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1.
PLoS One ; 14(10): e0223724, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31600351

RESUMO

A definitive endodermal cell lineage is a prerequisite for the efficient generation of mature endoderm derivatives that give rise to organs, such as the pancreas and liver. We previously reported that the induction of mesenchymal definitive endoderm cells depends on autocrine TGF-ß signaling and that pharmacological blockage of TGF-ß signaling by Repsox disrupts endoderm specification. The definitive endoderm arises from a primitive streak, which depends largely on TGF-ß signaling. If the TGF-ß pathway is blocked by Repsox, cell fate after the primitive streak induction is so-far unknown. We report here, that an induced primitive streak cell-population contained many T/SOX2 co-expressing cells, and subsequent inhibition of TGF-ß signaling by Repsox promoted neuroectodermal cell fate, which was characterized using single-cell qPCR analysis and immunostaining. The process of epithelial-to-mesenchymal transition, which is inherent to the process of definitive endoderm differentiation, was also disrupted upon Repsox treatment. Our findings may provide a new approach to produce neural progenitors.


Assuntos
Linhagem da Célula/efeitos dos fármacos , Endoderma/citologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Placa Neural/citologia , Reação em Cadeia da Polimerase , Pirazóis/farmacologia , Piridinas/farmacologia , Análise de Célula Única , Ativinas/farmacologia , Humanos , Linha Primitiva/citologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
2.
Stem Cell Reports ; 13(2): 322-337, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31378669

RESUMO

Kidney formation is regulated by the balance between maintenance and differentiation of nephron progenitor cells (NPCs). Now that directed differentiation of NPCs from human induced pluripotent stem cells (iPSCs) can be achieved, maintenance and propagation of NPCs in vitro should be beneficial for regenerative medicine. Although WNT and FGF signals were previously shown to be essential for NPC propagation, the requirement for BMP/TGFß signaling remains controversial. Here we reveal that activin has superior effects to BMP7 on maintenance efficiency of human iPSC-derived NPCs. Activin expanded ITGA8+/PDGFRA-/SIX2-GFP+ NPCs by 5-fold per week at 80%-90% efficiency, and the propagated cells possessed robust capacity for nephron formation both in vitro and in vivo. The expanded cells also maintained their nephron-forming potential after freezing. Furthermore, the protocol was applicable to multiple non-GFP-tagged iPSC lines. Thus, our activin-based protocol will be applicable to a variety of research fields including disease modeling and drug screening.


Assuntos
Ativinas/farmacologia , Proteína Morfogenética Óssea 7/farmacologia , Proliferação de Células/efeitos dos fármacos , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Reprogramação Celular , Edição de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Néfrons/citologia , Néfrons/metabolismo , Néfrons/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Podócitos/metabolismo , Podócitos/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo
3.
J Med Invest ; 66(1.2): 123-127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31064924

RESUMO

PURPOSE: This study aimed to investigate the effect of intraperitoneal administration of activin on the occurrence of endometriosis using a mouse model of endometriosis. METHODS: A mouse model of endometriosis was prepared by intraperitoneally administering endometrial tissue and blood collected from donor mice to C57BL/6J 7-8- week-old recipient mice. A total of 400 µg of activin A was intraperitoneally administered to model mice in the activin group for 5 days. Intraperitoneal endometriotic lesions were confirmed macroscopically and IL-6 and TNF-α levels in washed ascites were measured by ELISA. RESULTS: Endometriotic lesions were observed in all mice. In the activin group, the maximum diameter of endometriotic lesions was significantly larger than that in control group (4.7?1.3 vs 2.9?0.9 mm, p?0.01). The total area of the lesion was also significantly higher in the activin group than in the control group (21.1?9.9 vs 8.8?5.4 mm2,p?0.01). Furthermore, IL-6 and TNF-α levels in ascites were significantly higher in the activin group than in the control group (IL-6 : 85.8?15.3 vs 75.1?19.3 pg/ml, p?0.05 ; TNF-α : 629.8?15.4 vs 605.9?11.4 pg/ml, p?0.05). CONCLUSION: Activin promotes occurrence of endometriosis. Inflammatory cytokines are also elevated by activin administration,suggesting that they may contribute to progression of endometriosis J. Med. Invest. 66 : 123-127, February, 2019.


