RESUMO
Sequencing-based mapping of ensemble pairwise interactions among regulatory elements support the existence of topological assemblies known as promoter-enhancer hubs or cliques in cancer. Yet, prevalence, regulators, and functions of promoter-enhancer hubs in individual cancer cells remain unclear. Here, we systematically integrated functional genomics, transcription factor screening, and optical mapping of promoter-enhancer interactions to identify key promoter-enhancer hubs, examine heterogeneity of their assembly, determine their regulators, and elucidate their role in gene expression control in individual triple negative breast cancer (TNBC) cells. Optical mapping of individual SOX9 and MYC alleles revealed the existence of frequent multiway interactions among promoters and enhancers within spatial hubs. Our single-allele studies further demonstrated that lineage-determining SOX9 and signaling-dependent NOTCH1 transcription factors compact MYC and SOX9 hubs. Together, our findings suggest that promoter-enhancer hubs are dynamic and heterogeneous topological assemblies, which are controlled by oncogenic transcription factors and facilitate subtype-restricted gene expression in cancer.
Assuntos
Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição SOX9 , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Humanos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Oncogenes , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMO
BACKGROUND: The role of miR-145-5p in non-small cell lung cancer (NSCLC) has been studied, however, the regulation of hBMSCs-derived exosomes (Exo) transmitted miR-145-5p in NSCLC was still unknown. This study aimed to investigate the role of hBMSCs-derived exosomes (Exo) in the progression of NSCLC. METHODS: The Exo was extracted from hBMSCs and added to A549 and H1299 cell culture, followed by the detection of cell proliferation, migration, and invasion. The correlation between the expression of miR-145-5p and SOX9, as well as their binding relationship was determined by correlation analysis, luciferase gene reporter assay and RNA pull-down assays. The in vivo animal model was established to further verify the impact of hBMSCs-Exo. RESULTS: It showed that miR-145-5p was downregulated and SOX9 was upregulated in NSCLC tissues. HBMSCs-derived Exo, and hBMSCs-Exo with overexpression of miR-145-5p could inhibit cell proliferation, migration, and invasion of both A549 and H1299 cells, and prevent against tumor progression in vivo. MiR-145-5p and SOX9 were found to be able to bind to each other, and a negative correlation were observed between the expression of them in NSCLC tissues. Furthermore, inhibition of SOX9 could reversed the suppressed role of miR-145-5p in vitro and in vivo. CONCLUSION: Therefore, HBMSCs-Exo effectively transmitted miR-145-5p, leading to the suppression of malignant development in NSCLC through the regulation of SOX9.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Movimento Celular , Proliferação de Células , Exossomos , Neoplasias Pulmonares , Células-Tronco Mesenquimais , MicroRNAs , Fatores de Transcrição SOX9 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Animais , Exossomos/metabolismo , Exossomos/genética , Camundongos , Proliferação de Células/genética , Células-Tronco Mesenquimais/metabolismo , Movimento Celular/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Masculino , Feminino , Células A549 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Human intervertebral disk degeneration (IVDD) is a sophisticated degenerative pathological process. A key cause of IVDD progression is nucleus pulposus cell (NPC) degeneration, which contributes to excessive endoplasmic reticulum stress in the intervertebral disk. However, the mechanisms underlying IVDD and NPC degeneration remain unclear. METHODS: We used interleukin (IL)-1ß stimulation to establish an NPC-degenerated IVDD model and investigated whether human urine-derived stem cell (USC) exosomes could prevent IL-1ß-induced NPC degeneration using western blotting, quantitative real-time polymerase chain reaction, flow cytometry, and transcriptome sequencing techniques. RESULTS: We successfully extracted and identified USCs and exosomes from human urine. IL-1ß substantially downregulated NPC viability and induced NPC degeneration while modulating the expression of SOX-9, collagen II, and aggrecan. Exosomes from USCs could rescue IL-1ß-induced NPC degeneration and restore the expression levels of SOX-9, collagen II, and aggrecan. CONCLUSIONS: USC-derived exosomes can prevent NPCs from degeneration following IL-1ß stimulation. This finding can aid the development of a potential treatment strategy for IVDD.
