RESUMEN
The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.
Asunto(s)
Bortezomib/farmacología , Proliferación Celular/efectos de los fármacos , Curación de Fractura/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Fracturas de la Tibia/enzimología , Animales , Proliferación Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Modelos Animales de Enfermedad , Curación de Fractura/genética , Masculino , Ratones , Ratones Transgénicos , Osteoblastos/enzimología , Complejo de la Endopetidasa Proteasomal/genética , Fracturas de la Tibia/tratamiento farmacológico , Fracturas de la Tibia/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Human aortic valve interstitial cells (hAVICs) are a key factor in the pathogenesis of calcific aortic valve disease (CAVD). This research examines the role and mechanism of microRNA miR-138-5p in osteogenic differentiation of hAVICs. METHODS: RT-qPCR analysis was applied for detecting miR-138-5p and RUNX2 expression in valve tissues of CAVD patients and controls. On completion of induction of osteogenic differentiation of hAVICs, and after overexpression or interference of miR-138-5p expression, the condition of osteogenic differentiation and calcification of hAVICs was confirmed using alkaline phosphatase staining and alizarin red staining. Subsequently, western blot was utilized to detect the expression of osteogenesis-related proteins OPN and ALP, and Wnt/ß-catenin signaling pathway-related proteins. Finally, the relationship between miR-138-5p and RUNX2 was validated by dual-luciferase reporter assay and Pearson's correlation test. RESULTS: Down-regulation of miR-138-5p was found in CAVD patients and during osteogenic differentiation of hAVICs. Overexpression of miR-138-5p contribute to the inhibition of osteoblast differentiation and calcium deposition in hAVICs, and of ALP and OPN protein expression. RUNX2 was a target gene of miR-138-5p, and it was negatively correlated with miR-138-5p in CAVD. Additionally, overexpression of RUNX2 could reverse the inhibitory effect of miR-138-5p on osteogenic differentiation of hAVICs. CONCLUSION: miR-138-5p can act as a positive regulator of osteogenic differentiation in CAVD patients to involve in inhibiting valve calcification, which is achieved through RUNX2 and Wnt/ß-catenin signaling pathway.
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Estenosis de la Válvula Aórtica/genética , Válvula Aórtica/patología , Calcinosis/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Regulación de la Expresión Génica , MicroARNs/genética , Osteogénesis/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Adulto , Válvula Aórtica/diagnóstico por imagen , Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/diagnóstico , Estenosis de la Válvula Aórtica/metabolismo , Calcinosis/diagnóstico , Calcinosis/metabolismo , Diferenciación Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Femenino , Humanos , Masculino , MicroARNs/biosíntesis , beta Catenina/biosíntesisRESUMEN
BACKGROUND: Type 2-high asthma is a prominent endotype of asthma which is characterized by airway eosinophilic inflammation. Airway epithelial cells play a critical role in the pathogenesis of asthma. Our previous miRNA profiling data showed that miR-30a-3p was downregulated in bronchial epithelial cells from asthma patients. We hypothesize that epithelial miR-30a-3p plays a role in asthma airway inflammation. METHODS: We measured miR-30a-3p expression in bronchial brushings of asthma patients (n = 51) and healthy controls (n = 16), and analyzed the correlations between miR-30a-3p expression and airway eosinophilia. We examined whether Runt-related transcription factor 2 (RUNX2) was a target of miR-30a-3p and whether RUNX2 bound to the promoter of high mobility group box 1 (HMGB1) by using luciferase reporter assay and chromatin immunoprecipitation (ChIP)-PCR. The role of miR-30a-3p was also investigated in a murine model of allergic airway inflammation. RESULTS: We found that miR-30a-3p expression were significantly decreased in bronchial brushings of asthma patients compared to control subjects. Epithelial miR-30a-3p expression was negatively correlated with parameters reflecting airway eosinophilia including eosinophils in induced sputum and bronchial biopsies, and fraction of exhaled nitric oxide in asthma patients. We verified that RUNX2 is a target of miR-30a-3p. Furthermore, RUNX2 bound to the promoter of HMGB1 and upregulated HMGB1 expression. RUNX2 and HMGB1 expression was both enhanced in airway epithelium and was correlated with each other in asthma patients. Inhibition of miR-30a-3p enhanced RUNX2 and HMGB1 expression, and RUNX2 overexpression upregulated HMGB1 in BEAS-2B cells. Intriguingly, airway overexpression of mmu-miR-30a-3p suppressed Runx2 and Hmgb1 expression, and alleviated airway eosinophilia in a mouse model of allergic airway inflammation. CONCLUSIONS: Epithelial miR-30a-3p could possibly target RUNX2/HMGB1 axis to suppress airway eosinophilia in asthma.