Assuntos
Ativinas/farmacologia , Modelos Animais de Doenças , Endometriose/induzido quimicamente , Ativinas/administração & dosagem , Animais , Endometriose/imunologia , Feminino , Injeções Intraperitoneais , Interleucina-6/análise , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/análise
4.
J Med Invest ; 66(1.2): 165-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31064932

RESUMO

As the follicular environment transits from being activin dominant to inhibin dominant during folliculogenesis, it is assumed that activin plays an important role in the early stage of follicular growth. We examined the effects of activin on morphological, biochemical and molecular changes in isolated preantral follicles. Preantral follicles were mechanically isolated from 14-day old female C57BL/6 mice. Each follicle was cultured and observed for 14 days usingan in vitro follicle culture system containing FSH, FSH + activin A and FSH + inhibin in the culture medium. We subsequently examined FSH receptor (FSH-R) mRNA expression in isolated follicle cultures with or without activin on days 0 and 2. Activin was observed to significantly stimulate follicle enlargement on days 2, 4, 6 and 8, accelerate morphological changes and increase estradiollevels in culture medium on days 4, 12 and 14. In contrast, inhibin did not alter follicular growth. Additionally, activin stimulated the expression of FSH-R mRNA in isolated granulosa cells. It was demonstrated that activin stimulated the growth of preantral follicles, mainly during the early stage of folliculogenesis, by inducing FSH-R expression, in an isolated follicle culture system. J. Med. Invest. 66 : 165-171, February, 2019.


Assuntos
Ativinas/farmacologia , Folículo Ovariano/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Hormônio Foliculoestimulante/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Folículo Ovariano/fisiologia , Receptores do FSH/análise , Receptores do FSH/genética
5.
Sci Transl Med ; 11(489)2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-31019025

RESUMO

Bone morphogenetic protein (BMP)/carriers approved for orthopedic procedures achieve efficacy superior or equivalent to autograft bone. However, required supraphysiological BMP concentrations have been associated with potential local and systemic adverse events. Suboptimal BMP/receptor binding and rapid BMP release from approved carriers may contribute to these outcomes. To address these issues and improve efficacy, we engineered chimeras with increased receptor binding by substituting BMP-6 and activin A receptor binding domains into BMP-2 and optimized a carrier for chimera retention and tissue ingrowth. BV-265, a BMP-2/BMP-6/activin A chimera, demonstrated increased binding affinity to BMP receptors, including activin-like kinase-2 (ALK2) critical for bone formation in people. BV-265 increased BMP intracellular signaling, osteogenic activity, and expression of bone-related genes in murine and human cells to a greater extent than BMP-2 and was not inhibited by BMP antagonist noggin or gremlin. BV-265 induced larger ectopic bone nodules in rats compared to BMP-2 and was superior to BMP-2, BMP-2/6, and other chimeras in nonhuman primate bone repair models. A composite matrix (CM) containing calcium-deficient hydroxyapatite granules suspended in a macroporous, fenestrated, polymer mesh-reinforced recombinant human type I collagen matrix demonstrated improved BV-265 retention, minimal inflammation, and enhanced handling. BV-265/CM was efficacious in nonhuman primate bone repair models at concentrations ranging from 1/10 to 1/30 of the BMP-2/absorbable collagen sponge (ACS) concentration approved for clinical use. Initial toxicology studies were negative. These results support evaluations of BV-265/CM as an alternative to BMP-2/ACS in clinical trials for orthopedic conditions requiring augmented healing.


Assuntos
Ativinas/química , Proteína Morfogenética Óssea 6/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Ativinas/farmacologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 6/farmacologia , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
6.
J Vet Med Sci ; 81(5): 646-652, 2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-30880304

RESUMO

Activin E, a secreted peptide encoded by the inhibin/activin ßE subunit gene, is a member of the transforming growth factor-ß superfamily, which is predominantly expressed in the liver. Recent reports have suggested that activin E plays a role in energy homeostasis as a hepatokine. Here, using transgenic mice overexpressing activin E under the control of the ß-actin promoter, we demonstrate that activin E controls energy metabolism through brown/beige adipocytes. The glucose tolerance test and insulin tolerance test showed that the insulin sensitivity was improved in the transgenic mice. Furthermore, the mice had a high body temperature compared with wild-type mice. The transgenic brown adipose tissue and mesenteric white adipose tissue showed upregulation of uncoupling protein 1, which enables energy dissipation as heat by uncoupling oxidative phosphorylation from ATP production. Present results indicate that activin E activates energy expenditure through brown/beige adipocyte activation, suggesting that activin E has high potential for obesity therapy.