Assuntos
Exossomos , Interleucina-1beta , Degeneração do Disco Intervertebral , Núcleo Pulposo , Fatores de Transcrição SOX9 , Humanos , Interleucina-1beta/metabolismo , Exossomos/metabolismo , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/terapia , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Animais , Células-Tronco/metabolismo , Células Cultivadas , Agrecanas/metabolismo , Agrecanas/genética , Masculino , Urina/citologia , Urina/química , Feminino , Colágeno Tipo II/metabolismoRESUMO
BACKGROUND: Understanding how healthy articular cartilage responds to mechanical loading is critical. Moderate mechanical loading has positive effects on the cartilage, such as maintaining cartilage homeostasis. The degree of mechanical loading is determined by a combination of intensity, frequency, and duration; however, the best combination of these parameters for knee cartilage remains unclear. This study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical loading on healthy knee articular cartilage in vitro and in vivo. METHODS AND RESULTS: In this study, 33 male mice were used. Chondrocytes isolated from mouse knee joints were subjected to different cyclic tensile strains (CTSs) and assessed by measuring the expression of cartilage matrix-related genes. Furthermore, the histological characteristics of mouse tibial cartilages were quantified using different treadmill exercises. Chondrocytes and mice were divided into the control group and eight intervention groups: high-intensity, high-frequency, and long-duration; high-intensity, high-frequency, and short-duration; high-intensity, low-frequency, and long-duration; high-intensity, low-frequency, and short-duration; low-intensity, high-frequency, and long-duration; low-intensity, high-frequency, and short-duration; low-intensity, low-frequency, and long-duration; low-intensity, low-frequency, and short-duration. In low-intensity CTSs, chondrocytes showed anabolic responses by altering the mRNA expression of COL2A1 in short durations and SOX9 in long durations. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises minimized chondrocyte hypertrophy and enhanced aggrecan synthesis in tibial cartilages. CONCLUSION: Low-intensity, low-frequency, and long-duration mechanical loading is the best combination for healthy knee cartilage to maintain homeostasis and activate anabolic responses. Our findings provide a significant scientific basis for exercise and lifestyle instructions.
Assuntos
Cartilagem Articular , Condrócitos , Estresse Mecânico , Suporte de Carga , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/fisiologia , Camundongos , Condrócitos/metabolismo , Masculino , Suporte de Carga/fisiologia , Condicionamento Físico Animal/fisiologia , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo II/genética , Articulação do Joelho/metabolismo , Articulação do Joelho/fisiologia , Camundongos Endogâmicos C57BLRESUMO
Chondrocyte apoptosis is recognized as one of the pathological features involved in cartilage degeneration driving the onset and progression of knee osteoarthritis (OA). This study aimed to determine the molecular mechanism underlying the effect of clusterin (CLU), anti-apoptotic molecule, in human knee OA chondrocytes. Primary knee OA chondrocytes were isolated from the cartilage of knee OA patients and divided into five groups: (1) the cells treated with interleukin (IL)-1ß, (2) CLU alone, (3) a combination of IL-1ß and CLU, (4) LY294002 (PI3K inhibitor) along with IL-1ß and CLU, and (5) the untreated cells. Production of apoptotic, inflammatory, anabolic, and catabolic mediators in knee OA chondrocytes was determined after treatment for 24 h. Our in vitro study uncovered that CLU significantly suppressed the production of inflammatory mediators [nitric oxide (NO), IL6, and tumor necrosis factor (TNF)-α] and apoptotic molecule (caspase-3, CASP3). CLU significantly upregulated messenger ribonucleic acid (mRNA) expressions of anabolic factors [SRY-box transcription factor-9 (SOX9) and aggrecan (ACAN)], but significantly downregulated mRNA expressions of IL6, nuclear factor kappa-B (NF-κB), CASP3, and matrix metalloproteinase-13 (MMP13). Anti-apoptotic and anti-inflammatory effects of CLU were mediated through activating PI3K/Akt signaling pathway. The findings suggest that CLU might have beneficial effects on knee OA chondrocytes by exerting anti-apoptotic and anti-inflammatory functions via PI3K/Akt pathway, making CLU a promising target for potential therapeutic interventions in knee OA.
