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Osteoblastic Cells (osteoblastic + cell)
Kinds of Osteoblastic Cells Terms modified by Osteoblastic Cells Selected AbstractsCyclin D1 as a Target for the Proliferative Effects of PTH and PTHrP in Early Osteoblastic CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2007Nabanita S Datta MS Abstract PTHrP induced a proliferative cyclin D1 activation in low-density osteoblastic cells. The process was PKA and MAPK dependent and involved both AP-1 and CRE sites. In ectopic ossicles generated from implanted bone marrow stromal cells, PTH upregulated cyclin D1 after acute or intermittent anabolic treatment. These data suggest a positive role of PTH and PTHrP in the cell cycle of early osteoblasts. Introduction: The mechanisms underlying the actions of PTH and its related protein (PTHrP) in osteoblast proliferation, differentiation, and bone remodeling remain unclear. The action of PTH or PTHrP on the cell cycle during osteoblast proliferation was studied. Materials and Methods: Mouse calvarial MC3T3-E1 clone 4 cells were synchronized by serum starvation and induced with 100 nM PTHrP for 2,24 h under defined low serum conditions. Western blot, real-time PCR, EMSAs, and promoter/luciferase assays were performed to evaluate cyclin D1 expression. Pharmacological inhibitors were used to determine the relevant signaling pathways. Ectopic ossicles generated from implanted bone marrow stromal cells were treated with acute (a single 8- or 12-h injection) or intermittent anabolic PTH treatment for 7 days, and RNA and histologic analysis were performed. Results: PTHrP upregulated cyclin D1 and CDK1 and decreased p27 expression. Cyclin D1 promoter/luciferase assays showed that the PTHrP regulation involved both activator protein-1 (AP-1) and cyclic AMP response element binding protein (CRE) sites. AP-1 and CRE double mutants completely abolished the PTHrP effect of cyclin D1 transcription. Upregulation of cyclin D1 was found to be protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) dependent in proliferating MC3T3-E1 cells. In vivo expression of cyclin D1 in ectopic ossicles was upregulated after a single 12-h PTH injection or intermittent anabolic PTH treatment for 7 days in early developing ossicles. Conclusions: These data indicate that PTH and PTHrP induce cyclin D1 expression in early osteoblastic cells and their action is developmental stage specific. [source] Alendronate Interacts With the Inhibitory Effect of 1,25(OH)2D3 on Parathyroid Hormone-Related Protein Expression In Human Osteoblastic Cells,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2003L Gómez-García Abstract The bisphosphonate alendronate is a potent inhibitor of bone resorption by its direct action on osteoclasts. In addition, there is some data suggesting that alendronate could also inhibit bone resorption indirectly by interacting with osteoblasts. Parathyroid hormone-related protein (PTHrP) produced by osteoblasts and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are regulators of bone remodeling, which have interrelated actions in these cells. In this study, we assessed whether alendronate can affect PTHrP expression in the presence or absence of 1,25(OH)2D3 in human primary osteoblastic (hOB) cells from trabecular bone. Cell total RNA was isolated, and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out using human PTHrP-specific primers. PTHrP in the hOB cell-conditioned medium was analyzed by a specific immunoradiometric assay. We found that PTHrP mRNA and secreted PTHrP were maximally inhibited by 10,8 -10,6 M of 1,25(OH)2D3 treatment within 8,72 h in hOB cells. Alendronate (10,14 -10,8 M) modified neither PTHrP mRNA nor PTHrP secretion, although it consistently abrogated the decrease in PTHrP production induced by 1,25(OH)2D3 in these cells. On the other hand, alendronate within the same dose range did not affect either the vitamin D receptor (VDR) mRNA or osteocalcin secretion, with or without 1,25(OH)2D3, in hOB cells. The inhibitory effect of alendronate on the 1,25(OH)2D3 -induced decrease in PTHrP in these cells was mimicked by the calcium ionophore A23187 (5 × 10,6 M), while it was eliminated by 5 × 10,5 M of nifedipine. Furthermore, although alendronate alone failed to affect [Ca2+]i in these cells, it stimulated [Ca2+]i after pretreatment of hOB cells with 10,8 M of 1,25(OH)2D3, an effect that was abolished by 5 × 10,5 M of nifedipine. These results show that alendronate disrupts the modulatory effect of 1,25(OH)2D3 on PTHrP production in hOB cells. Our findings indicate that an increase in calcium influx appears to be involved in the mechanism mediating this effect of alendronate. [source] Induction of Transcriptional Activity of the Cyclic Adenosine Monophosphate Response Element Binding Protein by Parathyroid Hormone and Epidermal Growth Factor in Osteoblastic Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002John T. Swarthout Abstract Previously, we have shown that parathyroid hormone (PTH) transactivation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) requires both serine 129 (S129) and serine 133 (S133) in rat osteosarcoma cells UMR 106-01 (UMR) cells. Furthermore, although protein kinase A (PKA) is responsible for phosphorylation at S133, glycogen synthase kinase 3, (GSK-3,) activity is required and may be responsible for phosphorylation of CREB at S129. Here, we show, using the GAL4-CREB reporter system, that epidermal growth factor (EGF) can transactivate CREB in UMR cells in addition to PTH. Additionally, treatment of UMR cells with both PTH and EGF results in greater than additive transactivation of CREB. Furthermore, using mutational analysis we show that S129 and S133 are required for EGF-induced transcriptional activity. EGF activates members of the MAPK family including p38 and extracellular signal,activated kinases (ERKs), and treatment of UMR cells with either the p38 inhibitor (SB203580) or the MEK inhibitor (PD98059) prevents phosphorylation of CREB at S133 by EGF but not by PTH. Treatment of cells with either SB203580 or PD98059 alone or together significantly inhibits transactivation of CREB