Assuntos
Ativinas/farmacologia , Adipócitos Bege/metabolismo , Adipócitos Marrons/metabolismo , Resistência à Insulina , Actinas/genética , Actinas/metabolismo , Ativinas/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Temperatura Corporal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Intolerância à Glucose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Termogênese/efeitos dos fármacos , Proteína Desacopladora 1/metabolismo
7.
Theriogenology ; 129: 168-177, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30856402

RESUMO

Understanding regulators of folliculogenesis remains limited in the domestic dog (Canis familiaris), which challenges our ability to develop in vitro follicle culture systems for canid genome rescue efforts. Here, we investigated the influence of activin on dog follicle development and survival, oocyte quality, and FSH receptor expression in culture. Preantral (150 - ≤230 µm diameter), early antral (231 - ≤330 µm), and antral (>330-550 µm) stage follicles were encapsulated in a fibrin-alginate hydrogel with 0, 100, or 200 ng/ml rhActivin plus 0, 0.1, 1, or 10 µg/ml FSH for 12 or 21 d of in vitro culture. All follicle groups increased in diameter (P < 0.05) with activin acting synergistically with FSH to improve (P < 0.05) growth and antral cavity expansion (to >630 µm) in early antral and antral cohorts. This complementary effect was not linked to changes in FSHR mRNA expression (P > 0.05). Although not influencing (P > 0.05) follicle survival or transzonal projection (TZP) density in shorter term 12 d culture, activin in the presence of 1 ng/ml FSH maintained TZP density from the 12-21 d interval. Activin also increased oocyte diameter and improved nuclear integrity compared to un-supplemented controls. These results indicate that activin acts synergistically with FSH to promote growth and antral cavity expansion of the dog follicle in vitro, information useful to formulating an effective culture microenvironment for this species.


Assuntos
Ativinas/farmacologia , Cães/fisiologia , Folículo Ovariano/efeitos dos fármacos , Animais , Técnicas de Cultura de Células/veterinária , Feminino , Hormônio Foliculoestimulante/farmacologia , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/crescimento & desenvolvimento , Receptores do FSH/metabolismo , Regulação para Cima
8.
Proc Natl Acad Sci U S A ; 116(11): 4989-4998, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30819898

RESUMO

WNT/ß-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem cell niches in adults, and is dysregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of ß-catenin, the primary transducer of canonical WNT signals, using quantitative live cell imaging. We show that ß-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained on differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell lines. Signal attenuation in pluripotent cells is observed even at saturating doses, where ligand stability does not affect the dynamics. TGFß superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the ß-catenin response in a manner independent of their ability to induce new WNT ligand production. Our results reveal how variables external to the pathway, including differentiation status and cross-talk with other pathways, dramatically alter WNT/ß-catenin dynamics.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt , Ativinas/farmacologia , Adaptação Biológica/efeitos dos fármacos , Proteína Morfogenética Óssea 4/farmacologia , Sistemas CRISPR-Cas/genética , Diferenciação Celular/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ligantes , Células-Tronco Pluripotentes/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
9.
BMC Neurosci ; 20(1): 5, 2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760214

RESUMO

BACKGROUND: Accurately assessing promising therapeutic interventions for human diseases depends, in part, on the reproducibility of preclinical disease models. With the development of transgenic mice, the rapid adaptation of a 6-OHDA mouse model of Parkinson's disease that was originally described for the use in rats has come with a lack of a comprehensive characterization of lesion progression. In this study we therefore first characterised the time course of neurodegeneration in the substantia nigra pars compacta and striatum over a 4 week period following 6-OHDA injection into the medial forebrain bundle of mice. We then utilised the model to assess the anti-dyskinetic efficacy of recombinant activin A, a putative neuroprotectant and anti-inflammatory that is endogenously upregulated during the course of Parkinson's disease. RESULTS: We found that degeneration of fibers in the striatum was fully established within 1 week following 6-OHDA administration, but that the loss of neurons continued to progress over time, becoming fully established 3 weeks after the 6-OHDA injection. In assessing the anti-dyskinetic efficacy of activin A using this model we found that treatment with activin A did not significantly reduce the severity, or delay the time-of-onset, of dyskinesia. CONCLUSION: First, the current study concludes that a 3 week duration is required to establish a complete lesion of the nigrostriatal tract following 6-OHDA injection into the medial forebrain bundle of mice. Second, we found that activin A was not anti-dyskinetic in this model.


