RESUMO
Extracellular matrix (ECM) stiffness is a major driver of stem cell fate. However, the involvement of the three-dimensional (3D) genomic reorganization in response to ECM stiffness remains unclear. Here, we generated comprehensive 3D chromatin landscapes of mesenchymal stem cells (MSCs) exposed to various ECM stiffness. We found that there were more long-range chromatin interactions, but less compartment A in MSCs cultured on stiff ECM than those cultured on soft ECM. However, the switch from compartment B in MSCs cultured on soft ECM to compartment A in MSCs cultured on stiff ECM included genes encoding proteins primarily enriched in cytoskeleton organization. At the topologically associating domains (TADs) level, stiff ECM tends to have merged TADs on soft ECM. These merged TADs on stiff ECM include upregulated genes encoding proteins enriched in osteogenesis, such as SP1, ETS1, and DCHS1, which were validated by quantitative real-time polymerase chain reaction and found to be consistent with the increase of alkaline phosphatase staining. Knockdown of SP1 or ETS1 led to the downregulation of osteogenic marker genes, including COL1A1, RUNX2, ALP, and OCN in MSCs cultured on stiff ECM. Our study provides an important insight into the stiff ECM-mediated promotion of MSC differentiation towards osteogenesis, emphasizing the influence of mechanical cues on the reorganization of 3D genome architecture and stem cell fate.
Assuntos
Diferenciação Celular , Matriz Extracelular , Células-Tronco Mesenquimais , Osteogênese , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Matriz Extracelular/metabolismo , Diferenciação Celular/genética , Humanos , Células Cultivadas , AnimaisRESUMO
This study aimed to investigate the effects of Shuanglongjiegu pill (SLJGP) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explore its mechanism based on miR-217/RUNX2 axis. Results found that drug-containing serum of SLJGP promoted BMSCs viability with a dose-dependent effect. Under osteogenic differentiation conditions, SLJGP promoted the expression of ALP, OPN, BMP2, RUNX2, and the osteogenic differentiation ability of BMSCs. In addition, SLJGP significantly reduced miR-217 expression, and miR-217 directly targeted RUNX2. After treatment with miR-217 mimic, the promoting effects of SLJGP on proliferation and osteogenic differentiation of BMSCs were significantly inhibited. MiR-217 mimic co-treated with pcDNA-RUNX2 further confirmed that the miR-217/RUNX2 axis was involved in SLJGP to promote osteogenic differentiation of BMSCs. In addition, analysis of Wnt/ß-catenin pathway protein expression showed that SLJGP activated the Wnt/ß-catenin pathway through miR-217/RUNX2. In conclusion, SLJGP promoted osteogenic differentiation of BMSCs by regulating miR-217/RUNX2 axis and activating Wnt/ß-catenin pathway.
Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Medicamentos de Ervas Chinesas , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Via de Sinalização Wnt , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Medicamentos de Ervas Chinesas/farmacologia , Células Cultivadas , Humanos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate whether bone marrow mesenchymal stem cells (BMMSCs) modulate periodontal bone repair through the hydroxylase domain-containing protein 2 (PHD2)/hypoxia- inducible factor-1 (HIF-1) signalling pathway in response to inflammatory conditions. METHODS: Osteogenic differentiation of PHD2 shRNA-modified BMMSCs and the possible mechanism were explored in an inflammatory microenvironment stimulated by porphyromonas gingivalis lipopolysaccharide (Pg-LPS) in vitro. The effect of PHD2 gene-modified BMMSCs on periodontal bone loss was evaluated with experimental periodontitis. RESULTS: Pg-LPS stimulation greatly impaired the osteogenic differentiation of BMMSCs, whereas the silence of PHD2 significantly enhanced the osteogenesis of BMMSCs. More importantly, increased level of vascular endothelial growth factor (VEGF) was detected under Pg-LPS stimulation, which was verified to be associated with the augmented osteogenesis. In experimental periodontitis, PHD2-modified BMMSCs transplantation elevated osteogenic parameters and the expression of VEGF in periodontal tissue. CONCLUSION: This study highlighted that PHD2 gene silencing could be a feasible approach to combat inflammatory bone loss by rescuing the dysfunction of seed cells.
Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia , Células-Tronco Mesenquimais , Osteogênese , RNA Interferente Pequeno , Animais , RNA Interferente Pequeno/genética , Osteogênese/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Porphyromonas gingivalis , Periodontite/terapia , Periodontite/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Diferenciação Celular , Lipopolissacarídeos , Perda do Osso Alveolar , Camundongos , Masculino , Células da Medula Óssea , Regeneração Óssea/genéticaRESUMO
OBJECTIVE: To investigate the biological regulatory function of Gremlin1 (GREM1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH) in dental pulp stem cells (DPSCs), and determine the underlying molecular mechanism involved. METHODS: Alkaline phosphatase (ALP) activity, alizarin red staining, scratch migration assays and in vitro and in vivo osteo-/dentinogenic marker detection of bone-like tissue generation in nude mice were used to assess osteo-/dentinogenic differentiation. Coimmunoprecipitation and polypeptide microarray assays were employed to detect the molecular mechanisms involved. RESULTS: The data revealed that knockdown of GREM1 promoted ALP activity, mineralisation in vitro and the expression of osteo-/dentinogenic differentiation markers and enhanced osteo-/ dentinogenesis of DPSCs in vivo. GREM1 bound to YWHAH in DPSCs, and the binding site was also identified. Knockdown of YWHAH suppressed the osteo-/dentinogenesis of DPSCs in vitro, and overexpression of YWHAH promoted the osteo-/dentinogenesis of DPSCs in vitro and in vivo. CONCLUSION: Taken together, the findings highlight the critical roles of GREM1-YWHAH in the osteo-/dentinogenesis of DPSCs.
Assuntos
Diferenciação Celular , Polpa Dentária , Peptídeos e Proteínas de Sinalização Intercelular , Osteogênese , Células-Tronco , Animais , Humanos , Camundongos , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Dentinogênese/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos Nus , Osteogênese/genética , Células-Tronco/metabolismoRESUMO
Age-related osteoporosis is a prevalent bone metabolic disorder distinguished by an aberration in the equilibrium between bone formation and resorption. The reduction in the stemness of Bone Marrow Mesenchymal Stem Cells (BMSCs) plays a pivotal role in the onset of this ailment. Comprehending the molecular pathways that govern BMSCs stemness is imperative for delineating the etiology of age-related osteoporosis and devising efficacious treatment modalities. The study utilized single-cell RNA sequencing and miRNA sequencing to investigate the cellular heterogeneity and stemness of BMSCs. Through dual-luciferase reporter assays and functional experiments, the regulatory effect of miR-183 on CTNNB1 (ß-catenin) was confirmed. Overexpression and knockdown studies were conducted to explore the impact of miR-183 and ß-catenin on stemness-related transcription factors Oct4, Nanog, and Sox2. Cell proliferation assays and osteogenic differentiation experiments were carried out to validate the influence of miR-183 and ß-catenin on the stemness properties of BMSCs. Single-cell analysis revealed that ß-catenin is highly expressed in both high stemness clusters and terminal differentiation clusters of BMSCs. Overexpression of ß-catenin upregulated stemness transcription factors, while its suppression had the opposite effect, indicating a dual regulatory role of ß-catenin in maintaining BMSCs stemness and promoting bone differentiation. Furthermore, the confluence of miRNA sequencing analyses and predictions from online databases revealed miR-183 as a potential modulator of BMSCs stemness and a novel upstream regulator of ß-catenin. The overexpression of miR-183 effectively diminished the stemness characteristics of BMSCs by suppressing ß-catenin, whereas the inhibition of miR-183 augmented stemness. These outcomes align with the observed alterations in the expression levels and functional assessments of transcription factors associated with stemness. This study provides evidence for the essential involvement of ß-catenin in preserving the stemness of BMSCs, as well as elucidating the molecular mechanism through which miR-183 selectively targets ß-catenin to modulate stemness. These results underscore the potential of miR-183 and ß-catenin as molecular targets for augmenting the stemness of BMSCs. This strategy is anticipated to facilitate the restoration of bone microarchitecture and facilitate bone tissue regeneration by addressing potential cellular dysfunctions, thereby presenting novel targets and perspectives for the management of age-related osteoporosis.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Osteoporose , beta Catenina , MicroRNAs/genética , MicroRNAs/metabolismo , beta Catenina/metabolismo , beta Catenina/genética , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Animais , Diferenciação Celular/genética , Humanos , Proliferação de Células/genética , Análise de Célula Única , Regulação da Expressão Gênica , CamundongosRESUMO