by EGF but not by PTH, indicating that EGF regulates CREB phosphorylation and transactivation through p38 and ERKs and PTH does not. Finally, the greater than additive transactivation of CREB by PTH and EGF is significantly inhibited by the PKA inhibitor H-89 or by cotreatment with SB203580 and PD98059. Thus, several different signaling pathways in osteoblastic cells can converge on and regulate CREB activity. This suggests, in vivo, that circulating agents such as PTH and EGF are acting in concert to exert their effects. [source] A Role for G Protein-Coupled Lysophospholipid Receptors in Sphingolipid-Induced Ca2+ Signaling in MC3T3-E1 Osteoblastic CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2001Jeremy M. Lyons Abstract Sphingolipids have been proposed to modulate cell function by acting as intracellular second messengers and through binding to plasma membrane receptors. Exposure of MC3T3-E1 osteoblastic cells to sphingosine (SPH), sphingosine-1-phosphate (SPP), or sphingosylphosphorylcholine (SPC) led to the release of Ca2+ from the endoplasmic reticulum (ER) and acute elevations in cytosolic-free Ca2+ ([Ca2+]i). Desensitization studies suggest that SPP and SPC bind plasma membrane endothelial differentiation gene (Edg) receptors for lysophosphatidic acid (LPA). Consistent with the coupling of Edg receptors to G proteins, SPP- and SPC-induced Ca2+ signaling was inhibited by pretreatment of the cells with pertussis toxin (PTx). Of the Edg receptors known to bind SPH derivatives in other cell types, MC3T3-E1 cells were found to express transcripts encoding Edg -1 and Edg -5 but not Edg -3, Edg -6, or Edg -8. In contrast to SPP and SPC, the ability of SPH to elicit [Ca2+]i elevations was affected neither by prior exposure of cells to LPA nor by PTx treatment. However, LPA-induced Ca2+ signaling was blocked in MC3T3-E1 cells previously exposed to SPH. Elevations in [Ca2+]i were not evoked by SPP or SPC in cells treated with 2-aminoethoxydiphenylborate (2-APB), an inhibitor of inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ channels in the ER. No effect of 2-APB was observed on SPH- or LPA-induced [Ca2+]i elevations. The data support a model in which SPP and SPC bind Edg -1 and/or Edg -5 receptors in osteoblasts leading to the release of Ca2+ from the ER through IP3 -gated channels. [source] Dual Mechanism of Intercellular Communication in HOBIT Osteoblastic Cells: A Role for Gap-Junctional HemichannelsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2001Milena Romanello Abstract Intercellular communication allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks, which favors the intercellular exchange of nutrients and second messengers. Intercellular Ca2+ signaling was investigated in human osteoblast-like initial transfectant (HOBIT) cells, a human osteoblastic cell line in which cells retain most of the osteoblastic differentiation markers. HOBIT cells express connexin43 (Cx43) clustered at the cell-to-cell boundary and display functional intercellular coupling as assessed by the intercellular transfer of Lucifer yellow. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading and dye transfer were inhibited by 18,-glycyrrhetinic acid (18-GA), showing the involvement of gap junctions in signal propagation. Pretreatment of cells with suramin or with apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signaling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake, and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate (InsP3) from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18-GA. A role for Cx hemichannels in adenosine triphosphate (ATP) release and paracrine stimulation is suggested. [source] Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependentJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Grasiele E. Crippa Abstract It has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13,16 years old), young adults (18,35 years old), and adults (36,49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents. J. Cell. Biochem. 104: 369,376, 2008. © 2007 Wiley-Liss, Inc. [source] Transforming growth factor- ,3 (Tgf- ,3) down-regulates Tgf- , receptor type I (T,r-I) during rescue of cranial sutures from osseous obliterationORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 1 2002LA Opperman Abstract Appropriate biochemical regulation of intramembranous bone growth from sutures is necessary to achieve correct craniofacial morphology. Failure to form sutures (agenesis) or to maintain sutures in their unossified state (craniosynostosis) can result in severe facial dysmorphology. Several factors such as Twist, Msx2, fibroblast growth factors (Fgfs), bone morphogenetic proteins (Bmps) and transforming growth factors- , (Tgf- ,s) regulate suture patency, likely by interacting with one another. Tgf- ,2 and Tgf- ,3 use the same cell surface receptors, yet have opposite effects on suture patency, cellular proliferation and apoptosis within the suture. One possible mechanism by which Tgf- ,3 rescues sutures from obliteration is by regulating the ability of suture cells to respond to Tgf- ,2. As Tgf- ,3 does not regulate protein levels of Tgf- ,2 in sutures, Tgf- ,3 could regulate tissue responsiveness to Tgf- ,2 by regulating Tgf- ,2 access to receptors. Tgf- ,3 is a more potent competitor than Tgf- ,2 for cell surface receptors, so it is proposed that Tgf- ,3 binds to and down-regulates Tgf- , receptor type I (T,r-I) expression by suture cells. This down-regulation would limit the ability of cells to respond to all Tgf- ,s, including Tgf- ,2. To test this hypothesis, an in vitro culture model was used in which fetal rat sutures either remain patent or are induced to fuse when cultured in the presence or absence of dura mater, respectively. Tgf- ,3 was added to cultured calvaria and changes in the number of receptor positive cells within the suture were established. Data were compared with that seen in control sutures and in normal sutures in vivo. It was found that the numbers of cells expressing T,r-I within the suture matrix increased over time in sutures remaining patent. Osteoblastic cells lining the bone fronts on either