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Frontiers in Bioscience (Landmark... Oct 2022Recently, single-cell RNA sequencing (scRNA-seq) technology was increasingly used to study transcriptomics at a single-cell resolution, scRNA-seq analysis was...
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 ( and ). 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.
Topics: Humans; RNA-Seq; Osteoblasts; Transcriptome; Cell Differentiation; Osteogenesis; Gene Expression Profiling
PubMed: 36336853
DOI: 10.31083/j.fbl2710295 -
Archivum Immunologiae Et Therapiae... Oct 2014Vascular endothelial growth factor-A (VEGF) is a key regulator of physiological hemangiogenesis during development, postnatal growth, and homeostasis. It is well known... (Review)
Review
Vascular endothelial growth factor-A (VEGF) is a key regulator of physiological hemangiogenesis during development, postnatal growth, and homeostasis. It is well known that VEGF is required for effective coupling of angiogenesis to endochondral and membranous bone formation during skeletal development. However, less well known are the roles of VEGF in regulating the differentiation and/or functions of skeletal cells such as chondrocytes, osteoblasts, and osteoclasts. In this review, we discuss some of these functions. During early skeletal development, VEGF is important for the survival of chondrocytes in the hypoxic regions of the cartilage models of future bones, the vascularization of developing bones and proliferation and differentiation of osteoblastic cells. Postnatally, osteoblast-derived VEGF is critical for maintaining bone homeostasis by stimulating the differentiation of mesenchymal stem cells to osteoblasts and repressing their differentiation to adipocytes. Recent data indicate that these effects of VEGF on osteogenic/adipogenic stem cell fates are based on an intracellular (intracrine) mechanism. In contrast, osteoblast-derived VEGF is also known to stimulate the differentiation of monocytes to osteoclasts by a paracrine mechanism. Mice with VEGF-deficient osteoblastic lineage cells exhibit age-dependent loss of bone mass and an increase in bone marrow fat. These changes are similar to the changes associated with osteoporosis in humans. Thus, a better understanding of the intracellular mechanisms by which VEGF regulates osteoblastic and adipogenic differentiation may lead to the identification of new targets for therapies to prevent osteoporotic bone loss.
Topics: Animals; Cell Differentiation; Chondrocytes; Homeostasis; Humans; Mesenchymal Stem Cells; Mice; Neovascularization, Physiologic; Osteoblasts; Osteoclasts; Osteogenesis; Vascular Endothelial Growth Factor A
PubMed: 24699630
DOI: 10.1007/s00005-014-0285-y -
Bone May 2020Non-traditional bisphosphonates, that is, bisphosphonates that do not inhibit osteoclast viability or function, were initially reported in the 1990s by Socrates...
Non-traditional bisphosphonates, that is, bisphosphonates that do not inhibit osteoclast viability or function, were initially reported in the 1990s by Socrates Papapoulos' group. Originally designed to study the role of the R1 residue of aminobisphosphonates on bisphosphonate affinity for hydroxyapatite, these modified bisphosphonates retained similar affinity for mineralized bone as their parent compounds, but they lacked the potential to inhibit the mevalonate pathway or bone resorption. We found that, similar to classical bisphosphonates, these non-traditional compounds prevented osteoblast and osteocyte apoptosis in vitro through a pathway that requires the expression of the gap junction protein connexin 43, and the activation of the Src/MEK/ERK signaling pathway. Furthermore, one of those compounds named IG9402 (also known as amino-olpadronate or lidadronate), was able to inhibit osteoblast and osteocyte apoptosis, without affecting osteoclast number or bone resorption in vivo in a model of glucocorticoid-induced osteoporosis. IG9402 administration also ameliorated the decrease in bone mass and in bone mechanical properties induced by glucocorticoids. Similarly, IG9402 prevented apoptosis of osteoblastic cells in a model of immobilization due to hindlimb unloading. However, in this case, the bisphosphonate was not able to preserve the bone mass, and only partially prevented the decrease in bone mechanical properties induced by immobilization. The effect of IG9402 administration was also tested in a mouse model of masticatory hypofunction through the induction of masseter muscle atrophy by unilateral injection of botulinum toxin type A (BoNTA). IG9402 partially inhibited the loss of trabecular bone microstructure in the mandibular condyle, but not the decrease in masseter muscle mass induced by BoNTA administration. In summary, these non-traditional bisphosphonates that lack anti-resorptive activity but are able to preserve osteoblast and osteocyte viability could constitute useful tools to study the consequences of preventing apoptosis of osteoblastic cells in animal models. Furthermore, they could be used to treat conditions associated with reduced bone mass and increased bone fragility in which a reduction of bone remodeling is not desirable.