OBJECTIVE: This study was designed to evaluate TFAP2A-AS1 expression in the dental pulp of teeth with or without pulpitis and to determine the function of TFAP2A-AS1 in pulp cells. METHODS: GSE92681 was analyzed to filter out differentially expressed lncRNAs. Pulp samples from teeth with pulpitis and healthy teeth (control) were examined using real-time (RT) quantitative polymerase chain reaction (qPCR). Human dental pulp stem cells (hDPSCs) were cultured in a specific medium for osteogenic induction, or treated with lipopolysaccharide (LPS) to simulate inflammation. The viability and apoptosis of human DPSCs (hDPSCs) were determined by XTT assay and apoptosis detection kit. Inflammation was induced by LPS and assessed by measuring the expression and release of inflammatory cytokines after TFAP2A-AS1 knockdown. Osteogenic differentiation of hDPSCs was investigated by determining expression levels of osteogenic markers and alkaline phosphatase (ALP) activity after TFAP2A-AS1 overexpression. The downstream microRNA (miRNA) was predicted. Dual-luciferase reporter was used to confirm the binding between miR-32-5p and TFAP2A-AS1. RESULTS: The expression of TFAP2A-AS1 was evaluated in inflamed pulp using RT-qPCR. TFAP2A-AS1 had a discriminatory ability for healthy individuals and patients with pulpitis. The expression of TFAP2A-AS1 decreased upon the osteogenic differentiation of hDPSCs, and increased upon the LPS induction. TFAP2A-AS1 can reverse the osteogenic differentiation of hDPSCs, as evidenced by decreased levels of dentine sialophosphoprotein, dentin matrix protein-1, and ALP activity. TFAP2A-AS1 knockdown can promote cell proliferation of hDPSCs and relieve LPS-induced inflammation, as evidenced by decreased levels of TNF-α, IL-1ß, and IL-6. miR-32-5p was identified as a downstream miRNA of TFAP2A-AS1. CONCLUSION: This study demonstrated the expression and potential function of TFAP2A-AS1 in the human dental pulp. TFAP2A-AS1 can inhibit odontogenic differentiation but promote inflammation in pulp cells.
Assuntos
Polpa Dentária , MicroRNAs , Pulpite , RNA Longo não Codificante , Fator de Transcrição AP-2 , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , Polpa Dentária/metabolismo , Polpa Dentária/patologia , Pulpite/metabolismo , Pulpite/genética , Pulpite/patologia , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , Diferenciação Celular/genética , Osteogênese/genética , Apoptose/genética , Regulação da Expressão Gênica , Células Cultivadas , Lipopolissacarídeos , Células-Tronco/metabolismoRESUMO
Background: As a powerful tool, bioinformatics analysis is playing an increasingly important role in many fields. Osteogenic differentiation is a complex biological process involving the fine regulation of numerous genes and signaling pathways. Method: Osteogenic differentiation-related genes are collected from the online databases. Then, we proposed two indexes Jaccard similarity and Sorensen-Dice similarity to measure the topological relevance of genes in the human PPI network. Furthermore, we selected three pathways involving osteoblast-related transcription factors, osteoblast differentiation, and RUNX2 regulation of osteoblast differentiation for investigation. Subsequently, we performed functional a enrichment analysis of these top-ranked genes to check whether these candidate genes identified by similarity-based metrics are enriched in some specific biological functions and states. we performed a permutation test to investigate the similarity score with four well-known osteogenic differentiation-related pathways including hedgehog signaling pathway, BMP signaling, ERK pathway, and Wnt signaling pathway to check whether these osteogenic differentiation-related pathways can be regulated by FOXA1. Lentiviral transfection was used to knockdown and overexpress gene FOXA1 in human bone mesenchymal stem cells (hBMSCs). Alkaline phosphatase (ALP) staining and Alizarin Red staining (ARS) were employed to investigate osteogenic differentiation of hBMSCs. Result: After data collection, human PPI network involving 19,344 genes is included in our analysis. After simplifying, we used Jaccard and Sorensen-Dice similarity to identify osteogenic differentiation-related genes and integrated into a final similarity matrix. Furthermore, we calculated the sum of similarity scores with these osteogenic differentiation-related genes for each gene and found 337 osteogenic differentiation-related genes are involved in our analysis. We selected three pathways involving osteoblast-related transcription factors, osteoblast differentiation, and RUNX2 regulation of osteoblast differentiation for investigation and performed functional enrichment analysis of these top-ranked 50 genes. The results collectively demonstrate that these candidate genes can indeed capture osteogenic differentiation-related features of hBSMCs. According to the novel analyzing method, we found that these four pathways have significantly higher similarity with FOXA1 than random noise. Moreover, knockdown FOXA1 significantly increased the ALP activity and mineral deposits. Furthermore, overexpression of FOXA1 dramatically decreased the ALP activity and mineral deposits. Conclusion: In summary, this study showed that FOXA1 is a novel significant osteogenic differentiation-related transcription factor. Moreover, our study has tightly integrated bioinformatics analysis with biological knowledge, and developed a novel method for analyzing the osteogenic differentiation regulatory network.