side of sutures were T,r-I positive during early morphogenesis, but these numbers declined as sutures fused, both in vivo and in vitro. Addition of Tgf- ,3 to calvaria in culture decreased the number of T,r-I expressing cells in both fusing and non-fusing sutures, with dramatic decreases in the numbers of osteoblasts expressing T,r-I. [source] Positively Charged Material Surfaces Generated by Plasma Polymerized Allylamine Enhance Vinculin Mobility in Vital Human Osteoblastss,ADVANCED ENGINEERING MATERIALS, Issue 8 2010Henrike Rebl Abstract Several studies suggest that the modification of an implant surface by chemical means plays an important role in bone tissue engineering. Previously we have shown that osteoblast cell adhesion and spreading can strongly be increased by a positively charged surface. Cell adhesion and migration are two vital processes that are completely dependent on coordinated formation of focal adhesions. Changes in the organization of the actin cytoskeleton and the focal adhesions are essential for numerous cellular processes including cell motility and tissue morphogenesis. We examined the mobility of the cytoskeletally associated protein vinculin on functionalized surfaces using plasma polymerized allylamine (PPAAm), a homogenous plasma polymer layer with randomly distributed amino groups. In living, GFP,vinculin transfected osteoblastic cells we determined a significant increase in vinculin mobility and vinculin contact length on PPAAm compared to collagen I coated surfaces during the initial adhesion phase. We suggest that positive charges control the cell physiology which seems to be dominant over the integrin receptor binding to collagen I. The results emphasize the role of the surface charge for the design of artificial scaffolds in bone repair. [source] Diamond Seeding and Growth of Hierarchically Structured Films for Tissue Engineering,ADVANCED ENGINEERING MATERIALS, Issue 7 2009Alexander Kromka The importance of using optimized diamond powder suspension for an efficient seeding process is shown to be crucial for the growth of hierarchically structured nanocrystalline diamond (NCD) films. The morphology of freshly adhered human osteoblastic cells (SAOS-2) on such structured NCD films is significantly influenced by the substrate roughness. The hierarchically structured NCD films can thus find application in cell cultivation implants and tissue engineering. [source] Bumetanide, the Specific Inhibitor of Na+ -K+ -2Cl, Cotransport, Inhibits 1,,25-Dihydroxyvitamin D3 -Induced Osteoclastogenesis in a Mouse co-culture SystemEXPERIMENTAL PHYSIOLOGY, Issue 5 2003Hyun-A Lee The Na+ -K+ -2Cl, cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-,B ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 µM bumetanide reduced RANKL mRNA expression induced by 10 nM 1,,25-dihydroxyvitamin D3 (1,,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2 -terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1,,25(OH)2D3 -induced osteoclastogenesis partly via the phosphorylation of JNK. [source] Association of a single nucleotide polymorphism in the steroid and xenobiotic receptor (SXR) gene (IVS1-579A/G) with bone mineral densityGERIATRICS & GERONTOLOGY INTERNATIONAL, Issue 2 2007Tomohiko Urano Vitamin K2 plays an important role in the bone metabolism. The steroid and xenobiotic receptor (SXR) as a nuclear receptor activated by vitamin K2 as well as rifampicin could increase bone markers such as alkaline phosphatase in human osteoblastic cells. Thus, the SXR could mediate vitamin K2 signaling pathway in bone cells. Therefore, we analyzed expression of the SXR mRNA in human primary osteoblasts and chondrocytes. We also studied association of a single nucleotide polymorphism (SNP) in the SXR gene with bone mineral density (BMD). Expression levels of the SXR mRNA were analyzed during the culture course of human primary osteoblasts and chondrocytes. Association of a SNP in the SXR gene in intron 1 (IVS1-579A>G) with BMD was examined in 294 healthy postmenopausal Japanese women. The SXR mRNA increased at day 5 and then decreased at day 10 in human primary osteoblasts. Its mRNA gradually increased in human primary chondrocytes until day 10. As an association study of a SNP in the SXR gene (IVS1-579A/G), the subjects without the A allele (GG; n = 47) had significantly higher total BMD than the subjects bearing at least one A allele (AA + AG; n = 247) (Z score ± SD; 0.635 ± 1.031 versus 0.268 ± 1.061; P = 0.0298). The SXR mRNA was expressed and regulated in primary human osteoblasts and chondrocytes. A genetic variation at the SXR gene locus is associated with BMD, suggesting an involvement of the SXR gene in human bone metabolism. [source] Functional behaviour of bone around dental implants,GERODONTOLOGY, Issue 2 2004Clark M. Stanford Achieving a long-term stable implant interface is a significant clinical issue when there is insufficient cortical bone stabilisation at implant placement. Clinical outcomes studies suggest that the higher risk implants are those placed in compromised cortical bone (thin, porous, etc.) in anatomical sites with minimal existing trabecular bone (characterised as type IV bone). In establishing and maintaining an implant interface in such an environment, one needs to consider the impact of masticatory forces, the response of bone to these forces and the impact of age on the adaptive capacity of bone. These forces, in turn, have the potential to create localised changes in interfacial stiffness through viscoelastic changes at the interface. Changes in bone as a function of age (e.g. localised hypermineralised osteopetrosis and localised areas of osteopenia) will alter the communication between osteocytes and osteoblasts creating the potential for differences in response of osteoblastic cells in the older population. A key to understanding the biomechanical and functional behaviour of implants in the older population is to control the anticipated modelling and remodelling behaviour through implant design that takes into account how tissues respond to the mechanically active environment. [source] Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone-forming capacityJOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2010Kenneth H Larsen Abstract The ability of bone marrow stromal cells (BMSCs) to differentiate into osteoblasts is being exploited in cell-based therapy for repair of bone defects. However, the phenotype of ex vivo cultured BMSCs predicting their bone-forming capacity is not known. Thus we employed DNA microarrays comparing two human bone marrow stromal cell (hBMSC) populations: One is capable of in vivo heterotopic bone formation (hBMSC-TERT+Bone), and the other is not (hBMSC-TERT,Bone). Compared with hBMSC-TERT,Bone, the hBMSC-TERT+Bone cells had an increased overrepresentation of extracellular matrix genes (17% versus 5%) and a larger percentage of genes with predicted SP3 transcription factor,binding sites in their promoter region (21% versus 8%). On the other hand, hBMSC-TERT,Bone cells expressed a larger number of immune-response-related genes (26% versus 8%). In order to test for the predictive value of these markers, we studied the correlation between their expression levels in six different hBMSC-derived clones and the ability to form bone in vivo. We found a significant correlation for decorin, lysyl oxidase-like 4, natriuretic peptide receptor C, and tetranectin. No significant positive correlation was found for canonical osteoblastic markers Runx2, alkaline phosphatase, collagen type I, osteopontin, and bone sialoprotein. Prospective isolation of four additional hBMSC clones based on their expression levels of the molecular markers correlated with their in vivo bone-formation ability. In conclusion, our data suggest an in vitro molecular signature predictive for hBMSCs' in vivo bone-formation ability. Identifying more of these predictive markers would be very useful in the quality control of osteoblastic cells before use in therapy. © 2010 American Society for Bone and Mineral Research [source] Connexin 43 Is Required for the Anti-Apoptotic Effect of Bisphosphonates on Osteocytes and Osteoblasts In Vivo,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2008Lilian I Plotkin Abstract Connexin (Cx)43 is required for inhibition of osteocyte and osteoblast apoptosis by bisphosphonates in vitro. Herein, we evaluated its requirement for the in vivo actions of bisphosphonates using mice in which Cx43 was deleted specifically from osteocytes and osteoblasts (Cx43,Ob,Ot/, mice). Effective removal of Cx43 was confirmed by the presence of the deleted form of the gene and by reduced mRNA and protein expression in osteoblastic cells and bones obtained from Cx43,Ob,Ot/, mice. The amino-bisphosphonate alendronate (2.3 ,mol/kg/d) was injected daily into 5-mo-old female mice (n = 6,11) for 31 days, starting 3 days before implantation of pellets releasing the glucocorticoid prednisolone (2.1 mg/kg/d). Cx43,Ob,Ot/, mice and their littermates (Cx43fl/,, Cx43,Ob,Ot/+, and Cx43fl/+) gained bone with similar kinetics and exhibited identical bone mass from 2 to 4.5 mo of age, indicating that Cx43 deletion from osteocytes and mature osteoblasts does not impair bone acquisition. In addition, prednisolone induced a similar increase in osteocyte and osteoblast apoptosis in Cx43,Ob,Ot/, or in control Cx43fl/, littermates. However, whereas alendronate prevented prednisolone-induced apoptosis in control Cx43fl/, mice, it was ineffective in Cx43,Ob,Ot/, mice. In contrast, alendronate inhibited glucocorticoid-induced bone loss in both type of animals, suggesting that inhibition of resorption is the predominant effect of alendronate against the early phase of glucocorticoid-induced bone loss. Taken together with earlier in vitro evidence, these findings show that Cx43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts. [source] Maspin Is Involved in Bone Matrix Maturation by Enhancing the Accumulation of Latent TGF-,,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2007Reiko Tokuyama Abstract Maspin, a serine protease inhibitor, is expressed by formative osteoblasts. The repression of maspin expression in osteoblastic cells decreased the level of latent TGF-, in the extracellular matrix, whereas the overexpression of maspin increased latent TGF-,. These findings suggest that maspin plays an important role in bone matrix formation, particularly in the accumulation of latent TGF-,. Introduction: Maspin is a serine protease inhibitor that exhibits tumor suppressive and anti-angiogenic activities. This study was performed to elucidate a possible role for maspin in bone formation. Materials and Methods: We performed immunohistochemical analysis of the expression of maspin during endochondral ossification. We evaluated the expression of maspin mRNA and protein in ROS 17/2.8 cells and primary rat osteoblastic cells by RT-PCR, immunocytochemistry, and Western blot analysis. We also examined the accumulation of TGF-, in the extracellular matrix of cultured ROS 17/2.8 cells after transfection with vectors expressing either maspin or maspin antisense. Results: We observed expression of maspin by active osteoblasts in vivo. Rat osteoblastic cells also expressed maspin mRNA and protein in vitro. Moreover, the accumulation of latent TGF-, in the extracellular matrix significantly decreased in cultures exposed to an anti-maspin antibody and when cells were transfected with a maspin antisense-expressing vector. In contrast, accumulation of latent TGF-, in the extracellular matrix increased after transfection of cells with a vector expressing maspin. Conclusions: These findings suggest that maspin expressed in active osteoblasts plays an important physiological role during maturation of the bone matrix, and in particular, during the process of accumulation of latent TGF-, in the extracellular matrix. [source] Cyclin D1 as a Target for the Proliferative Effects of PTH and PTHrP in Early Osteoblastic CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2007Nabanita S Datta MS Abstract PTHrP induced a proliferative cyclin D1 activation in low-density osteoblastic cells. The process was PKA and MAPK dependent and involved both AP-1 and CRE sites. In ectopic ossicles generated from implanted bone marrow stromal cells, PTH