-
Journal of Orthopaedic Research :... Jan 2013Connexin43 (Cx43) plays an important role in osteoblastic differentiation in vitro, and bone formation in vivo. Mice with osteoblast/osteocyte-specific loss of Cx43...
Connexin43 (Cx43) plays an important role in osteoblastic differentiation in vitro, and bone formation in vivo. Mice with osteoblast/osteocyte-specific loss of Cx43 display decreased gap junctional intercellular communication (GJIC), bone density, and cortical thickness. To determine the role of Cx43 in fracture healing, a closed femur fracture was induced in Osteocalcin-Cre+; Cx43(flox/flox) (Cx43cKO) and Cre-; Cx43(flox/flox) (WT) mice. We tested the hypothesis that loss of Cx43 results in decreased bone formation and impaired healing following fracture. Here, we show that osteoblast and osteocyte-specific deletion of Cx43 results in decreased bone formation, bone remodeling, and mechanical properties during fracture healing. Cx43cKO mice display decreased bone volume, total volume, and fewer TRAP+ osteoclasts. Furthermore, loss of Cx43 in mature osteoblasts and osteocytes results in a significant decrease in torsional rigidity between 21 and 35 days post-fracture, compared to WT mice. These studies identify a novel role for the gap junction protein Cx43 during fracture healing, suggesting that loss of Cx43 can result in both decreased bone formation and bone resorption. Therefore, enhancing Cx43 expression or GJIC may provide a novel means to enhance bone formation during fracture healing.
Topics: Animals; Calcification, Physiologic; Cell Differentiation; Connexin 43; Femoral Fractures; Fracture Healing; Gap Junctions; Mice; Mice, Knockout; Osteoblasts; Osteocytes; Osteogenesis; Torsion, Mechanical
PubMed: 22718243
DOI: 10.1002/jor.22178 -
Biometals : An International Journal on... Oct 2018The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, β-thalassemia and sickle cell anemia with multiple...
The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, β-thalassemia and sickle cell anemia with multiple blood transfusion. One of the contributing factors is iron toxicity to osteoblasts. Some studies showed the negative effects of iron on osteoblasts; however, the effects of two biological available iron species, i.e., ferric and ferrous, on osteoblasts are elusive. Since most intracellular ionized iron is ferric, osteoblasts was hypothesized to be more responsive to ferric iron. Herein, ferric ammonium citrate (FAC) and ferrous ammonium sulfate (FAS) were used as ferric and ferrous donors. Our results showed that both iron species suppressed cell survival and proliferation. Both also induced osteoblast cell death consistent with the higher levels of cleaved caspase 3 and caspase 7 in osteoblasts, indicating that iron induced osteoblast apoptosis. Iron treatments led to the elevated intracellular iron in osteoblasts as determined by atomic absorption spectrophotometry, thereby leading to a decreased expression of genes for cellular iron import and increased expression of genes for cellular iron export. Effects of FAC and FAS on osteoblast differentiation were determined by the activity of alkaline phosphatase (ALP). The lower ALP activity from osteoblast with iron exposure was found. In addition, ferric and ferrous differentially induced osteoblastic and osteoblast-derived osteoclastogenic gene expression alterations in osteoblast. Even though both iron species had similar effects on osteoblast cell survival and differentiation, the overall effects were markedly stronger in FAC-treated groups, suggesting that osteoblasts were more sensitive to ferric than ferrous.
Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Ferric Compounds; Ferrous Compounds; Osteoblasts; Rats; Structure-Activity Relationship
PubMed: 30014351
DOI: 10.1007/s10534-018-0130-6 -
European Journal of Orthodontics Dec 1997Identifying the biological properties of the cells residing within the periodontal ligament (PDL) will help in understanding the role that these cells play in the...
Identifying the biological properties of the cells residing within the periodontal ligament (PDL) will help in understanding the role that these cells play in the various functions of the periodontal ligament, and will improve the success of clinical procedures such as orthodontic tooth movement. For this purpose, fibroblasts isolated from human periodontium were cultured and characterized both histochemically and biochemically with respect to their putative osteoblast-like properties. Histochemically, cultured PDL fibroblasts showed an intense staining for alkaline phosphatase (ALP). Biochemically, the basal ALP activity increased in culture over time. ALP levels after stimulation with 1 alpha, 25-dihydroxyvitamin D3 were significantly higher than those of control cultures. Moreover, immunofluorescence against osteocalcin (a highly reliable osteoblastic marker) was strongly positive. Von Kossa staining of the cell cultures revealed the formation of mineral-like nodules. These results indicate that human PDL fibroblasts exhibit in vitro phenotypic characteristics consistent with osteoblast-like cells, thus suggesting that such cells have the potential to differentiate into osteoblasts and/or cementoblasts.
