RESUMO
The lungs have a remarkable ability to regenerate damaged tissues caused by acute injury. Many lung diseases, especially chronic lung diseases, are associated with a reduced or disrupted regeneration potential of the lungs. Therefore, understanding the underlying mechanisms of the regenerative capacity of the lungs offers the potential to identify novel therapeutic targets for these diseases. R-spondin2, a co-activator of WNT/ß-catenin signaling, plays an important role in embryonic murine lung development. However, the role of Rspo2 in adult lung homeostasis and regeneration remains unknown. The aim of this study is to determine Rspo2 function in distal lung stem/progenitor cells and adult lung regeneration. In this study, we found that robust Rspo2 expression was detected in different epithelial cells, including airway club cells and alveolar type 2 (AT2) cells in the adult lungs. However, Rspo2 expression significantly decreased during the first week after naphthalene-induced airway injury and was restored by day 14 post-injury. In ex vivo 3D organoid culture, recombinant RSPO2 promoted the colony formation and differentiation of both club and AT2 cells through the activation of canonical WNT signaling. In contrast, Rspo2 ablation in club and AT2 cells significantly disrupted their expansion capacity in the ex vivo 3D organoid culture. Furthermore, mice lacking Rspo2 showed significant defects in airway regeneration after naphthalene-induced injury. Our results strongly suggest that RSPO2 plays a key role in the adult lung epithelial stem/progenitor cells during homeostasis and regeneration, and therefore, it may be a potential therapeutic target for chronic lung diseases with reduced regenerative capability.
Assuntos
Pneumopatias , Via de Sinalização Wnt , Animais , Células Epiteliais/metabolismo , Pulmão/metabolismo , Pneumopatias/genética , Camundongos , Células-Tronco/metabolismo , beta Catenina/metabolismoRESUMO
The cell-cell/cell-matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Heparina/química , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Atrofia Muscular/patologia , Mioblastos/citologia , Polímeros/administração & dosagem , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Fusão Celular , Perfilação da Expressão Gênica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fenótipo , Polímeros/químicaRESUMO
Outgrowth and fusion of the lateral and medial nasal processes and of the maxillary process of the first branchial arch are integral to lip and primary palate development. Wnt9b mutations are associated with cleft lip and cleft palate in mice; however, the cause of these defects remains unknown. Here, we report that Wnt9b(-/-) mice show significantly retarded outgrowth of the nasal and maxillary processes due to reduced proliferation of mesenchymal cells, which subsequently results in a failure of physical contact between the facial processes that leads to cleft lip and cleft palate. These cellular defects in Wnt9b(-/-) mice are mainly caused by reduced FGF family gene expression and FGF signaling activity resulting from compromised canonical WNT/ß-catenin signaling. Our study has identified a previously unknown regulatory link between WNT9B and FGF signaling during lip and upper jaw development.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Arcada Osseodentária/metabolismo , Lábio/metabolismo , Mucosa Nasal/metabolismo , Proteínas Wnt/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Fenda Labial/genética , Fenda Labial/metabolismo , Fissura Palatina/genética , Fissura Palatina/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Hibridização In Situ , Arcada Osseodentária/embriologia , Lábio/embriologia , Maxila/embriologia , Maxila/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Nariz/embriologia , Técnicas de Cultura de Órgãos , Palato/embriologia , Palato/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMO
R-spondin (Rspo)s proteins are a new group of Wnt/beta-catenin signaling agonists. These signaling molecules are known to be involved in the developmental stages of skeletal system. Recent studies in various murine osteoblast models have proposed that Rspo 1 may interact with Wnt signaling pathway to induce differentiation in osteoblasts. Though findings in murine osteoblasts implicate a synergestic role of Rspo 1 with Wnt signaling, still no study has addressed the similar role in more clinically applicable osteoblast models i.e., human cell lines or primary cells. Therefore, in the present study, we investigated the possible role of Rspo 1 during differentiation process of human in vitro osteoblast cell models like primary osteoblasts or human osteoprogenitor cell line hFOB 1.19 along with murine preosteoblast cell line MC3T3 E-1. Our results showed increase in Rspo 1 at transcript level during differentiating phase of human primary osteoblasts and human FOB 1.19 cells. We also found that Rspo 1 (100 ng/mL) acts additively with Wnt3a to activate Wnt signaling, as confirmed by luciferase activity after transfection of TOPFLASH construct to hFOB 1.19 cells. Similar additive role of Rspo 1 and Wnt3a was apparent in alkaline phosphatase (ALP) activity analysis of human primary cells. Moreover, a reduction in ALP activity was observed with knock-down of Rspo 1 by transfected shRNA in hFOB 1.19 cells. These results suggested the possibility of autocrine regulation by Rspo 1 on the osteogenic activities in human in vitro osteoblast models. Furthermore, these results were corroborated in MC3T3-E1, murine osteoblast cell model. Osteoblastic differentiation was induced by transfection of Rspo 1 which was confirmed by increased ALP staining and qRT-PCR analysis of osteogenic markers, such as Runx2 and osteocalcin. In conclusion, present study highlights the role of Rspo 1 in bone remodeling where it activates Wnt signaling to induce differentiation, as shown in human as well murine in vitro osteoblast cell models.