Assuntos
Apoptose , Condrócitos , Clusterina , Interleucina-1beta , Osteoartrite do Joelho , Humanos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Osteoartrite do Joelho/patologia , Osteoartrite do Joelho/metabolismo , Apoptose/efeitos dos fármacos , Clusterina/metabolismo , Clusterina/genética , Interleucina-1beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Cultivadas , Masculino , Pessoa de Meia-Idade , Idoso , Inflamação/metabolismo , Inflamação/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Feminino , Fosfatidilinositol 3-Quinases/metabolismo , Morfolinas/farmacologia , Cromonas/farmacologia , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Metaloproteinase 13 da Matriz/metabolismo , Mediadores da Inflamação/metabolismo , Óxido Nítrico/metabolismoRESUMO
To evaluate Tribulus terrestris and Mucuna pruriens for inducing all-male tilapia, mixed-sex Nile tilapia, Oreochromis niloticus, (mean weight 0.025 ± 0.009 g; mean length 1.25 ± 0.012 cm), were given a meal supplemented with either T. terrestris powder (commercial fish feed, 40% crude protein) (TT group), M. pruriens seed extract (MP group), MP + TT (mixed group), 17α-methyl testosterone (MT, control positive), or without supplements (control negative). The MP extracts significantly increased (P < 0.05) the final weight, weight gain, weight gain rate, and specific growth rate while feed conversion ratio was significantly decreased (P < 0.05). Plant extracts markedly improved (P < 0.05) the survival rate, proportion of males, and total testosterone compared to control and MT. Estrogen levels were lower in groups with plant extract than other groups. Fifteen days post-feeding, the Amh gene was expressed in the brain of O. niloticus fries with higher levels in MP, TT, and MT groups. Additionally, the expression of the Sox9 and Dmrt1 genes as a male related genes in fish fry gonads revealed significantly (P < 0.05) higher levels in groups fed on MP, TT, and MT compared to control after 30-day post-feeding, whereas; Foxl2 gene expression as a female related gene was significantly (P < 0.05) lower in fish fed on MP, TT, and MT compared to other groups after 30 days post feeding. Histologically, MT, MP, TT, and the mixture all exhibited solely male reproductive traits without noticeable abnormalities. This study concluded that each of the TT or MP extracts can induce sex reversal in tilapia while having no negative health impact compared to MT as the growth and survival rate in the treated groups with TT and MP were higher than control and group treated with MT.
Assuntos
Ração Animal , Ciclídeos , Suplementos Nutricionais , Metiltestosterona , Mucuna , Tribulus , Animais , Masculino , Tribulus/química , Metiltestosterona/farmacologia , Ração Animal/análise , Mucuna/química , Ciclídeos/crescimento & desenvolvimento , Ciclídeos/genética , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Hormônio Antimülleriano/genética , Hormônio Antimülleriano/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dieta/veterinária , Proteína Forkhead Box L2/genética , Feminino , Testosterona/sangueRESUMO
The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5' upstream) and E160 (located 160 kb 5' upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2-dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.
Assuntos
Displasia Campomélica , Diferenciação Celular , Condrócitos , Condrogênese , Fatores de Transcrição SOX9 , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Animais , Condrócitos/metabolismo , Camundongos , Displasia Campomélica/genética , Displasia Campomélica/patologia , Displasia Campomélica/metabolismo , Condrogênese/genética , Diferenciação Celular/genética , Elementos Facilitadores Genéticos/genética , Cromatina/metabolismo , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Knockout , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/genética , Humanos , Desenvolvimento Ósseo/genéticaRESUMO
The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We generated heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of SOX9 in critical developmental processes.
