SEARCH · PubMed Health

Results for “OSTEOBLASTS”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 703 records · Page 39Linked to original sources

PubMed2001

In situ forming lactic acid based orthopaedic biomaterials: influence of oligomer chemistry on osteoblast attachment and function.

The ability of osteoblasts to attach and function normally on scaffolds fabricated from synthetic materials is essential for musculoskeletal tissue engineering applications. In this study, the osteoconductivity of polymer networks formed from multifunctional lactic acid oligomers was assessed. These oligomers form highly crosslinked networks via a photoinitiated polymerization, which provides potential advantages for many orthopaedic applications. Depending on the initial oligomer chemistry and the resultant polymer hydrophobicity, protein adsorption and osteoblast function varied significantly between the various lactic acid based polymer chemistries. Results were compared to control polymers of tissue culture polystyrene (TCPS) and 50:50 poly(lactic-co-glycolic acid) (PLGA). The viability of osteoblasts attached to poly(2EG10LA) and poly(2EG6LA) was close to the TCPS and PLGA after 7 and 14 days of culture, whereas cell viability was approximately 50% lower on poly(8EG6LA). Additionally, the alkaline phosphatase activity and mineralization of attached osteoblasts were similar on poly(2EG10LA) and PLGA, whereas these markers of bone formation were significantly lower for poly(2EG6LA) and poly(8EG6LA). For example, the alkaline phosphatase activity of rat calvarial osteoblasts attached to poly(2EG10LA) was 0.048 +/- 0.006 micromol mg(-1) protein-min, but only 0.030 +/- 0.003 micromol mg(-1) protein-min for osteoblasts attached to poly(8EG6LA) after 14 days of culture. Finally, osteoblasts were seeded onto three-dimensional scaffolds to demonstrate the applicability of the scaffolds for bone tissue engineering.

Adsorption↗

PubMed1999

The developmental control of osteoblast-specific gene expression: role of specific transcription factors and the extracellular matrix environment.

Bone formation is a carefully controlled developmental process involving morphogen-mediated patterning signals that define areas of initial mesenchyme condensation followed by induction of cell-specific differentiation programs to produce chondrocytes and osteoblasts. Positional information is conveyed via gradients of molecules, such as Sonic Hedgehog that are released from cells within a particular morphogenic field together with region-specific patterns of hox gene expression. These, in turn, regulate the localized production of bone morphogenetic proteins and related molecules which initiate chondrocyte- and osteoblast-specific differentiation programs. Differentiation requires the initial commitment of mesenchymal stem cells to a given lineage, followed by induction of tissue-specific patterns of gene expression. Considerable information about the control of osteoblast-specific gene expression has come from analysis of the promoter regions of genes encoding proteins like osteocalcin that are selectively expressed in bone. Both general and tissue-specific transcription factors control this promoter. Osf2/Cbfa1, the first osteoblast-specific transcription factor to be identified, is expressed early in the osteoblast lineage and interacts with specific DNA sequences in the osteocalcin promoter essential for its selective expression in osteoblasts. The OSF2/CBFA1 gene is necessary for the development of mineralized tissues, and its mutation causes the human disease, cleidocranial dysplasia. Committed osteoprogenitor cells already expressing Osf2/Cbfa1 must synthesize a collagenous ECM before they will differentiate. A cell:ECM interaction mediated by integrin-type cell-surface receptors is essential for the induction of osteocalcin and other osteoblast-related proteins. This interaction stimulates the binding of Osf2/Cbfa1 to the osteocalcin promoter through an as-yet-undefined mechanism.

Animals↗

PubMed1984

Separation of osteoblast-like cells from bone marrow by fluorescence-activated cell sorting.

The purification of the osteoblast-like cells (2-3%) among the bone marrow cells (BMC) of C57BL/6 mice using a specific anti-osteoblast serum and a fluorescence-activated cell sorter is described. The antiserum was raised against osteoblast cells isolated from calvaria from neonatal mice. The majority of the cells of the osteoblast-enriched fraction from bone marrow showed a parathormone-induced increase in cyclic adenine monophosphate but no response to calcitonin. This is similar to the response of osteoblast cells obtained from the calvaria. Electron microscopic studies of the extracellular matrix of cultured osteoblast-like cells purified from bone marrow showed the deposition of apatite crystals within and in close apposition to the vesicles. These findings suggest that the isolated cell population was enriched in osteoblasts. Such a cell system from bone marrow might provide an experimental system for investigating the mechanism of bone formation.

Animals↗

PubMed2003

Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts.