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Asma/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Eosinofilia/genética , Regulación de la Expresión Génica , Proteína HMGB1/genética , Inflamación/genética , MicroARNs/genética , Animales , Asma/complicaciones , Asma/patología , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Modelos Animales de Enfermedad , Eosinofilia/complicaciones , Eosinofilia/patología , Femenino , Proteína HMGB1/biosíntesis , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , MicroARNs/biosíntesis , Esputo/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Calcium accumulation and fibrotic activities are principal mechanisms for calcific aortic valve disease (CAVD). Active complement products are observed in human stenotic aortic valves. Runt-related transcription factor 2 (Runx-2) is involved in tissue calcification. We hypothesized that complement upregulates Runx-2 to induce profibrogenic change in human aortic valve interstitial cells (AVICs). METHODS: AVICs were isolated from 6 normal and 6 CAVD donor valves. Cells were treated with complement cocktails. Profibrogenic activities and associated signaling molecules were analyzed by Western blot assay and collagen staining. RESULTS: Complement time and dose dependently enhanced profibrogenic activities in AVICs, and complement exposure also induced total collagen deposition in AVICs. Complement-induced profibrogenic responses were associated with increased Runx-2 expression and phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Genetic silencing of Runx-2 decreased both matrix metalloproteinase 9 (MMP-9) and collagen I levels. Pharmacological inhibition of ERK1/2 decreased complement-mediated MMP-9, collagen I, and Runx-2 expression as well as total collagen deposition in human AVICs. Further, treating AVICs with heat-deactivated complement resulted in reduced MMP-9, collagen I, and Runx-2 levels compared with active complement treatment. CONCLUSIONS: Complement induced profibrogenic activities in AVICs by activation of ERK1/2-mediated Runx-2 signaling pathways. This study demonstrates a potential role for complement-mediated CAVD pathogenesis, establishing a possible therapeutic target to limit CAVD progression.
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Estenosis de la Válvula Aórtica/genética , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Regulación de la Expresión Génica , Anciano , Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/patología , Western Blotting , Calcinosis/metabolismo , Calcinosis/patología , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN/genética , Transducción de SeñalRESUMEN
Our previous studies found that prenatal dexamethasone exposure could cause abnormal follicular development in fetal rats. This study intends to observe the transgenerational inheritance effects of ovarian estrogen inhibition in offspring exposed to dexamethasone (0.2 mg/kg ⢠d) from gestational day 9 (GD9) to GD20 in Wistar rats, and explore the intrauterine programming mechanisms. Prenatal dexamethasone exposure reduced the expression of ovarian cytochrome P450 aromatase (P450arom), the level of serum estradiol (E2) and the number of primordial follicles, while increased the number of atresia follicles before and after birth in F1 offspring rats. At the same time, the expression of miRNA320a-3p in F1 ovaries was down-regulated, and RUNX2 expression increased significantly. These changes were continued to F2 and F3 generations, accompanied by consistently down-regulated miRNA320a-3p expression in oocyte of F1 and F2 adult offspring. In vitro, fetal rat ovaries and KGN human ovarian granulosa cells were treated with dexamethasone. It showed that dexamethasone decreased miRNA320a-3p and P450arom expression, as well as E2 synthesis, and increased RUNX2 expression. All these effects could be reversed by the GR antagonist RU486. The overexpression of miRNA320a-3p in vitro could also reverse the effects of dexamethasone on RUNX2, P450arom, and E2 levels. The dual-luciferase reporter gene experiment further confirmed the direct targeted regulation of miRNA320a-3p on RUNX2. These results indicate that prenatal dexamethasone exposure induces ovarian E2 synthesis inhibition mediated by the GR/miRNA320a-3p/RUNX2/P450arom cascade signal in fetal rat ovary, which has transgenerational inheritance effects and may related to the inhibited miRNA320a-3p expression in oocyte.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Dexametasona/toxicidad , Estrógenos/biosíntesis , MicroARNs/sangre , Ovario/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Animales , Animales Recién Nacidos , Línea Celular Tumoral , Células Cultivadas , Femenino , Glucocorticoides/toxicidad , Humanos , MicroARNs/antagonistas & inhibidores , Ovario/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Ratas , Ratas Wistar , Inhibidores de la Síntesis de Esteroides/toxicidadRESUMEN