Assuntos
Heterozigoto , Homozigoto , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição SOX9 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Linhagem Celular , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Edição de Genes , Diferenciação CelularRESUMO
Osteogenesis imperfecta (OI) type V is the second most common form of OI, distinguished by hyperplastic callus formation and calcification of the interosseous membranes, in addition to the bone fragility. It is caused by a recurrent, dominant pathogenic variant (c.-14C>T) in interferon-induced transmembrane protein 5 (IFITM5). Here, we generated a conditional Rosa26-knockin mouse model to study the mechanistic consequences of the recurrent mutation. Expression of the mutant Ifitm5 in osteo-chondroprogenitor or chondrogenic cells resulted in low bone mass and growth retardation. Mutant limbs showed impaired endochondral ossification, cartilage overgrowth, and abnormal growth plate architecture. The cartilage phenotype correlates with the pathology reported in patients with OI type V. Surprisingly, expression of mutant Ifitm5 in mature osteoblasts caused no obvious skeletal abnormalities. In contrast, earlier expression in osteo-chondroprogenitors was associated with an increase in the skeletal progenitor cell population within the periosteum. Lineage tracing showed that chondrogenic cells expressing the mutant Ifitm5 had decreased differentiation into osteoblastic cells in diaphyseal bone. Moreover, mutant IFITM5 disrupted early skeletal homeostasis in part by activating ERK signaling and downstream SOX9 protein, and inhibition of these pathways partially rescued the phenotype in mutant animals. These data identify the contribution of a signaling defect altering osteo-chondroprogenitor differentiation as a driver in the pathogenesis of OI type V.
Assuntos
Diferenciação Celular , Sistema de Sinalização das MAP Quinases , Osteoblastos , Osteogênese Imperfeita , Fatores de Transcrição SOX9 , Animais , Feminino , Masculino , Camundongos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Transgênicos , Mutação , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/genética , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/patologia , Osteogênese Imperfeita/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/metabolismo , Células-Tronco/patologia , MAP Quinases Reguladas por Sinal ExtracelularRESUMO
Osteoarthritis (OA) is a painful and debilitating disease affecting over 500 million people worldwide. Intraarticular injection of mesenchymal stromal cells (MSCs) shows promise for the clinical treatment of OA, but the lack of consistency in MSC preparation and application makes it difficult to further optimize MSC therapy and to properly evaluate the clinical outcomes. In this study, we used Sox9 activation and RelA inhibition, both mediated by the CRISPR-dCas9 technology simultaneously, to engineer MSCs with enhanced chondrogenic potential and downregulated inflammatory responses. We found that both Sox9 and RelA could be fine-tuned to the desired levels, which enhances the chondrogenic and immunomodulatory potentials of the cells. Intraarticular injection of modified cells significantly attenuated cartilage degradation and palliated OA pain compared with the injection of cell culture medium or unmodified cells. Mechanistically, the modified cells promoted the expression of factors beneficial to cartilage integrity, inhibited the production of catabolic enzymes in osteoarthritic joints, and suppressed immune cells. Interestingly, a substantial number of modified cells could survive in the cartilaginous tissues including articular cartilage and meniscus. Together, our results suggest that CRISPR-dCas9-based gene regulation is useful for optimizing MSC therapy for OA.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Mesenquimais , Osteoartrite , Fatores de Transcrição SOX9 , Fator de Transcrição RelA , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Osteoartrite/terapia , Osteoartrite/genética , Osteoartrite/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Camundongos , Humanos , Modelos Animais de Doenças , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Transplante de Células-Tronco Mesenquimais/métodos , Condrogênese/genética , Edição de Genes , Terapia Baseada em Transplante de Células e Tecidos/métodos , Condrócitos/metabolismoRESUMO
