Primary Osteoblastic Cells Research Articles

Osteoporosis Prevention by Adlay (薏苡 Yì Yǐ: The Seeds of Coix Lachryma-Jobi L. var. ma-yuen Stapf) in a Mouse Model

Osteoporosis is characterized by reduced bone mass and quality due to an imbalanced bone remodeling. A grass crop, adlay (Coix lachryma-jobi), is a kind of nourishing food, which has also been used in traditional Chinese medicine. In this study, we investigated the effect of adlay (C. lachryma-jobi L. var. ma-yuen Stapf) on osteoporosis using an ovariectomized mouse model. The adlay diet (10% and 30% adlay in mouse diet) or water extract of adlay (0.3 g/kg/day) was given to ovariectomized mice for 4 weeks. In some experiments, the primary rat osteoblast cells were used to test the possible mechanism of adlay on osteoporosis. The body weight was slightly increased and uterus weight was markedly decreased in ovariectomized mice, which were not affected by adlay treatment. Adlay diet (30%) and adlay extract feedings significantly reversed the decreased bone alkaline phosphatase activity and calcium contents and bone mineral density in ovariectomized mice. Moreover, adlay extracts increased the osteoblast cell proliferation in a dose-dependent manner. Adlay extracts also increased the protein expressions of proliferating cell nuclear antigen and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 in osteoblast cells. ERK inhibitor PD98059 significantly reversed the increased osteoblast cell proliferation by adlay extracts. Taken together, these findings indicate that adlay effectively alleviates the osteoporotic status in ovariectomized mice. Adlay is capable of increasing the proliferation of osteoblast cells via an ERK-regulated signaling pathway. Adlay may be a helpful healthy food for osteoporosis prevention.

Cathepsin K secretion in human primary osteoblastic cell cultures and its influence by different glucose concentrations

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Synthesis of a biodegradable polymer in gas expanded solution: effect of the process on cytocompatibility

The aim of this study was to investigate the feasibility of eliminating organic solvent consumption for the synthesis of biodegradable poly(lactide ethylene oxide fumarate) (PLEOF). A gas expanded solution comprised of high pressure carbon dioxide (CO2) and dichloromethane (DCM) or poly(ethylene glycol) (PEG) was used as an alternative medium for the synthesis of PLEOF. The effect of pressure (50–150 bar), temperature (25–50 °C) and the amount of DCM on the yield, molecular weight and thermal properties of PLEOF was examined. The results of 1H NMR and ATR-FTIR analysis confirmed that it is possible to conduct the PLEOF synthesis in a high pressure CO2 expanded solution as a reaction medium and acquire similar characteristics compared to the conventional method using neat DCM. A comparable yield was achieved in a high pressure CO2 expanded solution process even when DCM was decreased to only 14% of the amount used in the conventional method. Increasing the pressure from 50 to 150 bar had no significant effect on the yield. In addition, DCM could be completely eliminated from the polymerisation by increasing the temperature to 50 °C and pressure to 100 bar. Under these conditions, the synthesis was carried out in PEG expanded solution due to the melting point depression effect of CO2. It is, therefore, viable to conduct the polymerisation of PLEOF in gas expanded solution as a benign solvent and can either substantially reduce or eliminate the consumption of volatile organic solvents. The synthesised PLEOF was also crosslinked with poly(ethylene glycol) diacrylate (PEGDA) as a non-toxic crosslinker to produce an injectable hydrogel. The hydrogel prepared from PLEOF that was synthesised by gas expanded solution had better mechanical properties and less toxicity to primary human osteoblast cells. This study demonstrated that the synthesis process can have a significant impact on improving the physical properties and biological activity of the PLEOF polymer.

