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1.
Methods Mol Biol ; 2582: 255-268, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370355

RESUMO

Although two-dimensional (2D) cultures from bone lineage cells are often used, it is well-known that this culture system is completely different from the in vivo bone matrix environment. In this paper, we describe a 3D culture method using both the mouse osteocytic cell line, MLO-Y4, and an osteocyte-enriched population of the cells isolated from mice. These cells are embedded in collagen gel with recombinant cellular communication network (CCN) factor proteins; then, osteoblasts or osteoclasts are inoculated and cultured on the collagen gel. Because this method mimics the in vitro bone matrix environment, it is useful for understanding the detailed mechanism of actions of CCN proteins in the bone matrix.


Assuntos
Osteoblastos , Osteócitos , Camundongos , Animais , Diferenciação Celular , Osteoblastos/metabolismo , Remodelação Óssea , Colágeno/metabolismo , Bioensaio
2.
Dis Markers ; 2022: 9872243, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36340581

RESUMO

Purpose: Osteoporosis is a complication of type 2 diabetes, and it is characterized by reduced bone mass, augmented bone fragility, and increased risk of fracture, thus reducing patient quality of life, especially in the elderly. Ferroptosis has been implicated in the pathological process of type 2 diabetic osteoporosis (T2DOP), but the specific underlying mechanisms remain largely unknown. This study clarified the role of activating transcription factor 3 (ATF3) in T2DOP and explored its specific regulatory mechanism, providing a new treatment target for T2DOP. Methods: We cultured hFob1.19 cells in high glucose (HG, 35 mM) and knocked down ATF3 using short hairpin RNA (shRNA). We then measured cell viability, assessed morphology, quantified the expression of ATF3 and glutathione peroxidase 4 (GPX4), detected the levels of reactive oxygen species (ROS) and lipid peroxides, and determined the osteogenic function of osteoblasts. Cystine/glutamate antiporter (system Xc-) activity was evaluated by determining the expression of SLC7A11 and the levels of glutathione (GSH) and extracellular glutamate. We constructed a T2DOP rat model and observed the effect of ATF3 on ferroptosis and T2DOP by knocking down ATF3 using small interfering RNA (siRNA). Then, we evaluated the levels of iron metabolism, lipid peroxidation, and bone turnover in serum, detected the expression of ATF3, SLC7A11, and GPX4 in bone tissues, and assessed bone microstructure using microcomputed tomography. Results: ATF3 expression was increased in osteoblasts under HG condition and in T2DOP rats. Inhibiting the function of ATF3 increased GPX4 levels and reduced the accumulation of ROS and lipid peroxides. These changes inhibited the ferroptosis of osteoblasts and improved osteogenic function. In addition, HG induced ATF3 upregulation, resulting in decreased SLC7A11 expression and lower levels of intracellular GSH and extracellular glutamate. Conclusion: Osteoblast ferroptosis under HG conditions is induced by ATF3-mediated inhibition of system Xc- activity, and these events contribute to T2DOP pathogenesis.


Assuntos
Diabetes Mellitus Tipo 2 , Ferroptose , Osteoporose , Ratos , Animais , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peróxidos Lipídicos , Diabetes Mellitus Tipo 2/complicações , Microtomografia por Raio-X , Qualidade de Vida , Osteoblastos/metabolismo , Osteoporose/genética , Glutamatos
3.
Acta Chir Orthop Traumatol Cech ; 89(5): 370-375, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36322038