Topics: Alkaline Phosphatase; Biochemical Phenomena; Biochemistry; Biomarkers; Calcification, Physiologic; Calcitriol; Cell Differentiation; Cells, Cultured; Coloring Agents; Dental Cementum; Fibroblasts; Fluorescent Antibody Technique, Direct; Histocytochemistry; Humans; Minerals; Osteoblasts; Osteocalcin; Periodontal Ligament; Phenotype; Stem Cells; Tooth Movement Techniques; Treatment Outcome
PubMed: 9458594
DOI: 10.1093/ejo/19.6.615 -
The Kaohsiung Journal of Medical... May 1999Primary osteoblast cultures, which reflect more phenotypic properties of normal osteoblasts than osteoblastic cell lines, can be used as an experimental tool for...
Primary osteoblast cultures, which reflect more phenotypic properties of normal osteoblasts than osteoblastic cell lines, can be used as an experimental tool for investigating the osteoblastic functions in vitro. Primary osteoblast cultures were obtained from the parietal bones of calvaria of fetal rats in this study. Differential characteristics of osteoblasts in our culture system were examined and fibroblast cultures were also tested for comparison. We tested the alkaline phosphatase (ALP) and von Kossa stains on osteoblast and fibroblast cultures to examine the expression of ALP and the subsequent matrix mineralization occurred at 2 and 3 weeks after cell confluence respectively. The results showed that osteoblast cultures revealed obvious positive stains of ALP and von Kossa, while fibroblast cultures revealed negative stains, suggesting the osteoblast culture system used in this study reflects the typical phenotypes of primary osteoblasts but not fibroblasts. We tested the ALP activities following various doses of PGE2 or ketorolac treatments in primary osteoblast and fibroblast cultures. The results showed that PGE2 and ketorolac stimulated intracellular ALP activities of osteoblasts in dose dependent fashions, while very low ALP activities were detected in either the control or agents treated cultures of fibroblast. These results suggest that PGE2 may be involved in osteoblastic differentiation and the stimulatory effect of ketorolac on osteoblastic ALP activity may not be PGE2 mediated. The responses of osteoblasts to both agents can be as the characteristics of primary osteoblast derived from rat calvaria.
Topics: Alkaline Phosphatase; Animals; Cells, Cultured; Dinoprostone; Female; Fetus; Ketorolac; Osteoblasts; Pregnancy; Rats; Rats, Sprague-Dawley; Skull; Tolmetin
PubMed: 10375866
DOI: No ID Found -
Biochimie Mar 2019Parathyroid hormone (PTH) acts as a regulator of calcium homeostasis and bone remodeling. Runx2, an essential transcription factor in bone, is required for osteoblast...
Parathyroid hormone (PTH) acts as a regulator of calcium homeostasis and bone remodeling. Runx2, an essential transcription factor in bone, is required for osteoblast differentiation. Noncoding RNAs such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play crucial roles in regulating gene expression in osteoblasts. In this study, we investigated the effects of PTH on osteoblast differentiation via Runx2, lncRNA, and miRNA expression in human bone marrow stromal cells (hBMSCs) and human osteoblastic cells (MG63). PTH-treatment of hBMSCs for 24 h, 7 days, and 14 days stimulated Runx2 mRNA expression. Using bioinformatics tools, we identified 17 lncRNAs originating from human Runx2 gene. Among these, lnc-SUPT3H-1:16 (RUNX2-AS1:32) expression was highly up-regulated by the 7 d PTH-treatment in hBMSCs. We also identified miR-6797-5p as the putative target of lnc-SUPT3H-1:16 and Runx2 using bioinformatics tools. PTH-treatment increased the expression of miR-6797-5p in hBMSCs, and overexpression of miR-6797-5p decreased osteoblast differentiation in MG63 cells, suggesting a role for lnc-SUPT3H-1:16 as sponge molecule. A luciferase gene reporter assay identified direct targeting of miR-6797-5p with lnc-SUPT3H-1:16 and 3'UTR Runx2 in MG63 cells. Thus, PTH stimulated the expression of lnc-SUPT3H-1:16, miR-6797-5p and Runx2, and due to the sponging mechanism of lnc- SUPT3H-1:16 towards miR-6797-5p, Runx2 was protected, resulting in the promotion of osteoblast differentiation.
