RESUMO
Adipose-derived stem cells (ASCs) from diabetic osteoporosis (DOP) mice showed impaired osteogenic differentiation capacity. Recent studies have shown that in addition to antidiabetic drugs, sodium-glucose co-transporter inhibitor-2 (SGLT-2), empagliflozin, can play multipotent roles through various mechanisms of action. In this study, we aimed to investigate the effects and underlying mechanisms of empagliflozin on osteogenic differentiation of ASCs in DOP mice. Our results showed that osteogenic differentiation potential and autophagy activity weakened in DOP-ASCs when compared to controls. However, empagliflozin enhanced autophagy flux by promoting the formation of autophagosomes and acidification of autophagic lysosomes, resulting in an increase in LC3-II expression and a decrease in SQSTM1 expression. Furthermore, empagliflozin contributed to the reversal of osteogenesis inhibition in DOP-ASCs induced by a diabetic microenvironment. When 3-methyladenine was used to block autophagy activity, empagliflozin could not exert its protective effect on DOP-ASCs. Nonetheless, this study demonstrated that the advent of cellular autophagy attributed to the administration of empagliflozin could ameliorate the impaired osteogenic differentiation potential of ASCs in DOP mice. This finding might be conducive to the application of ASCs transplantation for promoting bone fracture healing and bone regeneration in patients with DOP.
Assuntos
Autofagia , Compostos Benzidrílicos , Diferenciação Celular , Glucosídeos , Osteogênese , Osteoporose , Animais , Glucosídeos/farmacologia , Autofagia/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Compostos Benzidrílicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Camundongos , Osteoporose/patologia , Osteoporose/tratamento farmacológico , Células-Tronco/metabolismo , Células-Tronco/efeitos dos fármacos , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/citologia , Camundongos Endogâmicos C57BL , MasculinoRESUMO
Posttranslational modifications (PTMs) are crucial regulatory mechanisms for cellular differentiation and organismal development. Acylation modification is one of the main PTMs that plays a pivotal role in regulating the osteogenic differentiation of mesenchymal stem cells and is a focal point of research in bone tissue regeneration. However, its mechanism remains incompletely understood. This article aims to investigate the impact of protein crotonylation on osteogenic differentiation in periodontal ligament stem cells (PDLSCs) and elucidate its underlying mechanisms. Western blot analysis identified that the modification level of acetylation, crotonylation, and succinylation were significantly upregulated after osteogenic induction of PDLSCs. Subsequently, sodium crotonate (NaCr) was added to the medium and acyl-CoA synthetase short-chain family member 2 (ACSS2) was knocked down by short hairpin RNA plasmids to regulate the total level of protein crotonylation. The results indicated that treatment with NaCr promoted the expression of osteogenic differentiation-related factors in PDLSCs, whereas silencing ACSS2 had the opposite effect. In addition, mass spectrometry analysis was used to investigate the comprehensive analysis of proteome-wide crotonylation in PDLSCs under osteogenic differentiation. The analysis revealed that the level of protein crotonylation related to the PI3K-AKT signaling pathway was significantly upregulated in PDLSCs after osteogenic induction. Treatment with NaCr and silencing ACSS2 affected the activation of the PI3K-AKT signaling pathway. Collectively, our study demonstrates that protein crotonylation promotes osteogenic differentiation of PDLSCs via the PI3K-AKT pathway, providing a novel targeting therapeutic approach for bone tissue regeneration.