Liver fibrosis is a significant health concern globally due to its association with severe liver conditions like cirrhosis and liver cancer. Histone lactylation has been implicated in the progression of hepatic fibrosis, but its specific role in liver fibrosis, particularly regarding H3K18 lactylation, remained unclear. To investigate this, we established in vivo and in vitro models of liver fibrosis using carbon tetrachloride (CCl4) injection in rats and stimulation of hepatic stellate cells (HSCs) with TGF-ß1, respectively. We found that histone lactylation, particularly H3K18 lactylation, was upregulated in both CCl4-induced rats and TGF-ß1-activated HSCs, indicating its potential involvement in liver fibrosis. Further experiments revealed that lactate dehydrogenase A (LDHA) knockdown inhibited H3K18 lactylation and had a beneficial effect on liver fibrosis by suppressing HSC proliferation, migration, and extracellular matrix (ECM) deposition. This suggests that H3K18 lactylation promotes liver fibrosis progression. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that H3K18 lactylation facilitated the transcription of SOX9, a transcription factor associated with fibrosis. Importantly, overexpression of SOX9 counteracted the effects of LDHA silencing on activated HSCs, indicating that SOX9 is downstream of H3K18 lactylation in promoting liver fibrosis. In summary, this study uncovers a novel mechanism by which H3K18 lactylation contributes to liver fibrosis by activating SOX9 transcription. This finding opens avenues for exploring new therapeutic strategies for hepatic fibrosis targeting histone lactylation pathways.
Assuntos
Progressão da Doença , Células Estreladas do Fígado , Histonas , Cirrose Hepática , Ratos Sprague-Dawley , Fatores de Transcrição SOX9 , Animais , Humanos , Masculino , Ratos , Tetracloreto de Carbono , Movimento Celular/genética , Proliferação de Células , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Histonas/metabolismo , Histonas/genética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Cirrose Hepática/induzido quimicamente , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Transcrição Gênica , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.
Assuntos
Actinas , Desdiferenciação Celular , Condrócitos , Fibras de Estresse , Tropomiosina , Condrócitos/metabolismo , Condrócitos/citologia , Fibras de Estresse/metabolismo , Animais , Actinas/metabolismo , Tropomiosina/metabolismo , Tropomiosina/genética , Fenótipo , Células Cultivadas , Proteína cdc42 de Ligação ao GTP/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Transativadores/metabolismo , Transativadores/genéticaRESUMO
Hyaline cartilage regeneration will bring evangel to millions of people suffered from cartilage diseases. However, uncontrollable cartilage fibrosis and matrix mineralization are the primary causes of cartilage regeneration failure in many tissue engineering scaffolds. This study presents a new attempt to avoid endochondral ossification or fibrosis in cartilage regeneration therapy by establishing biochemical regulatory area. Here, SOX9 expression plasmids are assembled in cellulose gels by chitosan gene vectors to fabricate SOX9+ functionalized scaffolds. RT-qPCR, western blot and biochemical analysis all show that the SOX9 reinforcement strategy can enhance chondrogenic specific proteins expression and promote GAG production. Notably, the interference from SOX9 has resisted osteogenic inducing significantly, showing an inhibition of COL1, OPN and OC production, and the inhibition efficiency was about 58.4â¯%, 22.8â¯% and 76.9â¯% respectively. In vivo study, implantation of these scaffolds with BMSCs can induce chondrogenic differentiation and resist endochondral ossification effectively. Moreover, specific SOX9+ functionalized area of the gel exhibited the resistance to matrix mineralization, indicating the special biochemical functional area for cartilage regeneration. These results indicate that this strategy is effective for promoting the hyaline cartilage regeneration and avoiding cartilage fibrosis, which provides a new insight to the future development of cartilage regeneration scaffolds.