BACKGROUND: Staphylococcus aureus infection of normal osteoblasts induces expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). RESULTS: Normal osteoblasts were incubated in the presence of purified bacterial products over a range of concentrations. Results demonstrate that purified surface structures and a selected superantigen present in the extracellular environment are not capable of inducing TRAIL expression by osteoblasts. Osteoblasts were co-cultured with S. aureus at various multiplicities of infection utilizing cell culture chamber inserts. Results of those experiments suggest that direct contact between bacteria and osteoblasts is necessary for optimal TRAIL induction. Finally, S. aureus infection of osteoblasts in the presence of anti-TRAIL antibody demonstrates that TRAIL mediates caspase-8 activation and apoptosis of infected cells. CONCLUSIONS: Collectively, these findings suggest a mechanism whereby S. aureus mediates bone destruction via induction of osteoblast apoptosis.

Animals↗

PubMed2006

Age of donor alters the effect of cyclic hydrostatic pressure on production by human macrophages and osteoblasts of sRANKL, OPG and RANK.

BACKGROUND: Cyclic hydrostatic pressure within bone has been proposed both as a stimulus of aseptic implant loosening and associated bone resorption and of bone formation. We showed previously that cyclical hydrostatic pressure influenced macrophage synthesis of several factors linked to osteoclastogenesis. The osteoprotegerin/soluble receptor activator of NF-kappa beta ligand /receptor activator of NF-kappa beta (OPG/ RANKL/ RANK) triumvirate has been implicated in control of bone resorption under various circumstances. We studied whether cyclical pressure might affect bone turnover via effects on OPG/ sRANKL/ RANK. METHODS: In this study, cultures of human osteoblasts or macrophages (supplemented with osteoclastogenic factors) or co-cultures of macrophages and osteoblasts (from the same donor), were subjected to cyclic hydrostatic pressure. Secretion of OPG and sRANKL was assayed in the culture media and the cells were stained for RANK and osteoclast markers. Data were analysed by nonparametric statistics. RESULTS: In co-cultures of macrophages and osteoblasts, pressure modulated secretion of sRANKL or OPG in a variable manner. Examination of the OPG:sRANKL ratio in co cultures without pressurisation showed that the ratio was greater in donors <70 years at the time of operation (p < 0.05 Mann Whitney U) than it was in patients >70 years. However, with pressure the difference in the OPG:sRANKL ratios between young and old donors was not significant. It was striking that in some patients the OPG:sRANKL ratio increased with pressure whereas in some it decreased. The tendency was for the ratio to decrease with pressure in patients younger than 70 years, and increase in patients > or = 70 years (Fishers exact p < 0.01). Cultures of osteoblasts alone showed a significant increase in both sRANKL and OPG with pressure, and again there was a decrease in the ratio of OPG:RANKL. Secretion of sRANKL by cultures of macrophages alone was not modulated by pressure. Only sRANKL was assayed in this study, but transmembrane RANKL may also be important in this system. Macrophages subjected to pressure (both alone and in co-culture) stained more strongly for RANK on immunohistochemstry than non-pressurized controls and 1,25-dihydroxyvitamin D3 (1,25 D3) further increased this. Immunocytochemical staining also demonstrated that more cells in pressurized co-cultures exhibited osteoclast markers (tartrate-resistant acid phosphatase, vitronectin receptor and multinuclearity) than did unpressurized controls. CONCLUSION: These data show that in co-cultures of osteoblasts and macrophages the ratio of OPG : sRANKL was decreased by pressure in younger patients but increased in older patients. As falls in this ratio promote bone resorption, this finding may be important in explaining the relatively high incidence of osteolysis around orthopaedic implants in young patients. The finding that secretion of OPG and sRANKL by osteoblasts in monoculture was sensitive to hydrostatic pressure, and that hydrostatic pressure stimulated the differentiation of macrophages into cells exhibiting osteoclast markers indicates that both osteoblasts and preosteoclasts are sensitive to cyclic pressure. However, the effects of pressure on cocultures were not simply additive and coculture appears useful to examine the interaction of these cell types. These findings have implications for future therapies for aseptic loosening and for the development of tests to predict the development of this condition.

Acid Phosphatase↗

PubMed2002

The phospholipids sphingosine-1-phosphate and lysophosphatidic acid prevent apoptosis in osteoblastic cells via a signaling pathway involving G(i) proteins and phosphatidylinositol-3 kinase.