AIM: Vinpocetine (Vin) has long been used as a medicine to treat cerebrovascular disorders and as a dietary supplement to improve cognitive functions. Previous studies have revealed that the transcription factor nuclear factor kappa B (NF-κB) activity plays an important role in osteogenic differentiation of mesenchymal stem cells (MSC). Vin inhibits NF-κB-dependent inflammatory responses; however, the effect of Vin on the osteogenic differentiation of MSCs has not been reported. In this study, we aimed to the investigate effect of Vin on the osteogenic differentiation of rat bone marrow-derived MSCs (BMSCs). METHODS: We treated BMSCs with clinical plasma (0.17 µM) or higher concentrations (5 and 20 µM) of Vin with no significant effect on the cell viability. Alizarin Red S and alkaline phosphatase (ALP) stainings were used to evaluate mineralizations on days 14 and 21. Moreover, expressions of target genes were detected using qRT-PCR analysis. RESULTS: Osteogenic differentiation of BMSCs did not significantly change with Vin's clinical plasma concentration, but significantly decreased with higher concentrations. Calcium mineralization, ALP staining and mRNA gene expressions of Runx2 and ALP were decreased significantly with high concentrations of Vin, paticularly on day 21. CONCLUSION: Our in vitro findings suggest that clinically relevant concentration of Vin seems safe to use in elderly patients with respect to osteoporosis. On the other hand, Vin at high concentrations appears to be harmful to bone homeostasis.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Alcaloides de la Vinca/sangre , Alcaloides de la Vinca/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Células de la Médula Ósea/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Regulación de la Expresión Génica/efectos de los fármacos , FN-kappa B/efectos de los fármacos , RatasRESUMEN
This study evaluated the response of a nano-hydroxyapatite coating implant through gene expression analysis (runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), osteopontin (Opn), osteocalcin (Oc), receptor activator of nuclear factor-kappa B (Rank), receptor activator of nuclear factor-kappa B ligand (Rank-L), and osteoprotegerin (Opg)). Three-dimensional evaluation (percent bone volume (BV/TV); percent intersection surface (BIC); bone surface/volume ratio (BS/BV); and total porosity (To.Po)) were also analyzed. Mini implants were surgically placed in tibias of both healthy and diabetic rats. The animals were euthanized at 7 and 30 days. Evaluating all factors the relative expression of Rank showed that NANO surface presented the best results at 7 days (diabetic rats). Furthermore the levels of Runx2, Alp, Oc, and Opn suggest an increase in osteoblasts proliferation, especially in early stages of osseointegration. %BIC in healthy and diabetic (7 days) depicted statistically significant differences for NANO group. BV/TV, BS/BV and To.Po demonstrated higher values for NANO group in all evaluated time point and irrespective of systemic condition, but BS/BV 30 days (healthy rat) and 7 and 30 days (diabetic rat). Microtomographic and gene expression analyses have shown the benefits of nano-hydroxyapatite coated implants in promoting new bone formation in diabetic rats.
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Regeneración Ósea/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Durapatita/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Implantes Experimentales , Nanopartículas , Osteogénesis/efectos de los fármacos , Microtomografía por Rayos X , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Diabetes Mellitus Experimental , Durapatita/uso terapéutico , Masculino , Microscopía Electrónica de Rastreo , Nanopartículas/uso terapéutico , Oseointegración , Osteocalcina/biosíntesis , Osteocalcina/genética , Osteogénesis/genética , Osteopontina/biosíntesis , Osteopontina/genética , Osteoprotegerina/biosíntesis , Osteoprotegerina/genética , Ligando RANK/biosíntesis , Ligando RANK/genética , Ratas , Ratas Wistar , Tibia/cirugíaRESUMEN
BACKGROUND: Osteoporosis, characterized by bone loss, usually occurs with the increased bone resorption and decreased bone formation. H2O2-induced MC3T3-E1 cells are commonly used for the study of osteoblastic activities, which play a crucial role in bone formation. OBJECTIVE: This study aimed to investigate the effects of Phosphocreatine (PCr) on the osteoblastic activities in H2O2-induced MC3T3-E1 cells and elaborate on the possible molecular mechanism. METHODS: The Osteoprotegerin (OPG)/Receptor Activator of NF-κB Ligand (RANKL) ratio and osteogenic markers were detected to investigate the effects of PCr on osteoblastic activities, and the osteoblastic apoptosis was detected using Hochest staining. Moreover, oxidative stress, Adenosine Triphosphate (ATP) generation and the expression of Sirtuin 1 (SIRT1), Forkhead Box O 1 (FOXO1) and Peroxisome Proliferator-Activated Receptor Γ Coactivator-1α (PGC-1α) were also examined to uncover the possible molecular mechanism in H2O2-induced MC3T3-E1 cells. RESULT: The results showed that PCr promoted the osteoblastic differentiation by increasing the expression levels of osteogenic markers of Alkaline Phosphatase (ALP) and Runt-related transcription factor 2 (Runx2), as well as increased the OPG/RANKL ratio and suppressed the osteoblastic apoptosis in H2O2-induced MC3T3-E1 cells. Moreover, treatment with PCr suppressed reactive oxygen species (ROS) over-generation and promoted the ATP production as well as increased the PGC-1α, FOXO1 and SIRT1 protein expression levels in H2O2-induced MC3T3-E1 cells. CONCLUSION: PCr treatment could promote osteoblastic activities via suppressing oxidative stress and increasing the ATP generation in H2O2-induced MC3T3-E1 cells. In addition, the positive effects of PCr on osteoblasts might be regulated by SIRT1/FOXO1/ PGC-1α signaling pathway.