Assuntos
Diferenciação Celular , Fator 3-alfa Nuclear de Hepatócito , Células-Tronco Mesenquimais , Osteogênese , Humanos , Osteogênese/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Osteoblastos/metabolismo , Osteoblastos/citologia , Biologia Computacional/métodos , Transdução de Sinais/genéticaRESUMO
Endochondral ossification is the process by which cartilage is mineralized into bone, and is essential for the development of long bones. Osteocalcin (OCN), a protein abundant in bone matrix, also exhibits high expression in chondrocytes, especially hypertrophic chondrocytes, while its role in endochondral ossification remains unclear. Utilizing a new CRISPR/Cas9-mediated bglap-bglap2 deficiency (OCNem) mouse model generated in our laboratory, we provide the first evidence of OCN's regulatory function in chondrocyte differentiation and endochondral ossification. The OCNem mice exhibited significant delays in primary and secondary ossification centers compared to wild-type mice, along with increased cartilage length in growth plates and hypertrophic zones during neonatal and adolescent stages. These anomalies indicated that OCN deficiency disturbed endochondral ossification during embryonic and postnatal periods. Mechanism wise, OCN deficiency was found to increase chondrocyte differentiation and postpone vascularization process. Furthermore, bone marrow mesenchymal stromal cells (BMSCs) from OCNem mice demonstrated an increased capacity for chondrogenic differentiation. Transcriptional network analysis implicated that BMP and TGF-ß signaling pathways were highly affected in OCNem BMSCs, which is closely associated with cartilage development and maintenance. This elucidation of OCN's function in chondrocyte differentiation and endochondral ossification contributes to a more comprehensive understanding of its impact on skeletal development and homeostasis.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Condrócitos , Condrogênese , Osteocalcina , Osteogênese , Animais , Condrócitos/metabolismo , Condrócitos/citologia , Osteogênese/genética , Camundongos , Osteocalcina/metabolismo , Osteocalcina/genética , Diferenciação Celular/genética , Condrogênese/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Cartilagem/metabolismo , Transdução de Sinais , Camundongos KnockoutRESUMO
Regular physical activity is widely recognized for reducing the risk of various disorders, with skeletal muscles playing a key role by releasing biomolecules that benefit multiple organs and tissues. However, many individuals, particularly the elderly and those with clinical conditions, are unable to engage in physical exercise, necessitating alternative strategies to stimulate muscle cells to secrete beneficial biomolecules. Histone acetylation and deacetylation significantly influence exercise-induced gene expression, suggesting that targeting histone deacetylases (HDACs) could mimic some exercise responses. In this study, we explored the effects of the HDAC inhibitor Trichostatin A (TSA) on human skeletal muscle myoblasts (HSMMs). Our findings showed that TSA-induced hyperacetylation enhanced myotube fusion and increased the secretion of extracellular vesicles (EVs) enriched with miR-873-3p. These TSA-EVs promoted osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs) by targeting H2 calponin (CNN2). In vivo, systemic administration of TSA-EVs to osteoporosis mice resulted in significant improvements in bone mass. Moreover, TSA-EVs mimicked the osteogenic benefits of exercise-induced EVs, suggesting that HDAC inhibition can replicate exercise-induced bone health benefits. These results demonstrate the potential of TSA-induced muscle-derived EVs as a therapeutic strategy to enhance bone formation and prevent osteoporosis, particularly for individuals unable to exercise. Given the FDA-approved status of various HDAC inhibitors, this approach holds significant promise for rapid clinical translation in osteoporosis treatment.