Assuntos
Diferenciação Celular , Osteogênese , Ligamento Periodontal , Transdução de Sinais , Células-Tronco , Humanos , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologiaRESUMO
This study aimed to explore how mechanical stress affects osteogenic differentiation via the miR-187-3p/CNR2 pathway. To conduct this study, 24 female C57BL/6 mice, aged 8 weeks, were used and divided into four groups. The Sham and OVX groups did not undergo treadmill exercise, while the Sham + EX and OVX + EX groups received a 8-week treadmill exercise. Post-training, bone marrow and fresh femur samples were collected for further analysis. Molecular biology analysis, histomorphology analysis, and micro-CT analysis were conducted on these samples. Moreover, primary osteoblasts were cultured under osteogenic conditions and divided into GM group and CTS group. The cells in the CTS group underwent a sinusoidal stretching regimen for either 3 or 7 days. The expression of early osteoblast markers (Runx2, OPN, and ALP) was measured to assess differentiation. The study findings revealed that mechanical stress has a regulatory impact on osteoblast differentiation. The expression of miR-187-3p was observed to decrease, facilitating osteogenic differentiation, while the expression of CNR2 increased significantly. These observations suggest that mechanical stress, miR-187-3p, and CNR2 play crucial roles in regulating osteogenic differentiation. Both in vivo and in vitro experiments have confirmed that mechanical stress downregulates miR-187-3p and upregulates CNR2, which leads to the restoration of distal femoral bone mass and enhancement of osteoblast differentiation. Therefore, mechanical stress promotes osteoblasts, resulting in improved osteoporosis through the miR-187-3p/CNR2 signaling pathway. These findings have broad prospect and provide molecular biology guidance for the basic research and clinical application of exercise in the prevention and treatment of PMOP.
Assuntos
Diferenciação Celular , MicroRNAs , Osteoblastos , Osteogênese , Osteoporose Pós-Menopausa , Estresse Mecânico , Animais , Feminino , Humanos , Camundongos , Células Cultivadas , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/terapia , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/patologia , Transdução de SinaisRESUMO
Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.
Assuntos
Diferenciação Celular , Flavonoides , Metiltransferases , Osteogênese , Ratos Sprague-Dawley , Animais , Osteogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ratos , Feminino , Flavonoides/farmacologia , Metiltransferases/metabolismo , Metiltransferases/genética , Humanos , Osteoporose/patologia , Osteoporose/metabolismo , Osteoporose/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Ovariectomia , Regulação para Cima/efeitos dos fármacos , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Adenosina/análogos & derivados , Adenosina/metabolismoRESUMO
Alveolar bone loss is a main manifestation of periodontitis. Human periodontal ligament stem cells (PDLSCs) are considered as optimal seed cells for alveolar bone regeneration due to its mesenchymal stem cell like properties. Osteogenic potential is the premise for PDLSCs to repair alveolar bone loss. However, the mechanism regulating osteogenic differentiation of PDLSCs remain elusive. In this study, we identified Neuron-derived orphan receptor 1 (NOR1), was particularly expressed in PDL tissue in vivo and gradually increased during osteogenic differentiation of PDLSCs in vitro. Knockdown of NOR1 in hPDLSCs inhibited their osteogenic potential while NOR1 overexpression reversed this effect. In order to elucidate the downstream regulatory network of NOR1, RNA-sequencing was used. We found that downregulated genes were mainly enriched in TGF-ß, Hippo, Wnt signaling pathway. Further, by western blot analysis, we verified that the expression level of phosphorylated-SMAD2/3 and phosphorylated-SMAD4 were all decreased after NOR1 knockdown. Additionally, ChIP-qPCR and dual luciferase reporter assay indicated that NOR1 could bind to the promoter of TGFBR1 and regulate its activity. Moreover, overexpression of TGFBR1 in PDLSCs could rescue the damaged osteogenic potential after NOR1 knockdown. Taken together, our results demonstrated that NOR1 could activate TGF-ß/SMAD signaling pathway and positively regulates the commitment of osteoblast lineages of PDLSCs by targeting TGFBR1 directly.
Assuntos
Diferenciação Celular , Osteoblastos , Osteogênese , Ligamento Periodontal , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Diferenciação Celular/genética , Células Cultivadas , Osteoblastos/metabolismo , Osteoblastos/citologia , Osteogênese/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
After epiphyseal fracture, the epiphyseal plate is prone to ischemia and hypoxia, leading to the formation of bone bridge and deformity. However, the exact mechanism controlling the bone bridge formation remains unclear. Notch/RBPJ signaling axis has been indicated to regulate angiogenesis and osteogenic differentiation. Our study aims to investigate the mechanism of bone bridge formation after epiphyseal plate injury, and to provide a theoretical basis for new therapeutic approaches to prevent the bone bridge formation. The expression of DLL4 and RBPJ was significantly up-regulated in HUVECs after ischemia and hypoxia treatment. Notch/RBPJ pathway positively regulated the osteogenic differentiation of BMSCs. HUVECs can induce osteogenic differentiation of BMSCs under ischemia and hypoxia. Notch/RBPJ pathway is involved in the regulation of the trans-epiphyseal bridge formation. Notch/RBPJ in HUVECs is associated with osteogenic differentiation of BMSCs and may participate in the regulation of the bone bridge formation across the epiphyseal plate.