Assuntos
Condrogênese , Fibrose , Fatores de Transcrição SOX9 , Alicerces Teciduais , Animais , Humanos , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular , Células Cultivadas , Quitosana/química , Osteogênese/efeitos dos fármacos , Regeneração , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Engenharia Tecidual , Alicerces Teciduais/química , Masculino , CoelhosRESUMO
OBJECTIVE: Understanding the mechanisms of hip disease, such as osteoarthritis (OA), is crucial to advance their treatment. Such hip diseases often involve specific morphological changes. Genetic variations, called single nucleotide polymorphisms (SNPs), influence various hip morphological parameters. This study investigated the biological relevance of SNPs correlated to hip morphology in genome-wide association studies (GWAS). The SNP-associated genes were compared to genes associated with OA in other joints, aiming to see if the same genes play a role in both hip development and the risk of OA in other lower limb joints. METHODOLOGY: A systematic literature review was conducted to identify SNPs correlated with hip morphology, based on the Population, Intervention, Comparison, Outcome, and Study (PICOS) framework. Afterwards, Gene Ontology (GO) analysis was performed, using EnrichR, on the SNP-associated genes and compared with non-hip OA-associated genes, across different databases. RESULTS: Reviewing 49 GWAS identified 436 SNPs associated with hip joint morphology, encompassing variance in bone size, structure and shape. Among the SNP-associated genes, SOX9 plays a pivotal role in size, GDF5 impacts bone structure, and BMP7 affects shape. Overall, skeletal system development, regulation of cell differentiation, and chondrocyte differentiation emerged as crucial processes influencing hip morphology. Eighteen percent of GWAS-identified genes related to hip morphology were also associated with non-hip OA. CONCLUSION: Our findings indicate the existence of multiple shared genetic mechanisms across hip morphology and OA, highlighting the necessity for more extensive research in this area, as in contrast to the hip, the genetic background on knee or foot morphology remains largely understudied.
Assuntos
Estudo de Associação Genômica Ampla , Fator 5 de Diferenciação de Crescimento , Articulação do Quadril , Osteoartrite do Quadril , Polimorfismo de Nucleotídeo Único , Humanos , Osteoartrite do Quadril/genética , Osteoartrite do Quadril/patologia , Fator 5 de Diferenciação de Crescimento/genética , Articulação do Quadril/patologia , Proteína Morfogenética Óssea 7/genética , Fatores de Transcrição SOX9/genética , Predisposição Genética para DoençaRESUMO
Osteoblast-derived semaphorin3A (Sema3A) has been reported to be involved in bone protection, and Sema3A knockout mice have been reported to exhibit chondrodysplasia. From these reports, Sema3A is considered to be involved in chondrogenic differentiation and skeletal formation, but there are many unclear points about its function and mechanism in chondrogenic differentiation. This study investigated the pharmacological effects of Sema3A in chondrogenic differentiation. The amount of Sema3A secreted into the culture supernatant was measured using an enzyme-linked immunosorbent assay. The expression of chondrogenic differentiation-related factors, such as Type II collagen (COL2A1), Aggrecan (ACAN), hyaluronan synthase 2 (HAS2), SRY-box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), and Type X collagen (COL10A1) in ATDC5 cells treated with Sema3A (1,10 and 100 ng/mL) was examined using real-time reverse transcription polymerase chain reaction. Further, to assess the deposition of total glycosaminoglycans during chondrogenic differentiation, ATDC5 cells were stained with Alcian Blue. Moreover, the amount of hyaluronan in the culture supernatant was measured by enzyme-linked immunosorbent assay. The addition of Sema3A to cultured ATDC5 cells increased the expression of Sox9, Runx2, COL2A1, ACAN, HAS2, and COL10A1 during chondrogenic differentiation. Moreover, it enhanced total proteoglycan and hyaluronan synthesis. Further, Sema3A was upregulated in the early stages of chondrogenic differentiation, and its secretion decreased later. Sema3A increases extracellular matrix production and promotes chondrogenic differentiation. To the best of our knowledge, this is the first study to demonstrate the role of Sema3A on chondrogenic differentiation.