RESUMO

PURPOSE OF THE STUDY Nitinol (NiTi) is a biomaterial widely used in medicine based on super-elastic and shape memory properties. miR-124 has a key role in inflammatory process, osteoblasts differentiation, and mineralization. The aim of study was evaluating the differences in gene expression of miR-124 of human physiological osteoblasts (HOB) and human osteoarthritic osteoblasts (OSBA) as a response to NiTi alloy in different heat treatments. MATERIAL AND METHODS The cells were cultivated with NiTi discs with/without addition of bacterial lipopolysaccharide (LPS) for 72 hours. MicroRNAs were isolated, underwent reverse transcription and were analyzed by RT-PCR. RESULTS As a response to LPS, HOB overexpressed miR-124, while in OSBA expression change did not occur. Overexpression was also observed in both cell lines as a response to hydrogen and helium treated NiTi discs. HOB expressed significantly higher amount of miR-124 than OSBA as a response to hydrogen treatment of NiTi discs. In addition, hydrogen treatment caused significantly higher expression in HOB than LPS. The combination of NiTi disc and LPS treatment in HOB didn't cause any expression changes. Comparing to LPS-only treatment, the expression in HOB with combination of LPS and alloy was significantly lower. In OSBA, the expression was increased by the combination of LPS and hydrogen disc, in case of helium disc, the expression was decreased. CONCLUSIONS In conclusion, human physiological and osteoarthritic osteoblasts respond to NiTi alloy with both surface (hydrogen and helium atmosphere) treatment by overexpression of miR-124. The effect of LPS as inflammatory modulator suggests the presence of an "anti-inflammatory preconditioning" in osteoarthritic osteoblasts, as physiological osteoblasts overexpression was significantly higher. Key words: nitinol, osteoblast, miR-124, lipopolysaccharide.


Assuntos
Lipopolissacarídeos , MicroRNAs , Humanos , Ligas/metabolismo , Ligas/farmacologia , Hélio/metabolismo , Hélio/farmacologia , Hidrogênio/metabolismo , Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Osteoblastos/metabolismo , Titânio , Osteoartrite/genética
4.
Front Biosci (Landmark Ed) ; 27(10): 295, 2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36336853

RESUMO

BACKGROUND: Recently, single-cell RNA sequencing (scRNA-seq) technology was increasingly used to study transcriptomics at a single-cell resolution, scRNA-seq analysis was complicated by the "dropout", where the data only captures a small fraction of the transcriptome. This phenomenon can lead to the fact that the actual expressed transcript may not be detected. We previously performed osteoblast subtypes classification and dissection on freshly isolated human osteoblasts. MATERIALS AND METHODS: Here, we used the scImpute method to impute the missing values of dropout genes from a scRNA-seq dataset generated on freshly isolated human osteoblasts. RESULTS: Based on the imputed gene expression patterns, we discovered three new osteoblast subtypes. Specifically, these newfound osteoblast subtypes are osteoblast progenitors, and two undetermined osteoblasts. Osteoblast progenitors showed significantly high expression of proliferation related genes (FOS, JUN, JUNB and JUND). Analysis of each subtype showed that in addition to bone formation, these undetermined osteoblasts may involve osteoclast and adipocyte differentiation and have the potential function of regulate immune activation. CONCLUSIONS: Our findings provided a new perspective for studying the osteoblast heterogeneity and potential biological functions of these freshly isolated human osteoblasts at the single-cell level, which provides further insight into osteoblasts subtypes under various (pathological) physiological conditions.


Assuntos
Osteoblastos , Transcriptoma , Humanos , RNA-Seq , Osteoblastos/metabolismo , Diferenciação Celular/genética , Osteogênese/genética , Perfilação da Expressão Gênica
5.
Cells ; 11(21)2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36359752

RESUMO

The culture of osteoblasts (OB) of human origin is a useful experimental model in studying bone biology, osteogenic differentiation, functions of bone proteins, oncological processes in bone tissue, testing drugs against bone desires, and many other fields. The purpose of the present study is to share a workflow that has established the conditions to efficiently isolate and grow OB cells obtained from surgically removed bones from human donors. The protocol described here also shows how to determine cell phenotype. Here we provide characteristics of cells isolated by this protocol that might help researchers to decide if such OB are suitable for the purposes of their study. Osteoblasts isolated from collagenase-treated explants of adult bones are able to proliferate and keep their phenotype in culture. OB cells have high synthetic properties. They express osteomarkers, such as RUNX2, osteocalcin, BMP2, and osteopontin both in control conditions and in an osteogenic medium that could be estimated by qPCR and immunocytochemical staining and by Western blotting. Induction of osteogenic differentiation does not dramatically influence the synthetic properties of OB cells, while the cells gain the ability to extracellular mineralization only in an osteogenic medium.


