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Acta Biomaterialia Aug 2023Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and...
Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2-deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration. STATEMENT OF SIGNIFICANCE: A method for stimulating osteoblast differentiation of skeletal progenitor cells is described that uses culture surfaces coated with a collagen-derived triple-helical peptide to selectively activate discoidin domain receptors. When this peptide is combined with an integrin-activating peptide, synergistic stimulation of differentiation is seen. This approach of combining collagen-derived peptides to stimulate the two main collagen receptors in bone (DDR2 and collagen-binding integrins) provides a route for developing a new class of tissue engineering scaffolds for bone regeneration.
Topics: Animals; Mice; Cell Line; Collagen; Peptides; Discoidin Domain Receptor 2; Integrins; Stem Cells; Osteoblasts; Cell Differentiation; Phosphorylation; Signal Transduction
PubMed: 37245640
DOI: 10.1016/j.actbio.2023.05.039 -
Phytomedicine : International Journal... Jun 2024Recently, osteoblast pyroptosis has been proposed as a potential pathogenic mechanism underlying osteoporosis, although this remains to be confirmed. Luteolin (Lut), a...
BACKGROUND
Recently, osteoblast pyroptosis has been proposed as a potential pathogenic mechanism underlying osteoporosis, although this remains to be confirmed. Luteolin (Lut), a flavonoid phytochemical, plays a critical role in the anti-osteoporosis effects of many traditional Chinese medicine prescriptions. However, its protective impact on osteoblasts in postmenopausal osteoporosis (PMOP) has not been elucidated.
PURPOSE
This research aimed to determine the effect of Lut in ameliorating PMOP by alleviating osteoblast pyroptosis and sustaining osteogenesis.
STUDY DESIGN
This research was designed to investigate the novel mechanism of Lut in alleviating PMOP both in cell and animal models.
METHODS
Ovariectomy-induced PMOP models were established in mice with/without daily gavaged of 10 or 20 mg/kg body weight Lut. The impact of Lut on bone microstructure, metabolism and oxidative stress was evaluated with 0.104 mg/kg body weight Estradiol Valerate Tablets daily gavaged as positive control. Network pharmacological analysis and molecular docking were employed to investigate the mechanisms of Lut in PMOP treatment. Subsequently, the impacts of Lut on the PI3K/AKT axis, oxidative stress, mitochondria, and osteoblast pyroptosis were assessed. In vitro, cultured MC3T3-E1(14) cells were exposed to HO with/without Lut to examine its effects on the PI3K/AKT signaling pathway, osteogenic differentiation, mitochondrial function, and osteoblast pyroptosis.
RESULTS
Our findings demonstrated that 20 mg/kg Lut, similar to the positive control drug, effectively reduced systemic bone loss and oxidative stress, and enhanced bone metabolism induced by ovariectomy. Network pharmacological analysis and molecular docking indicated that the PI3K/AKT axis was a potential target, with oxidative stress response and nuclear membrane function being key mechanisms. Consequently, the effects of Lut on the PI3K/AKT axis and pyroptosis were investigated. In vivo data revealed that the PI3K/AKT axis was deactivated following ovariectomy, and Lut restored the phosphorylation of key proteins, thereby reactivating the axis. Additionally, Lut alleviated osteoblast pyroptosis and mitochondrial abnormalities induced by ovariectomy. In vitro, Lut intervention mitigated the inhibition of the PI3K/AKT axis and osteogenesis, as well as HO-induced pyroptosis. Furthermore, Lut attenuated ROS accumulation and mitochondrial dysfunction. The effects of Lut, including osteogenesis restoration, anti-pyroptosis, and mitochondrial maintenance, were all reversed with LY294002 (a PI3K/AKT pathway inhibitor).
CONCLUSION
In summary, Lut could improve mitochondrial dysfunction, alleviate GSDME-mediated pyroptosis and maintain osteogenesis via activating the PI3K/AKT axis, offering a new therapeutic strategy for PMOP.