Silica Aerogel Improves the Biocompatibility in a Poly-ε-Caprolactone Composite Used as a Tissue Engineering Scaffold

Poly-ε-caprolactone (PCL) is a biodegradable polyester that has received great attentions in clinical and biomedical applications as sutures, drug delivery tool, and implantable scaffold material. Silica aerogel is a material composed of SiO2that has excellent physical properties for use in drug release formulations and biomaterials for tissue engineering. The current study addresses a composite of silica aerogel with PCL as a potential bone scaffold material for bone tissue engineering. The biocompatibility evaluation of this composite indicates that the presence of silica aerogel effectively prevented any cytotoxic effects of the PCL membrane during extended tissue culture periods and improved the survival, attachment, and growth of 3T3 cells and primary mouse osteoblastic cells. The beneficial effect of silica aerogel may be due to neutralization of the acidic condition that develops during PCL degradation. Specifically, it appears that silica aerogel to PCL wt/wt ratio at 0.5 : 1 maintains a constant pH environment for up to 4 weeks and provides a better environment for cell growth.

Fe–Pd based ferromagnetic shape memory actuators for medical applications: Biocompatibility, effect of surface roughness and protein coatings

Ferromagnetic shape memory (FMSM) alloys constitute an exciting new class of smart materials that can yield magnetically switchable strains of several percent at constant temperatures and frequencies from quasi-static up to some kilohertz. In addition to their FMSM properties, these alloys can still be operated as conventional shape memory materials and also exhibit related superelasticity, which are both important features for use in medical devices. In this study, extensive in vitro assessments demonstrate for the first time that vapor-deposited single crystalline Fe70Pd30 thin films and roughness graded polycrystalline splats of the same stoichiometry exhibit excellent biocompatibility and even bioactivity in contact with different cell types—a prerequisite for medical applications. The present study shows that fibroblast and epithelial cell lines, as well as primary osteoblast cells, proliferate well on Fe–Pd. The number of focal contacts, important for strong tissue bonding, can be improved with different binding agents from the extracellular matrix. However, even without coating, there is clear evidence that cells on Fe–Pd substrates behave similarly to control experiments. Additionally, cytotoxic effects of polycrystalline surfaces with various roughness profiles can be excluded, giving another tunable parameter for applying Fe–Pd magnetically switchable membranes in, e.g., stents and valves.

Transcriptome correlation analysis identifies two unique craniosynostosis subtypes associated with IRS1 activation

The discovery of causal mechanisms associated with nonsyndromic craniosynostosis has proven to be a difficult task due to the complex nature of the disease. In this study, differential transcriptome correlation analysis was used to identify two molecularly distinct subtypes of nonsyndromic craniosynostosis, termed subtype A and subtype B. In addition to unique correlation structure, subtype A was also associated with high IGF pathway expression, whereas subtype B was associated with high integrin expression. To identify a pathologic link between altered gene correlation/expression and the disease state, phosphorylation assays were performed on primary osteoblast cell lines derived from cases within subtype A or subtype B, as well as on primary osteoblast cell lines with novel IGF1R variants previously reported by our lab (Cunningham ML, Horst JA, Rieder MJ, Hing AV, Stanaway IB, Park SS, Samudrala R, Speltz ML. Am J Med Genet A 155A: 91-97, 2011). Elevated IRS1 (pan-tyr) and GSK3β (ser-9) phosphorylation were observed in two novel IGF1R variants with receptor L domain mutations. In subtype A, a hypomineralization phenotype coupled with decreased phosphorylation of IRS1 (ser-312), p38 (thr-180/tyr-182), and p70S6K (thr-412) was observed. In subtype B, decreased phosphorylation of IRS1 (ser-312) as well as increased phosphorylation of Akt (ser-473), GSK3β (ser-9), IGF1R (tyr-1135/tyr-1136), JNK (thr-183/tyr-187), p70S6K (thr-412), and pRPS6 (ser-235/ser-236) was observed, thus implicating the activation of IRS1-mediated Akt signaling in potentiating craniosynostosis in this subtype. Taken together, these results suggest that despite the stimulation of different pathways, activating phosphorylation patterns for IRS1 were consistent in cell lines from both subtypes and the IGF1R variants, thus implicating a key role for IRS1 in the pathogenesis of nonsyndromic craniosynostosis.

