resulted in its increased expression. Finally, simultaneous knockdown of YAP and TAZ resulted in cell death. CONCLUSION: Our findings suggest that YAP and TAZ function as distinct modulators of TGFß1 induced myofibroblast transformation and have different roles in signalling. Specifically, TAZ limits YAP's ability to mediate KFM transformation via Smad proteins. The data also suggest that while having distinct effects, YAP and TAZ have redundant or combinatorial functions critical to cell survival. These results suggest that a loss of TAZ may help drive corneal haze and fibrosis and that the balance between YAP/TAZ is essential in controlling myofibroblast differentiation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Transdiferenciação Celular/fisiologia , Ceratócitos da Córnea/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Miofibroblastos/fisiologia , Fosfoproteínas/fisiologia , Actinas/genética , Actinas/metabolismo , Western Blotting , Transdiferenciação Celular/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Inativação Gênica/fisiologia , Humanos , Fosforilação , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fatores de Transcrição , Transfecção , Fator de Crescimento Transformador beta1/farmacologia
12.
Exp Eye Res ; 180: 146-154, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30578789

RESUMO

In this study, we investigated the pro-proliferative, pro-transdifferentiation effects and mechanisms of nerve growth factor (NGF), internal limiting membrane (ILM), and NGF+ILM on Müller cells. The Müller cells cultured with NGF, ILM or both were mediated with tyrosine kinase A (TrkA) (a high affinity receptor for NGF) inhibitor, phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) (an intracellular signaling pathway important in regulating the cell cycle) inhibitor, or LIN28 (a RNA-binding protein and a posttranscriptional regulator of genes involved in developmental timing and self-renewal in embryonic stem cells) siRNA. Immunofluorescence (IF), western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect the expression of related genes. As a result, NGF, ILM and NGF+ILM promoted cell proliferation, increased the ratio of 5-bromo-2-deoxyuridine (BrdU)-positive cells, and were correlated with TrkA and PI3K/Akt signaling. NGF alone promoted cell dedifferentiation and redifferentiation mainly towards neurons rather than towards glial cells, related to TrkA and PI3K/Akt signals. The expression of p-Akt and cyclinD1 was increased by the intervention of NGF, ILM or NGF+ILM via TrkA and PI3K/Akt signals. NGF alone promoted the expression of paired box 6 (PAX6) (a transcription factor present during embryonic development), sex-determining region Y-box 2 (SOX2) (a transcription factor essential for the self-renewal, or pluripotency, of undifferentiated embryonic stem cells) and LIN28, and inhibited the expression of lethal-7 (Let-7b), Let-7d, Let-7i and miR-98 (microRNAs, key developmental regulators). The expression of Achaete-scute homolog 1 (Ascl1, also called MASH1) (a neurodevelopmental gene) and endogenous NGF (closely related to neurogenesis) was also promoted by exogenous NGF and related to TrkA and PI3K/Akt signaling. The down-regulation of LIN28 significantly antagonized the effects of NGF on the transdifferentiation of Müller cells. Over all, our results showed that NGF promoted the proliferation and transdifferentiation of Müller cells towards photoreceptor neurons--not towards glial cells, which was related to the LIN28/Let-7 pathway through TrkA and PI3K/Akt signals. Additionally, ILM promoted Müller cells to enter the cell cycle and enhanced cell proliferation, since NGF+ILM promoted the proliferation of Müller cell more significantly than NGF alone.


Assuntos
Membrana Basal/fisiologia , Proliferação de Células/fisiologia , Transdiferenciação Celular/fisiologia , Células Ependimogliais/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Células Ependimogliais/citologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Reação em Cadeia da Polimerase em Tempo Real
13.
Mol Med Rep ; 19(1): 468-476, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30483803