Topics: Animals; Female; Pyroptosis; Osteoporosis, Postmenopausal; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Mice; Osteoblasts; Ovariectomy; Signal Transduction; Oxidative Stress; Luteolin; Osteogenesis; Molecular Docking Simulation; Disease Models, Animal; Humans; Mice, Inbred C57BL; Mitochondria; Network Pharmacology; Cell Line
PubMed: 38547625
DOI: 10.1016/j.phymed.2024.155516 -
Cell Chemical Biology Sep 2023Type I diabetes (T1D) impairs bone accrual in patients, but the mechanism is unclear. Here in a murine monogenic model for T1D, we demonstrate that diabetes suppresses...
Type I diabetes (T1D) impairs bone accrual in patients, but the mechanism is unclear. Here in a murine monogenic model for T1D, we demonstrate that diabetes suppresses bone formation resulting in a rapid loss of both cortical and trabecular bone. Single-cell RNA sequencing uncovers metabolic dysregulation in bone marrow osteogenic cells of diabetic mice. In vivo stable isotope tracing reveals impaired glycolysis in diabetic bone that is highly responsive to insulin stimulation. Remarkably, deletion of the insulin receptor reduces cortical but not trabecular bone. Increasing glucose uptake by overexpressing Glut1 in osteoblasts exacerbates bone defects in T1D mice. Conversely, activation of glycolysis by Pfkfb3 overexpression preserves both trabecular and cortical bone mass in the face of diabetes. The study identifies defective glucose metabolism in osteoblasts as a pathogenic mechanism for osteopenia in T1D, and furthermore implicates boosting osteoblast glycolysis as a potential bone anabolic therapy.
Topics: Humans; Mice; Animals; Diabetes Mellitus, Type 1; Diabetes Mellitus, Experimental; Osteoblasts; Bone Density; Glycolysis
PubMed: 37562406
DOI: 10.1016/j.chembiol.2023.07.003 -
Frontiers in Endocrinology 2023Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover,...
INTRODUCTION
Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts.
METHODS
We first examined the effect of the calciotropic hormone 1,25-dihydroxyvitamin D [1,25(OH)D] on transcription in osteoblasts. We next generated mice with a conditional deletion of in the osteoblast cell lineage under control of the paired related homeobox 1 promoter and mice with a conditional knockdown in osteoclasts under control of the Lysozyme promoter. Mice were examined under basal conditions or after treatment with either the bone anabolic vitamin D analog WY 1048 or with 1,25(OH)D.
RESULTS AND DISCUSSION
We show that expression is induced by 1,25(OH)D in osteoblasts and is associated with enrichment of the vitamin D receptor in an intronic region of the Nrp2 gene. In male mice, conditional deletion of in osteoblast precursors and mature osteoblasts recapitulated the bone phenotype of global knockout mice, with a reduced cortical cross-sectional tissue area and lower trabecular bone content. However, female mice with reduced osteoblastic expression display a reduced cross-sectional tissue area but have a normal trabecular bone mass. Treatment with the vitamin D analog WY 1048 (0.4 μg/kg/d, 14 days, ip) resulted in a similar increase in bone mass in both genotypes and genders. Deleting from the osteoclast lineage did not result in a bone phenotype, even though osteoclastogenesis of hematopoietic cells derived from mutant mice was significantly increased. Moreover, treatment with a high dose of 1,25(OH)D (0.5 μg/kg/d, 6 days, ip), to induce osteoclast-mediated bone resorption, resulted in a similar reduction in trabecular and cortical bone mass. In conclusion, osteoblastic expression is suggested to regulate bone homeostasis in a sex-specific manner.
Topics: Animals; Female; Male; Mice; Cancellous Bone; Cholecalciferol; Cross-Sectional Studies; Neuropilin-2; Osteoblasts; Vascular Endothelial Growth Factor A; Calcitriol
PubMed: 37600714
DOI: 10.3389/fendo.2023.1223021 -
Journal of Endocrinological... Jun 2024Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of...
PURPOSE
Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly.
METHODS
We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae.
RESULTS
We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (- 30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5 days post-fertilization.