Topics: Adult; Cell Differentiation; Cell Line; Core Binding Factor Alpha 1 Subunit; Female; Humans; MicroRNAs; Osteoblasts; Parathyroid Hormone; RNA, Long Noncoding; Up-Regulation
PubMed: 30562548
DOI: 10.1016/j.biochi.2018.12.006 -
Hormone and Metabolic Research =... Mar 2011Menin promotes the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage by interacting with the BMP-2 signaling molecules Smad1/5, and Runx2....
Menin promotes the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage by interacting with the BMP-2 signaling molecules Smad1/5, and Runx2. However, the relationship between menin and the Wnt-β-catenin pathway in bone is unclear. Reduction of menin expression by transfection of a menin antisense construct did not alter the levels of β-catenin in mouse mesenchymal C2C12 and osteoblastic MC3T3-E1 cells. However, menin co-immunoprecipitated with β-catenin as well as LEF-1 in C2C12 and MC3T3-E1 cells. Reduction of menin expression by antisense menin transfection antagonized β-catenin-induced transcriptional activity of the pGL3-OT luciferase reporter construct in C2C12 and MC3T3-E1 cells. Antisense menin transfection antagonized the BMP-2 and β-catenin-stimulated increases in Runx2 and alkaline phosphatase levels in C2C12 cells. The data show that menin interacts with β-catenin in mouse mesenchymal and osteoblastic cells, and suggest that the interaction is important for osteoblast differentiation.
Topics: Animals; Bone Morphogenetic Protein 2; Cell Differentiation; Cells, Cultured; Mesenchymal Stem Cells; Mice; Osteoblasts; Protein Binding; Proto-Oncogene Proteins; beta Catenin
PubMed: 21264795
DOI: 10.1055/s-0030-1270527 -
Immunology Jan 1990We studied the ability of the rat osteosarcoma derived cell-line with osteoblastic properties ROS-17/2.8 (ROS) to maintain in vitro rat peritoneal mast cells (MC) in a...
We studied the ability of the rat osteosarcoma derived cell-line with osteoblastic properties ROS-17/2.8 (ROS) to maintain in vitro rat peritoneal mast cells (MC) in a functional state. Highly purified (greater than 95%) MC were seeded on confluent ROS cells. The MC adhered tightly to the monolayers within a few hours and remained viable for at least 2 weeks, but did not proliferate. The MC retained their typical appearance, exhibiting highly granulated resting morphology when stained with alcian blue followed by safranin or with acidic toluidine blue. Furthermore, after 2 weeks, the MC were fully responsive to activation with compound 48/80 (3 micrograms/ml), releasing 75% of their histamine content, as compared to 3% in the absence of the secretagogue. Utilizing metabolically inactive ROS cells and prevention of contact between ROS and MC, we found that both release of factor(s) and cell-cell contact were required by ROS to exhibit their MC supporting activity. Various other cells and cell lines were unable to support MC viability. On the other hand, as demonstrated before, 3T3 fibroblasts were capable of promoting MC viability. Thus, MC viability and functional activity are specifically maintained by fibroblastic and osteoblastic cells. The abundance of MC in bone, and their participation in bone remodelling raise the possibility of physiological and pathological significance to interactions between MC and osteoblasts.
Topics: Animals; Cell Adhesion; Cell Communication; Cell Line; Cell Survival; Mast Cells; Osteoblasts; Peritoneal Cavity; Rats
PubMed: 2312152
DOI: No ID Found -
Food and Chemical Toxicology : An... Aug 2013Age-related osteoblast dysfunction is the main cause of age-related bone loss in both men and women. In the present study, the effect of sciadopitysin, a type of...