Topics: Animals; Apoptosis; Bone Resorption; Diphosphonates; Mice; Osteoblasts; Osteoclasts; Osteocytes
PubMed: 32112989
DOI: 10.1016/j.bone.2020.115301 -
TheScientificWorldJournal Apr 2010Bone tissue renovation is a dynamic event in which osteoblasts and osteoclasts are responsible for the turnover between bone formation and bone resorption, respectively.... (Review)
Review
Bone tissue renovation is a dynamic event in which osteoblasts and osteoclasts are responsible for the turnover between bone formation and bone resorption, respectively. During bone development, extracellular matrix remodeling is required for osteoblast differentiation and the process is largely mediated by the proteolytic activity of extracellular matrix metalloproteinases (MMPs), which play a fundamental role in osteoblast migration, unmineralized matrix degradation, and cell invasion. The recent advances towards investigation in osteogenesis have provided significant information about the transcriptional regulation of several genes, including MMPs, by the expression of crucial transcription factors like NFAT, ATF4, osterix, TAZ, and Cbfa-1-responsive elements. Evidence from gene knock-out studies have shown that bone formation is, at least in part, mediated by nitric oxide (NO), since mice deficient in endothelial nitric oxide synthase (eNOS) and mice deficient in the eNOS downstream effector (cGMP)-dependent protein kinase (PKG) show bone abnormalities, while inducible NOS (iNOS) null mice also show imbalances in bone osteogenesis and abnormalities in bone healing. Recently, in vitro data showed that Cbfa-1 and the MAPK pathways were crucial for osteoblastic cell differentiation, and NO was found to play a significant role. This article sheds light on some of the mechanisms that may influence NO-mediated actions in bone development.
Topics: Animals; Bone Development; Cell Differentiation; Cell Proliferation; Mice; Nitric Oxide; Osteoblasts; Transcription Factors
PubMed: 20419275
DOI: 10.1100/tsw.2010.58 -
Reumatologia Clinica Sep 2011Bone tissue is constantly renewed by the coordinated action of osteoclasts and osteoblasts. Osteocytes have a prominent role in the onset of bone remodeling at selected...
Bone tissue is constantly renewed by the coordinated action of osteoclasts and osteoblasts. Osteocytes have a prominent role in the onset of bone remodeling at selected sites of the skeleton. These and other osteoblastic cells produce a variety of mediators that modulate the differentiation of osteoclast precursors, a critical step for bone resorption to occur. Among them, M-CSF and RANKL are especially important.
Topics: Bone Remodeling; Cell Communication; Humans; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B
PubMed: 21924211
DOI: 10.1016/j.reuma.2011.03.003 -
FEBS Open Bio Aug 2020Osteoblasts release adenosine triphosphate (ATP) out of the cell following mechanical stress. Although it is well established that extracellular ATP affects bone...
Osteoblasts release adenosine triphosphate (ATP) out of the cell following mechanical stress. Although it is well established that extracellular ATP affects bone metabolism via P2 receptors [such as purinergic receptor P2X7 (P2X7R) and purinergic receptor P2Y2 (P2Y2R)], the mechanism of ATP release from osteoblasts remains unknown. Recently, a vesicular nucleotide transporter [VNUT, solute carrier family 17 member 9 (SLC17A9)] that preserves ATP in vesicles has been identified. The purpose of this study was to elucidate the role of VNUT in osteoblast bone metabolism. mRNA and protein expression of VNUT were confirmed in mouse bone and in osteoblasts by quantitative real-time PCR (qPCR) and immunohistochemistry. Next, when compressive force was applied to MC3T3-E1 cells by centrifugation, the expression of Slc17a9, P2x7r, and P2y2r was increased concomitant with an increase in extracellular ATP levels. Furthermore, compressive force decreased the osteoblast differentiation capacity of MC3T3-E1 cells. shRNA knockdown of Slc17a9 in MC3T3-E1 cells reduced levels of extracellular ATP and also led to increased osteoblast differentiation after the application of compressive force as assessed by qPCR analysis of osteoblast markers such as Runx2, Osterix, and alkaline phosphatase (ALP) as well as ALP activity. Consistent with these observations, knockdown of P2x7r or P2y2r by siRNA partially rescued the downregulation of osteoblast differentiation markers, caused by mechanical loading. In conclusion, our results demonstrate that VNUT is expressed in osteoblasts and that VNUT inhibits osteoblast differentiation in response to compressive force by mechanisms related to ATP release and P2X7R and/or P2Y2R activity.
