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Laboratory Investigation; a Journal of... Feb 2019Osteoblast differentiation plays a critical role in bone formation and maintaining balance in bone remodeling. Runt-related transcription factor 2 (Runx2) is a central...
Osteoblast differentiation plays a critical role in bone formation and maintaining balance in bone remodeling. Runt-related transcription factor 2 (Runx2) is a central transcription factor regulating osteoblast differentiation and promoting bone mineralization. Until now, the molecular regulatory basis and especially the gene regulatory network of osteogenic differentiation have been unclear. Krüppel-like factor 2 (KLF2) is a zinc finger structure and DNA-binding transcription factor. The current study aimed to investigate the physiological function of KLF2 in osteoblast differentiation. Our results indicate that KLF2 is expressed in pre-osteoblast MC3T3-E1 cells and primary osteoblasts. Interestingly, KLF2 expression is increased in osteoblasts during the osteoblastic differentiation process. Overexpression of KLF2 in MC3T3-E1 cells promoted the expression of the osteoblastic differentiation marker genes Alp, Osx, and Ocn, and stimulated mineralization by increasing Runx2 expression at both the mRNA and protein levels. In contrast, knockdown of KLF2 produced the opposite effects. Importantly, we found that KLF2 could physically interact with Runx2. KLF2 promoted osteoblast differentiation by regulating Runx2 and physically interacting with Runx2. Taken together, the findings of this study identify KLF2 as a novel regulator of osteoblast differentiation. Our findings suggest that KLF2 might be a new therapeutic target for bone disease.
Topics: Animals; Cell Differentiation; Cell Line; Core Binding Factor Alpha 1 Subunit; Gene Knockdown Techniques; Human Umbilical Vein Endothelial Cells; Humans; Kruppel-Like Transcription Factors; Mice; Osteoblasts
PubMed: 30429507
DOI: 10.1038/s41374-018-0149-x -
Arbutin ameliorates glucocorticoid-induced osteoporosis through activating autophagy in osteoblasts.Experimental Biology and Medicine... Jul 2021Chronic long-term glucocorticoid use causes osteoporosis partly by interrupting osteoblast homeostasis and exacerbating bone loss. Arbutin, a natural hydroquinone...
Chronic long-term glucocorticoid use causes osteoporosis partly by interrupting osteoblast homeostasis and exacerbating bone loss. Arbutin, a natural hydroquinone glycoside, has been reported to have biological activities related to the differentiation of osteoblasts and osteoclasts. However, the role and underlying mechanism of arbutin in glucocorticoid-induced osteoporosis are elusive. In this study, we demonstrated that arbutin administration ameliorated osteoporotic disorders in glucocorticoid dexamethasone (Dex)-induced mouse model, including attenuating the loss of bone mass and trabecular microstructure, promoting bone formation, suppressing bone resorption, and activating autophagy in bone tissues. Furthermore, Dex-stimulated mouse osteoblastic MC3T3-E1 cells were treated with arbutin. Arbutin treatment rescued Dex-induced repression of osteoblast differentiation and mineralization, the downregulation of osteogenic gene expression, reduced autophagic marker expression, and decreased autophagic puncta formation. The application of autophagy inhibitor 3-MA decreased autophagy, differentiation, and mineralization of MC3T3-E1 cells triggered by arbutin. Taken together, our findings suggest that arbutin treatment fends off glucocorticoid-induced osteoporosis, partly through promoting differentiation and mineralization of osteoblasts by autophagy activation.
Topics: Animals; Arbutin; Autophagy; Cell Line; Dexamethasone; Glucocorticoids; Male; Mice; Mice, Inbred C57BL; Osteoblasts; Osteoporosis
PubMed: 33757338
DOI: 10.1177/15353702211002136 -
International Journal of Molecular... May 2021has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to...
has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 μM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 μM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 μM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 μM) increased the bone morphogenetic protein (BMP)-Smad1/5/8 and Wnt-β-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 μM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.