RESUMO

Subconjunctival fibrosis represents the primary cause of postoperative failure of trabeculectomy, and at present there is a lack of effective intervention strategies. The present study aimed to investigate the effect of the mitogen­activated protein kinase kinase (MEK) inhibitor U0126 on human tenon fibroblast (HTF) myofibrosis transdifferentiation, and to illuminate the underlying molecular mechanisms involved. It was demonstrated that U0126 significantly inhibited the proliferation, migration and collagen contraction of HTFs stimulated with TGF­ß1. In addition, U0126 largely attenuated the TGF­ß1­induced conversion of HTFs into myofibroblasts, as indicated by a downregulation of the mRNA and protein expression of α­smooth muscle actin and zinc finger protein SNAI1, and by ameliorating the 3D­collagen contraction response. Mechanistically, U0126 suppressed the TGF­ß1­stimulated phosphorylation of mothers against decapentaplegic homolog 2/3, P38 mitogen­activated protein kinase and extracellular signal­regulated kinase 1/2, indicating that U0126 may inhibit HTF activation through the canonical and non­canonical signaling pathways of TGF­ß1. Therefore, U0126 exhibits a potent anti­fibrotic effect among HTFs, and the inhibition of MEK signaling may serve as an alternative intervention strategy for the treatment of trabeculectomy­associated fibrosis.


Assuntos
Transdiferenciação Celular/fisiologia , Fibroblastos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Actinas/metabolismo , Butadienos/farmacologia , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Humanos , Miofibroblastos/efeitos dos fármacos , Nitrilos/farmacologia , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição da Família Snail/metabolismo
14.
Int J Mol Sci ; 19(12)2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30501048

RESUMO

Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colestase/metabolismo , Regeneração Hepática/fisiologia , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ductos Biliares/metabolismo , Ductos Biliares/fisiologia , Western Blotting , Proteínas de Ciclo Celular , Transdiferenciação Celular/genética , Transdiferenciação Celular/fisiologia , Células Cultivadas , Colestase/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Regeneração Hepática/genética , Masculino , Camundongos , Fosfoproteínas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
15.
Invest Ophthalmol Vis Sci ; 59(15): 5785-5794, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30521667

RESUMO

Purpose: Posterior capsule opacification (PCO) is a complication after cataract surgery, particularly in children. Epithelial-mesenchymal transition (EMT) of lens epithelial cells, mediated by transforming growth factor beta (TGFß), contributes to PCO. However, its pathogenesis in children is poorly understood. We correlated cell growth in culture with patient characteristics, studied gene expression of pediatric lens epithelial cells (pLEC), and examined the effects of TGFß-2 on these cells in vitro. Methods: Clinical characteristics of children with cataracts correlated with growth behavior of pLEC in vitro. mRNA expression of epithelial (αB-crystallin, connexin-43) and mesenchymal (αV-integrin, α-smooth muscle actin, collagen-Iα2, fibronectin-1) markers was quantified in pLEC and in cell line HLE-B3 in the presence and absence of TGFß-2. Results: Fifty-four anterior lens capsules from 40 children aged 1 to 180 months were obtained. Cell outgrowth occurred in 44% of the capsules from patients ≤ 12 months and in 33% of capsules from children aged 13 to 60 months, but in only 6% of capsules from children over 60 months. TGFß-2 significantly upregulated expression of αB-crystallin (HLE-B3), αV-integrin (HLE-B3), collagen-Iα2, and fibronectin-1 (in pLEC and HLE-B3 cells). Conclusions: Patient characteristics correlated with growth behavior of pLEC in vitro, paralleling a higher clinical incidence of PCO in younger children. Gene expression profiles of pLEC and HLE-B3 suggest that upregulation of αV-integrin, collagen-Iα2, and fibronectin-1 are involved in EMT.