Topics: 3T3 Cells; Animals; Cell Differentiation; Cells, Cultured; Mice; Nucleotide Transport Proteins; Osteoblasts
PubMed: 32592329
DOI: 10.1002/2211-5463.12918 -
Scientific Reports Mar 2017The present study aimed to investigate the role of magnitude in adaptive response of osteoblasts exposed to compressive stress. Murine primary osteoblasts and MC3T3-E1...
The present study aimed to investigate the role of magnitude in adaptive response of osteoblasts exposed to compressive stress. Murine primary osteoblasts and MC3T3-E1 cells were exposed to compressive stress (0, 1, 2, 3, 4, and 5 g/cm) in 3D culture. Cell viability was evaluated, and expression levels of Runx2, Alp, Ocn, Rankl, and Opg were examined. ALP activity in osteoblasts and TRAP activity in RAW264.7 cells co-cultured with MC3T3-E1 cells were assayed. Results showed that compressive stress within 5.0 g/cm did not influence cell viability. Both osteoblastic and osteoblast-regulated osteoclastic differentiation were enhanced at 2 g/cm. An increase in stress above 2 g/cm did not enhance osteoblastic differentiation further but significantly inhibited osteoblast-regualted osteoclastic differentiation. This study suggested that compressive stress regulates osteoblastic and osteoclastic differentiation through osteoblasts in a magnitude-dependent manner.
Topics: 3T3 Cells; Adaptation, Physiological; Animals; Cell Culture Techniques; Cell Differentiation; Cell Survival; Mice; Osteoblasts; Spheroids, Cellular; Stress, Mechanical
PubMed: 28317941
DOI: 10.1038/srep44925 -
Endocrine Journal Apr 2024Lipopolysaccharide (LPS) and Receptor Activator of Nuclear Factor-κB Ligand (RANKL) are the two important factors causing bone loss, which is an important pathogenesis...
Lipopolysaccharide (LPS) and Receptor Activator of Nuclear Factor-κB Ligand (RANKL) are the two important factors causing bone loss, which is an important pathogenesis for osteoporosis. However, the relationship between LPS and RANKL is not yet clear. LPS can be involved in the weakened osteoblast formation as an autophagy regulator, and osteoblasts and their precursors are the source cells for RANKL production. Our study aimed to explore the relationship between autophagy changes and RANKL production during LPS-regulated osteoblasts. Our results showed that LPS inhibited autophagy (LC3 conversion and autophagosome formation) and enhanced the protein and mRNA expression of RANKL in MC3T3-E1 osteoblast precursor line. Autophagy upregulation with Rapamycin over BECN1 overexpression rescued LPS-inhibited osteoblast formation and -promoted RANKL protein production in MC3T3-E1 cells. In vivo experiments supported that damaged bone mass, bone microstructure, osteoblastic activity (ALP and P1NP production by ELISA assays) and enhanced RANKL production by LPS administration were partially rescued by Rapamycin application. In conclusion, LPS can inhibit autophagy in osteoblast precursors, thereby inhibiting osteoblast formation and RANKL autophagic degradation.
Topics: Lipopolysaccharides; Osteoblasts; Animals; Autophagy; RANK Ligand; Mice; Cell Line; Osteogenesis; Cell Differentiation; Sirolimus; Male; Beclin-1
PubMed: 38355125
DOI: 10.1507/endocrj.EJ23-0484 -
International Journal of Molecular... Jun 2023Periprosthetic osteolysis (PPO) induced by wear particles is the most severe complication of total joint replacement; however, the mechanism behind PPO remains elusive....