Assuntos
Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/fisiologia , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Humanos , CamundongosRESUMO
R-spondins are a recently characterized family of secreted proteins that activate Wnt/ß-catenin signaling. Herein, we determine R-spondin2 (Rspo2) function in craniofacial development in mice. Mice lacking a functional Rspo2 gene exhibit craniofacial abnormalities such as mandibular hypoplasia, maxillary and mandibular skeletal deformation, and cleft palate. We found that loss of the mouse Rspo2 gene significantly disrupted Wnt/ß-catenin signaling and gene expression within the first branchial arch (BA1). Rspo2, which is normally expressed in BA1 mesenchymal cells, regulates gene expression through a unique ectoderm-mesenchyme interaction loop. The Rspo2 protein, potentially in combination with ectoderm-derived Wnt ligands, up-regulates Msx1 and Msx2 expression within mesenchymal cells. In contrast, Rspo2 regulates expression of the Dlx5, Dlx6, and Hand2 genes in mesenchymal cells via inducing expression of their upstream activator, Endothelin1 (Edn1), within ectodermal cells. Loss of Rspo2 also causes increased cell apoptosis, especially within the aboral (or caudal) domain of the BA1, resulting in hypoplasia of the BA1. Severely reduced expression of Fgf8, a survival factor for mesenchymal cells, in the ectoderm of Rspo2(-/-) embryos is likely responsible for increased cell apoptosis. Additionally, we found that the cleft palate in Rspo2(-/-) mice is not associated with defects intrinsic to the palatal shelves. A possible cause of cleft palate is a delay of proper palatal shelf elevation that may result from the small mandible and a failure of lowering the tongue. Thus, our study identifies Rspo2 as a mesenchyme-derived factor that plays critical roles in regulating BA1 patterning and morphogenesis through ectodermal-mesenchymal interaction and a novel genetic factor for cleft palate.
Assuntos
Padronização Corporal , Região Branquial/embriologia , Ectoderma/embriologia , Mesoderma/embriologia , Transdução de Sinais , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animais , Apoptose , Padronização Corporal/genética , Desenvolvimento Ósseo/genética , Região Branquial/metabolismo , Região Branquial/patologia , Fissura Palatina/embriologia , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Ectoderma/metabolismo , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Face/embriologia , Face/patologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Modelos Biológicos , Especificidade de Órgãos/genética , Crânio/embriologia , Crânio/metabolismo , Crânio/patologia , Trombospondinas/deficiência , Trombospondinas/genética , beta Catenina/metabolismoRESUMO
R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/ß-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/ß-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/ß-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/ß-catenin signaling pathway.