Assuntos
Osteoblastos , Osteogênese , Humanos , Osteogênese/genética , Osteoblastos/metabolismo , Diferenciação Celular , Osteocalcina/metabolismo , Osso e Ossos/metabolismo
6.
Elife ; 112022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36342465

RESUMO

Pathological obesity and its complications are associated with an increased propensity for bone fractures. Humans with certain genetic polymorphisms at the kinase suppressor of ras2 (KSR2) locus develop severe early-onset obesity and type 2 diabetes. Both conditions are phenocopied in mice with Ksr2 deleted, but whether this affects bone health remains unknown. Here we studied the bones of global Ksr2 null mice and found that Ksr2 negatively regulates femoral, but not vertebral, bone mass in two genetic backgrounds, while the paralogous gene, Ksr1, was dispensable for bone homeostasis. Mechanistically, KSR2 regulates bone formation by influencing adipocyte differentiation at the expense of osteoblasts in the bone marrow. Compared with Ksr2's known role as a regulator of feeding by its function in the hypothalamus, pair-feeding and osteoblast-specific conditional deletion of Ksr2 reveals that Ksr2 can regulate bone formation autonomously. Despite the gains in appendicular bone mass observed in the absence of Ksr2, bone strength, as well as fracture healing response, remains compromised in these mice. This study highlights the interrelationship between adiposity and bone health and provides mechanistic insights into how Ksr2, an adiposity and diabetic gene, regulates bone metabolism.


Our bones are living tissues which constantly reshape and renew themselves. This ability relies on stem cells present in the marrow cavity, which can mature into the various types of cells needed to produce new bone material, marrow fat, or other components. Obesity and associated conditions such as type 2 diabetes are often linked to harmful changes in the skeleton. In particular, these metabolic conditions are associated with weight-bearing bones becoming more prone to facture and healing poorly. Mice genetically modified to model obesity and diabetes could help researchers to study exactly how these conditions ­ and the genetic changes that underlie them ­ impact bone health. Gomez et al. aimed to address this question by focusing on KSR2, a gene involved in energy consumption and feeding behavior. Children who carry certain KSR2 mutations are prone to obesity and type 2 diabetes; mice lacking the gene also develop these conditions due to uncontrolled eating. Closely examining mutant mice in which Ksr2 had been deactivated in every cell revealed that the weight-bearing bones of these animals were also more likely to break, and the fractures then healed more slowly. This was the case even though these bones had higher mass and less marrow fat compared to healthy mice. Non-weight bearing bones (such as the spine) did not exhibit these changes. Further experiments revealed that, when expressed normally in the skeleton, Ksr2 skews the stem cell maturation process towards marrow fat cells instead of bone-creating cells. This suggests a new role for Ksr2, which therefore seems to independently regulate both feeding behavior and bone health. In addition, the work by Gomez et al. demonstrate that Ksr2 mutant mice could be a useful model to better understand how obesity and diabetes affect human bones, and to potentially develop new therapies.


Assuntos
Adiposidade , Medula Óssea , Osso Esponjoso , Animais , Humanos , Camundongos , Adiposidade/genética , Medula Óssea/metabolismo , Osso Esponjoso/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Camundongos Knockout , Obesidade/metabolismo , Osteoblastos/metabolismo , Proteínas Serina-Treonina Quinases
7.
Int J Mol Sci ; 23(21)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36362346

RESUMO

Suffruticosol B (Suf-B) is a stilbene found in Paeonia suffruticosa ANDR., which has been traditionally used in medicine. Stilbenes and their derivatives possess various pharmacological effects, such as anticancer, anti-inflammatory, and anti-osteoporotic activities. This study aimed to explore the bone-forming activities and mechanisms of Suf-B in pre-osteoblasts. Herein, >99.9% pure Suf-B was isolated from P. suffruticosa methanolic extracts. High concentrations of Suf-B were cytotoxic, whereas low concentrations did not affect cytotoxicity in pre-osteoblasts. Under zero levels of cytotoxicity, Suf-B exhibited bone-forming abilities by enhancing alkaline phosphatase enzyme activities, bone matrix calcification, and expression levels with non-collagenous proteins. Suf-B induces intracellular signal transduction, leading to nuclear RUNX2 expression. Suf-B-stimulated differentiation showed increases in autophagy proteins and autophagosomes, as well as enhancement of osteoblast adhesion and transmigration on the ECM. These results indicate that Suf-B has osteogenic qualities related to differentiation, autophagy, adhesion, and migration. This also suggests that Suf-B could have a therapeutic effect as a phytomedicine in skeletal disorders.