Assuntos
Ativinas/farmacologia , Discinesia Induzida por Medicamentos/tratamento farmacológico , Feixe Prosencefálico Mediano/fisiopatologia , Doenças Neurodegenerativas/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Transtornos Parkinsonianos/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antiparkinsonianos/efeitos adversos , Antiparkinsonianos/farmacologia , Progressão da Doença , Discinesia Induzida por Medicamentos/patologia , Discinesia Induzida por Medicamentos/fisiopatologia , Levodopa/efeitos adversos , Levodopa/farmacologia , Masculino , Feixe Prosencefálico Mediano/efeitos dos fármacos , Feixe Prosencefálico Mediano/patologia , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Oxidopamina , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/fisiopatologia , Distribuição Aleatória , Falha de Tratamento
10.
Int J Mol Sci ; 20(4)2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30781441

RESUMO

Activins and their receptors play important roles in the control of hair follicle morphogenesis, but their role in vibrissae follicle growth remains unclear. To investigate the effect of Activin B on vibrissae follicles, the anagen induction assay and an in vitro vibrissae culture system were constructed. Hematoxylin and eosin staining were performed to determine the hair cycle stages. The 5-ethynyl-2'-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assays were used to examine the cell proliferation. Flow cytometry was used to detect the cell cycle phase. Inhibitors and Western blot analysis were used to investigate the signaling pathway induced by Activin B. As a result, we found that the vibrissae follicle growth was accelerated by 10 ng/mL Activin B in the anagen induction assay and in an organ culture model. 10 ng/mL Activin B promoted hair matrix cell proliferation in vivo and in vitro. Moreover, Activin B modulates hair matrix cell growth through the ERK⁻Elk1 signaling pathway, and Activin B accelerates hair matrix cell transition from the G1/G0 phase to the S phase through the ERK⁻Cyclin D1 signaling pathway. Taken together, these results demonstrated that Activin B may promote mouse vibrissae growth by stimulating hair matrix cell proliferation and cell cycle progression through ERK signaling.


Assuntos
Ativinas/farmacologia , Ciclo Celular , Folículo Piloso/citologia , Sistema de Sinalização das MAP Quinases , Vibrissas/citologia , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/crescimento & desenvolvimento , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Técnicas de Cultura de Órgãos , Fosforilação/efeitos dos fármacos , Proteínas Elk-1 do Domínio ets/metabolismo
11.
Cell Mol Life Sci ; 76(1): 179-192, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30310934

RESUMO

Human pluripotent stem cells (hPSCs) provide a source for the generation of defined kidney cells and renal organoids applicable in regenerative medicine, disease modeling, and drug screening. These applications require the provision of hPSC-derived renal cells by reproducible, scalable, and efficient methods. We established a chemically defined protocol by application of Activin A, BMP4, and Retinoic acid followed by GDNF, which steered hPSCs to the renal lineage and resulted in populations of SIX2+/CITED1+ metanephric mesenchyme- (MM) and of HOXB7+/GRHL2+ ureteric bud (UB)-like cells already by 6 days. Transcriptome analysis corroborated that the PSC-derived cell types at day 8 resemble their renal vesicle and ureteric epithelial counterpart in vivo, forming tubular and glomerular renal cells 6 days later. We demonstrate that starting from hPSCs, our in vitro protocol generates a pool of nephrogenic progenitors at the renal vesicle stage, which can be further directed into specialized nephronal cell types including mesangial-, proximal tubular-, distal tubular, collecting duct epithelial cells, and podocyte precursors after 14 days. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, which are applicable for drug testing or cell-based regenerative therapies.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Néfrons/citologia , Células-Tronco Pluripotentes/citologia , Ativinas/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Humanos , Néfrons/efeitos dos fármacos , Néfrons/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Transcriptoma/efeitos dos fármacos , Tretinoína/farmacologia
12.
Stem Cells ; 37(1): 150-162, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30358011