Asunto(s)
Proteína Forkhead Box O1/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/efectos de los fármacos , Fosfocreatina/farmacología , Transducción de Señal/efectos de los fármacos , Sirtuina 1/efectos de los fármacos , Células 3T3 , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/efectos de los fármacos , Ratones , Osteoprotegerina/efectos de los fármacos , Osteoprotegerina/metabolismo , Estrés Oxidativo , Ligando RANK/efectos de los fármacos , Ligando RANK/metabolismo , Especies Reactivas de OxígenoRESUMEN
Temporal and/or spatial alteration of collagen family gene expression results in bone defects. However, how collagen expression controls bone size remains largely unknown. The basic helix-loop-helix transcription factor HAND1 is expressed in developing long bones and is involved in their morphogenesis. To understand the functional role of HAND1 and collagen in the postnatal development of long bones, we overexpressed Hand1 in the osteochondroprogenitors of model mice and found that the bone volumes of cortical bones decreased in Hand1Tg/+;Twist2-Cre mice. Continuous Hand1 expression downregulated the gene expression of type I, V, and XI collagen in the diaphyses of long bones and was associated with decreased expression of Runx2 and Sp7/Osterix, encoding transcription factors involved in the transactivation of fibril-forming collagen genes. Members of the microRNA-196 family, which target the 3' untranslated regions of COL1A1 and COL1A2, were significantly upregulated in Hand1Tg/+;Twist2-Cre mice. Mass spectrometry revealed that the expression ratios of alpha 1(XI), alpha 2(XI), and alpha 2(V) in the diaphysis increased during postnatal development in wild-type mice, which was delayed in Hand1Tg/+;Twist2-Cre mice. Our results demonstrate that HAND1 regulates bone size and morphology through osteochondroprogenitors, at least partially by suppressing postnatal expression of collagen fibrils in the cortical bones.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Colágeno/biosíntesis , Hueso Cortical/crecimiento & desarrollo , Regulación de la Expresión Génica , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Diáfisis/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Tamaño de los Órganos , Factor de Transcripción Sp7/biosíntesis , Factor de Transcripción Sp7/genéticaRESUMEN
Calcium deposition in vascular smooth muscle cells (VSMCs) is a form of ectopic ossification in blood vessels. It can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that the microRNA miR-134-5p potentiates inorganic phosphate (Pi)-induced calcium deposition in VSMCs by inhibiting histone deacetylase 5 (HDAC5). Using miRNA microarray analysis of Pi-treated rat VSMCs, we first selected miR-134-5p for further evaluation. Quantitative RT-PCR confirmed that miR-134-5p was increased in Pi-treated A10 cells, a rat VSMC line. Transfection of miR-134-5p mimic potentiated the Pi-induced increase in calcium contents. miR-134-5p increased the amounts of bone runt-related transcription factor 2 (RUNX2) protein and bone morphogenic protein 2 (BMP2) mRNA in the presence of Pi but decreased the expression of osteoprotegerin (OPG). Bioinformatic analysis showed that the HDAC5 3'untranslated region (3'UTR) was one of the targets of miR-134-5p. The luciferase construct containing the 3'UTR of HDAC5 was down-regulated by miR-134-5p mimic in a dose-dependent manner in VSMCs. Overexpression of HDAC5 mitigated the calcium deposition induced by miR-134-5p. Our results suggest that a Pi-induced increase of miR-134-5p may cause vascular calcification through repression of HDAC5.