upregulated cyclin D1 after acute or intermittent anabolic treatment. These data suggest a positive role of PTH and PTHrP in the cell cycle of early osteoblasts. Introduction: The mechanisms underlying the actions of PTH and its related protein (PTHrP) in osteoblast proliferation, differentiation, and bone remodeling remain unclear. The action of PTH or PTHrP on the cell cycle during osteoblast proliferation was studied. Materials and Methods: Mouse calvarial MC3T3-E1 clone 4 cells were synchronized by serum starvation and induced with 100 nM PTHrP for 2,24 h under defined low serum conditions. Western blot, real-time PCR, EMSAs, and promoter/luciferase assays were performed to evaluate cyclin D1 expression. Pharmacological inhibitors were used to determine the relevant signaling pathways. Ectopic ossicles generated from implanted bone marrow stromal cells were treated with acute (a single 8- or 12-h injection) or intermittent anabolic PTH treatment for 7 days, and RNA and histologic analysis were performed. Results: PTHrP upregulated cyclin D1 and CDK1 and decreased p27 expression. Cyclin D1 promoter/luciferase assays showed that the PTHrP regulation involved both activator protein-1 (AP-1) and cyclic AMP response element binding protein (CRE) sites. AP-1 and CRE double mutants completely abolished the PTHrP effect of cyclin D1 transcription. Upregulation of cyclin D1 was found to be protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) dependent in proliferating MC3T3-E1 cells. In vivo expression of cyclin D1 in ectopic ossicles was upregulated after a single 12-h PTH injection or intermittent anabolic PTH treatment for 7 days in early developing ossicles. Conclusions: These data indicate that PTH and PTHrP induce cyclin D1 expression in early osteoblastic cells and their action is developmental stage specific. [source] Thyroid-Stimulating Hormone Restores Bone Volume, Microarchitecture, and Strength in Aged Ovariectomized Rats*,,§JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2007T Kuber Sampath PhD Abstract We show the systemic administration of low levels of TSH increases bone volume and improves bone microarchitecture and strength in aged OVX rats. TSH's actions are mediated by its inhibitory effects on RANKL-induced osteoclast formation and bone resorption coupled with stimulatory effects on osteoblast differentiation and bone formation, suggesting TSH directly affects bone remodeling in vivo. Introduction: Thyroid-stimulating hormone (TSH) receptor haploinsufficient mice with normal circulating thyroid hormone levels have reduced bone mass, suggesting that TSH directly affects bone remodeling. We examined whether systemic TSH administration restored bone volume in aged ovariectomized (OVX) rats and influenced osteoclast formation and osteoblast differentiation in vitro. Materials and Methods: Sprague-Dawley rats were OVX at 6 months, and TSH therapy was started immediately after surgery (prevention mode; n = 80) or 7 mo later (restoration mode; n = 152). Hind limbs and lumbar spine BMD was measured at 2- or 4-wk intervals in vivo and ex vivo on termination at 8,16 wk. Long bones were subjected to ,CT, histomorphometric, and biomechanical analyses. The direct effect of TSH was examined in osteoclast and osteoblast progenitor cultures and established rat osteosarcoma-derived osteoblastic cells. Data were analyzed by ANOVA Dunnett test. Results: In the prevention mode, low doses (0.1 and 0.3 ,g) of native rat TSH prevented the progressive bone loss, and importantly, did not increase serum triiodothyroxine (T3) and thyroxine (T4) levels in aged OVX rats. In restoration mode, animals receiving 0.1 and 0.3 ,g TSH had increased BMD (10,11%), trabecular bone volume (100,130%), trabecular number (25,40%), trabecular thickness (45,60%), cortical thickness (5,16%), mineral apposition and bone formation rate (200,300%), and enhanced mechanical strength of the femur (51,60%) compared with control OVX rats. In vitro studies suggest that TSH's action is mediated by its inhibitory effects on RANKL-induced osteoclast formation, as shown in hematopoietic stem cells cultivated from TSH-treated OVX rats. TSH also stimulates osteoblast differentiation, as shown by effects on alkaline phosphatase activity, osteocalcin expression, and mineralization rate. Conclusions: These results show for the first time that systemically administered TSH prevents bone loss and restores bone mass in aged OVX rats through both antiresorptive and anabolic effects on bone remodeling. [source] Differential Expression of Estrogen Receptor-Related Receptor , and Estrogen Receptors , and , in Osteoblasts In Vivo and In Vitro,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002Edith Bonnelye Abstract The orphan nuclear estrogen receptor-related receptor (ERR) , is expressed by osteoblastic cells, is known to transactivate at least one osteoblast-associated gene osteopontin (OPN) and plays a functional role in osteoprogenitor cell proliferation and differentiation. To dissect further the role of ERR-, in bone formation, we compared its expression to that of the estrogen receptor (ER) , and ER-, in rat calvaria (RC) and fetal tibia in vivo and in RC and rat bone marrow (RBM) cells in vitro. We found that ERR-, is highly and widely expressed in most, if not all, cells in RC cell cultures from early proliferation stages through mineralized nodule formation; ER-, was localized similarly but at lower levels and ER-,, although present, was barely detectable. These patterns of expression in vitro correlated with what we observed in vivo in sections of 21-day fetal RC, in which ERR-, appeared to be more highly expressed than either of the ERs. Interestingly, ERR-, also is highly expressed in RBM cells, while ER-, and ER-, mRNA is expressed, but at lower levels. Moreover, we found that ERR-,, ER-,, and ER-, were all expressed in osteoblasts in fetal and adult tibia whereas they were expressed differentially in calvaria in vivo in subsets of osteoblasts, supporting the hypothesis that ERR-, may interact with one or both of the ERs in those osteoblasts in which they are coexpressed and that all three receptors