Assuntos
Paeonia , Estilbenos , Osteogênese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Autofagia , Paeonia/metabolismo , Estilbenos/farmacologia , Diferenciação Celular
8.
Int J Mol Sci ; 23(21)2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36362086

RESUMO

RUNX proteins, such as RUNX2, regulate the proliferation and differentiation of chondrocytes and osteoblasts. Haploinsufficiency of RUNX2 causes cleidocranial dysplasia, but a detailed analysis of Runx2+/- mice has not been reported. Furthermore, CBFB is required for the stability and DNA binding of RUNX family proteins. CBFB has two isoforms, and CBFB2 plays a major role in skeletal development. The calvaria, femurs, vertebrae and ribs in Cbfb2-/- mice were analyzed after birth, and compared with those in Runx2+/- mice. Calvarial development was impaired in Runx2+/- mice but mildly delayed in Cbfb2-/- mice. In femurs, the cortical bone but not trabecular bone was reduced in Cbfb2-/- mice, whereas both the trabecular and cortical bone were reduced in Runx2+/- mice. The trabecular bone in vertebrae increased in Cbfb2-/- mice but not in Runx2+/- mice. Rib development was impaired in Cbfb2-/- mice but not in Runx2+/- mice. These differences were likely caused by differences in the indispensability of CBFB and RUNX2, the balance of bone formation and resorption, or the number and maturation stage of osteoblasts. Thus, different amounts of CBFB and RUNX2 were required among the bone tissues for proper bone development and maintenance.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Osteoblastos , Animais , Camundongos , Diferenciação Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Costelas/metabolismo , Crânio/metabolismo , Coluna Vertebral/metabolismo
9.
Int J Oral Sci ; 14(1): 53, 2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376275

RESUMO

Bone regeneration remains a great clinical challenge. Low intensity near-infrared (NIR) light showed strong potential to promote tissue regeneration, offering a promising strategy for bone defect regeneration. However, the effect and underlying mechanism of NIR on bone regeneration remain unclear. We demonstrated that bone regeneration in the rat skull defect model was significantly accelerated with low-intensity NIR stimulation. In vitro studies showed that NIR stimulation could promote the osteoblast differentiation in bone mesenchymal stem cells (BMSCs) and MC3T3-E1 cells, which was associated with increased ubiquitination of the core circadian clock protein Cryptochrome 1 (CRY1) in the nucleus. We found that the reduction of CRY1 induced by NIR light activated the bone morphogenetic protein (BMP) signaling pathways, promoting SMAD1/5/9 phosphorylation and increasing the expression levels of Runx2 and Osterix. NIR light treatment may act through sodium voltage-gated channel Scn4a, which may be a potential responder of NIR light to accelerate bone regeneration. Together, these findings suggest that low-intensity NIR light may promote in situ bone regeneration in a CRY1-dependent manner, providing a novel, efficient and non-invasive strategy to promote bone regeneration for clinical bone defects.


Assuntos
Regeneração Óssea , Relógios Circadianos , Criptocromos , Animais , Ratos , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Criptocromos/metabolismo , Osteoblastos/metabolismo , Osteogênese , Fatores de Transcrição/metabolismo
10.
Proc Natl Acad Sci U S A ; 119(45): e2212178119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322718

RESUMO

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.