Assuntos
Diferenciação Celular , Células Endoteliais da Veia Umbilical Humana , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Neovascularização Fisiológica , Osteogênese , Receptores Notch , Transdução de Sinais , Humanos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Receptores Notch/metabolismo , Receptores Notch/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Hipóxia Celular , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Células Cultivadas , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , AngiogêneseRESUMO
The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.
Assuntos
Diferenciação Celular , Colágeno , Matriz Extracelular , Osteoblastos , Osteogênese , Matriz Extracelular/metabolismo , Osteoblastos/metabolismo , Osteoblastos/citologia , Colágeno/metabolismo , Osteogênese/fisiologia , Animais , Técnicas de Cultura de Células em Três Dimensões/métodos , Camundongos , Osteoclastos/metabolismo , Osteoclastos/citologiaRESUMO
Single immobilization theory cannot fully account for the extensive bone loss observed after spinal cord injury (SCI). Bone marrow mesenchymal stem cells (BMSCs) are crucial in bone homeostasis because they possess self-renewal capabilities and various types of differentiation potential. This study aimed to explore the molecular mechanism of long non-coding RNA H19 in osteoporosis after SCI and provide new research directions for existing prevention strategies. We used small interfering RNA to knockdown H19 expression and regulated miR-29b-2p expression using miR-29b-3p mimetics and inhibitors. Western blotting, real-time fluorescence quantitative PCR, Alizarin red staining, alkaline phosphatase staining and double-luciferase reporter gene assays were used to assess gene expression, osteogenic ability and binding sites. lncRNA H19 was upregulated in BMSCs from the osteoporosis group, whereas miR-29b-3p was downregulated. We identified the binding sites between miR-29b-3p and lncRNAs H19 and DKK1. H19 knockdown promoted BMSCs' osteogenic differentiation, whereas miR-29b-3p inhibition attenuated this effect. We discovered potential binding sites for miR-29b-3p in lncRNAs H19 and DKK1. Our findings suggest that long non-coding RNA H19 mediates BMSCs' osteogenic differentiation in osteoporosis after SCI through the miR-29b-3p/DKK1 axis and by directly inhibiting the ß-catenin signalling pathway.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Células-Tronco Mesenquimais , Osteogênese , RNA Longo não Codificante , Animais , Humanos , Masculino , Ratos , Diferenciação Celular , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoporose/genética , Osteoporose/patologia , Osteoporose/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Sensory nerves play a crucial role in maintaining bone homeostasis by releasing Semaphorin 3A (Sema3A). However, the specific mechanism of Sema3A in regulation of bone marrow mesenchymal stem cells (BMMSCs) during bone remodelling remains unclear. The tibial denervation model was used and the denervated tibia exhibited significantly lower mass as compared to sham operated bones. In vitro, BMMSCs cocultured with dorsal root ganglion cells (DRGs) or stimulated by Sema3A could promote osteogenic differentiation through the Wnt/ß-catenin/Nrp1 positive feedback loop, and the enhancement of osteogenic activity could be inhibited by SM345431 (Sema3A-specific inhibitor). In addition, Sema3A-stimulated BMMSCs or intravenous injection of Sema3A could promote new bone formation in vivo. To sum up, the coregulation of bone remodelling is due to the ageing of BMMSCs and increased osteoclast activity. Furthermore, the sensory neurotransmitter Sema3A promotes osteogenic differentiation of BMMSCs via Wnt/ß-catenin/Nrp1 positive feedback loop, thus promoting osteogenesis in vivo and in vitro.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Osteogênese/genética , Semaforina-3A/genética , Retroalimentação , beta Catenina , Gânglios Espinais , Neuropilina-1/genéticaRESUMO