Assuntos
Diferenciação Celular , Condrogênese , Semaforina-3A , Animais , Diferenciação Celular/efeitos dos fármacos , Semaforina-3A/metabolismo , Condrogênese/efeitos dos fármacos , Camundongos , Condrócitos/metabolismo , Condrócitos/citologia , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Linhagem Celular , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo II/genética , Agrecanas/metabolismo , Agrecanas/genética , Hialuronan Sintases/metabolismo , Hialuronan Sintases/genética , Glicosaminoglicanos/metabolismo , Colágeno Tipo X/metabolismo , Colágeno Tipo X/genéticaRESUMO
Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor's self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.
Assuntos
Lipoproteínas LDL , Fatores de Transcrição SOX9 , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Animais , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Camundongos , Humanos , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/genética , Transição Epitelial-Mesenquimal , Camundongos Endogâmicos C57BL , Masculino , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/citologia , Autorrenovação Celular , Células Endoteliais/metabolismoRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood. OBJECTIVE: This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress. METHODS: The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot. RESULTS: High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway. CONCLUSION: This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II , Diferenciação Celular , Condrogênese , Células-Tronco Mesenquimais , MicroRNAs , Transdução de Sinais , Estresse Mecânico , Membrana Sinovial , Humanos , Agrecanas/metabolismo , Agrecanas/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Condrogênese/fisiologia , Colágeno Tipo II/metabolismo , Colágeno Tipo II/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Membrana Sinovial/citologia , Membrana Sinovial/metabolismoRESUMO
Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.
Assuntos
Células Epiteliais Alveolares , Proteínas Estimuladoras de Ligação a CCAAT , Plasticidade Celular , Animais , Camundongos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/citologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular , Plasticidade Celular/genética , Proliferação de Células , Epigênese Genética , Pulmão/metabolismo , Lesão Pulmonar/patologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração , Vírus Sendai/genética , Vírus Sendai/fisiologia , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Fator Nuclear 1 de Tireoide/metabolismo , Fator Nuclear 1 de Tireoide/genéticaRESUMO
Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.
Assuntos
Linhagem da Célula , Cromatina , Gônadas , Fatores de Transcrição SOX9 , Análise de Célula Única , Animais , Cromatina/metabolismo , Cromatina/genética , Camundongos , Linhagem da Célula/genética , Feminino , Masculino , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Gônadas/metabolismo , Gônadas/citologia , Gônadas/embriologia , Fator Esteroidogênico 1/genética , Fator Esteroidogênico 1/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Testículo/metabolismo , Testículo/citologia , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Ovário/metabolismo , Ovário/citologiaRESUMO
Osteoarthritis (OA) is a common joint disorder characterized by cartilage degeneration, often leading to pain and functional impairment. Minced cartilage implantation (MCI) has emerged as a promising one-step alternative for large cartilage defects. However, the source of chondrocytes for MCI remains a challenge, particularly in advanced OA, as normal cartilage is scarce. We performed in vitro studies to evaluate the feasibility of MCI using osteophyte cartilage, which is present in patients with advanced OA. Osteophyte and articular cartilage samples were obtained from 22 patients who underwent total knee arthroplasty. Chondrocyte migration and proliferation were assessed using cartilage fragment/atelocollagen composites to compare the characteristics and regenerative potential of osteophytes and articular cartilage. Histological analysis revealed differences in cartilage composition between osteophytes and articular cartilage, with higher expression of type X collagen and increased chondrocyte proliferation in the osteophyte cartilage. Gene expression analysis identified distinct gene expression profiles between osteophytes and articular cartilage; the expression levels of COL2A1, ACAN, and SOX9 were not significantly different. Chondrocytes derived from osteophyte cartilage exhibit enhanced proliferation, and glycosaminoglycan production is increased in both osteophytes and articular cartilage. Osteophyte cartilage may serve as a viable alternative source of MCI for treating large cartilage defects in OA.