Asunto(s)
Calcio/metabolismo , Histona Desacetilasas/efectos de los fármacos , MicroARNs/fisiología , Miocitos del Músculo Liso/metabolismo , Calcificación Vascular/etiología , Regiones no Traducidas 3' , Animales , Aorta Torácica/citología , Línea Celular , Simulación por Computador , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/inmunología , Regulación hacia Abajo , Regulación de la Expresión Génica , Genes Reporteros , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/genética , MicroARNs/genética , Análisis por Micromatrices , Músculo Liso Vascular/citología , Osteoprotegerina/biosíntesis , Osteoprotegerina/genética , Fosfatos/toxicidad , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Calcificación Vascular/genética , Calcificación Vascular/metabolismo , Calcificación Vascular/prevención & controlRESUMEN
The recurring association of specific genetic lesions with particular types of cancer is a fascinating and largely unexplained area of cancer biology. This is particularly true of clear cell renal cell carcinoma (ccRCC) where, although key mutations such as loss of VHL is an almost ubiquitous finding, there remains a conspicuous lack of targetable genetic drivers. In this study, we have identified a previously unknown protumorigenic role for the RUNX genes in this disease setting. Analysis of patient tumor biopsies together with loss-of-function studies in preclinical models established the importance of RUNX1 and RUNX2 in ccRCC. Patients with high RUNX1 (and RUNX2) expression exhibited significantly poorer clinical survival compared with patients with low expression. This was functionally relevant, as deletion of RUNX1 in ccRCC cell lines reduced tumor cell growth and viability in vitro and in vivo. Transcriptional profiling of RUNX1-CRISPR-deleted cells revealed a gene signature dominated by extracellular matrix remodeling, notably affecting STMN3, SERPINH1, and EPHRIN signaling. Finally, RUNX1 deletion in a genetic mouse model of kidney cancer improved overall survival and reduced tumor cell proliferation. In summary, these data attest to the validity of targeting a RUNX1-transcriptional program in ccRCC. SIGNIFICANCE: These data reveal a novel unexplored oncogenic role for RUNX genes in kidney cancer and indicate that targeting the effects of RUNX transcriptional activity could be relevant for clinical intervention in ccRCC.
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Carcinoma de Células Renales/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/biosíntesis , Neoplasias Renales/metabolismo , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Procesos de Crecimiento Celular , Línea Celular Tumoral , Movimiento Celular/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Femenino , Técnicas de Inactivación de Genes , Células HEK293 , Xenoinjertos , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Ratones , Ratones Desnudos , Pronóstico , TranscriptomaRESUMEN
OBJECTIVE: To elucidate the role of telomerase RNA elements (TERC) in alleviating osteoporosis (OP) by absorbing microRNA-217 (miRNAs-217) to regulate runt-related transcription factor 2 (RUNX2) level. MATERIALS AND METHODS: The serum levels of TERC and miRNA-217 in OP patients and healthy controls were determined. During the osteogenic process, the relative levels of alkaline phosphatase (ALP), RUNX2, and Osterix were determined in hMSCs. The regulatory effects of TERC, miRNA-217, and RUNX2 on ALP and RUNX2 levels in hMSCs were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. In addition, the changes in ALP activity and calcification ability in hMSCs influenced by them were assessed through ALP activity determination and alizarin red staining, respectively. The interaction of TERC/miRNA-217/RUNX2 regulatory loop and its role in influencing hMSCs osteogenesis were assessed by Dual-Luciferase Reporter Gene Assay and a series of rescue experiments, respectively. RESULTS: The downregulated TERC and upregulated miRNA-217 were identified in the serum of the OP patients. Consistently, the downregulated TERC and upregulated miRNA-217 were discovered in in vitro osteogenic process of hMSCs. The silence of TERC, or RUNX2 downregulated ALP and RUNX2 levels, decreased ALP activity and attenuated the calcification ability in hMSCs. The overexpression of miRNA-217 gave similar results. The binding relationship in TERC/miRNA-217/RUNX2 regulatory loop was verified. At last, rescue experiments suggested that TERC accelerated hMSCs osteogenesis by absorbing miRNA-217 to upregulate RUNX2. CONCLUSIONS: The serum level of TERC is lowly expressed in OP patients. TERC influences hMSCs osteogenesis by absorbing miRNA-217 to upregulate RUNX2, thus alleviating the progression of OP.