Assuntos
Embrião de Mamíferos/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Trombospondinas/biossíntese , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Mutantes , Fibras Musculares Esqueléticas/citologia , Fator Regulador Miogênico 5/biossíntese , Fator Regulador Miogênico 5/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Trombospondinas/genética , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
BACKGROUND: The effects of some drugs, aging, cancers, and other diseases can cause muscle wasting. Currently, there are no effective drugs for treating muscle wasting. In this study, the effects of ginsenoside Rd (GRd) on muscle wasting were studied. METHODS: Tumour necrosis factor-alpha (TNF-α)/interferon-gamma (IFN-γ)-induced myotube atrophy in mouse C2C12 and human skeletal myoblasts (HSkM) was evaluated based on cell thickness. Atrophy-related signalling, reactive oxygen species (ROS) level, mitochondrial membrane potential, and mitochondrial number were assessed. GRd (10 mg/kg body weight) was orally administered to aged mice (23-24 months old) and tumour-bearing (Lewis lung carcinoma [LLC1] or CT26) mice for 5 weeks and 16 days, respectively. Body weight, grip strength, inverted hanging time, and muscle weight were assessed. Histological analysis was also performed to assess the effects of GRd. The evolutionary chemical binding similarity (ECBS) approach, molecular docking, Biacore assay, and signal transducer and activator of transcription (STAT) 3 reporter assay were used to identify targets of GRd. RESULTS: GRd significantly induced hypertrophy in the C2C12 and HSkM myotubes (average diameter 50.8 ± 2.6% and 49.9% ± 3.7% higher at 100 nM, vs. control, P ≤ 0.001). GRd treatment ameliorated aging- and cancer-induced (LLC1 or CT26) muscle atrophy in mice, which was evidenced by significant increases in grip strength, hanging time, muscle mass, and muscle tissue cross-sectional area (1.3-fold to 4.6-fold, vs. vehicle, P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). STAT3 was found to be a possible target of GRd by the ECBS approach and molecular docking assay. Validation of direct interaction between GRd and STAT3 was confirmed through Biacore analysis. GRd also inhibited STAT3 phosphorylation and STAT3 reporter activity, which led to the inhibition of STAT3 nuclear translocation and the suppression of downstream targets of STAT3, such as atrogin-1, muscle-specific RING finger protein (MuRF-1), and myostatin (MSTN) (29.0 ± 11.2% to 84.3 ± 30.5%, vs. vehicle, P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). Additionally, GRd scavenged ROS (91.7 ± 1.4% reduction at 1 nM, vs. vehicle, P ≤ 0.001), inhibited TNF-α-induced dysregulation of ROS level, and improved mitochondrial integrity (P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). CONCLUSIONS: GRd ameliorates aging- and cancer-induced muscle wasting. Our findings suggest that GRd may be a novel therapeutic agent or adjuvant for reversing muscle wasting.
Assuntos
Carcinoma Pulmonar de Lewis , Mioblastos Esqueléticos , Fator de Transcrição STAT3 , Animais , Humanos , Camundongos , Caquexia/etiologia , Carcinoma Pulmonar de Lewis/complicações , Simulação de Acoplamento Molecular , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/farmacologia , Fator de Necrose Tumoral alfaRESUMO
Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%-40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5-8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission.
Assuntos
Técnicas de Cultura de Células/métodos , Neurônios/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Rombencéfalo/citologia , Serotonina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Humanos , Imuno-Histoquímica/métodos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/metabolismo , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rombencéfalo/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Tretinoína/farmacologiaRESUMO
Development of the teeth requires complex signaling interactions between the mesenchyme and the epithelium mediated by multiple pathways. For example, canonical WNT signaling is essential to many aspects of odontogenesis, and inhibiting this pathway blocks tooth development at an early stage. R-spondins (RSPOs) are secreted proteins, and they mostly augment WNT signaling. Although RSPOs have been shown to play important roles in the development of many organs, their role in tooth development is unclear. A previous study reported that mutating Rspo2 in mice led to supernumerary lower molars, while teeth forming at the normal positions showed no significant anomalies. Because multiple Rspo genes are expressed in the orofacial region, it is possible that the relatively mild phenotype of Rspo2 mutants is due to functional compensation by other RSPO proteins. We found that inactivating Rspo3 in the craniofacial mesenchyme caused the loss of lower incisors, which did not progress beyond the bud stage. A simultaneous deletion of Rspo2 and Rspo3 caused severe disruption of craniofacial development from early stages, which was accompanied with impaired development of all teeth. Together, these results indicate that Rspo3 is an important regulator of mammalian dental and craniofacial development.
RESUMO
Extracellular vesicles (EVs) are 50-300 nm vesicles secreted by eukaryotic cells. They can carry cargo (including miRNA) from the donor cell to the recipient cell. miRNAs in EVs can change the translational profile of the recipient cell and modulate cellular morphology. This endogenous mechanism has attracted the attention of the drug-delivery community in the last few years. EVs can be enriched with exogenous therapeutic miRNAs and used for treatment of diseases by targeting pathological recipient cells. However, there are some obstacles that need to be addressed before introducing therapeutic miRNA-enriched EVs in clinics. Here, we focused on the progress in the field of therapeutic miRNA enriched EVs, highlighted important areas where research is needed, and discussed the potential to use them as therapeutic miRNA carriers in the future.