Osteogenic differentiation is important for fracture healing. Microfibrial-associated glycoprotein 2 (MAGP2) is found to function as a proangiogenic regulator in bone formation; however, its role in osteogenic differentiation during bone repair is not clear. Here, a mouse model of critical-sized femur fracture was constructed, and the adenovirus expressing MAGP2 was delivered into the fracture site. Mice with MAGP2 overexpression exhibited increased bone mineral density and bone volume fraction (BV/TV) at Day 14 postfracture. Within 7 days postfracture, overexpression of MAGP2 increased collagen I and II expression at the fracture callus, with increasing chondrogenesis. MAGP2 inhibited collagen II level but elevated collagen I by 14 days following fracture, accompanied by increased endochondral bone formation. In mouse osteoblast precursor MC3T3-E1 cells, MAGP2 treatment elevated the expression of osteoblastic factors (osterix, BGLAP and collagen I) and enhanced ALP activity and mineralization through activating ß-catenin signaling after osteogenic induction. Besides, MAGP2 could interact with lipoprotein receptor-related protein 5 (LRP5) and upregulated its expression. Promotion of osteogenic differentiation and ß-catenin activation mediated by MAGP2 was partially reversed by LRP5 knockdown. Interestingly, ß-catenin/transcription factor 4 (TCF4) increased MAGP2 expression probably by binding to MAGP2 promoter. These findings suggest that MAGP2 may interact with ß-catenin/TCF4 to enhance ß-catenin/TCF4's function and activate LRP5-activated ß-catenin signaling pathway, thus promoting osteogenic differentiation for fracture repair. mRNA sequencing identified the potential targets of MAGP2, providing novel insights into MAGP2 function and the directions for future research.
Assuntos
Fraturas Ósseas , Osteogênese , Animais , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Diferenciação Celular/genética , Colágeno/metabolismo , Consolidação da Fratura , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Osteoblastos/metabolismo , Via de Sinalização Wnt , Masculino , Camundongos Endogâmicos C57BL , Linhagem CelularRESUMO
Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.
Assuntos
Calcinose , Catarata , Subunidade alfa 1 de Fator de Ligação ao Core , Glucose , Hiperglicemia , Fator 1 Induzível por Hipóxia , Cristalino , Humanos , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/genética , Calcinose/etiologia , Calcinose/metabolismo , Calcinose/patologia , Catarata/etiologia , Catarata/metabolismo , Catarata/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Glucose/metabolismo , Hiperglicemia/complicações , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Cristalino/metabolismo , Cristalino/patologia , Osteocalcina/metabolismo , Osteocalcina/genética , Transdução de Sinais , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismoRESUMO
The long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) plays a crucial role in tumorigenesis and is frequently employed as a prognostic biomarker. However, its involvement in the osteogenic differentiation of oral stem cells, particularly human dental follicle stem cells (hDFSCs), remains unclear. Our investigation revealed that the absence of SNHG1 enhances the osteogenic differentiation of hDFSCs. Furthermore, the downregulation of SNHG1 induces autophagy in hDFSCs, leading to a reduction in intracellular oxidative stress levels. Notably, this effect is orchestrated through the epigenetic regulation of EZH2. Our study unveils a novel function of SNHG1 in governing the osteogenic differentiation of hDFSCs, offering fresh insights for an in-depth exploration of the molecular mechanisms underlying dental follicle development. These findings not only provide a foundation for advancing the understanding of SNHG1 but also present innovative perspectives for promoting the repair and regeneration of periodontal supporting tissue, ultimately contributing to the restoration of periodontal health and tooth function.