Effect of Pulsed Electromagnetic Fields on Human Osteoblast Cultures

Exogenous electromagnetic fields (EMFs) affect bone metabolism, but the mechanisms responsible for this phenomenon are unclear. Pulsed EMFs (PEMFs) can be effective in the management of congenital pseudarthrosis or delayed union or non-union of fractures. We investigated the effects of PEMFs used in clinical practice on human osteoblast cultures. Primary osteoblastic cells were isolated from a human femoral head. Cultures were exposed to the PEMF stimulation for 72 hours, 7 and 10 days and compared with a control group of primary osteoblastic cells non-exposed to PEMF. Cell growth and alkaline phosphatase activity were evaluated in the osteoblast cell cultures at each observation time. At each observation time, the differences in cell numbers between PEMF-exposed cells and control group were statistically significant (p < 0.05). The alkaline phosphatase-specific activity of PEMF-exposed osteoblast cultures showed a statistically significant (p < 0.05) increase when compared with the control group after 7 and 10 days of exposure. The application of PEMF stimulation on human osteoblasts accelerates cellular proliferation when compared with a control group of non-PEMF-exposed cells.

Epigallocatechin-3-gallate diminishes cytokine-stimulated Cyr61 expression in human osteoblastic cells: a therapeutic potential for arthritis

To assess the effects of epigallocatechin-3-gallate (EGCG) on cytokine-induced Cyr61 synthesis in human osteoblastic cells and the associated signalling pathways. The therapeutic effect of EGCG on CIA in rats was also studied. The expression of Cyr61 and NF-κB pathway molecules was examined by western blotting. CCL2 expression was assessed by northern blotting and ELISA. Interaction between NF-κB and Cyr61 promoter was evaluated by electrophoretic mobility shift assay. In rat CIA, osteoblastic expression of Cyr61 was examined by immunohistochemistry and disease progression was assessed by clinical, radiographic and histological examinations. EGCG inhibited Cyr61 expression stimulated by cytokines in primary human osteoblasts and human osteoblastic cell line U2OS. In U2OS, oncostatin M (OSM) induced IκB-α degradation through the mTOR/rictor/Akt pathway, and EGCG attenuated the action. Electrophoretic mobility shift assay revealed that the OSM-enhanced NF-κB/DNA binding was reduced by EGCG, possibly through abrogating nucleus localization of p65 and p50. Cyr61 enhanced OSM-induced expression of CCL2. Moreover, EGCG diminished OSM-stimulated CCL2 expression at least partially via suppressing Cyr61 induction. Co-distribution of CD68(+) macrophages and Cyr61(+) osteoblasts in osteolytic areas was obvious in the CIA model. Clinical, radiographic and immunohistochemical analyses revealed that administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the number of Cyr61-synthesizing osteoblasts. By modulating the mTOR/rictor/Akt/NF-κB pathway, EGCG attenuated Cyr61 production in osteoblastic cells and in turn diminished macrophage chemotaxis. Our data support the therapeutic potential of EGCG on arthritis.

In search of an osteoblast cell model for in vitro research

The process of bone formation, remodelling and healing involves a coordinated action of various cell types. Advances in understanding the biology of osteoblast cells during these processes have been enabled through the use of various in vitro culture models from different origins. In an era of intensive bone tissue engineering research, these cell models are more and more often applied due to limited availability of primary human osteoblast cells. While they are a helpful tool in developing novel therapies or biomaterials; concerns arise regarding their phenotypic state and differences in relation to primary human osteoblast cells. In this review we discuss the osteoblastic development of some of the available cell models; such as primary human, rat, mouse, bovine, ovine and rabbit osteoblast cells; as well as MC3T3-E1, MG-63 and SaOs-2 cell lines, together with their advantages and disadvantages. Through this, we provide suggestions on the selection of the appropriate and most relevant osteoblast model for in vitro studies, with specific emphasis on cell-material based studies.

Expression of BMP-2 and Ets1 in BMP-2-stimulated mouse pre-osteoblast differentiation is regulated by microRNA-370

MicroRNAs (miRs) regulate several biological functions such as cell growth, cell differentiation, and carcinogenesis, by binding to the 3′-untranslated regions (3′-UTR) of specific target genes, in order to repress translation or promote degradation of the transcribed mRNAs. In the present study, using microRNA array and in silico analyses, we found that miR-370 regulates the expression of bone morphogenetic protein-2 (BMP-2) and V-ets Erythroblastosis Virus E26 Oncogene Homolog 1 (Ets1) in BMP-2-stimulated murine pre-osteoblast MC3T3-E1 cell differentiation. The enforced expression of mature miR-370 in MC3T3-E1 cells or primary osteoblast cells remarkably attenuated BMP-2-induced pre-osteoblast differentiation. To ascertain the mechanisms underlying the regulation of osteoblast differentiation by miR-370, we hypothesized a BMP-2-Ets1-PTHrP feed-forward loop regulatory mechanism.