Assuntos
Ácido Cítrico , Simportadores , Animais , Camundongos , Ácido Cítrico/metabolismo , Simportadores/metabolismo , Durapatita/metabolismo , Citratos , Ciclo do Ácido Cítrico , Osteoblastos/metabolismo , Mamíferos/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo
11.
Elife ; 112022 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-36326085

RESUMO

The spatiotemporal blood vessel formation and specification at the osteogenic and angiogenic interface of murine cranial bone defect repair were examined utilizing a high-resolution multiphoton-based imaging platform in conjunction with advanced optical techniques that allow interrogation of the oxygen microenvironment and cellular energy metabolism in living animals. Our study demonstrates the dynamic changes of vessel types, that is, arterial, venous, and capillary vessel networks at the superior and dura periosteum of cranial bone defect, suggesting a differential coupling of the vessel type with osteoblast expansion and bone tissue deposition/remodeling during repair. Employing transgenic reporter mouse models that label distinct types of vessels at the site of repair, we further show that oxygen distributions in capillary vessels at the healing site are heterogeneous as well as time- and location-dependent. The endothelial cells coupling to osteoblasts prefer glycolysis and are less sensitive to microenvironmental oxygen changes than osteoblasts. In comparison, osteoblasts utilize relatively more OxPhos and potentially consume more oxygen at the site of repair. Taken together, our study highlights the dynamics and functional significance of blood vessel types at the site of defect repair, opening up opportunities for further delineating the oxygen and metabolic microenvironment at the interface of bone tissue regeneration.


Assuntos
Células Endoteliais , Microscopia , Camundongos , Animais , Osteogênese , Crânio/diagnóstico por imagem , Osteoblastos/metabolismo , Camundongos Transgênicos , Oxigênio/metabolismo , Diferenciação Celular
12.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 36(11): 1428-1433, 2022 Nov 15.
Artigo em Chinês | MEDLINE | ID: mdl-36382463

RESUMO

Objective: To summarize the characteristics of the occurrence and development of osteonecrosis of the femoral head (ONFH), and to review the important regulatory role of immune cells in the progression of ONFH. Methods: The domestic and foreign literature on the immune regulation of ONFH was reviewed, and the relationship between immune cells and the occurrence and development of ONFH was analyzed. Results: The ONFH region has a chronic inflammatory reaction and an imbalance between osteoblast and osteoclast, while innate immune cells such as macrophages, neutrophils, dendritic cells, and immune effector cells such as T cells and B cells are closely related to the maintenance of bone homeostasis. Conclusion: Immunotherapy targeting the immune cells in the ONFH region and the key factors and proteins in their regulatory pathways may be a feasible method to delay the occurrence, development, and even reverse the pathology of ONFH.


Assuntos
Necrose da Cabeça do Fêmur , Células-Tronco Mesenquimais , Osteonecrose , Humanos , Cabeça do Fêmur/patologia , Necrose da Cabeça do Fêmur/etiologia , Necrose da Cabeça do Fêmur/patologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo
13.
Proc Natl Acad Sci U S A ; 119(48): e2209231119, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36417434

RESUMO

The shaping of bone structures relies on various cell types and signaling pathways. Here, we use the zebrafish bifurcating fin rays during regeneration to investigate bone patterning. We found that the regenerating fin rays form via two mineralization fronts that undergo an osteoblast-dependent fusion/stitching until the branchpoint, and that bifurcation is not simply the splitting of one unit into two. We identified tartrate-resistant acid phosphatase-positive osteolytic tubular structures at the branchpoints, hereafter named osteolytic tubules (OLTs). Chemical inhibition of their bone-resorbing activity strongly impairs ray bifurcation, indicating that OLTs counteract the stitching process. Furthermore, by testing different osteoactive compounds, we show that the position of the branchpoint depends on the balance between bone mineralization and resorption activities. Overall, these findings provide a unique perspective on fin ray formation and bifurcation, and reveal a key role for OLTs in defining the proximo-distal position of the branchpoint.


Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Osso e Ossos/metabolismo
14.
Int J Mol Sci ; 23(21)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36361965

RESUMO

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.