Assuntos
Autofagia , Diferenciação Celular , Saco Dentário , Proteína Potenciadora do Homólogo 2 de Zeste , Osteogênese , Estresse Oxidativo , RNA Longo não Codificante , Células-Tronco , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Autofagia/genética , Estresse Oxidativo/genética , Osteogênese/genética , Diferenciação Celular/genética , Células-Tronco/metabolismo , Saco Dentário/metabolismo , Saco Dentário/citologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética , Células Cultivadas , Técnicas de Silenciamento de GenesRESUMO
Vascular calcification is a pathological process commonly associated with atherosclerosis, chronic kidney disease, and diabetes. Paraspeckle protein NONO is a multifunctional RNA/DNA binding protein involved in many nuclear biological processes but its role in vascular calcification remains unclear. Here, we observed that NONO expression was decreased in calcified arteries of mice and patients with CKD. We generated smooth muscle-specific NONO-knockout mice and established three different mouse models of vascular calcification by means of 5/6 nephrectomy, adenine diet to induce chronic kidney failure, or vitamin D injection. The knockout mice were more susceptible to the development of vascular calcification relative to control mice, as verified by an increased calcification severity and calcium deposition. Likewise, aortic rings from knockout mice showed more significant vascular calcification than those from control mice ex vivo. In vitro, NONO deficiency aggravated high phosphate-induced vascular smooth muscle cell osteogenic differentiation and apoptosis, whereas NONO overexpression had a protective effect. Mechanistically, we demonstrated that the regulation of vascular calcification by NONO was mediated by bone morphogenetic protein 2 (BMP2). NONO directly bound to the BMP2 promoter using its C-terminal region, exerting an inhibitory effect on the transcription of BMP2. Thus, our study reveals that NONO is a novel negative regulator of vascular calcification, which inhibits osteogenic differentiation of vascular smooth muscle cell and vascular calcification via negatively regulating BMP2 transcription. Hence, NONO may provide a promising target for the prevention and treatment of vascular calcification.
Assuntos
Proteína Morfogenética Óssea 2 , Modelos Animais de Doenças , Camundongos Knockout , Músculo Liso Vascular , Miócitos de Músculo Liso , Osteogênese , Insuficiência Renal Crônica , Transcrição Gênica , Calcificação Vascular , Animais , Humanos , Masculino , Camundongos , Doenças da Aorta/genética , Doenças da Aorta/prevenção & controle , Doenças da Aorta/patologia , Doenças da Aorta/metabolismo , Apoptose/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Regiões Promotoras Genéticas , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/prevenção & controle , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Calcificação Vascular/patologia , Calcificação Vascular/prevenção & controle , Calcificação Vascular/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/etiologiaRESUMO
With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1's modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss.
Assuntos
Diferenciação Celular , Proteínas com Domínio LIM , Células-Tronco Mesenquimais , Osteoblastos , Osteogênese , Osteoporose , Via de Sinalização Wnt , Osteogênese/genética , Animais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/genética , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/genética , Humanos , Osteoblastos/metabolismo , Osteoblastos/citologia , Feminino , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células Cultivadas , Pessoa de Meia-Idade , Idoso , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de TransporteRESUMO
In view of the increased levels of reactive oxygen species (ROS) that disturb the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), the repair of diabetic bone defects remains a great challenge. Herein, a factor-free hydrogel is reported with ROS scavenging and responsive degradation properties for enhanced diabetic bone healing. These hydrogels contain ROS-cleavable thioketal (TK) linkers and ultraviolet (UV)-responsive norbornene (NB) groups conjugated with 8-arm PEG macromers, which are formed via UV crosslinking-mediated gelation. Upon reacting with high levels of ROS in the bone defect microenvironment, ROS-cleavable TK linkers are destroyed, allowing the responsive degradation of hydrogels, which promotes the migration of BMSCs. Moreover, ROS levels are reduced through hydrogel-mediated ROS scavenging to reverse BMSC differentiation from adipogenic to osteogenic phenotype. As such, a favorable microenvironment is created after simultaneous ROS scavenging and hydrogel degradation, leading to the effective repair of bone defects in diabetic mouse models, even without the addition of growth factors. Thus, this study presents a responsive hydrogel platform that regulates ROS scavenging and stromal degradation in bone engineering.