RESUMO

In a previous study, we have shown that Activin B is a potent chemoattractant for bone marrow-derived mesenchymal stromal cells (BMSCs). As such, the combination of Activin B and BMSCs significantly accelerated rat skin wound healing. In another study, we showed that RhoA activation plays a key role in Activin B-induced BMSC migration. However, the role of the immediate downstream effectors of RhoA in this process is unclear. Here, we demonstrated that mammalian homolog of Drosophila diaphanous-1 (mDia1), a downstream effector of RhoA, exerts a crucial function in Activin B-induced BMSC migration by promoting membrane ruffling, microtubule morphology, and adhesion signaling dynamics. Furthermore, we showed that Activin B does not change Rac1 activity but increases Cdc42 activity in BMSCs. Inactivation of Cdc42 inhibited Activin B-stimulated Golgi reorientation and the cell migration of BMSCs. Furthermore, knockdown of mDia1 affected Activin B-induced BMSC-mediated wound healing in vivo. In conclusion, this study demonstrated that the RhoA-mDia1 and Cdc42 pathways regulate Activin B-induced BMSC migration. This study may help to optimize clinical MSC-based transplantation strategies to promote skin wound healing. Stem Cells 2019;37:150-162.


Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Forminas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Ativinas/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Forminas/genética , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Cicatrização , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
13.
Cancer Sci ; 110(1): 209-220, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30343527

RESUMO

Cyclin-dependent kinase (CDK) 4 and CDK6 inhibitors are effective therapeutic options for hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. Although CDK4/6 inhibitors mainly target the cyclin D-CDK4/6-retinoblastoma tumor suppressor protein (RB) axis, little is known about the clinical impact of inhibiting phosphorylation of other CDK4/6 target proteins. Here, we focused on other CDK4/6 targets, SMAD proteins. We showed that a CDK4/6 inhibitor palbociclib and activin-SMAD2 signaling cooperatively inhibited cell cycle progression of a luminal-type breast cancer cell line T47D. Palbociclib enhanced SMAD2 binding to the genome by inhibiting CDK4/6-mediated linker phosphorylation of the SMAD2 protein. We also showed that cyclin G2 plays essential roles in SMAD2-dependent cytostatic response. Moreover, comparison of the SMAD2 ChIP-seq data of T47D cells with those of Hs578T (triple-negative breast cancer cells) indicated that palbociclib augmented different SMAD2-mediated functions based on cell type, and enhanced SMAD2 binding to the target regions on the genome without affecting its binding pattern. In summary, palbociclib enhances the cytostatic effects of the activin-SMAD2 signaling pathway, whereas it possibly strengthens the tumor-promoting aspect in aggressive breast cancer.


Assuntos
Ativinas/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Receptores Estrogênicos/metabolismo , Proteína Smad2/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Citostáticos/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Smad2/genética
14.
J Cell Physiol ; 234(7): 10238-10247, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30417373

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation.


Assuntos
Ativinas/farmacologia , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Miosite Ossificante/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Receptores de Ativinas Tipo I/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Miosite Ossificante/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Ligamento Periodontal/metabolismo , Ligamento Periodontal/patologia , Fosforilação , Transdução de Sinais , Proteína Smad3/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Adulto Jovem
15.
Exp Cell Res ; 374(1): 114-121, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30458178

RESUMO

Activin A, a multifunctional cytokine of transforming growth factor-ß (TGF-ß) superfamily, can be produced by the diverse immune cells. NK cells in peripheral blood are one of the major immune cells applied to cancer therapy in recent years. However, whether activin A can be produced by natural killer (NK) cells and be involved in regulation of peripheral blood NK cells activities of mouse are not well characterized. Here, we found that activin type IIA and IIB receptors and signaling molecules Smad2, 3 were expressed in peripheral blood NK cells of mouse by flow cytometry and RT-PCR. The cultured blood NK cells of mouse not only produced activin ßA chain protein by intracellular cytokine staining, but also secreted mature activin A protein by enzyme-linked immunosorbent assay (ELISA), and the production was promoted by IL-2. In addition, IL-2 as a positive control obviously promoted IFNγ production of mouse blood NK cells in vitro. However, activin A suppressed IFNγ production, but enhanced IL-2 synthesis and did not alter IL-10 production. Moreover, we found that activin A significantly suppressed the ability of NK cells to lyse target cells. These data revealed that blood NK cells of mouse were not only the target cells in response to activin A, but also the source of activin A, suggesting that activin A may play an important role in regulation of NK cells activities of mouse in an autocrine / paracrine manner.