Topics: Acetophenones; Animals; Bone Morphogenetic Protein 2; Calcification, Physiologic; Cell Death; Cell Differentiation; Cell Line; Cell Movement; Core Binding Factor Alpha 1 Subunit; MAP Kinase Signaling System; Mice; Osteoblasts; Osteogenesis; Wnt3 Protein
PubMed: 34066458
DOI: 10.3390/ijms22094924 -
The FEBS Journal Oct 2020Gene expression in extant animals might reveal how skeletal cells have evolved over the past 500 million years. The cells that make up cartilage (chondrocytes) and bone... (Review)
Review
Gene expression in extant animals might reveal how skeletal cells have evolved over the past 500 million years. The cells that make up cartilage (chondrocytes) and bone (osteoblasts) express many of the same genes, but they also have important molecular differences that allow us to distinguish them as separate cell types. For example, traditional studies of later-diverged vertebrates, such as mouse and chick, defined the genes Col2a1 and sex-determining region Y-box 9 as cartilage-specific. However, recent studies have shown that osteoblasts of earlier-diverged vertebrates, such as frog, gar, and zebrafish, express these 'chondrogenic' markers. In this review, we examine the resulting hypothesis that chondrogenic gene expression became repressed in osteoblasts over evolutionary time. The amphibian is an underexplored skeletal model that is uniquely positioned to address this hypothesis, especially given that it diverged when life transitioned from water to land. Given the relationship between phylogeny and ontogeny, a novel discovery for skeletal cell evolution might bolster our understanding of skeletal cell development.
Topics: Animals; Chondrogenesis; Osteoblasts
PubMed: 31994313
DOI: 10.1111/febs.15228 -
Cells Sep 2021The complex multidimensional skeletal organization can adapt its structure in accordance with external contexts, demonstrating excellent self-renewal capacity. Thus,... (Review)
Review
The complex multidimensional skeletal organization can adapt its structure in accordance with external contexts, demonstrating excellent self-renewal capacity. Thus, optimal extracellular environmental properties are critical for bone regeneration and inextricably linked to the mechanical and biological states of bone. It is interesting to note that the microstructure of bone depends not only on genetic determinants (which control the bone remodeling loop through autocrine and paracrine signals) but also, more importantly, on the continuous response of cells to external mechanical cues. In particular, bone cells sense mechanical signals such as shear, tensile, loading and vibration, and once activated, they react by regulating bone anabolism. Although several specific surrounding conditions needed for osteoblast cells to specifically augment bone formation have been empirically discovered, most of the underlying biomechanical cellular processes underneath remain largely unknown. Nevertheless, exogenous stimuli of endogenous osteogenesis can be applied to promote the mineral apposition rate, bone formation, bone mass and bone strength, as well as expediting fracture repair and bone regeneration. The following review summarizes the latest studies related to the proliferation and differentiation of osteoblastic cells, enhanced by mechanical forces or supplemental signaling factors (such as trace metals, nutraceuticals, vitamins and exosomes), providing a thorough overview of the exogenous osteogenic agents which can be exploited to modulate and influence the mechanically induced anabolism of bone. Furthermore, this review aims to discuss the emerging role of extracellular stimuli in skeletal metabolism as well as their potential roles and provide new perspectives for the treatment of bone disorders.
Topics: Anabolic Agents; Animals; Bone Regeneration; Cell Differentiation; Humans; Mechanotransduction, Cellular; Osteoblasts; Signal Transduction
PubMed: 34572032
DOI: 10.3390/cells10092383 -
Molecular and Cellular Biology Apr 2015Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show that Runx3, like Runx2, is...
Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show that Runx3, like Runx2, is expressed in precommitted embryonic osteoblasts and that Runx3-deficient mice develop severe congenital osteopenia. Runx3-deficient osteoblast-specific (Runx3(fl/fl)/Col1α1-cre), but not chondrocyte-specific (Runx3(fl/fl)/Col1α2-cre), mice are osteopenic. This demonstrates that an osteoblastic cell-autonomous function of Runx3 is required for proper osteogenesis. Bone histomorphometry revealed that decreased osteoblast numbers and reduced mineral deposition capacity in Runx3-deficient mice cause this bone formation deficiency. Neonatal bone and cultured primary osteoblast analyses revealed a Runx3-deficiency-associated decrease in the number of active osteoblasts resulting from diminished proliferation and not from enhanced osteoblast apoptosis. These findings are supported by Runx3-null culture transcriptome analyses showing significant decreases in the levels of osteoblastic markers and increases in the levels of Notch signaling components. Thus, while Runx2 is mandatory for the osteoblastic lineage commitment, Runx3 is nonredundantly required for the proliferation of these precommitted cells, to generate adequate numbers of active osteoblasts. Human RUNX3 resides on chromosome 1p36, a region that is associated with osteoporosis. Therefore, RUNX3 might also be involved in human bone mineralization.