Assuntos
Apigenina , Calcificação Fisiológica , Humanos , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Anexina A6/efeitos dos fármacos , Anexina A6/metabolismo , Apigenina/farmacologia , Apigenina/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/fisiologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo
15.
Biomed Res Int ; 2022: 2898729, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36225981

RESUMO

Background: The microenvironment of bone defects displayed that M2 polarization of macrophagocyte could promote the osteoblast growth and benefit the wound healing. Bone scaffold transplantation is considered to be one of the most promising methods for repairing bone defects. The present research was aimed at constructing a kind of novel bone scaffold nanomaterial of MSN@IL-4 for treating bone defects responding to the wound microenvironment of bone defects and elucidating the mechanics of MSN@IL-4 treating bone defect via controlling release of IL-4, inducing M2 polarization and active factor release of macrophagocyte, and eventually relieving osteoblast injury. Methods: MSN@IL-4 was firstly fabricated and its release of IL-4 was assessed in vitro. Following, the effects of MSN@IL-4 nanocomplex on the release of active factors of macrophage were examined using Elisa assay and promoting M2 polarization of the macrophage by immunofluorescence staining. And then, the effects of active factors from macrophage supernatant induced by MSN@IL-4 on osteoblast growth were examined by CCK-8, flow cytometry, and western blot assay. Results: The release curve of IL-4 in vitro displayed that there was more than 80% release ratio for 30th day with a sustained manner in pH 5.5. Elisa assay data showed that MSN@IL-4 nanocomplex could constantly promote the release of proproliferative cytokine IL-10, SDF-1α, and BMP-2 in macrophagocyte compared to only IL-4 treatment, and immunofluorescent image showed that MSN@IL-4 could promote M2 polarization of macrophagocytes via inducing CD206 expression and suppressing CD86 expression. Osteoblast injury data showed that the supernatant from macrophagocyte treated by MSN@IL-4 could promote the osteoblast proliferation by MTT assay. Flow cytometry data showed that the supernatant from macrophagocyte treated by MSN@IL-4 could suppress the osteoblast apoptosis from 22.1% to 14.6%, and apoptosis-related protein expression data showed that the supernatant from macrophagocyte treated by MSN@IL-4 could suppress the expression of Bax, cleaved caspase 3, and cleaved caspase 8. Furthermore, the immunofluorescent image showed that the supernatant from macrophagocyte treated by MSN@IL-4 could inhibit nucleus location of p65, and western blot data showed that the supernatant from macrophagocyte treated by MSN@IL-4 could suppress the phosphorylation of IKK and induce the expression of IκB. Conclusion: MSN@IL-4 could control the sustaining release of IL-4, and it exerts the protective effect on osteoblast injury via inducing M2 polarization and proproliferative cytokine of macrophagocyte and following inhibiting the apoptosis and NF-κB pathway-associated inflammation of osteoblast.


Assuntos
Interleucina-4 , NF-kappa B , Apoptose , Caspase 3/metabolismo , Caspase 8/metabolismo , Quimiocina CXCL12/metabolismo , Citocinas/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , NF-kappa B/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Sincalida/metabolismo , Proteína X Associada a bcl-2/metabolismo
16.
Cell Death Dis ; 13(10): 866, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224171

RESUMO

Human mesenchymal stem cells (hMSCs) can be differentiated into osteoblasts and adipocytes. During these processes, super enhancers (SEs) play important roles. Here, we performed comprehensive characterization of the SEs changes associated with adipogenic and osteogenic differentiation of hMSCs, and revealed that SEs changed more dramatically compared with typical enhancers. We identified a set of lineage-selective SEs, whose target genes were enriched with cell type-specific functions. Functional experiments in lineage-selective SEs demonstrated their specific roles in directed differentiation of hMSCs. We also found that some key transcription factors regulated by lineage-selective SEs could form core regulatory circuitry (CRC) to regulate each other's expression and control the hMSCs fate determination. In addition, we found that GWAS SNPs of osteoporosis and obesity were significantly enriched in osteoblasts-selective SEs or adipocytes-selective SEs, respectively. Taken together, our studies unveiled important roles of lineage-selective SEs in hMSCs differentiation into osteoblasts and adipocytes.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Adipogenia/genética , Diferenciação Celular/genética , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Fatores de Transcrição/metabolismo
17.
Shanghai Kou Qiang Yi Xue ; 31(3): 237-242, 2022 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-36204949