Expression and stability of hypoxia inducible factor 1α in osteosarcoma

Hypoxia contributes to both physiological and pathological processes and its effects are mainly mediated through the transcription factors hypoxia-inducible factor 1α and 2α (HIF1α and HIF2α). The purpose of this study was to examine the role of these proteins in osteosarcoma progression. We developed a method to isolate primary human osteoblast cell lines. HIF1α and HIF2α expression were then compared in osteoblast and osteosarcoma cell lines under 21% oxygen (normoxia) and 1% oxygen (hypoxia). We also used hypoxia-responsive element (HRE)-driven reporter constructs in conjunction with siRNAs specific to HIF1α or HIF2α to determine the contribution of each protein to HRE-mediated transcription. Finally, we measured HIF1α expression in primary osteosarcoma tumors by immunohistochemistry. We found that mainly HIF1α transcript was significantly higher in osteosarcoma cell lines compared to normal osteoblasts under both normoxia and hypoxia. At the protein level, HIF1α was preferentially stabilized in osteosarcoma cell lines under both conditions. HIF1α expression was required for the observed increases in HRE activity. Finally, nuclear or nucleocytoplasmic HIF1α staining in osteosarcoma cases was associated with high-grade tumors. These findings point to a role for HIF1α in osteosarcoma progression and suggest that the observed differences in HIF1α oxygen dependent degradation may play an important pathophysiological role in this disease. Pediatr Blood Cancer 2012; 59: 1215-1222. © 2012 Wiley Periodicals, Inc.

The beneficial effect of Radix Dipsaci total saponins on bone metabolism in vitro and in vivo and the possible mechanisms of action

The purpose of this study is to investigate the anti-osteoporotic effects of Radix Dipsaci total saponins (RTS). We showed that RTS was able to improve bone properties by either an increase of osteoblastic activity or a decrease in osteoclastic activity. Radix Dipsaci has long been used as an anti-osteoporotic drug. The present study investigates the anti-osteoporotic effects of RTS. Three-month-old female rats were randomly assigned into a sham-operated group (sham) and five ovariectomy (OVX) subgroups, namely, OVX with vehicle (OVX), OVX with 17β-ethinylestradiol (E(2)), and OVX with graded doses of RTS (50, 100, or 200 mg/kg/d). RTS and E(2) were administered orally, daily from 1 week after OVX treatment for 4 months. Bone mass, turnover, and strength were evaluated by dual-energy X-ray absorptiometry, biochemical markers, and the three-point bending test. The trabecular bone microarchitecture was assessed by microCT. In vitro experiments were performed to determine the potential molecular mechanisms of the anti-osteoporotic effect of RTS. RTS prevented the loss of bone mass induced by OVX. The preventive effect on bone loss was primarily indicated by decreasing levels of bone turnover markers and confirmed by the changes in urinary calcium and phosphorus excretion. The treatment also enhanced the biomechanical strength of bone and prevented the deterioration of trabecular bone microarchitecture. RTS induced MC3T3-E1 and primary osteoblastic cell maturation and differentiation and increased bone formation by increasing BMP-2 synthesis. In addition, RTS inhibited osteoclastogenesis through an increase in osteoprotegrin and a decrease in NF-kB ligand expression in vitro. RTS treatment can effectively suppress the loss of bone mass induced by OVX and in vitro evidence suggests this could be through actions on both osteoblasts and osteoclasts.

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107–111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca2+. These findings provide an attractive strategy for bone tissue engineering.