Assuntos
Vesículas Extracelulares , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos , MicroRNAs , Osteogênese , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Camundongos , MicroRNAs/genética , Humanos , Animais , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ácidos Hidroxâmicos/farmacologia , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Osteoporose/genética , Osteoporose/patologia , Osteoporose/tratamento farmacológico , Osteoporose/metabolismoRESUMO
Electromagnetic fields (EMFs) have emerged as an effective treatment for osteoporosis. However, the specific mechanism underlying their therapeutic efficacy remains controversial. Herein, we confirm the pro-osteogenic effects of 15 Hz and 0.4-1 mT low-frequency sinusoidal EMFs (SEMFs) on rat bone marrow mesenchymal stem cells (BMSCs). Subsequent miRNA sequencing reveal that miR-34b-5p is downregulated in both the 0.4 mT and 1 mT SEMFs-stimulated groups. To clarify the role of miR-34b-5p in osteogenesis, BMSCs are transfected separately with miR-34b-5p mimic and inhibitor. The results indicate that miR-34b-5p mimic transfection suppress osteogenic differentiation, whereas inhibition of miR-34b-5p promote osteogenic differentiation of BMSCs. In vivo assessments using microcomputed tomography, H&E staining, and Masson staining show that miR-34b-5p inhibitor injections alleviate bone mass loss and trabecular microstructure deterioration in ovariectomy (OVX) rats. Further validation demonstrates that miR-34b-5p exerts its effects by regulating STAC2 expression. Modulating the miR-34b-5p/STAC2 axis attenuate the pro-osteogenic effects of low-frequency SEMFs on BMSCs. These studies indicate that the pro-osteogenic effect of SEMFs is partly due to the regulation of the miR-34b-5p/STAC2 pathway, which provides a potential therapeutic candidate for osteoporosis.
Assuntos
Diferenciação Celular , Campos Eletromagnéticos , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Ratos Sprague-Dawley , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Ratos , Feminino , Osteoporose/genética , Osteoporose/terapia , Osteoporose/metabolismo , Células CultivadasRESUMO
BACKGROUND: Osteoporosis results from decreased bone mass and disturbed bone structure. Human bone marrow mesenchymal stem cells (hBMSCs) demonstrate robust osteogenic differentiation, a critical process for bone formation. This research was designed to examine the functions of LINC01133 in osteogenic differentiation. METHODS: Differentially expressed lncRNAs affecting osteogenic differentiation in hBMSCs were identified from the GEO database. A total of 74 osteoporosis patients and 70 controls were enrolled. hBMSCs were stimulated to undergo osteogenic differentiation using an osteogenic differentiation medium (OM). RT-qPCR was performed to evaluate LINC01133 levels and osteogenesis-related genes such as osteocalcin, osteopontin, and RUNX2. An alkaline phosphates (ALP) activity assay was conducted to assess osteogenic differentiation. Cell apoptosis was detected using flow cytometry. Dual luciferase reporter assay and RIP assay were employed to investigate the association between miR-214-3p and LINC01133 or CTNNB1. Loss or gain of function assays were conducted to elucidate the impact of LINC01133 and miR-214-3p on osteogenic differentiation of hBMSCs. RESULTS: LINC01133 and CTNNB1 expression decreased in osteoporotic patients but increased in OM-cultured hBMSCs, whereas miR-214-3p showed an opposite trend. Depletion of LINC01133 suppressed the expression of genes associated with bone formation and ALP activity triggered by OM in hBMSCs, leading to increased cell apoptosis. Nevertheless, this suppression was partially counteracted by the reduced miR-214-3p levels. Mechanistically, LINC01133 and CTNNB1 were identified as direct targets of miR-214-3p. CONCLUSIONS: Our study highlights the role of LINC01133 in positively regulating CTNNB1 expression by inhibiting miR-214-3p, thereby promoting osteogenic differentiation of BMSCs. These findings may provide valuable insights into bone regeneration in osteoporosis.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Osteoporose , RNA Longo não Codificante , Regulação para Cima , beta Catenina , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Diferenciação Celular/genética , RNA Longo não Codificante/genética , beta Catenina/genética , beta Catenina/metabolismo , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Células Cultivadas , Feminino , Pessoa de Meia-Idade , Masculino , Apoptose/genética , Células da Medula Óssea/metabolismoRESUMO