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Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Progresión de la Enfermedad , MicroARNs/sangre , Osteoporosis/sangre , ARN/sangre , Telomerasa/sangre , Regulación hacia Arriba/fisiología , Células Cultivadas , Humanos , Osteogénesis/fisiología , Osteoporosis/patología , Osteoporosis/prevención & controlRESUMEN
OBJECTIVE: To study the influence of micro ribonucleic acid (miR)-137 on osteoporosis rats by regulating runt-related transcription factor 2 (RUNX2). MATERIALS AND METHODS: A total of 36 Sprague-Dawley rats were randomly assigned to the normal group (n=12), model group (n=12), and inhibitor group (n=12). No treatment was performed in the normal group. The osteoporosis model in rats was prepared in the model group, and miR-137 inhibitor was administered in osteoporosis rats of inhibitor group. Following 12 weeks of intervention, sampling was conducted. The expression of RUNX2 was detected via immunohistochemistry, and its protein expression level was determined via Western blotting. Quantitative Polymerase Chain Reaction (qPCR) was carried out to detect the mRNA level of miR-137. The contents of serum bone Gla protein (BGP) and total alkaline phosphatase (TALP) were measured using enzyme-linked immunosorbent assay (ELISA). Finally, bone mineral density was determined with a dual-energy X-ray absorptiometry instrument. RESULTS: According to the immunohistochemistry detection, the rats in model group and inhibitor group had a notably lower positive expression level of RUNX2 than normal group (p<0.05), and its expression level in the inhibitor group was substantially higher than that in the model group (p<0.05). Western blotting results showed that compared with that in the normal group, the protein expression level of RUNX2 was notably lowered in the model and inhibitor group (p<0.05), which was markedly higher in the inhibitor group than that in the model group (p<0.05). It was found through the qPCR that the expression level of miR-137 was remarkably raised in both model group and inhibitor group compared with that in the normal group, showing statistically significant differences (p<0.05). The rats in the inhibitor group had a remarkably lower expression level of miR-137 than the model group (p<0.05). ELISA results revealed that the model group and inhibitor group had substantially lower contents of serum BGP and TALP than the normal group (p<0.05), and that their contents rose dramatically in the inhibitor group compared with that in the model group (p<0.05). Additionally, based on the measurement of bone mineral density, compared with that in the normal group, bone mineral density declined considerably in the model group and inhibitor group (p<0.05). It was markedly elevated in inhibitor group in comparison with that in the model group (p<0.05). CONCLUSIONS: MiR-137 regulates RUNX2 to affect the bone mineral density of osteoporosis model rats.
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Densidad Ósea/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , MicroARNs/biosíntesis , Osteoporosis/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Dexametasona/toxicidad , Femenino , Osteoporosis/inducido químicamente , Osteoporosis/diagnóstico por imagen , Ratas , Ratas Sprague-DawleyRESUMEN
Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.
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Envejecimiento/metabolismo , Condrocitos/metabolismo , Condrogénesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Proteínas Fetales/biosíntesis , Degeneración del Disco Intervertebral/metabolismo , Factor de Transcripción Sp7/biosíntesis , Proteínas de Dominio T Box/biosíntesis , Envejecimiento/genética , Envejecimiento/patología , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Condrocitos/patología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Proteínas Fetales/genética , Regulación de la Expresión Génica , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/patología , Ratones , Ratones Transgénicos , Factor de Transcripción Sp7/genética , Proteínas de Dominio T Box/genéticaRESUMEN
Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.
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Lamina Tipo B/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , NADPH Deshidrogenasa/metabolismo , Actinas/metabolismo , Animales , Diferenciación Celular/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Técnicas de Silenciamiento del Gen , Ratones , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , NADPH Deshidrogenasa/deficiencia , NADPH Deshidrogenasa/genética , Membrana Nuclear/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , OsteogénesisRESUMEN
BACKGROUND: Calcium ions (Ca2+ ) influence natural bone healing, and calcium is frequently used in bone tissue engineering scaffolds and cements. Scaffolds can also incorporate gene delivery systems to further promote osteoblast differentiation. Thus, our goal was to identify if Ca2+ concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene-activated scaffolds designed to promote bone growth. METHODS: Bone marrow-derived mesenchymal stem cells (BMSCs) were cultured in media with Ca2+ concentrations ranging from 0 to 20 mM and transfected with polyethyleneimine-plasmid DNA (PEI-pDNA) complexes. Cell viability and transfection efficiency were determined using MTS assays and flow cytometry, respectively. PEI-pDNA complex localization in BMSCs was assessed using fluorescence microscopy. To determine BMSC differentiation, messenger RNA (mRNA) for osteocalcin and CBFA1 was quantified using real time-polymerase chain reaction (PCR). Calcium deposition was qualitatively assessed after three and 14 days using Alizarin Red staining. RESULT: Our results indicate that Ca2+ levels between 8 and 12 mM positively impacted transfection of BMSCs with PEI-pDNA complexes in terms of cell viability and transfection efficiency. A Ca2+ concentration of 10 mM also increased the expression of an osteogenic gene, osteocalcin, when the cells were transfected with plasmid DNA encoding bone morphogenetic protein 2 (BMP-2). CONCLUSION: Ca2+ at a 10 mM concentration can significantly reduce toxicity and enhance transfection efficiency when combined with PEI-pDNA complexes, and this combination can be specifically applied to further enhance the differentiation of BMSCs by using the combination of polyethyleneimine-plasmid bone morphogenetic protein 2 (PEI-pBMP-2) and 10 mM Ca2+ as compared with PEI-pBMP-2 alone.