Assuntos
Ativinas/farmacologia , Comunicação Autócrina , Células Matadoras Naturais/metabolismo , Comunicação Parácrina , Receptores de Activinas Tipo II/sangue , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animais , Comunicação Autócrina/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Folistatina/farmacologia , Subunidades beta de Inibinas/sangue , Subunidades beta de Inibinas/genética , Subunidades beta de Inibinas/metabolismo , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-2/biossíntese , Células Matadoras Naturais/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Comunicação Parácrina/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Smad/sangue , Proteínas Smad/genética , Proteínas Smad/metabolismo
16.
Nat Protoc ; 14(1): 28-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30470820

RESUMO

The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Both protocols begin with the differentiation of hPSCs into definitive endoderm (DE) using activin A, followed by the generation of free-floating 3D posterior foregut spheroids using FGF4, Wnt pathway agonist CHIR99021 (CHIR), BMP pathway antagonist Noggin, and retinoic acid. Embedding spheroids in Matrigel and continuing 3D growth in epidermal growth factor (EGF)-containing medium for 4 weeks results in antral hGOs (hAGOs). To obtain fundic hGOs (hFGOs), spheroids are additionally treated with CHIR and FGF10. Induced differentiation of acid-secreting parietal cells in hFGOs requires temporal treatment of BMP4 and the MEK inhibitor PD0325901 for 48 h on protocol day 30. In total, it takes ~34 d to generate hGOs from hPSCs. To date, this is the only approach that generates functional human differentiated gastric cells de novo from hPSCs.


Assuntos
Técnicas de Cultura de Células , Endoderma/citologia , Células Epiteliais/citologia , Fundo Gástrico/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Antro Pilórico/citologia , Ativinas/farmacologia , Benzamidas/farmacologia , Proteínas de Transporte/farmacologia , Diferenciação Celular , Colágeno/química , Meios de Cultura/química , Meios de Cultura/farmacologia , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Combinação de Medicamentos , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator 10 de Crescimento de Fibroblastos/farmacologia , Fator 4 de Crescimento de Fibroblastos/farmacologia , Fundo Gástrico/metabolismo , Humanos , Laminina/química , Especificidade de Órgãos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Proteoglicanas/química , Antro Pilórico/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Tretinoína/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
17.
J Cell Physiol ; 234(5): 7622-7633, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30317591

RESUMO

Uterine leiom yomas are benign tumors highly prevalent in reproductive women. In thecurrent study, initially, we aimed to screen five different strawberry cultivars (Alba, Clery, Portola, Tecla, and Romina) to identify efficient cultivars in terms of phytochemical characterization and biological properties by measuring phenolic and anthocyanin content as well as antioxidant capacity, and by measuring apoptotic rate and reactive oxygen species (ROS) production in uterine leiomyoma cells. Next, we focused on the most efficient ones, cultivar Alba (A) and Romina (R) as well as Romina anthocyanin (RA) fraction for their ability to regulate oxidative phosphorylation (oxygen consumption rate [OCR]) glycolysis (extracellular acidification rate [ECAR]), and also fibrosis. Leiomyoma and myometrial cells were treated with a methanolic extract of A and R (250 µg/ml) or with RA (50 µg/ml) for 48 hr to measure OCR and ECAR, as well as gene expression associated with fibrosis. In the leiomyoma cells, RA was more effective in inducing apoptosis and increasing intracellular ROS levels, followed by R and A. In myometrial cells, all strawberry treatments increased the cellular viability and decreased ROS concentrations. Leiomyoma cells showed also a significant decrease in ECAR, especially after RA treatment, while OCR was slightly increased in both myometrial and leiomyoma cells. R and RA treatment significantly decreased collagen 1A1, fibronectin, versican, and activin A messenger RNA expression in leiomyoma cells. In conclusion, this study suggests that Romina, or its anthocyanin fraction, can be developed as a therapeutic and/or preventive agent for uterine leiomyomas, confirming the healthy effects exerted by these fruits and their bioactive compounds.


Assuntos
Fragaria/química , Leiomioma/tratamento farmacológico , Preparações de Plantas/farmacologia , Neoplasias Uterinas/tratamento farmacológico , Ativinas/farmacologia , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Feminino , Fibronectinas/metabolismo , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Leiomioma/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Neoplasias Uterinas/metabolismo , Versicanas/farmacologia
18.
Nat Microbiol ; 4(2): 339-351, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30510168

RESUMO

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.