Heterotopic ossification (HO) is a pathological condition characterized by the formation of bone within soft tissues. The development of HO is a result of abnormal activation of the bone formation programs, where multiple signalling pathways, including Wnt/ß-catenin, BMP and hedgehog signalling, are involved. The Wnt/ß-catenin signalling pathway, a conserved pathway essential for various fundamental activities, has been found to play a significant role in pathological bone formation processes. It regulates angiogenesis, chondrocyte hypertrophy and osteoblast differentiation during the development of HO. More importantly, the crosstalk between Wnt signalling and other factors including BMP, Hedgehog signalling, YAP may contribute in a HO-favourable manner. Moreover, several miRNAs may also be involved in HO formation via the regulation of Wnt signalling. This review aims to summarize the role of Wnt/ß-catenin signalling in the pathogenesis of HO, its interactions with related molecules, and potential preventive and therapeutic measures targeting Wnt/ß-catenin signalling.
Assuntos
Ossificação Heterotópica , Via de Sinalização Wnt , Humanos , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Ossificação Heterotópica/genética , Animais , Osteogênese/genética , MicroRNAs/genética , MicroRNAs/metabolismo , beta Catenina/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Diferenciação CelularRESUMO
ZC4H2 encodes a C4H2-type zinc finger protein, mutations of which lead to a spectrum of diseases known as ZC4H2 associated rare disorders (ZARD). In addition to neurological phenotypes, the most typical symptoms of ZARD are multiple joint contractures of varying degrees, accompanied by abnormal development of muscles and bones, and osteoporosis in some cases. The pathogenic mechanisms of such bone related phenotypes, however, remain unclear. Here, we showed that ZC4H2 is expressed in the developing bones in mice. ZC4H2 knockout mice were neonatal-lethal and smaller in size, with reduced calcification of long bones. Upon induced loss of ZC4H2 postnatally, the femoral bones developed an osteoporosis-like phenotype, with reduced bone mineral density, bone-volume fraction, and trabecular bone number. Knockdown of ZC4H2 showed no clear effect on the expression of osteogenic differentiation genes in in vitro models using mesenchymal stem cells. Interestingly, ZC4H2 knockdown significantly enhanced osteoclast differentiation and bone resorption in induced bone marrow-derived macrophages. We further confirmed that the number of osteoclasts in the long bone of ZC4H2 knockout mice was increased, as well as the expression of the serum bone resorption/osteoporosis marker CTX-1. Our study unveils a new role of ZC4H2 in osteoclast differentiation and bone development, providing new clues on the pathology of ZARD.
Assuntos
Artrogripose , Camundongos Knockout , Osteoclastos , Osteogênese , Animais , Camundongos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/genética , Artrogripose/genética , Artrogripose/patologia , Diferenciação Celular/genética , Osteoporose/genética , Osteoporose/patologia , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Células-Tronco Mesenquimais/metabolismo , Densidade Óssea/genéticaRESUMO
BACKGROUND/OBJECTIVES: Glass, bone, and dentin are commonly applied substrates for osteoclast cultures; however, the impact of these substrates on osteoclastogenesis remains underexplored. This study aimed to address a significant gap in understanding how different substrates influence the process of osteoclastogenesis. METHODS: RAW 264.7 cells were cultured and induced with RANKL on glass, bone, and dentin slides. Histological and molecular techniques were used to identify patterns and differences in osteoclast behavior on each substrate. RESULTS: Osteoclasts cultured on glass slides possessed the greatest number of nuclei and the highest expression levels of ACP5 (TRAP) and CTSK, with osteoclasts on bone and dentin slides displaying progressively lower levels. Src expression was also most pronounced in osteoclasts on glass slides, with decreased levels observed on bone and dentin. This variation in Src expression likely contributed to differences in cytoskeletal remodeling and oxidative phosphorylation (OXPHOS), resulting in substrate-dependent divergences in osteoclastogenesis. CONCLUSIONS: Glass slides were the most favorable substrate for inducing osteoclastogenesis, while bone and dentin slides were less effective. The substrate-induced expression of Src played a fundamental role in shaping the phenotypic divergence of osteoclasts. These insights fill important knowledge gaps and have significant implications for the development and selection of in vitro models for bone-related diseases and drug screening platforms.