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Células de la Médula Ósea/metabolismo , Calcio/farmacología , Osteoblastos/metabolismo , Células Madre/metabolismo , Transfección , Células de la Médula Ósea/citología , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Células HEK293 , Humanos , Osteoblastos/citología , Osteocalcina/biosíntesis , Osteocalcina/genética , Células Madre/citología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD) and contributes to their high rate of cardiovascular mortality. Indoxyl sulfate (IS) is a representative protein-bound uremic toxin in CKD patients, which has been recognized as a major risk factor for VC. Recent studies have demonstrated that nuclear factor-kappa B (NK-κB) is highly activated in the chronic inflammation conditions of CKD patients and participated in the pathogenesis of VC. However, whether NK-κB is involved in the progression of IS-induced VC remains without elucidation. Here, we showed that NK-κB activity was increased in the IS-induced calcification of human aortic smooth muscle cells (HASMCs). Blocking the NK-κB with a selective inhibitor (Bay-11-7082) significantly relieved the osteogenic transdifferentiation of HASMCs, characterized by the downregulation of early osteogenic-specific marker, core-binding factor alpha subunit 1 (Cbfα1), and upregulation of smooth muscle α-actin (α-SMA), a specific vascular smooth muscle cell marker. Besides, IS stimulated the activation of PI3K/Akt signaling. Furthermore, LY294002, a specific inhibitor of PI3K/Akt pathway, attenuated the activation of NK-κB and osteogenic differentiation of HASMCs. Together, these results suggest that PI3K/Akt/NK-κB signaling plays an important role in the pathogenesis of osteogenic transdifferentiation induced by IS.
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Indicán/toxicidad , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Calcificación Vascular/patología , Actinas/biosíntesis , Línea Celular , Cromonas/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Regulación hacia Abajo/efectos de los fármacos , Humanos , Morfolinas/farmacología , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Insuficiencia Renal Crónica/patología , Transducción de Señal , Sulfonas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Estradiol (E2) serves an important role in the changes of postmenopausal bone turnover rate and the development of osteoporosis. The present study aimed to investigate the effects of E2 on high glucose (HG)induced osteoblast injury. Cell Counting Kit8 was used to determine cell viability. Reverse transcriptionquantitative PCR (RTqPCR) and western blotting was used to analyze the mRNA and protein expression levels of osteocalcin, Runtrelated transcription factor 2 (Runx2), nuclear factor E2related factor 2 (Nrf2) and heme oxygenase1 (HO1). Flow cytometry was performed to analyze apoptosis. The results revealed that cell viability was lower in cells treated with HG (100, 200 or 300 mg/dl) compared with the control group. Cell viability was decreased in cells treated with 200 mg/dl HG on days 3, 5 and 7. In addition, cell viability was increased by 0.1 µM E2. E2 with HG cotreatment increased cell viability, osteocalcin and Runx2 mRNA expression levels and nuclear Nrf2 and HO1 protein expression levels compared with the HGonly group. All these changes, with the exception of Runx2, were reversed by silencing Nrf2 expression using small interfering (si)RNA (siNrf2). Additionally, apoptosis was reduced by E2 in HGtreated cells, which was reversed by siNrf2 transfection. These results demonstrated that E2 may prevent HGinduced osteoblast injury by activating Nrf2/HO1 signaling pathways.