may be required for bone formation but at different times and for different functions. [source] Induction of Transcriptional Activity of the Cyclic Adenosine Monophosphate Response Element Binding Protein by Parathyroid Hormone and Epidermal Growth Factor in Osteoblastic Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002John T. Swarthout Abstract Previously, we have shown that parathyroid hormone (PTH) transactivation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) requires both serine 129 (S129) and serine 133 (S133) in rat osteosarcoma cells UMR 106-01 (UMR) cells. Furthermore, although protein kinase A (PKA) is responsible for phosphorylation at S133, glycogen synthase kinase 3, (GSK-3,) activity is required and may be responsible for phosphorylation of CREB at S129. Here, we show, using the GAL4-CREB reporter system, that epidermal growth factor (EGF) can transactivate CREB in UMR cells in addition to PTH. Additionally, treatment of UMR cells with both PTH and EGF results in greater than additive transactivation of CREB. Furthermore, using mutational analysis we show that S129 and S133 are required for EGF-induced transcriptional activity. EGF activates members of the MAPK family including p38 and extracellular signal,activated kinases (ERKs), and treatment of UMR cells with either the p38 inhibitor (SB203580) or the MEK inhibitor (PD98059) prevents phosphorylation of CREB at S133 by EGF but not by PTH. Treatment of cells with either SB203580 or PD98059 alone or together significantly inhibits transactivation of CREB by EGF but not by PTH, indicating that EGF regulates CREB phosphorylation and transactivation through p38 and ERKs and PTH does not. Finally, the greater than additive transactivation of CREB by PTH and EGF is significantly inhibited by the PKA inhibitor H-89 or by cotreatment with SB203580 and PD98059. Thus, several different signaling pathways in osteoblastic cells can converge on and regulate CREB activity. This suggests, in vivo, that circulating agents such as PTH and EGF are acting in concert to exert their effects. [source] Smad3 Promotes Alkaline Phosphatase Activity and Mineralization of Osteoblastic MC3T3-E1 Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2002Hideaki Sowa Abstract Transforming growth factor (TGF) , is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-, signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-, on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-, inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L -azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs. [source] Systemic Regulation of Distraction Osteogenesis: A Cascade of Biochemical Factors,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2002S. Weiss M.D. Abstract This study investigates the systemic biochemical regulation of fracture healing in distraction osteogenesis compared with rigid osteotomy in a prospective in vivo study in humans. To further clarify the influence of mechanical strain on the regulation of bone formation, bone growth factors (insulin-like growth factor [IGF] I, IGF binding protein [IGFBP] 3, transforming growth factor [TGF] ,1, and basic FGF [bFGF]), bone matrix degrading enzymes (matrix-metalloproteinases [MMPs] 1, 2, and 3), human growth hormone (hGH), and bone formation markers (ALP, bone-specific ALP [BAP], and osteocalcin [OC]) have been analyzed in serum samples from 10 patients in each group pre- and postoperatively. In the distraction group, a significant postoperative increase in MMP-1, bFGF, ALP, and BAP could be observed during the lengthening and the consolidation period when compared with the baseline levels. Osteotomy fracture healing without the traction stimulus failed to induce a corresponding increase in these factors. In addition, comparison of both groups revealed a significantly higher increase in TGF-,1, IGF-I, IGFBP-3, and hGH in the lengthening group during the distraction period, indicating key regulatory functions in mechanotransduction. The time courses of changes in MMP-1, bone growth factors (TGF-,1 and bFGF), and hGH, respectively, correlated significantly during the lengthening phase, indicating common regulatory pathways for these factors in distraction osteogenesis. Significant correlation between the osteoblastic marker BAP, TGF-,1, and bFGF suggests strain-activated osteoblastic cells as a major source of systemically increased bone growth factors during callus distraction. The systemic increase in bFGF and MMP-1 might reflect an increased local stimulation of angiogenesis during distraction osteogenesis. [source] A Role for G Protein-Coupled Lysophospholipid Receptors in Sphingolipid-Induced Ca2+ Signaling in MC3T3-E1 Osteoblastic CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2001Jeremy M. Lyons Abstract Sphingolipids have been proposed to modulate cell function by acting as intracellular second messengers and through binding to plasma membrane receptors. Exposure of MC3T3-E1 osteoblastic cells to sphingosine (SPH), sphingosine-1-phosphate (SPP), or sphingosylphosphorylcholine (SPC) led to the release of Ca2+ from the endoplasmic reticulum (ER) and acute elevations in cytosolic-free Ca2+ ([Ca2+]i). Desensitization studies suggest that SPP and SPC bind plasma membrane endothelial differentiation gene (Edg) receptors for lysophosphatidic acid (LPA). Consistent with the coupling of Edg receptors to G proteins, SPP- and SPC-induced Ca2+ signaling was inhibited by pretreatment of the cells with pertussis toxin (PTx). Of the Edg receptors known to bind SPH derivatives in other cell types, MC3T3-E1 cells were found to express transcripts encoding Edg -1 and Edg -5 but not Edg -3, Edg -6, or Edg -8. In contrast to SPP and SPC, the ability of SPH to elicit [Ca2+]i elevations was affected neither by prior exposure of cells to LPA nor by PTx treatment. However, LPA-induced Ca2+ signaling was blocked in MC3T3-E1 cells previously exposed to SPH. Elevations in [Ca2+]i were not evoked by SPP or SPC in cells treated with 2-aminoethoxydiphenylborate (2-APB), an inhibitor of inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ channels in the ER. No effect of 2-APB