Assuntos
Diferenciação Celular , Hidrogéis , Células-Tronco Mesenquimais , Osteogênese , Espécies Reativas de Oxigênio , Animais , Espécies Reativas de Oxigênio/metabolismo , Hidrogéis/química , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Camundongos , Diferenciação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental , Sequestradores de Radicais Livres/farmacologia , Sequestradores de Radicais Livres/química , Cicatrização/efeitos dos fármacos , Osso e Ossos , MasculinoRESUMO
Biomechanical signals in the extracellular niche are considered promising for programming the lineage specification of stem cells. Recent studies have reported that biomechanics, such as the microstructure of nanomaterials, can induce adipose-derived stem cells (ASCs) to differentiate into osteoblasts, mediating gene regulation at the epigenetic level. Therefore, in this study, transcriptome expression levels of histone demethylases in ASCs were screened after treatment with different matrix stiffnesses, and histone lysine demethylase 3B (KDM3B) was found to promote osteogenic differentiation of ASCs in response to matrix stiffness, indicating a positive modulatory effect on this biological process. ASCs exhibited widespread and polygonal shapes with a distinct bundle-like expression of vinculin parallel to the axial cytoskeleton along the cell margins on the stiff matrix rather than round shapes with a smeared and shorter expression on the soft matrix. Comparatively rigid polydimethylsiloxane material directed ASCs into an osteogenic phenotype in inductive culture media via the upregulation of osteocalcin, alkaline phosphatase, and runt-related transcription factor 2. Treatment with KDM3B-siRNA decreased the expression of osteogenic differentiation markers and impaired mitochondrial dynamics and mitochondrial membrane potential. These results illustrate the critical role of KDM3B in the biomechanics-induced osteogenic commitment of ASCs and provide new avenues for the further application of stem cells as potential therapeutics for bone regeneration.
Assuntos
Tecido Adiposo , Diferenciação Celular , Histona Desmetilases com o Domínio Jumonji , Osteogênese , Células-Tronco , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Dimetilpolisiloxanos/químicaRESUMO
Exogenous polyamines, including putrescine (PUT), spermidine (SPD), and spermine (SPM), and the irreversible inhibitor of the rate-limiting enzyme ornithine decarboxylase (ODC) of polyamine biosynthesis, α-difluoromethylornithine (DFMO), are implicated as stimulants for bone formation. We demonstrate in this study the osteogenic potential of exogenous polyamines and DFMO in human osteoblasts (hOBs), murine monocyte cell line RAW 264.7, and an ovariectomized rat model. The effect of polyamines and DFMO on hOBs and RAW 264.7 cells was studied by analyzing gene expression, alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) activity, and matrix mineralization. Ovariectomized rats were treated with polyamines and DFMO and analyzed by micro computed tomography (micro CT). The mRNA level of the early onset genes of osteogenic differentiation, Runt-related transcription factor 2 (Runx2) and ALP, was significantly elevated in hOBs under osteogenic conditions, while both ALP activity and matrix mineralization were enhanced by exogenous polyamines and DFMO. Under osteoclastogenic conditions, the gene expression of both receptor activator of nuclear factor-κB (RANK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) was reduced, and TRAP activity was suppressed by exogenous polyamines and DFMO in RAW 264.7 cells. In an osteoporotic animal model of ovariectomized rats, SPM and DFMO were found to improve bone volume in rat femurs, while trabecular thickness was increased in all treatment groups. Results from this study provide in vitro and in vivo evidence indicating that polyamines and DFMO act as stimulants for bone formation, and their osteogenic effect may be associated with the suppression of osteoclastogenesis.
Assuntos
Diferenciação Celular , Eflornitina , Osteoblastos , Osteoclastos , Osteogênese , Poliaminas , Animais , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Ratos , Humanos , Diferenciação Celular/efeitos dos fármacos , Eflornitina/farmacologia , Feminino , Poliaminas/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Células RAW 264.7 , Ovariectomia , Ratos Sprague-Dawley , Espermidina/farmacologiaRESUMO
Electrical stimulation (ES) plays an important role in regulating cell osteoblast differentiation. As a noninvasive rehabilitation therapy method, Es has a unique role in postoperative recovery. Bone morphogenetic protein-2 (BMP-2) is the most commonly used bioactive molecule in in situ tissue engineering scaffolds, and it plays an important regulatory role in the whole process of bone injury repair. In this study, the osteogenic regulation of MC-3T3-E1 cells was studied by combining pulsed electrical stimulation (PES) and different concentrations of BMP-2. The results showed that PES and BMP-2 could synergically promote the proliferation of MC-3T3-E1 cells. The qPCR results of osteoblast-related genes showed that PES was synergistic with BMP-2 to promote osteoblast differentiation mainly through the regulation of the Smad/BMP and insulin like growth factor 1 (IGF1) signaling pathways. The expression level of alkaline phosphatase (ALP) and alizarin red staining further demonstrated the synergistic effect of PES and BMP-2 on promoting osteogenic differentiation and mineralization of cells. PES and BMP-2 could also synergically promote cell proliferation, expression of collagen I (COL-I) and ALP, and cell mineralization on the 3D-printed polylactic acid scaffold. These results suggest that the use of PES can enhance the osteogenic effect of in situ bone repair scaffolds containing BMP-2, reduce the dose of BMP-2 alone, and reduce the possible side effects of high-dose BMP-2 in vivo.