Assuntos
Osteoclastos , Osteogênese , Animais , Camundongos , Osteoclastos/metabolismo , Osteoclastos/citologia , Células RAW 264.7 , Osteogênese/genética , Ligante RANK/metabolismo , Ligante RANK/genética , Quinases da Família src/metabolismo , Quinases da Família src/genética , Diferenciação Celular , Dentina/metabolismo , Vidro , Osso e Ossos/metabolismo , Osso e Ossos/citologiaRESUMO
Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Fator de Transcrição Sp7 , Animais , Humanos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fator de Transcrição Sp7/metabolismo , Fator de Transcrição Sp7/genética , Desenvolvimento Ósseo/genética , Osteoblastos/metabolismo , Osteoblastos/citologia , Diferenciação Celular , Osteogênese/genéticaRESUMO
Plastin-3 (PLS3) encodes T-plastin, an actin-bundling protein mediating the formation of actin filaments by which numerous cellular processes are regulated. Loss-of-function genetic defects in PLS3 are reported to cause X-linked osteoporosis and childhood-onset fractures. However, the molecular etiology of PLS3 remains elusive. Functional compensation by actin-bundling proteins ACTN1, ACTN4, and FSCN1 was investigated in zebrafish following morpholino-mediated pls3 knockdown. Primary dermal fibroblasts from six patients with a PLS3 variant were also used to examine expression of these proteins during osteogenic differentiation. In addition, Pls3 knockdown in the murine MLO-Y4 cell line was employed to provide insights in global gene expression. Our results showed that ACTN1 and ACTN4 can rescue the skeletal deformities in zebrafish after pls3 knockdown, but this was inadequate for FSCN1. Patients' fibroblasts showed the same osteogenic transdifferentiation ability as healthy donors. RNA-seq results showed differential expression in Wnt1, Nos1ap, and Myh3 after Pls3 knockdown in MLO-Y4 cells, which were also associated with the Wnt and Th17 cell differentiation pathways. Moreover, WNT2 was significantly increased in patient osteoblast-like cells compared to healthy donors. Altogether, our findings in different bone cell types indicate that the mechanism of PLS3-related pathology extends beyond actin-bundling proteins, implicating broader pathways of bone metabolism.
Assuntos
Diferenciação Celular , Glicoproteínas de Membrana , Proteínas dos Microfilamentos , Osteogênese , Peixe-Zebra , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Osteogênese/genética , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Fibroblastos/metabolismo , Osteoblastos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Técnicas de Silenciamento de GenesRESUMO
Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.
Assuntos
Diferenciação Celular , Proteínas Associadas aos Microtúbulos , Osteoblastos , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Linhagem Celular , Cílios/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteogênese/genética , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Both silicon (Si) and zinc (Zn) ions are essential elements to bone health and their mechanisms for promoting osteogenesis have aroused the extensive attention of researchers. Thereinto, the mechanism by which dual ions promote osteogenic differentiation remains to be elucidated. Herein, the effects of Si and Zn ions on the cytological behaviors of mBMSCs were firstly studied. Then, the molecular mechanism of Si-Zn dual ions regulating the osteogenic differentiation of mBMSCs was investigated via transcriptome sequencing technology. In the single-ion system, Si ion at the concentration of 1.5 mM (Si-1.5) had better comprehensive effects of cell proliferation, ALP activity and osteogenesis-related gene expression levels (ALP, Runx2, OCN, Col-I and BSP); Zn ion at the concentration of 50 µM (Zn-50) demonstrated better combining effects of cell proliferation, ALP activity and same osteogenic genes expression levels. In the dual-ion system, the Si (1.5 mM)-Zn (50 µM) group (Si1.5-Zn50) synthetically enhanced ALP activity and osteogenesis genes compared with single-ion groups. Analysis of the transcriptome sequencing results showed that Si ion had a certain effect on promoting the osteogenic differentiation of mBMSCs; Zn ion had a stronger effect of contributing to a better osteogenic differentiation of mBMSCs than that of Si ion; the Si-Zn dual ions had a synergistic enhancement on conducting to the osteogenic differentiation of mBMSCs compared to single ion (Si or Zn). This study offers a blueprint for exploring the regulation mechanism of osteogenic differentiation by dual ions.