Topics: Animals; Apoptosis; Bone Development; Bone Diseases, Metabolic; Bone and Bones; Cells, Cultured; Core Binding Factor Alpha 3 Subunit; Gene Deletion; Gene Expression Regulation, Developmental; Humans; Mice; Mice, Knockout; Osteoblasts; Osteogenesis; Transcriptome
PubMed: 25605327
DOI: 10.1128/MCB.01106-14 -
Stem Cell Research & Therapy Jun 2022Oncostatin M receptor (OSMR), as one of the receptors for oncostatin M (OSM), has previously been shown to mediate the stimulatory role of OSM in osteoclastogenesis and...
BACKGROUND
Oncostatin M receptor (OSMR), as one of the receptors for oncostatin M (OSM), has previously been shown to mediate the stimulatory role of OSM in osteoclastogenesis and bone resorption. However, it remains to be clarified whether and how OSMR affects the differentiation of osteoblasts.
METHODS
The expression level of OSMR during osteoblast and adipocyte differentiation was examined. The role of OSMR in the differentiation was investigated using in vitro gain-of-function and loss-of-function experiments. The mechanisms by which OSMR regulates bone cell differentiation were explored. Finally, in vivo function of OSMR in cell fate determination and bone homeostasis was studied after transplantation of OSMR-silenced bone marrow stromal cells (BMSCs) to the marrow of ovariectomized mice.
RESULTS
OSMR was regulated during osteogenic and adipogenic differentiation of marrow stromal progenitor cells and increased in the metaphysis of ovariectomized mice. OSMR suppressed osteogenic differentiation and stimulated adipogenic differentiation of progenitor cells. Mechanistic investigations showed that OSMR inhibited extracellular signal-regulated kinase (ERK) and autophagy signaling. The downregulation of autophagy, which was mediated by ERK inhibition, suppressed osteogenic differentiation of progenitor cells. Additionally, inactivation of ERK/autophagy signaling attenuated the stimulation of osteogenic differentiation induced by Osmr siRNA. Furthermore, transplantation of BMSCs in which OSMR was silenced to the marrow of mice promoted osteoblast differentiation, attenuated fat accumulation and osteoclast differentiation, and thereby relieved the osteopenic phenotype in the ovariectomized mice.
CONCLUSIONS
Our study has for the first time established the direct role of OSMR in regulating osteogenic differentiation of marrow stromal progenitor cells through ERK-mediated autophagy signaling. OSMR thus contributes to bone homeostasis through dual regulation of osteoblasts and osteoclasts. It also suggests that OSMR may be a potential target for the treatment of metabolic disorders such as osteoporosis.
Topics: Animals; Autophagy; Cell Differentiation; Extracellular Signal-Regulated MAP Kinases; MAP Kinase Signaling System; Mice; Oncostatin M Receptor beta Subunit; Osteoblasts; Osteogenesis
PubMed: 35765036
DOI: 10.1186/s13287-022-02958-1 -
Bone Jul 2018Glucocorticoid treatment, a major cause of drug-induced osteoporosis and fractures, is widely used to treat inflammatory conditions and diseases. By contrast, mechanical...
Glucocorticoid treatment, a major cause of drug-induced osteoporosis and fractures, is widely used to treat inflammatory conditions and diseases. By contrast, mechanical loading increases bone mass and decreases fracture risk. With these relationships in mind, we investigated whether mechanical loading interacts with GC treatment in bone. Three-month-old female C57BL/6 mice were treated with high-dose prednisolone (15 mg/60 day pellets/mouse) or vehicle for two weeks. During the treatment, right tibiae were subjected to short periods of cyclic compressive loading three times weekly, while left tibiae were used as physiologically loaded controls. The bones were analyzed using peripheral quantitative computed tomography, histomorphometry, real-time PCR, three-point bending and Fourier transform infrared micro-spectroscopy. Loading alone increased trabecular volumetric bone mineral density (vBMD), cortical thickness, cortical area, osteoblast-associated gene expression, osteocyte- and osteoclast number, and bone strength. Prednisolone alone decreased cortical area and thickness and osteoblast-associated gene expression. Importantly, prednisolone treatment decreased the load-induced increase in trabecular vBMD by 57% (p < 0.001) and expression of osteoblast-associated genes, while completely abolishing the load-induced increase in cortical area, cortical thickness, number of osteocytes and osteoclasts, and bone strength. When combined, loading and prednisolone decreased the collagen content. In conclusion, high-dose prednisolone treatment strongly inhibits the loading-induced increase in trabecular BMD, and abolishes the loading-induced increase in cortical bone mass. This phenomenon could be due to prednisolone inhibition of osteoblast differentiation and function.