Effects of Tinospora cordifolia (Menispermaceae) on the proliferation, osteogenic differentiation and mineralization of osteoblast model systems in vitro

Ethnopharmacological relevanceAncient Indian ayurvedic literature prescribes Tinospora cordifolia as a remedy to rheumatoid arthritis, inflammatory and allied diseases of musculo skeletal system. AimTo investigate the effects of the alcoholic extract of Tinospora cordifolia (TC) on the proliferation, differentiation and mineralization of bone like matrix on osteoblast model systems in vitro and hence its possible use as a potential anti-osteoporotic agent. Materials and methodsTwo in vitro osteoblast model systems were used in the study viz., human osteoblast-like cells MG-63 and primary osteoblast cells isolated from femur of rats. Cell growth and viability was assessed by standard colorimetric assays like MTT assay. The cell differentiation into osteoblastic lineage was evaluated by the activities of bone marker alkaline phosphatase. The effect of the extract on matrix mineralization was assessed by alizarin red-s staining and Von kossa staining. Cell morphology was studied by phase contrast microscopy and light microscopy (Giemsa/crystal violet staining). ResultsResults indicate that the alcoholic extract of TC at a dosage of 25μg/ml stimulated the growth of osteoblasts, increased the differentiation of cells into osteoblastic lineage and increased the mineralization of bone like matrix on both the osteoblast model systems used in the study. Cell morphology studies clearly indicated the increase in cell numbers and absence of adverse change in the cell morphology on treatment with the extract. ConclusionTC extract has a potential influence on osteogenesis and hence its use could be explored as a potential anti-osteoporotic agent.

Absence of Glutamine Supplementation Prevents Differentiation of Murine Calvarial Osteoblasts to a Mineralizing Phenotype

Osteoblasts in vitro differentiate from a proliferating to a mineralizing phenotype upon transfer to a medium rich in beta-glycerophosphate and ascorbic acid. The nutritional requirements of the cells at different stages of this differentiation process are not known. In other cell types, nutritional supplementation during surgery can improve the outcome in terms of speed of patient recovery and prognosis. There is therefore the potential for supplementation at the site of fracture repair or bone grafting with critical osteoblast nutritional factors to potentially accelerate healing. In this study we investigate which common cell nutrients are required for the proliferating and mineralizing stages of osteoblast differentiation. Medium containing 5.5mM glucose was sufficient to achieve maximal proliferation of primary calvarial osteoblasts and human osteoblast cell lines, with some added benefit of additional glutamine supplementation. However, when cells were stimulated to mineralize, glucose was insufficient to support their energetic requirements. Only when cells were supplemented with glucose together with glutamine were high levels of osteocalcin expression observed together with mineralized nodules in culture, suggesting that this would be a useful combination to assess in cultures of primary human osteoblasts to determine whether it may have beneficial effects during fracture surgery, bone grafting, and fixation of uncemented arthroplasty implants.

Metastatic breast cancer cells inhibit osteoblast differentiation through the Runx2/CBFβ-dependent expression of the Wnt antagonist, sclerostin

IntroductionBreast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction by stimulating osteoclasts to resorb bone and by preventing osteoblasts from producing new bone. The Runt-related transcription factor 2, Runx2, is an important determinant of bone metastasis in breast cancer. Runx2 is known to mediate activation of osteoclast activity and inhibition of osteoblast differentiation by metastatic breast cancer cells. However, while Runx2-regulated genes that mediate osteoclast activation have been identified, how Runx2 determines inhibition of osteoblasts is unknown.MethodsThe aim of this study was to determine how Runx2 mediates the ability of metastatic breast cancer cells to modulate the activity of bone cells. We have previously demonstrated that Runx2 requires the co-activator core binding factor beta (CBFβ) to regulate gene expression in breast cancer cells. We, therefore, performed independent microarray analyses to identify target genes whose expression is dependent upon both Runx2 and CBFβ. Common target genes, with a role in modulating bone-cell function, were confirmed using a combination of siRNA, quantitative reverse transcriptase PCR (qRT-PCR), ELISA, promoter reporter analysis, Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The function of Runx2/CBFβ-regulated genes in mediating the ability of MDA-MB-231 to inhibit osteoblast differentiation was subsequently established in primary bone marrow stromal cell cultures and MC-3T3 osteoblast cells.ResultsWe show that Runx2/CBFβ mediates inhibition of osteoblast differentiation by MDA-MB-231 cells through induction of the Wnt signaling antagonist, sclerostin. We demonstrate that MDA-MB-231 cells secrete sclerostin and that sclerostin-expression is critically dependent on both Runx2 and CBFβ. We also identified the osteoclast activators IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as new target genes of Runx2/CBFβ in metastatic breast cancer cells.ConclusionsThis study demonstrates that Runx2 and CBFβ are required for the expression of genes that mediate the ability of metastatic breast cancer cells to directly modulate both osteoclast and osteoblast function. We also show that Runx2-dependent inhibition of osteoblast differentiation by breast cancer cells is mediated through the Wnt antagonist, sclerostin.