Assuntos
Ativinas/farmacologia , Antivirais/farmacologia , Proteína Morfogenética Óssea 6/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Antivirais/metabolismo , Células Cultivadas , Endopeptidases/genética , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/metabolismo , Hepcidinas/genética , Humanos , Fatores Reguladores de Interferon/genética , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , RNA Viral/metabolismo , Transdução de Sinais/genética , Proteína Smad1/genética , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos
19.
Stem Cells Dev ; 28(4): 278-289, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30572803

RESUMO

There is a critical need to generate functional hepatocytes to aid in liver repair and regeneration upon availability of a renewable, and potentially personalized, source of human hepatocytes (hHEP). Currently, the vast majority of primary hHEP are obtained from human tissue through cadavers. Recent advances in stem cell differentiation have opened up the possibility to obtain fully functional hepatocytes from embryonic or induced pluripotent stem cells, or adult stem cells. With respect to the latter, human bone marrow mesenchymal stromal cells (hBMSCs) can serve as a source of autogenetic and allogenic multipotent stem cells for liver repair and regeneration. A major aspect of hBMSC differentiation is the extracellular matrix (ECM) composition and, in particular, the role of glycosaminoglycans (GAGs) in the differentiation process. In this study, we examine the influence of four distinct culture conditions/protocols (T1-T4) on GAG composition and hepatic markers. α-Fetoprotein and hepatocyte nuclear factor-4α were expressed continually over 21 days of differentiation, as indicated by real time quantitative PCR analysis, while albumin (ALB) expression did not begin until day 21. Hepatocyte growth factor (HGF) appears to be more effective than activin A in promoting hepatic-like cells through the mesenchymal-epithelial transition, perhaps due to the former binding to the HGF receptor to form a unique complex that diversifies the biological functions of HGF. Of the four protocols tested, uniform hepatocyte-like morphological changes, ALB secretion, and glycogen storage were found to be highest with protocol T2, which involves both early- and late-stage combinations of growth factors. The total GAG profile of the hBMSC ECM is rich in heparan sulfate (HS) and hyaluronan, both of which fluctuate during differentiation. The GAG profile of primary hHEP showed an HS-rich ECM, and thus, it may be possible to guide hBMSC differentiation to more mature hepatocytes by controlling the GAG profile expressed by differentiating cells.


Assuntos
Diferenciação Celular , Glicosaminoglicanos/metabolismo , Hepatócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Ativinas/farmacologia , Osso e Ossos/citologia , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Fator de Crescimento de Hepatócito/farmacologia , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo
20.
J Biomed Mater Res A ; 106(11): 2871-2880, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30367547

RESUMO

In several retinal degenerative disease pathologies, such as dry age-related macular degeneration (AMD), the retinal pigment epithelium (RPE) cell monolayer becomes dysfunctional. Promising tissue engineering treatment approaches implant RPE cells on scaffolds into the subretinal space. However, these approaches are not without challenges. Two major challenges that must be addressed are RPE dedifferentiation and the inflammatory response to cell/scaffold implantation. Design and optimization of scaffold cues for the purpose of RPE transplantation remain relatively unexplored, specifically the mechanical properties of the scaffolds. Prior work from our group indicated that by varying substrate moduli significant differences could be induced in cell cytoskeleton structure, cellular activity, and expression of inflammatory markers. We hypothesized that Activin A would provide rescue effects for cells demonstrating dedifferentiated characteristics. Results demonstrated that for cells on low modulus scaffolds, the mechanical environment was the dominating factor and Activin A was unable to rescue these cells. However, Activin A did demonstrate rescue effects for cells on high modulus scaffolds. This finding indicates that when cultured on scaffolds with an appropriate modulus, exogenous factors, such as Activin A, can improve RPE cell expression, morphology, and activity, while an inappropriate scaffold modulus can have devastating effects on RPE survival regardless of chemical stimulation. These findings have broad implications for the design and optimization of scaffolds for long-term successful RPE transplantation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2871-2880, 2018.


Assuntos
Ativinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Proteínas Imobilizadas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Tecidos Suporte/química , Ativinas/administração & dosagem , Ativinas/química , Materiais Biocompatíveis/química , Linhagem Celular , Células Cultivadas , Sistemas de Liberação de Medicamentos , Módulo de Elasticidade , Humanos , Hidrogéis/química , Proteínas Imobilizadas/administração & dosagem , Proteínas Imobilizadas/química , Teste de Materiais
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