Assuntos
Transdiferenciação Celular/fisiologia , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal/fisiologia , Cápsula do Cristalino/citologia , Actinas/genética , Extração de Catarata , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/genética , Conexina 43/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Fibronectinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Integrina alfaV/genética , Cápsula do Cristalino/metabolismo , Masculino , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta2/farmacologia , Cadeia B de alfa-Cristalina/genética
16.
Bioessays ; 40(11): e1800119, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30264410

RESUMO

We recently discovered a novel subset of beta cells that resemble immature beta cells during pancreas development. We named these "virgin" beta cells as they do not stem from existing mature beta cells. Virgin beta cells are found exclusively at the islet periphery in areas that we therefore designated as the "neogenic niche." As beta cells are our only source of insulin, their loss leads to diabetes. Islets also contain glucagon-producing alpha cells and somatostatin-producing delta cells, that are important for glucose homeostasis and form a mantle surrounding the beta cell core. This 3D architecture is important and determines access to blood flow and innervation. We propose that the distinctive islet architecture may also play an important, but hitherto unappreciated role in generation of new endocrine cells, including beta cells. We discuss several predictions to further test the contribution of the neogenic niche to beta cell regeneration.


Assuntos
Transdiferenciação Celular/fisiologia , Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Células Secretoras de Somatostatina/citologia , Diabetes Mellitus Tipo 1/patologia , Células Secretoras de Glucagon/metabolismo , Humanos , Células Secretoras de Insulina/classificação , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Células Secretoras de Somatostatina/metabolismo
17.
PLoS One ; 13(7): e0200210, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29979748

RESUMO

Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain. Although some spontaneous HC regeneration has been observed in neonatal mammals, the very small pool of putative progenitor cells that have been identified in the adult mammalian cochlea is not able to replace the damaged HCs, making any hearing impairment permanent. To date, guided differentiation of human cells to HC-like cells has only been achieved using either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, use of such cell types suffers from a number of important disadvantages, such as the risk of tumourigenicity if transplanted into the host´s tissue. We have obtained cells expressing hair cell markers from cultures of human fibroblasts by overexpression of GFI1, Pou4f3 and ATOH1 (GPA), three genes that are known to play a critical role in the development of HCs. Immunocytochemical, qPCR and RNAseq analyses demonstrate the expression of genes typically expressed by HCs in the transdifferentiated cells. Our protocol represents a much faster approach than the methods applied to ESCs and iPSCs and validates the combination of GPA as a set of genes whose activation leads to the direct conversion of human somatic cells towards the hair cell lineage. Our observations are expected to contribute to the development of future therapies aimed at the regeneration of the auditory organ and the restoration of hearing.


Assuntos
Transdiferenciação Celular/fisiologia , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Miosinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição Brn-3C/genética , Fator de Transcrição Brn-3C/metabolismo , Fatores de Transcrição/genética , Tretinoína/farmacologia
18.
Invest Ophthalmol Vis Sci ; 59(8): 3511-3520, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-30025094

RESUMO

Purpose: Transforming growth factor-beta (TGF-ß) isoform 1 (T1) is involved in corneal fibrotic wound healing by stimulating myofibroblast transformation and altering fibrotic gene expression. In this study, two specific inhibitors were used to dissect the relationship between myofibroblast generation and the TGF-ß/Smad- or TGF-ß/p38-signaling pathway in human corneal fibroblasts (HCF). Methods: In HCF, Trx-SARA (Smad-pathway inhibitor) was used to block the TGF-ß/Smad-signaling pathway, and the p38 inhibitor (p38inh, SB202190) was used to inhibit p38MAPK, thus blocking the TGF-ß/p38-signaling pathway. HCF ± Trx-SARA or Trx-GA (SARA control) were serum starved overnight in Eagle's minimum essential medium (EMEM) ± p38inh, grown in EMEM ± T1 ± p38inh for 24 hours, and then processed for indirect-immunofluorescence, Western blot, or quantitative real-time polymerase chain reaction to examine α-smooth muscle actin (αSMA) and other fibrotic genes, such as fibronectin, thrombospondin1, and type III collagen. In addition, the morphology and the effect of p38inh on myofibroblast phenotype after myofibroblast formation were examined. Results: We observed that Trx-SARA had little effect on αSMA expression, indicating that blocking the Smad pathway did not significantly inhibit myofibroblast formation. However, p38inh did significantly inhibit αSMA and other fibrotic genes, thus efficiently preventing the transition of HCFs to myofibroblasts. In addition, morphology changed and αSMA decreased in myofibroblasts exposed to p38inh medium, as compared with controls. Conclusions: HCF transition to myofibroblasts was mainly through the p38 pathway. Therefore, blocking the p38 pathway may be a potential therapeutic tool for human corneal fibrosis prevention/treatment, because it controls myofibroblast formation in human corneal cells, while leaving other functions of T1 unaffected.