RESUMO
BACKGROUND: Periodontitis is a chronic destructive inflammatory disease exacerbated by osteoblast dysfunction. Ferroptosis has emerged as a significant factor that could contribute to the pathological changes observed in periodontitis. However, the impact of ferroptosis on osteogenic differentiation and gene expression patterns of primary osteoblasts remain elusive. METHODS: In this study, osteoblasts were osteogenically induced for specific durations with and without the ferroptosis inducer erastin. Subsequently, cell proliferation, ferroptosis-related molecules, and osteogenic differentiation capacity were assessed. Furthermore, the differences in transcriptome expression following erastin treatment were analyzed by RNA sequencing. RESULTS: The results demonstrated that erastin treatment induced ferroptosis, resulting in suppressed cell proliferation and impaired osteogenic differentiation. Transcriptomic analysis revealed significant alterations in processes such as hydrogen peroxide catabolism, response to lipid peroxidation, and metal iron binding, as well as BMP receptor activity and collagen type XI trimer. CONCLUSION: The ferroptosis inducer erastin inhibited osteoblast proliferation and differentiation. Our study provides novel insights into the effect of ferroptosis on osteogenesis, suggesting that targeting ferroptosis may present a promising approach in the treatment of periodontitis.
Assuntos
Diferenciação Celular , Proliferação de Células , Ferroptose , Osteoblastos , Osteogênese , Piperazinas , Ferroptose/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Animais , Piperazinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Camundongos , Células CultivadasRESUMO
BACKGROUND: Previous research has identified a significant role of Thioredoxin-interacting protein (TXNIP) in bone loss. The purpose of this investigation was to assess the role and the underlying molecular mechanisms of TXNIP in the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) and pre-osteoblast MC3T3-E1 cells. METHODS: Human bone marrow stem cells (hBMSCs) and MC3T3-E1 cells were used to induce osteogenic differentiation. The expression of genes and proteins was assessed using RT-qPCR and western blot, respectively. ChIP assay was used to validate the interaction between genes. The osteogenic differentiation ability of cells was reflected using ALP staining and detection of ALP activity. The mineralization ability of cells was assessed using ARS staining. DCFCA staining was employed to evaluate the intracellular ROS level. RESULTS: Initially, downregulation of TXNIP and upregulation of EZH2 were observed during osteogenesis in hBMSCs and MC3T3-E1 cells. Additionally, it was discovered that EZH2 negatively regulates TXNIP expression in these cells. Furthermore, experiments indicated that the knockdown of TXNIP stimulated the activation of the PI3K/AKT/Nrf2 signaling pathway in hBMSCs and MC3T3- E1 cells, thus inhibiting the production of reactive oxygen species (ROS). Further functional experiments revealed that overexpression of TXNIP inhibited the osteogenic differentiation in hBMSCs and MC3T3-E1 cells by enhancing ROS produc-tion. On the other hand, knockdown of TXNIP promoted the osteogenic differentiation capacity of hBMSCs and MC3T3-E1 cells through the activation of the PI3K/AKT/Nrf2 pathway. CONCLUSION: In conclusion, this study demonstrated that TXNIP expression, under the regulation of EZH2, plays a crucial role in the osteogenic differentiation of hBMSCs and MC3T3-E1 cells by regulating ROS production and the PI3K/AKT/Nrf2 pathway.