was observed on SPH- or LPA-induced [Ca2+]i elevations. The data support a model in which SPP and SPC bind Edg -1 and/or Edg -5 receptors in osteoblasts leading to the release of Ca2+ from the ER through IP3 -gated channels. [source] A Dominant Negative Cadherin Inhibits Osteoblast Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Su-Li Cheng Abstract We have previously indicated that human osteoblasts express a repertoire of cadherins and that perturbation of cadherin-mediated cell-cell interaction reduces bone morphogenetic protein 2 (BMP-2) stimulation of alkaline phosphatase activity. To test whether inhibition of cadherin function interferes with osteoblast function, we expressed a truncated N-cadherin mutant (NCad,C) with dominant negative action in MC3T3-E1 osteoblastic cells. In stably transfected clones, calcium-dependent cell-cell adhesion was decreased by 50%. Analysis of matrix protein expression during a 4-week culture period revealed that bone sialoprotein, osteocalcin, and type I collagen were substantially inhibited with time in culture, whereas osteopontin transiently increased. Basal alkaline phosphatase activity declined in cells expressing NCad,C, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%). Finally,45Ca uptake, an index of matrix mineralization, was decreased by 35% in NCad,C-expressing cells compared with control cultures after 4 weeks in medium containing ascorbic acid and ,-glycerophosphate. Similarly, BMP-2 stimulation of alkaline phosphatase activity and bone sialoprotein and osteopontin expression also were curtailed in NCad,C cells. Therefore, expression of dominant negative cadherin results in decreased cell-cell adhesion associated with altered bone matrix protein expression and decreased matrix mineralization. Cadherin-mediated cell-cell adhesion is involved in regulating the function of bone-forming cells. [source] Direct and Indirect Actions of Fibroblast Growth Factor 2 on Osteoclastic Bone Resorption in CulturesJOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2000Hiroshi Kawaguchi M.D., Ph.D. Abstract Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF-2 at 10,9 M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10,8 M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction. COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage. To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF-2 (,10,12 M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10,11 M, and no further stimulation was observed at higher concentrations. However, FGF-2 at 10,9 M , 10,8 M stimulated resorbed pit formation by unfractionated bone cells up to 9.7-fold. NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF-2 at low concentrations (,10,12 M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (,10,9 M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently. [source] The notch-responsive transcription factor Hes-1 attenuates osteocalcin promoter activity in osteoblastic cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2009Ying Zhang Abstract Notch signaling plays a key role in osteoblast differentiation. A major transcriptional downstream regulator of this pathway is the helix,loop,helix (HLH) transcription factor Hairy/Enhancer of Split 1 (Hes-1). Here we investigated the function of Hes-1 in osteoblastic cells. Endogenous Hes-1 gene expression decreases during progression of bone cell phenotype development in MC3T3-E1 osteoblasts suggesting that it is a negative regulator of osteoblast differentiation. Forced expression of Hes-1 inhibits osteocalcin (OC) mRNA levels, and luciferase assays indicate that Hes-1 directly represses OC promoter activity. In vitro and in vivo protein/DNA interaction assays reveal that recombinant Hes-1 binds specifically to an E-box in the proximal promoter of the OC gene. Deletion of the Hes-1 WRPW domain (MHes-1) that recruits the co-repressor Groucho abrogates repression of OC promoter activity by Hes-1, but also blocks Hes-1 binding to the promoter. The latter result suggests that exogenous Hes-1 may be recruited to the OC promoter by both protein/DNA and protein/protein interactions. We conclude that the Notch-responsive Hes-1 protein is capable of repressing OC gene transcription in osteoblastic cells through an E-box in the proximal promoter. Hes-1 may contribute to osteoblast growth and differentiation by controlling basal bone-specific transcription directly through interactions with transcriptional regulators that are known to bind to the OC gene promoter. J. Cell. Biochem. 108: 651,659, 2009. © 2009 Wiley-Liss, Inc. [source] miR-29 suppression of osteonectin in osteoblasts: Regulation during differentiation and by canonical Wnt signalingJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2009Kristina Kapinas Abstract The matricellular protein osteonectin, secreted protein acidic and rich in cysteine (SPARC, BM-40), is the most abundant non-collagenous matrix protein in bone. Matricellular proteins play a fundamental role in the skeleton as regulators of bone remodeling. In the skeleton, osteonectin is essential for the maintenance of bone mass and for balancing bone formation and resorption in response to parathyroid hormone (PTH). It promotes osteoblast differentiation and cell survival. Mechanisms regulating the expression of osteonectin in the skeleton and in other tissues remain poorly understood. We found that the proximal region of the mouse osteonectin 3, untranslated region (UTR) contains a well-conserved, dominant regulatory motif that interacts with microRNAs (miRs)-29a and -29c. Transfection of osteoblastic cells with miR-29a inhibitors increased osteonectin protein levels, whereas transfection of miR-29a precursor RNA decreased osteonectin. miR-29a and -29c were increased during osteoblastic differentiation in vitro. The up-regulation of these miRNAs correlated with decreased osteonectin protein during the matrix maturation and mineralization phases of late differentiation. In contrast, osteonectin transcript levels remained relatively constant during this process, implying repression of translation. Treatment of osteoblasts with LiCl induced miR-29a and -29c expression and decreased osteonectin synthesis. When cells were treated with Dickkopf-1 (Dkk-1), miR-29a and -29c expression was repressed. These data suggest that canonical Wnt signaling, which is increased during osteoblastic differentiation, induces expression of miR-29. Osteonectin and miR-29 are co-expressed in extra-skeletal tissues, and the post-transcriptional mechanisms regulating osteonectin in osteoblasts are likely to be active in other cell systems. J. Cell. Biochem. 