RESUMO

PURPOSE: To investigate the effects of microRNA-31-5p (miR-31-5p) on the signal pathway of hypoxia inducible factor-1α (HIF-1α)/Bcl-2/adenovirus E1B 19-kDa-interacting protein 3(BNIP3) and the expression of osteoblast-related factors of dental pulp stem cells(DPSCs). METHODS: Human dental pulp stem cells (DPSCs) were cultured in vitro and divided into the control group (no transfection), mimic NC group (transfected with negative control-miR-31-5p), miR-31-5p mimic group (transfected with hsa-miR-31-5p mimic), siRNA NC group (transfected with nonsense siRNA) and miR-31-5p siRNA group (transfected with miR-31-5p siRNA).The expressions of miR-31-5p, HIF-1α, BNIP3, alkaline phosphatase(ALP) and Runt-related transcription factor-2(Runx2) mRNA in DPSCs were detected by real-time fluorescence quantitative PCR; the proliferation of DPSCs was detected by MTT; ALP activity of DPSCs was detected by ALP activity test kit; and the protein expressions of HIF-1α, BNIP3 and Runx2 in DPSCs were detected by Western blot. Statistical analysis was carried out with SPSS 24.0 software package. RESULTS: Compared with the control group and mimic NC group, the A value, ALP mRNA expression level and activity, Runx2 mRNA and protein expression levels of DPSCs in miR-31-5p mimic group were significantly lower (P<0.05), ALP staining decreased significantly, and the expression levels of miR-31-5p mRNA, HIF-1α, BNIP3 mRNA and HIF-1α, BNIP3, Beclin1 protein were significantly higher (P<0.05). Compared with the control group and siRNA NC group, the A value, ALP mRNA expression level and activity, Runx2 mRNA and protein expression levels of DPSCs in miR-31-5p siRNA group were significantly higher (P<0.05), ALP staining enhanced significantly, and the expression levels of miR-31-5p mRNA, HIF-1α, BNIP3 mRNA and HIF-1α, BNIP3, Beclin1 protein were significantly lower(P<0.05). CONCLUSIONS: MiR-31-5p may inhibit the expression of osteoblast-related factors of DPSCs, and activating HIF-1α/BNIP3 signaling pathway.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , MicroRNAs , Fosfatase Alcalina/metabolismo , Proteína Beclina-1/metabolismo , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Polpa Dentária/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo
18.
BMC Genomics ; 23(1): 695, 2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207684

RESUMO

BACKGROUND: Previous studies have shown that microtubule actin crosslinking factor 1 (MACF1) can regulate osteoblast proliferation and differentiation through non-coding RNA (ncRNA) in bone-forming osteoblasts. However, the role of MACF1 in targeting the competing endogenous RNA (ceRNA) network to regulate osteoblast differentiation remains poorly understood. Here, we profiled messenger RNA (mRNA), microRNA (miRNA), and long ncRNA (lncRNA) expression in MACF1 knockdown MC3TC­E1 pre­osteoblast cells. RESULTS: In total, 547 lncRNAs, 107 miRNAs, and 376 mRNAs were differentially expressed. Significantly altered lncRNAs, miRNAs, and mRNAs were primarily found on chromosome 2. A lncRNA-miRNA-mRNA network was constructed using a bioinformatics computational approach. The network indicated that mir-7063 and mir-7646 were the most potent ncRNA regulators and mef2c was the most potent target gene. Pathway enrichment analysis showed that the fluid shear stress and atherosclerosis, p53 signaling, and focal adhesion pathways were highly enriched and contributed to osteoblast proliferation. Importantly, the fluid shear stress and atherosclerosis pathway was co-regulated by lncRNAs and miRNAs. In this pathway, Dusp1 was regulated by AK079370, while Arhgef2 was regulated by mir-5101. Furthermore, Map3k5 was regulated by AK154638 and mir-466q simultaneously. AK003142 and mir-3082-5p as well as Ak141402 and mir-446 m-3p were identified as interacting pairs that regulate target genes. CONCLUSION: This study revealed the global expression profile of ceRNAs involved in the differentiation of MC3TC­E1 osteoblasts induced by MACF1 deletion. These results indicate that loss of MACF1 activates a comprehensive ceRNA network to regulate osteoblast proliferation.