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Apoptosis/efectos de los fármacos , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Osteoblastos/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Hemo-Oxigenasa 1/biosíntesis , Proteínas de la Membrana/biosíntesis , Ratones , Factor 2 Relacionado con NF-E2/biosíntesis , Osteoblastos/patología , Osteoporosis/metabolismo , Osteoporosis/patologíaRESUMEN
Transcription factors bind to cell-specific cis-regulatory elements, such as enhancers and promoters, to initiate much of the gene expression program of different biological process. Odontoblast differentiation is a necessary step for tooth formation and is also governed by a complex gene regulatory network. Our previous in vitro experiments showed that Krüppel-like factor 4 (KLF4) can promote odontoblastic differentiation of both mouse dental papillary cells (mDPCs) and human dental pulp cells; however, its mechanism remains unclear. We first used Wnt1-Cre; KLF4fx/fx (Klf4 cKO) mice to examine the role of KLF4 during odontoblast differentiation in vivo and demonstrated significantly impaired dentin mineralization and enlarged pulp/root canals. Additionally, combinatory analysis using RNA-seq and ATAC-seq revealed genomewide direct regulatory targets of KLF4 in mouse odontoblasts. We found that KLF4 can directly activate the TGF-ß signaling pathway at the beginning of odontoblast differentiation with Runx2 as a cofactor. Furthermore, we found that KLF4 can directly upregulate the expression levels of Dmp1 and Sp7, which are markers of odontoblastic differentiation, through binding to their promoters. Interestingly, as a transcription factor, KLF4 can also recruit histone acetylase as a regulatory companion to the downstream target genes to positively or negatively regulate transcription. To further investigate other regulatory companions of KLF4, we chose histone acetylase HDAC3 and P300. Immunoprecipitation demonstrated that KLF4 interacted with P300 and HDAC3. Next, ChIP analysis detected P300 and HDAC3 enrichment on the promoter region of KLF4 target genes Dmp1 and Sp7. HDAC3 mainly interacted with KLF4 on day 0 of odontoblastic induction, whereas P300 interacted on day 7 of induction. These temporal-specific interactions regulated Dmp1 and Sp7 transcription, thus regulating dentinogenesis. Taken together, these results demonstrated that KLF4 regulates Dmp1 and Sp7 transcription via the modulation of histone acetylation and is vital to dentinogenesis. © 2019 American Society for Bone and Mineral Research.
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Diferenciación Celular , Pulpa Dental/metabolismo , Histonas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Odontoblastos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Acetilación , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Pulpa Dental/citología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Histona Desacetilasa 2/biosíntesis , Histona Desacetilasa 2/genética , Histonas/genética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Noqueados , Odontoblastos/citología , Factor de Transcripción Sp7/biosíntesis , Factor de Transcripción Sp7/genética , Transcripción Genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Chronic cadmium (Cd) toxicity is a significant health concern, and the mechanism of long-term low-dose Cd exposure on bone has not been fully elucidated till date. This study aimed to assess the association between rat mesenchymal stem cells (MSCs) and long-term Cd exposure through 38-week intake of CdCl2 at 1 and 2â¯mg/kg body weight (bw). Increased gene expression of receptor activator of NF-κB ligand (RANKL) and decreased gene expression of osteoprotegerin (OPG) were observed. Fold change of RANKL gene expression (fold changeâ¯=â¯1.97) and OPG gene expression (fold changeâ¯=â¯1.72) showed statistically significant differences at dose 2â¯mg/kg bw. Decreased expression of key genes was observed during the early osteogenic differentiation of MSCs. The gene expression of Osterix in 1â¯mg/kg bw group was decreased by 3.70-fold, and the gene expressions of Osterix, Osteopontin, collagen type I alpha 2 chain (COL1a2) and runt-related transcription factor 2 (RUNX2) in 2â¯mg/kg bw group were decreased by 1.79, 1.67, 1.45 and 1.35-folds, respectively. Exposure to CdCl2 induced an increase in the renal Cd load, but only an adaptive response was observed, including increased expression of autophagy-related proteins LC3B and Beclin-1, autophagy receptor p62, and heme oxygenase 1 (HO-1), which is an inducible isoform that releases in response to stress. There were no significant changes in the urinary low molecular weight proteins including N-acetyl-b-D-glucosaminidase (NAG), ß2-microglobulin and albumin (U-Alb). Urinary calcium (Ca) excretion showed no increase, and no obvious renal histological changes. Taken together, these results indicated that the chronic CdCl2 exposure directly act on MSCs through RANKL/OPG pathway and downregulate the key genes involved in osteogenic differentiation of MSCs. The toxic effect of Cd on bone may occur in parallel to nephrotoxicity.