Topics: Anabolic Agents; Animals; Cancellous Bone; Collagen; Female; Gene Expression Regulation; Mice, Inbred C57BL; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Prednisolone; Weight-Bearing
PubMed: 29635039
DOI: 10.1016/j.bone.2018.04.002 -
International Journal of Molecular... May 2021Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, and therapeutic targeting of these cells is considered both for malignant and non-malignant diseases.... (Comparative Study)
Comparative Study
Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, and therapeutic targeting of these cells is considered both for malignant and non-malignant diseases. We analyzed global proteomic profiles for osteoblasts derived from ten and MSCs from six healthy individuals, and we quantified 5465 proteins for the osteoblasts and 5420 proteins for the MSCs. There was a large overlap in the profiles for the two cell types; 156 proteins were quantified only in osteoblasts and 111 proteins only for the MSCs. The osteoblast-specific proteins included several extracellular matrix proteins and a network including 27 proteins that influence intracellular signaling (Wnt/Notch/Bone morphogenic protein pathways) and bone mineralization. The osteoblasts and MSCs showed only minor age- and sex-dependent proteomic differences. Finally, the osteoblast and MSC proteomic profiles were altered by ex vivo culture in serum-free media. We conclude that although the proteomic profiles of osteoblasts and MSCs show many similarities, we identified several osteoblast-specific extracellular matrix proteins and an osteoblast-specific intracellular signaling network. Therapeutic targeting of these proteins will possibly have minor effects on MSCs. Furthermore, the use of ex vivo cultured osteoblasts/MSCs in clinical medicine will require careful standardization of the ex vivo handling of the cells.
Topics: Aged; Bone Marrow Cells; Female; Gene Expression Regulation; Humans; Male; Mesenchymal Stem Cells; Middle Aged; Osteoblasts; Proteomics; Wnt Signaling Pathway
PubMed: 34073480
DOI: 10.3390/ijms22115665 -
Tissue Engineering and Regenerative... Aug 2019Xanthine derivatives have been used to treat a variety of medical conditions including respiratory disease and neural degeneration. However, few studies have reported...
BACKGROUND
Xanthine derivatives have been used to treat a variety of medical conditions including respiratory disease and neural degeneration. However, few studies have reported their effects on bone regeneration. Therefore, we investigated the effects of KPR-A148, a synthetic xanthine derivative on osteoblast differentiation and bone regeneration .
METHODS
The cytotoxicity of KPR-A148 was evaluated using MC3T3-E1 cells by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltertrazolium bromide assay. The effects of KPR-A148 on osteoblast differentiation were examined by alkaline phosphatase staining, Alizarin red S staining, and real-time PCR of osteoblast differentiation marker genes. To investigate the effects of KPR-A148 on bone regeneration, a KPR-A148-containing collagen sponge was implanted into a mouse calvarial defect and KPR-A148 was injected twice, weekly. Bone regeneration was evaluated quantitatively by micro-CT and qualitatively by hematoxylin and eosin, as well as Masson's Trichrome staining.
RESULTS
KPR-A148 did not show toxicity in the MC3T3-E1 cells and promoted osteoblast differentiation in a concentration-dependent manner. 10 μM of KPR-A148 showed the most significant effect on alkaline phospatase staining and matrix mineralization. KPR-A148 increased the expression of osteoblast marker genes in both the early and late stages of differentiation. In addition, KPR-A148 significantly induced new bone formation in the calvarial defect model.
CONCLUSION
These results demonstrate that KPR-A148 strongly induces osteoblast differentiation and new bone formation. Therefore, it could be used as a potential therapeutic agent for regenerating bone following its destruction by disease or trauma.
Topics: Alkaline Phosphatase; Animals; Biomarkers; Bone Regeneration; Cell Differentiation; Cell Line; Collagen; Mice; Osteoblasts; Osteogenesis; Xanthine
PubMed: 31413944
DOI: 10.1007/s13770-019-00200-3