Assuntos
Aterosclerose , MicroRNAs , RNA Longo não Codificante , Actinas/genética , Actinas/metabolismo , Proliferação de Células/genética , Redes Reguladoras de Genes , Humanos , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Osteoblastos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína Supressora de Tumor p53/genética
19.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 30(5): 1348-1353, 2022 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-36208234

RESUMO

OBJECTIVE: To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells, and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1 (IL-1) in leukemia cell. METHODS: The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis (GSEA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition, qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell (BMMNC) from 43 patients with acute myeloid leukemia (AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1ß, colony forming ability of AE9a leukemia cells was determined by colony formation assay. RESULTS: In AE9a leukemia mouse, RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set, among them, IL-1α and IL-1ß were significantly higher expressed in AE9a leukemia cells that located osteoblast niche (IL-1α: P<0.001, IL-1ß:P<0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro, the expression of IL-1α and IL-1ß in leukemia cells were increased by 2.5 and 3.5 times respectively. In colony formation assay, the number of colonies was increased significantly after leukemia cells were co-cultured with osteoblasts (P<0.001). In addition, when AE9a leukemia cells were treated with IL-1ß, the number of colonies was also increased significantly (P<0.01). In AML patients, BMMNC with high percentage of CD34 positive cells exhibited higher level of IL-1 expression. CONCLUSION: Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1 in leukemia cells. In AML patients, the expression level of IL-1 was correlated to the percentage of CD34 positive cells in BMMNC.


Assuntos
Medula Óssea , Leucemia Mieloide Aguda , Animais , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Proliferação de Células , Leucemia Mieloide Aguda/metabolismo , Camundongos , Osteoblastos/metabolismo , Células-Tronco , Microambiente Tumoral
20.
Cell Mol Biol (Noisy-le-grand) ; 68(6): 124-129, 2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-36227667

RESUMO

This experiment was carried out to study the effects of estrogen on the proliferation and apoptosis of osteoblasts through regulating the G protein-coupled estrogen receptor (GPER)/protein kinase B (AKT) pathway. For this aim, osteoblasts were cultured in vitro and divided into control group, estrogen group and inhibitor group after passage. The osteoblasts in the control group were cultured normally, estrogen intervention was made in the estrogen group and G15 inhibitor intervention was made in the inhibitor group. After intervention for 24 h, osteoblasts were collected for detection. The positive expression of GPER and the double-positive expression of Tom20/Lamp2 were detected via immunofluorescence assay. The protein expressions of GPER, AKT and phosphorylated (p)-AKT were detected via Western blotting. The mRNA expression of GPER was detected via qPCR. Moreover, the autophagosomes were observed under a transmission electron microscope, and the apoptosis and cell proliferation were detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and cell counting kit-8 (CCK8) assay, respectively. Results of the immunofluorescence assay revealed that the positive expression of GPER in the estrogen group was higher than that in the control group and inhibitor group (p<0.05), while the double-positive expression of Tom20/Lamp2 in the estrogen group was lower than that in control group and inhibitor group (p<0.05). According to the results of Western blotting, the relative protein expression of AKT had no differences among the three groups (p>0.05), while the relative protein expressions of GPER and p-AKT in the estrogen group were higher than those in the control group and inhibitor group (p<0.05). The results of qPCR showed that the relative mRNA expression of GPER in the estrogen group was higher than that in the control group and inhibitor group (p<0.05). There were a small number of autophagosomes in osteoblasts in the control group and inhibitor group, while the number of autophagosomes in osteoblasts was smaller in the estrogen group. Besides, the estrogen group had a remarkably lower apoptosis rate of osteoblasts than the control group and inhibitor group and a remarkably higher proliferation rate than the control group and inhibitor group. Then estrogen can inhibit the mitochondrial autophagy of osteoblasts by regulating the GPER/AKT pathway, thereby inhibiting apoptosis and promoting cell proliferation.


Assuntos
Proteínas Proto-Oncogênicas c-akt , Receptores de Estrogênio , Apoptose , Proliferação de Células , DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidilexotransferase/farmacologia , Estrogênios/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
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