Periprosthetic osteolysis (PPO) induced by wear particles is the most severe complication of total joint replacement; however, the mechanism behind PPO remains elusive. Previous studies have shown that osteocytes play important roles in wear-particle-induced osteolysis. In this study, we investigated the effects of connexin 43 (Cx43) on the regulation of osteocyte-to-osteoblast differentiation. We established an in vivo murine model of calvarial osteolysis induced by titanium (Ti) particles. The osteolysis characteristic and osteogenesis markers in the and were observed. The calvarial osteolysis induced by Ti particles was partially attenuated in . The expression of β-catenin and osteogenesis markers increased significantly in . In vitro, the osteocytic cell line MLO-Y4 was treated with Ti particles. The co-culturing of MLO-Y4 cells with MC3T3-E1 osteoblastic cells was used to observe the effects of Ti-treated osteocytes on osteoblast differentiation. When Cx43 of MLO-Y4 cells was silenced or overexpressed, β-catenin was detected. Additionally, co-immunoprecipitation detection of Cx43 and β-catenin binding in MLO-Y4 cells and MC3T3-E1 cells was performed. Finally, β-catenin expression in MC3T3-E1 cells and osteoblast differentiation were evaluated after 18α-glycyrrhetinic acid (18α-GA) was used to block the intercellular communication of Cx43 between MLO-Y4 and MC3T3-E1 cells. Ti particles increased Cx43 expression and decreased β-catenin expression in MLO-Y4 cells. The silencing of Cx43 increased the β-catenin expression, and the over-expression of Cx43 decreased the β-catenin expression. In the co-culture model, Ti treatment of MLO-Y4 cells inhibited the osteoblastic differentiation of MC3T3-E1 cells and Cx43 silencing in MLO-Y4 cells attenuated the inhibitory effects on osteoblastic differentiation. With Cx43 silencing in the MLO-Y4 cells, the MC3T3-E1 cells, co-cultured alongside MLO-Y4, displayed decreased Cx43 expression, increased β-catenin expression, activation of Runx2, and promotion of osteoblastic differentiation in vitro co-culture. Finally, Cx43 expression was found to be negatively correlated to the activity of the Wnt signaling pathway, mostly through the Cx43 binding of β-catenin from its translocation to the nucleus. The results of our study suggest that Ti particles increased Cx43 expression in osteocytes and that osteocytes may participate in the regulation of osteoblast function via the Cx43 during PPO.
Topics: Mice; Animals; Osteocytes; beta Catenin; Connexin 43; Titanium; Osteolysis; Cell Differentiation; Osteoblasts
PubMed: 37446062
DOI: 10.3390/ijms241310864 -
Bone Jun 2007Intermittent administration of parathyroid hormone (PTH) stimulates bone formation by increasing osteoblast number, but the molecular and cellular mechanisms underlying... (Review)
Review
Intermittent administration of parathyroid hormone (PTH) stimulates bone formation by increasing osteoblast number, but the molecular and cellular mechanisms underlying this effect are not completely understood. In vitro and in vivo studies have shown that PTH directly activates survival signaling in osteoblasts; and that delay of osteoblast apoptosis is a major contributor to the increased osteoblast number, at least in mice. This effect requires Runx2-dependent expression of anti-apoptotic genes like Bcl-2. PTH also causes exit of replicating progenitors from the cell cycle by decreasing expression of cyclin D and increasing expression of several cyclin-dependent kinase inhibitors. Exit from the cell cycle may set the stage for pro-differentiating and pro-survival effects of locally produced growth factors and cytokines, the level and/or activity of which are known to be influenced by PTH. Observations from genetically modified mice suggest that the anabolic effect of intermittent PTH requires insulin-like growth factor-I (IGF-I), fibroblast growth factor-2 (FGF-2), and perhaps Wnts. Attenuation of the negative effects of PPAR gamma may also lead to increased osteoblast number. Daily injections of PTH may add to the pro-differentiating and pro-survival effects of locally produced PTH related protein (PTHrP). As a result, osteoblast number increases beyond that needed to replace the bone removed by osteoclasts during bone remodeling. The pleiotropic effects of intermittent PTH, each of which alone may increase osteoblast number, may explain why this therapy reverses bone loss in most osteoporotic individuals regardless of the underlying pathophysiology.
Topics: Animals; Apoptosis; Cell Differentiation; Humans; Mice; Models, Biological; Osteoblasts; Osteogenesis; Parathyroid Hormone
PubMed: 17517365
DOI: 10.1016/j.bone.2007.03.017