108: 216,224, 2009. © 2009 Wiley-Liss, Inc. [source] FIAT represses bone matrix mineralization by interacting with ATF4 through its second leucine zipperJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2008Vionnie W.C. Yu Abstract We have characterized FIAT, a 66 kDa leucine zipper (LZ) protein that dimerizes with activating transcription factor 4 (ATF4) to form inactive dimers that cannot bind DNA. Computer analysis identifies three putative LZ motifs within the FIAT amino acid sequence. We have used deletion- and/or site-specific mutagenesis to individually inactivate these motifs in order to identify the functional LZ that mediates the FIAT,ATF4 interaction. Amino acids 194,222 that encode the FIAT LZ2 were deleted (mutant FIAT ZIP2 DEL). We inactivated each zipper individually by replacing two or three leucine residues within each zipper by alanine residues. The engineered mutations were L142A/L149A (mutant M1, first zipper), L208A/L215A/L222A (mutant M2, second zipper), and L441A/L448A (mutant M3, third zipper). MC3T3-E1 osteoblastic cells with an integrated 1.3 kb mouse osteocalcin gene promoter fragment driving expression of luciferase were transfected with expression vectors for ATF4 and the various FIAT deletion- or site-specific mutants. Inhibition of ATF4-mediated transcription was compared between wild-type (WT) and LZ FIAT mutants. The deletion mutant FIAT ZIP2 DEL and the sequence-specific M2 mutant did not interact with ATF4 and were unable to inhibit ATF4-mediated transcription. The M1 or M3 mutations did not affect the ability of FIAT to contact ATF4 or to inhibit its transcriptional activity. Stable expression of WT FIAT in osteoblastic cells inhibited mineralization, but not expression of the FIAT ZIP2 DEL and M2 mutants. This structure,function analysis reveals that FIAT interacts with ATF4 and modulates its activity through its second leucine zipper motif. J. Cell. Biochem. 105: 859,865, 2008. © 2008 Wiley-Liss, Inc. [source] Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependentJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Grasiele E. Crippa Abstract It has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13,16 years old), young adults (18,35 years old), and adults (36,49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents. J. Cell. Biochem. 104: 369,376, 2008. © 2007 Wiley-Liss, Inc. [source] The classic receptor for 1,,25-dihydroxy vitamin D3 is required for non-genomic actions of 1,,25-dihydroxy vitamin D3 in osteosarcoma cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2006Soraya Bravo Abstract 1,,25-dihydroxy vitamin D3 has a major role in the regulation of the bone metabolism as it promotes the expression of key bone-related proteins in osteoblastic cells. In recent years it has become increasingly evident that in addition to its well-established genomic actions, 1,,25-dihydroxy vitamin D3 induces non-genomic responses by acting through a specific plasma membrane-associated receptor. Results from several groups suggest that the classical nuclear 1,,25-dihydroxy vitamin D3 receptor (VDR) is also responsible for these non-genomic actions of 1,,25-dihydroxy vitamin D3. Here, we have used siRNA to suppress the expression of VDR in osteoblastic cells and assessed the role of VDR in the non-genomic response to 1,,25-dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1,,25-dihydroxy vitamin D3-mediated increase in the intracellular Ca2+ concentration, a hallmark of the non-genomic actions of 1,,25-dihydroxy vitamin D3 in these cells. J. Cell. Biochem. 99: 995,1000, 2006. © 2006 Wiley-Liss, Inc. [source] ,-cryptoxanthin stimulates cell differentiation and mineralization in osteoblastic MC3T3-E1 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Satoshi Uchiyama Abstract The effect of ,-cryptoxanthin, a kind of carotenoid, on cell differentiation and mineralization in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 72 h in a minimum essential medium containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or ,-cryptoxanthin (10,8 to 10,6 M) without FBS. Cells were cultured for 3 to 21 days. Gene expression in osteoblastic cells was determined using reverse transcription-polymerase chain reaction (RT-PCR). Culture with ,-cryptoxanthin (10,7 or 10,6 M) for 3 days caused a significant increase in Runx2 type 1, Runx2 type 2, ,1 (I) collagen, and alkaline phosphatase mRNA levels in osteoblastic cells. These increases were completely blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6-dichloro-1-,- D -ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. Meanwhile, vitamin A (10,6 M) did not have a significant effect on Runx2 type 1 mRNA expression in the cells. The effect of ,-cryptoxanthin (10,6 M) in stimulating Runx2 type 1 and ,1 (I) collagen mRNA levels, protein content, and alkaline phosphatase activity in the cells was also seen in the presence of vitamin A (10,6 M), suggesting that the mode of ,-cryptoxanthin action differs from that of vitamin A. Prolonged culture with ,-cryptoxanthin (10,6 M) for 3 to 21 days caused a significant increase in cell number, deoxyribonucleic acid (DNA) content, protein content, and alkaline phosphatase activity in osteoblastic cells, suggesting that ,-cryptoxanthin stimulates cell proliferation and differentiation. Moreover, culture with ,-cryptoxanthin (10,7 or 10,6 M) for 5 to 21 days caused a remarkable increase in mineralization. This study demonstrates that ,-cryptoxanthin has a stimulatory effect on cell differentiation and mineralization due to enhancing gene expression of proteins, which involve in bone formation in osteoblastic MC3T3-E1 cells. © 2005 Wiley-Liss, Inc. [source] | ||||||||||||||||