Assuntos
Vesículas Extracelulares/transplante , Técnicas de Transferência de Genes/tendências , MicroRNAs/uso terapêutico , Transporte Biológico , Vesículas Extracelulares/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , ProteômicaRESUMO
The lung has a vital function in gas exchange between the blood and the external atmosphere. It also has a critical role in the immune defense against external pathogens and environmental factors. While the lung is classified as a relatively quiescent organ with little homeostatic turnover, it shows robust regenerative capacity in response to injury, mediated by the resident stem/progenitor cells. During regeneration, regionally distinct epithelial cell populations with specific functions are generated from several different types of stem/progenitor cells localized within four histologically distinguished regions: trachea, bronchi, bronchioles, and alveoli. WNT signaling is one of the key signaling pathways involved in regulating many types of stem/progenitor cells in various organs. In addition to its developmental role in the embryonic and fetal lung, WNT signaling is critical for lung homeostasis and regeneration. In this minireview, we summarize and discuss recent advances in the understanding of the role of WNT signaling in lung regeneration with an emphasis on stem/progenitor cells.
Assuntos
Pulmão/patologia , Regeneração/genética , Via de Sinalização Wnt/genética , Animais , CamundongosRESUMO
The R-spondin (RSPO) family of proteins potentiate canonical WNT/ß-catenin signaling and may provide a mechanism to fine-tune the strength of canonical WNT signaling. Although several in vitro studies have clearly demonstrated the potentiation of canonical WNT signaling by RSPOs, whether this potentiation actually occurs in normal development and tissue function in vivo still remains poorly understood. Here, we provide clear evidence of the potentiation of canonical WNT signaling by RSPO during mouse facial development by analyzing compound Wnt9b and Rspo2 gene knockout mice and utilizing ex vivo facial explants. Wnt9b;Rspo2 double mutant mice display facial defects and dysregulated gene expression pattern that are significantly more severe than and different from those of Wnt9b or Rspo2 null mutant mice. Furthermore, we found suggestive evidence that the LGR4/5/6 family of the RSPO receptors may play less critical roles in WNT9b:RSPO2 cooperation. Our results suggest that RSPO-induced cooperation is a key mechanism for fine-tuning canonical WNT/ß-catenin signaling in mouse facial development.
RESUMO
Ectonucleotide pyrophosphate phosphodiesterase type II (ENPP2), also known as Autotaxin (ATX), is an enzyme present in blood circulation that converts lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA). While LPA has been demonstrated to play diverse roles in skeletal myogenesis, mainly through in vitro studies, the role of ENPP2 in skeletal myogenesis has not been determined. We previously found that Enpp2 is induced by a positive WNT/ß-Catenin signaling regulator, R-spondin2 (RSPO2), in C2C12 myoblast cells. As RSPO2 promotes myogenic differentiation via the WNT/ß-Catenin signaling pathway, we hypothesized that ENPP2 may act as a key mediator for the crosstalk between WNT and LPA signaling during myogenic differentiation. Herein, we found that ENPP2 function is essential for myogenic differentiation in C2C12 cells. Pharmacological ENPP2 inhibitors or RNAi-mediated Enpp2 gene knockdown severely impaired the myogenic differentiation, including the cell fusion process, whereas administration of the recombinant ENPP2 protein enhanced myogenic differentiation. Consistent with the in vitro results, mice lacking the Enpp2 gene showed a disrupted muscle regeneration after acute muscle injury. The size of newly regenerated myofibers in Enpp2 mutant muscle was significantly reduced compared with wild-type regenerated muscle. Modified expression patterns of myogenic markers in Enpp2 mutant muscle further emphasized the impaired muscle regeneration process. Finally, we convincingly demonstrate that the Enpp2 gene is a direct transcriptional target for WNT/ß-Catenin signaling. Functional TCF/LEF1 binding sites within the upstream region of Enpp2 gene were identified by chromatin immunoprecipitation using anti-ß-Catenin antibodies and reporter assay. Our study reveals that ENPP2 is regulated by WNT/ß-Catenin signaling and plays a key positive role in myogenic differentiation.