Assuntos
Ceratócitos da Córnea/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Miofibroblastos/citologia , Fator de Crescimento Transformador beta/metabolismo , Actinas/genética , Western Blotting , Linhagem Celular , Transdiferenciação Celular/fisiologia , Células Cultivadas , Ceratócitos da Córnea/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imidazóis/farmacologia , Miofibroblastos/metabolismo , Piridinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Smad/metabolismo
19.
Biochim Biophys Acta Rev Cancer ; 1870(2): 151-157, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29997040

RESUMO

Metastasis is the leading cause of mortality among cancer patients. Dissemination enabled by an epithelial-to-mesenchymal transition (EMT) of carcinoma cells has long been considered to be the predominant mechanism for carcinoma metastasis, based on overexpression studies of many EMT-inducing transcription factors. Individual CTCs - and a binary framework of EMT - have been long considered to be sufficient and necessary condition for metastasis. However, recent studies have shown that collective migration and invasion through tumor buds and clusters of Circulating Tumor Cells (CTCs) as possibly being the prevalent mode of metastasis, although individual CTCs may still contribute to metastasis. These strands and clusters have been proposed to often exhibit a hybrid epithelial/mesenchymal (E/M) phenotype where cells retain epithelial traits of cell-cell adhesion and simultaneously gain mesenchymal characteristics of migration and invasion. To highlight the crucial questions regarding metastasis, we define EMT in a non-binary and context-specific manner, suggest that it can be viewed as a trans-differentiation process, and illustrate the implications of hybrid E/M phenotype(s) and cluster-based dissemination in metastasis.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Invasividade Neoplásica/patologia , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Animais , Transdiferenciação Celular/fisiologia , Humanos , Fenótipo
20.
J Clin Invest ; 128(7): 3024-3040, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29889103

RESUMO

Medial vascular calcification, associated with enhanced mortality in chronic kidney disease (CKD), is fostered by osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Here, we describe that serum- and glucocorticoid-inducible kinase 1 (SGK1) was upregulated in VSMCs under calcifying conditions. In primary human aortic VSMCs, overexpression of constitutively active SGK1S422D, but not inactive SGK1K127N, upregulated osteo-/chondrogenic marker expression and activity, effects pointing to increased osteo-/chondrogenic transdifferentiation. SGK1S422D induced nuclear translocation and increased transcriptional activity of NF-κB. Silencing or pharmacological inhibition of IKK abrogated the osteoinductive effects of SGK1S422D. Genetic deficiency, silencing, and pharmacological inhibition of SGK1 dissipated phosphate-induced calcification and osteo-/chondrogenic transdifferentiation of VSMCs. Aortic calcification, stiffness, and osteo-/chondrogenic transdifferentiation in mice following cholecalciferol overload were strongly reduced by genetic knockout or pharmacological inhibition of Sgk1 by EMD638683. Similarly, Sgk1 deficiency blunted vascular calcification in apolipoprotein E-deficient mice after subtotal nephrectomy. Treatment of human aortic smooth muscle cells with serum from uremic patients induced osteo-/chondrogenic transdifferentiation, effects ameliorated by EMD638683. These observations identified SGK1 as a key regulator of vascular calcification. SGK1 promoted vascular calcification, at least partly, via NF-κB activation. Inhibition of SGK1 may, thus, reduce the burden of vascular calcification in CKD.


Assuntos
Proteínas Imediatamente Precoces/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Calcificação Vascular/metabolismo , Animais , Benzamidas/farmacologia , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Transdiferenciação Celular/fisiologia , Células Cultivadas , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Condrogênese/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Hidrazinas/farmacologia , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Knockout para ApoE , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteogênese/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , Transdução de Sinais , Calcificação Vascular/etiologia , Calcificação Vascular/patologia
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