Osthol, a Coumarin Isolated from Common Cnidium Fruit, Enhances the Differentiation and Maturation of Osteoblasts in vitro

The effect of osthol on osteoblasts was investigated in primary osteoblastic cells isolated from newborn Wistar rats. Osthol was supplemented into cultured medium at 10^–7, 10^–6, 10^–5 and 10^–4 mol/l, respectively. No stimulating effect was found on cell proliferation, but 10^–5 mol/l osthol caused a significant increase in alkaline phosphatase (ALP) activity. Osteogenic differentiation markers were examined over a period of time at this concentration, and compared with control cells that were not supplemented with osthol. The results showed that the ALP activity, osteocalcin secretion and calcium deposition level in cells treated with osthol were 1.52, 2.74 and 2.0 times higher, respectively, than in the control cells. Results of ALP histochemical staining and mineralized bone nodule assays both showed that the number and area achieved in osthol-treated cells were 1.53-fold higher than in control cells. The gene expression of the growth and transcription factors basic fibroblast growth factor, insulin-like growth factor I, bone morphogenetic protein 2 (BMP-2), runt-related gene 2 (Runx-2) and osterix, which are associated with bone development, were also investigated. The increase in mRNA expression was 1.94, 1.74, 1.68, 1.83 and 2.31 times, respectively, higher compared to the control. Furthermore, osthol increased the protein expression of p38 mitogen-activated protein kinase (MAPK) and type I collagen. p38MAPK protein and collagen in osthol-treated cells were 1.42 and 1.58 times higher in osthol-treated cells compared to the control. The results of these studies support the conclusion that osthol significantly enhances the osteogenic differentiation of cultured osteoblasts. The results also indicated that osthol could stimulate the osteoblastic differentiation of rat calvarial osteoblast cultures by the BMP-2/p38MAPK/Runx-2/osterix pathway and that osthol may be used as an important compound in the development of new antiosteoporosis drugs.

The Oncofetal Gene Survivin Promotes Cell Proliferation and Survival in Primary Human Osteoblastic Cells

Survivin, the smallest member of the inhibitor of apoptosis gene family, is critical for the regulation of mitosis and maintenance of cell viability during embryonic development and cancer, while not being detectable in most adult differentiated tissues. We know little about whether survivin plays any physiological or pathophysiological role in the adult musculoskeletal system. We studied the expression of survivin in primary human osteoblastic cells and its biological functions in vitro. Survivin was detected by immunoblotting and real-time PCR. Subcellular localization was analyzed by immunofluorescence. Transfection of siRNA and plasmids coding for wild-type survivin was performed to study survivin function, i.e., proliferation and apoptosis assays. Survivin mRNA and protein are expressed in primary human osteoblastic cells. During interphase survivin localizes predominantly to the cytoplasmic compartment, which is relevant for the organization of the spindle apparatus during mitosis. Survivin knockdown resulted in an arrest of the cell cycle at the G(2)/M phase and increased rates of apoptosis. Elevated levels of survivin in primary human osteoblasts enhanced proliferation and cell viability. Taken together, we demonstrate for the first time that survivin is expressed in primary human osteoblastic cells on the mRNA and protein levels. Our results indicate that survivin is a critical factor for cell division and cell viability in primary human osteoblastic cells. Learning more about survivin's role in human osteoblasts could be an important step toward understanding the complex processes involved in bone homeostasis and remodeling.

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 μM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

Transcriptional Regulation of BMP2 Expression by the PTH-CREB Signaling Pathway in Osteoblasts

Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.