Assuntos
Diferenciação Celular/genética , Desenvolvimento Muscular/genética , Diester Fosfórico Hidrolases/genética , beta Catenina/genética , Animais , Camundongos , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Mioblastos/citologia , Regeneração/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/genéticaRESUMO
Under physiological and pathological conditions, mechanical forces generated from cells themselves or transmitted from extracellular matrix (ECM) through focal adhesions (FAs) and adherens junctions (AJs) are known to play a significant role in regulating various cell behaviors. Substantial progresses have been made in the field of mechanobiology towards novel methods to understand how cells are able to sense and adapt to these mechanical forces over the years. To address these issues, this review will discuss recent advancements of traction force microscopy (TFM), intracellular force microscopy (IFM), and monolayer stress microscopy (MSM) to measure multiple aspects of cellular forces exerted by cells at cell-ECM and cell-cell junctional intracellular interfaces. We will also highlight how these methods can elucidate the roles of mechanical forces at interfaces of cell-cell/cell-ECM in regulating various cellular functions. [BMB Reports 2020; 53(2): 74-81].
Assuntos
Matriz Extracelular/fisiologia , Mecanotransdução Celular/fisiologia , Microscopia de Força Atômica/métodos , Biopolímeros , Adesão Celular/fisiologia , Matriz Extracelular/química , Adesões Focais/química , Adesões Focais/fisiologia , Hidrogéis , Junções Intercelulares/química , Junções Intercelulares/fisiologia , Estresse Mecânico , TraçãoRESUMO
R-spondins (RSPOs) are secreted cysteine-rich glycoproteins that belong to a superfamily of thrombospondin type 1 repeat-containing proteins. RSPOs together with WNT proteins potentiate canonical WNT/ß-catenin signaling activity. Over the last several years, the understanding of the regulatory mechanisms and functional roles of RSPOs in many biological contexts has increased. Particularly, because a leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), a stem cell marker originally identified as a marker for intestinal stem cells, and two closely related proteins, LGR4 and LGR6, were identified as cognate receptors for RSPOs, significant research progress has been made in understanding the functional roles of RSPO/LGR signaling in stem cell biology. Given the crucial roles of canonical WNT signaling in self-renewal and differentiation of various types of stem cells, examination of RSPO function and underlying mechanism in these stem cells has provided new insight into the regulatory roles of WNT signaling in stem cell behavior. In this review, we summarize and discuss recent advances in the understanding of the signaling mechanism and roles of RSPOs in different stem cell contexts.
Assuntos
Células-Tronco Adultas/metabolismo , Trombospondinas/metabolismo , Via de Sinalização Wnt/fisiologia , Adulto , Células-Tronco Adultas/citologia , Humanos , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Lysophosphatidic acid (LPA) is an endogenous lysophospholipid with signaling properties outside of the cell and it signals through specific G protein-coupled receptors, known as LPA1-6. For one of its receptors, LPA1 (gene name Lpar1), details on the cis-acting elements for transcriptional control have not been defined. Using 5'RACE analysis, we report the identification of an alternative transcription start site of mouse Lpar1 and characterize approximately 3,500 bp of non-coding flanking sequence 5' of mouse Lpar1 gene for promoter activity. Transient transfection of cells derived from mouse neocortical neuroblasts with constructs from the 5' regions of mouse Lpar1 gene revealed the region between -248 to +225 serving as the basal promoter for Lpar1. This region also lacks a TATA box. For the region between -761 to -248, a negative regulatory element affected the basal expression of Lpar1. This region has three E-box sequences and mutagenesis of these E-boxes, followed by transient expression, demonstrated that two of the E-boxes act as negative modulators of Lpar1. One of these E-box sequences bound the HeLa E-box binding protein (HEB), and modulation of HEB levels in the transfected cells regulated the transcription of the reporter gene. Based on our data, we propose that HEB may be required for a proper regulation of Lpar1 expression in the embryonic neocortical neuroblast cells and to affect its function in both normal brain development and disease settings.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Elementos E-Box/genética , Neurônios/metabolismo , Regiões Promotoras Genéticas , Receptores de Ácidos Lisofosfatídicos/genética , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Neocórtex , Ligação Proteica/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Deleção de Sequência/genética , Sítio de Iniciação de TranscriçãoRESUMO
The robust capacity of skeletal muscle stem cells (SkMSCs, or satellite cells) to regenerate into new muscles in vivo has offered promising therapeutic options for the treatment of degenerative muscle diseases. However, the practical use of SkMSCs to treat muscle diseases is limited, owing to their inability to expand in vitro under defined cultivation conditions without loss of engraftment efficiency. To develop an optimal cultivation condition for SkMSCs, we investigated the behavior of SkMSCs on synthetic maltose-binding protein (MBP)-fibroblast growth factor 2 (FGF2)-immobilized matrix in vitro. We found that the chemically well-defined, xeno-free MBP-FGF2-immobilized matrix effectively supports SkMSC growth without reducing their differentiation potential in vitro. Our data highlights the possible application of the MBP-FGF2 matrix for SkMSC expansion in vitro.