CONCLUSION
This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.
Topics: Osteoblasts; Animals; Mice; Cell Proliferation; Zebrafish; Bone Development; Cell Differentiation; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Apoptosis
PubMed: 38159174
DOI: 10.1007/s40618-023-02281-3 -
Life Sciences in Space Research Aug 2023Zebrafish cultured scales have been used effectively to study cellular and molecular responses of bone cells. In order to expose zebrafish scales to simulated...
Zebrafish cultured scales have been used effectively to study cellular and molecular responses of bone cells. In order to expose zebrafish scales to simulated microgravity (SMG) and/or vibration, we first determined via apoptosis staining whether cells of the scale survive in culture for two days and hence, we restricted our analyses to two-day durations. Next, we measured the effects of SMG and vibration on cell death, osteoclast tartrate-resistant acid phosphatase, and osteoblast alkaline phosphatase activity and on the number of Runx2a positive cells. We found that during the SMG treatment, osteoclast tartrate-resistant acid phosphatase activity increased on average, while the number of Runx2a positive cells decreased significantly. In contrast, SMG exposure caused a decrease in osteoblast activity. The vibration treatment showed an increase, on average, in the osteoblast alkaline phosphatase activity. This study demonstrates the effect of SMG and vibration on zebrafish scales and the effects of SMG on bone cells. We also show that zebrafish scales can be used to examine the effects of SMG on bone maintenance.
Topics: Animals; Osteoclasts; Zebrafish; Alkaline Phosphatase; Tartrate-Resistant Acid Phosphatase; Vibration; Weightlessness; Osteoblasts
PubMed: 37481306
DOI: 10.1016/j.lssr.2023.05.002 -
Arthritis & Rheumatology (Hoboken, N.J.) Dec 2023IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of...
OBJECTIVE
IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of bone-forming osteoblasts and bone-resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology.
METHODS
To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6-14) lacking BCL3 (Bcl3 ) and wild-type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3 mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3-7) were assessed. Osteoclast differentiation and function in Bcl3 mice (n = 3-5) was assessed. Adult 20-week Bcl3 and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3 mice (n = 11-13).
RESULTS
Evaluation of Bcl3 mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3 cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis-induced osteophytogenesis, Bcl3 mice exhibited decreased pathological osteophyte formation (P < 0.05).
CONCLUSION
Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology.
Topics: Animals; Mice; Bone and Bones; Bone Density; Cell Differentiation; NF-kappa B; Osteoblasts; Osteoclasts; Osteogenesis; B-Cell Lymphoma 3 Protein
PubMed: 37410754
DOI: 10.1002/art.42639 -
Anatomical Science International Sep 2023Osteoblasts alignment and migration are involved in the directional formation of bone matrix and bone remodeling. Many studies have demonstrated that mechanical...
Osteoblasts alignment and migration are involved in the directional formation of bone matrix and bone remodeling. Many studies have demonstrated that mechanical stretching controls osteoblast morphology and alignment. However, little is known about its effects on osteoblast migration. Here, we investigated changes in the morphology and migration of preosteoblastic MC3T3-E1 cells after the removal of continuous or cyclic stretching. Actin staining and time-lapse recording were performed after stretching removal. The continuous and cyclic groups showed parallel and perpendicular alignment to the stretch direction, respectively. A more elongated cell morphology was observed in the cyclic group than in the continuous group. In both stretch groups, the cells migrated in a direction roughly consistent with the cell alignment. Compared to the other groups, the cells in the cyclic group showed an increased migration velocity and were almost divided in the same direction as the alignment. To summarize, our study showed that mechanical stretching changed cell alignment and morphology in osteoblasts, which affected the direction of migration and cell division, and velocity of migration. These results suggest that mechanical stimulation may modulate the direction of bone tissue formation by inducing the directional migration and cell division of osteoblasts.