Assuntos
Diferenciação Celular , Proliferação de Células , Íons , Células-Tronco Mesenquimais , Osteogênese , RNA Mensageiro , Silício , Zinco , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Silício/química , Silício/farmacologia , Diferenciação Celular/efeitos dos fármacos , Zinco/química , Zinco/farmacologia , Proliferação de Células/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNARESUMO
Splicing and endoplasmic reticulum (ER)-proteostasis are two key processes that ultimately regulate the functional proteins that are produced by a cell. However, the extent to which these processes interact remains poorly understood. Here, we identify SNRPB and other components of the Sm-ring, as targets of the unfolded protein response and novel regulators of export from the ER. Mechanistically, The Sm-ring regulates the splicing of components of the ER export machinery, including Sec16A, a component of ER exit sites. Loss of function of SNRPB is causally linked to cerebro-costo-mandibular syndrome (CCMS), a genetic disease characterized by bone defects. We show that heterozygous deletion of SNRPB in mice resulted in bone defects reminiscent of CCMS and that knockdown of SNRPB delays the trafficking of type-I collagen. Silencing SNRPB inhibited osteogenesis in vitro, which could be rescued by overexpression of Sec16A. This rescue indicates that the role of SNRPB in osteogenesis is linked to its effects on ER-export. Finally, we show that SNRPB is a target for the unfolded protein response, which supports a mechanistic link between the spliceosome and ER-proteostasis. Our work highlights components of the Sm-ring as a novel node in the proteostasis network, shedding light on CCMS pathophysiology.
Assuntos
Desenvolvimento Ósseo , Retículo Endoplasmático , Splicing de RNA , Resposta a Proteínas não Dobradas , Animais , Camundongos , Retículo Endoplasmático/metabolismo , Humanos , Desenvolvimento Ósseo/genética , Camundongos Knockout , Osteogênese/genéticaRESUMO
OBJECTIVE: Delayed fracture healing increases the suffering of patients. An in-depth investigation of the pathogenesis of delayed fracture healing may offer new direction for the prevention and treatment. METHODS: The study included 63 normal healing tibial fractures and 58 delayed healing tibial fractures patients. Long non-coding RNA (lncRNA)TRPM2-AS, microRNA-545-3p (miR-545-3p), bone morphogenetic protein 2 (Bmp2) mRNA and osteogenic differentiation markers, including runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), and alkaline phosphatase (Alp) mRNA expression were determined by Real-time quantitative reverse transcription-polymerase chain reaction in serum and MC3T3-E1 cells. The prediction potential of TRPM2-AS in delayed healing fracture patients was verified by receiver operating characteristic curves. The binding relationship of TRPM2-AS/miR-545-3p/Bmp2 was evaluated by dual luciferase reporter gene assay. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry. RESULTS: TRPM2-AS was remarkably down-regulated in patients with delayed fracture healing and could better predict the fracture healing status. TRPM2-AS downregulation inhibited osteogenic markers mRNA expression, restrained proliferation, and promoted apoptosis of MC3T3-E1 cells (p < 0.05). In delayed fracture healing, miR-545-3p was dramatically up-regulated and was negatively regulated by TRPM2-AS. Reducing miR-545-3p eliminate the negative effect of TRPM2-AS down-regulation on osteoblast proliferation and differentiation (p < 0.05). miR-545-3p targets Bmp2, which plays a positive role in osteoblast differentiation (p < 0.05). CONCLUSION: This study found that TRPM2-AS has the potential to be a diagnostic marker for delayed fracture healing and revealed that the TRPM2-AS/miR-545-3p/Bmp2 axis affects fracture healing by regulating osteoblast.