RESUMO
The R-spondin (Rspo) family of proteins consists of secreted cysteine-rich proteins that can activate beta-catenin signaling via the Frizzled/LRP5/6 receptor complex. Here, we report that targeted inactivation of the mouse Rspo2 gene causes developmental limb defects, especially in the hindlimb. Although the initiation of the expression of apical ectodermal ridge (AER)-specific genes, including fibroblast growth factor 8 (FGF8) and FGF4 occurred normally, the maintenance of these marker expressions was significantly defective in the hindlimb of Rspo2(-/-) mice. Consistent with the ligand role of R-spondins in the Wnt/beta-catenin signaling pathway, expression of Axin2 and Sp8, targets for beta-catenin signaling, within AER was greatly reduced in Rspo2(-/-) embryos. Furthermore, sonic hedgehog (Shh) signaling within the hindlimbs of Rspo2(-/-) mice was also significantly decreased. Rspo2 is expressed in the AER of all limb buds, however the stunted phenotype is significantly more severe in the hindlimbs than the forelimbs and strongly biased to the left side. Our findings strongly suggest that Rspo2 expression in the AER is required for AER maintenance likely by regulating Wnt/beta-catenin signaling.
Assuntos
Ectoderma/metabolismo , Membro Posterior/embriologia , Trombospondinas/metabolismo , Animais , Cateninas/metabolismo , Feminino , Proteínas Hedgehog/metabolismo , Masculino , Camundongos , Mutação , Transdução de Sinais , Trombospondinas/genética , Proteínas Wnt/metabolismoRESUMO
Embryonic stem (ES) cells have the potential to develop into various cell lineages including hemangioblasts (Flk1+), a common progenitor for hematopoietic and vascular endothelial cells. Previous studies indicate that Flk1+ cells, a marker for hemangioblast, can be derived from ES cell and that Flk1+ can be differentiated into hematopoietic or endothelial cells depending on culture conditions. We developed an improved in vitro system to generate Flk1+-enriched cultures from mouse ES cells and used this in vitro system to study the role of Wnt signalling in early endothelial progenitor cells. We determined the expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells. RT-PCR analyses identified significantly higher expression of non-canonical Wnt5a and Wnt11 genes in Flk1+ cells compared to Flk1- cells. In contrast, expression of canonical Wnt3a gene was reduced in Flk1+ cells. In addition, Frizzled2, Frizzled5 and Frizzled7 genes were also expressed at a higher level in Flk1+ cells. The differential expression of Wnt and Frizzled genes in Flk1+ cells provides a novel insight into the role of non-canonical Wnt signalling in vascular endothelial fate determination.
Assuntos
Células-Tronco Embrionárias/metabolismo , Receptores Frizzled/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Proteínas Wnt/genética , Sequência de Aminoácidos , Animais , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Receptores Frizzled/biossíntese , Receptores Frizzled/fisiologia , Camundongos , Dados de Sequência Molecular , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/genética , Proteínas Wnt/biossíntese , Proteínas Wnt/fisiologia , Proteína Wnt-5aRESUMO
R-spondins (Rspo) are a recently discovered secretory protein family with four members in human and mouse. We and others demonstrated that R-spondins can activate canonical Wnt signaling and beta-catenin-dependent gene expression. Our study further demonstrated that R-spondins are novel ligands for the Frizzled8 and LRP6 (LDL-receptor-related protein 6) receptors. To gain insight into their biological functions, the RNA expression pattern of the mouse R-spondin family genes was analyzed during mouse development. Our study shows that R-spondin gene transcripts are widely expressed with distinct patterns in mouse at different developmental stages.