Topics: Actins; Osteoblasts; Bone and Bones; Cell Division
PubMed: 37022568
DOI: 10.1007/s12565-023-00716-8 -
International Journal of Molecular... Oct 2023Deubiquitinases (DUBs) are essential for bone remodeling by regulating the differentiation of osteoblast and osteoclast. USP17 encodes for a deubiquitinating enzyme,...
Deubiquitinases (DUBs) are essential for bone remodeling by regulating the differentiation of osteoblast and osteoclast. USP17 encodes for a deubiquitinating enzyme, specifically known as ubiquitin-specific protease 17, which plays a critical role in regulating protein stability and cellular signaling pathways. However, the role of USP17 during osteoblast differentiation has not been elusive. In this study, we initially investigated whether USP17 could regulate the differentiation of osteoblasts. Moreover, overexpression experiments were conducted to assess the impact on osteoblast differentiation induced by bone morphogenetic protein 4 (BMP4). The positive effect was confirmed through alkaline phosphatase (ALP) expression and activity studies since ALP is a representative marker of osteoblast differentiation. To confirm this effect, knockdown was performed, and its impact on BMP4-induced osteoblast differentiation was examined. As expected, knockdown of led to the suppression of both ALP expression and activity. Mechanistically, it was observed that USP17 interacted with Osterix (Osx), which is a key transcription factor involved in osteoblast differentiation. Furthermore, overexpression of led to an increase in Osx protein levels. Thus, to investigate whether this effect was due to the intrinsic function of USP17 in deubiquitination, protein stabilization experiments and ubiquitination analysis were conducted. An increase in Osx protein levels was attributed to an enhancement in protein stabilization via USP17-mediated deubiquitination. In conclusion, USP17 participates in the deubiquitination of Osx, contributing to its protein stabilization, and ultimately promoting the differentiation of osteoblasts.
Topics: Sp7 Transcription Factor; Osteogenesis; Osteoblasts; Cell Differentiation; Protein Stability; Deubiquitinating Enzymes
PubMed: 37894935
DOI: 10.3390/ijms242015257 -
Oral Diseases Apr 2024Cell pyroptosis is implicated in progressive bone loss in dental inflammatory diseases. We induced caspase-3/Gasdermin E (GSDME)-mediated pyroptosis in osteoblast-like...
OBJECTIVE
Cell pyroptosis is implicated in progressive bone loss in dental inflammatory diseases. We induced caspase-3/Gasdermin E (GSDME)-mediated pyroptosis in osteoblast-like cells and evaluated the effects on osteogenesis.
MATERIALS AND METHODS
Osteoblast-like cells were treated with various concentrations of sodium butyrate (NaB) to identify the most appropriate for inducing caspase-3/GSDME-mediated pyroptosis. Cells were divided into control, NaB and NaB+Ac-DEVD-CHO (specific caspase-3 inhibitor) groups. Pyroptosis level was evaluated by immunofluorescence, morphological observation, flow cytometry, lactate dehydrogenase (LDH) release assays, mRNA and protein levels of pyroptosis-related markers. Then, inflammation level, osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression and osteogenic function were detected.
RESULTS
Treatment with 10 mM NaB increased caspase-3 expression, GSDME cleavage, LDH release and the number of pyroptotic cells, with morphologic changes, indicating GSDME-mediated pyroptosis induction. The pyroptosis-related changes were abolished by caspase-3 inhibition. Caspase-3/GSDME-mediated pyroptosis triggered the expression of inflammatory cytokines and RANKL, downregulated alkaline phosphatase (ALP) activity, mineralisation level, mRNA and protein levels of multiple osteogenic markers. These effects were partly reversed by Ac-DEVD-CHO.
CONCLUSION
Caspase-3/GSDME-mediated pyroptosis induced by NaB activated the inflammatory response, reduced osteogenic differentiation and disturbed OPG/RANKL axis, leading to osteogenic dysfunction in osteoblast-like cells.
Topics: Osteoblasts; Pyroptosis; Osteogenesis; Osteoprotegerin; Humans; Caspase 3; RANK Ligand; Butyric Acid; Cells, Cultured; Gasdermins
PubMed: 37004144
DOI: 10.1111/odi.14579