Age-related osteoblast dysfunction is the main cause of age-related bone loss in both men and women. In the present study, the effect of sciadopitysin, a type of biflavonoids, on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. Sciadopitysin caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, mineralization, and glutathione content in the cells (P<0.05). Sciadopitysin also decreased the production of tumor necrosis factor-a (TNF-α) induced by antimycin A, a mitochondrial electron transport inhibitor. We investigated the protective effects of sciadopitysin on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused a significant reduction in osteoblast dysfunction. However, pretreatment with sciadopitysin prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, adenosine triphosphate (ATP) loss, reactive oxygen species (ROS) release, and nitrotyrosine increase, suggesting that sciadopitysin may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, sciadopitysin increased phosphorylation of cAMP-response element-binding protein (CREB) inhibited by antimycin A. Our results demonstrate that sciadopitysin may reduce or prevent osteoblasts degeneration.
Topics: 3T3 Cells; Animals; Antimycin A; Antioxidants; Biflavonoids; Cyclic AMP Response Element-Binding Protein; Mice; Osteoblasts; Phosphorylation
PubMed: 23624148
DOI: 10.1016/j.fct.2013.04.028 -
Calcified Tissue International Sep 1991Although androgens exert major effects on bone remodeling, the mechanisms by which they exert their effects remain unclear. Recently, it has become apparent that...
Although androgens exert major effects on bone remodeling, the mechanisms by which they exert their effects remain unclear. Recently, it has become apparent that receptors for sex steroids may be present in osteoblastic cells. We have examined several cell lines with osteoblastic phenotypes to determine if specific, high affinity androgen receptors are present. Two cell lines of human origin (Saos-2 and U2-OS) and one of rat origin (UMR-106.01) were studied. Androgen binding sites were present in all cell lines. Binding affinities were high (KD = 1.6 - 2.5 x 10(-10) M), and similar to those in classical androgen target tissues (prostate, kidney). Concentrations were greater in the human cell lines (1277 and 1605 sites/cell) than in the rodent line (74 sites/cell). In the human cell lines androgen binding was also specific and typical of androgen receptors in other tissues. Specific estrogen binding was not present in the UMR-106.01 cells, and no estrogen receptors were detectable in the human cell lines using an enzyme-linked receptor immunoassay. Specific binding for progesterone was also absent in the UMR-106.01 cells, but progesterone receptors were detected immunologically in the Saos-2 (119 sites/cell) and U2-OS (118 sites/cell) lines. These findings indicate the presence of androgen receptors that are of similar character to those in classical androgen target tissues, and suggest that the study of these cell lines may be useful in the study of the regulation of androgen effects in osteoblasts.
Topics: Androgens; Animals; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Female; Humans; Osteoblasts; Protein Binding; Rats; Receptors, Androgen; Receptors, Progesterone
PubMed: 1933584
DOI: 10.1007/BF02556115 -
Biochemical and Biophysical Research... Sep 2015The osteoblastic expression of RANKL, which is essential for the communication between osteoblastic cells and osteoclastogenic cells, is stimulated by locally acting or...
The osteoblastic expression of RANKL, which is essential for the communication between osteoblastic cells and osteoclastogenic cells, is stimulated by locally acting or circulating osteotropic cytokines and hormones such as PTH and 1,25-(OH)2-D3 during the bone remodeling process. However, mechanisms those control subcellular trafficking events, membrane expression and extracellular secretion of the newly synthesized RANKL are still not well understood. In our previous study, we have found that the deficiency of osteoblastic Arl6ip5 (ADP-ribosylation-like factor 6 interacting protein 5), an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family, enhanced osteoclastogenesis by increasing RANKL transcription in an ER stress dependent signaling. Here we found that over-expression of hemagglutinin (HA)-tagged Arl6ip5 in UAMS32 stromal/osteoblastic cells inhibited osteoclastogenesis, decreased the amount of soluble RANKL in culture supernatant and increased RANKL retention in ER. Moreover, Arl6ip5 bound with RANKL and disturbed the RANKL-OPG complex in UAMS-32 cells. Finally, 1 to 36 amino acid deletion on the NH2 lumen terminus of Arl6ip5 impaired the interaction between Arl6ip5 and RANKL, restored the level of soluble RANKL and the osteoclastogenic ability. These findings indicated that Arl6ip5 was an anti-catabolic factor by binding with RANKL and disturbing its subcellular trafficking in osteoblast.
Topics: Animals; Carrier Proteins; Cell Line; Gene Expression Regulation; Heat-Shock Proteins; Membrane Transport Proteins; Mice; Osteoblasts; RANK Ligand; Subcellular Fractions; Up-Regulation
PubMed: 26220341
DOI: 10.1016/j.bbrc.2015.07.119