The naturally occurring phospholipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have recently emerged as bioactive compounds that exert mitogenic effects in many cell types, including osteoblasts. In the current study, we examined the ability of each of these compounds to influence osteoblast survival. Using terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick-end labeling and DNA fragmentation assays, we found that both LPA and S1P dose-dependently inhibited (by at least 50% and 40%, respectively) the apoptosis induced by serum withdrawal in cultures of primary calvarial rat osteoblasts and SaOS-2 cells. The antiapoptotic effects were inhibited by pertussis toxin, wortmannin, and LY294002, implicating G(i) proteins and phosphatidylinositol-3 kinase (PI-3 kinase) in the signaling pathway that mediates phospholipid-induced osteoblast survival. Specific inhibitors of p42/44 MAPK signaling did not block LPA- or S1P-induced osteoblast survival. LPA and S1P induced PI-3 kinase-dependent activation of p70 S6 kinase, but rapamycin, a specific inhibitor of p70 S6 kinase activation, did not prevent phospholipid-induced osteoblast survival. LPA and S1P also inhibited apoptosis in Swiss 3T3 fibroblastic cells in a G(i) protein-dependent fashion. In fibroblastic cells, however, the antiapoptotic effects of S1P were sensitive to inhibition of both PI-3 kinase and p42/44 MAPK signaling, whereas those of LPA were partially abrogated by inhibitors of p42/44 MAPK signaling but not by PI-3 kinase inhibitors. These data demonstrate that LPA and S1P potently promote osteoblast survival in vitro, and that cell-type specificity exists in the antiapoptotic signaling pathways activated by phospholipids.

Animals↗

PubMed2003

Activation of p38 mitogen-activated protein kinase is required for osteoblast differentiation.

p38 MAPK is a conserved subfamily of MAPKs involved in inflammatory response, stress response, cell growth and survival, as well as differentiation of a variety of cell types. In this report we demonstrated that p38 MAPK played an important role in osteoblast differentiation using primary calvarial osteoblast, bone marrow osteoprecursor culture, and a murine cell line, MC3T3-E1. We found that p38 MAPK was activated as calvarial osteoblast differentiates along with extracellular signal-regulated kinases (ERKs). When p38 MAPK is inhibited with a specific inhibitor, the expression of differentiation markers, such as alkaline phosphatase and mineral deposition, were significantly reduced. MC3T3-E1 cells expressing dominant negative p38 MAPK also displayed signs of delay in ALP and mineral deposition. Differentiation of the bone marrow osteoprecursors was also impeded by the p38 MAPK inhibitor, justified by the same markers. Yet the inhibitory effects observed in calvarial osteoblasts and bone marrow osteoprogenitor cells could be partially prevailed by bone morphogenetic protein-2. Inhibition of ERKs with a specific drug did not significantly affect osteoblast differentiation even though ERK1/2 were also activated during osteoblast differentiation. These results taken together indicate that p38 MAPK, but not ERKs, is necessary for osteoblast differentiation.

Alkaline Phosphatase↗

PubMed2003

Osteonectin-null mutation compromises osteoblast formation, maturation, and survival.

Osteonectin, also known as SPARC (secreted protein acidic and rich in cysteine) or BM-40, is one of the most abundant noncollagenous proteins in bone. Analysis of osteonectin-null mice revealed that osteonectin is necessary for the maintenance of bone mass and normal remodeling, as osteonectin-null mice have decreased osteoblast number and bone formation rate. Cultures of bone marrow stromal cells and osteoblasts from control and osteonectin-null mice were used to determine the cellular basis for the mutant phenotype. We found that marrow stroma from osteonectin-null mice contains fewer osteoblastic precursors than that of control mice, and the osteonectin-null mutation did not affect the proliferation rate of stromal cells or osteoblasts. Whereas osteonectin-null cells could adopt an osteoblastic phenotype, a smaller proportion of these cells expressed markers of a fully differentiated osteoblast. Mutant cells exhibited decreased formation of mineralized nodules, as well as diminished expression of osteocalcin mRNA and response to PTH. Furthermore, osteonectin-null cells showed an increased tendency to form adipocytes, with enhanced expression of the adipocytic markers adipsin and CCAAT/enhancer binding protein delta. Osteonectin-null cells were also more susceptible to environmental stresses. These data indicate that osteonectin is important for osteoblast formation, maturation, and survival.

Animals↗

PubMed2005

Mathematical model of paracrine interactions between osteoclasts and osteoblasts predicts anabolic action of parathyroid hormone on bone.

To restore falling plasma calcium levels, PTH promotes calcium liberation from bone. PTH targets bone-forming cells, osteoblasts, to increase expression of the cytokine receptor activator of nuclear factor kappaB ligand (RANKL), which then stimulates osteoclastic bone resorption. Intriguingly, whereas continuous administration of PTH decreases bone mass, intermittent PTH has an anabolic effect on bone, which was proposed to arise from direct effects of PTH on osteoblastic bone formation. However, antiresorptive therapies impair the ability of PTH to increase bone mass, indicating a complex role for osteoclasts in the process. We developed a mathematical model that describes the actions of PTH at a single site of bone remodeling, where osteoclasts and osteoblasts are regulated by local autocrine and paracrine factors. It was assumed that PTH acts only to increase the production of RANKL by osteoblasts. As a result, PTH stimulated osteoclasts upon application, followed by compensatory osteoblast activation due to the coupling of osteoblasts to osteoclasts through local paracrine factors. Continuous PTH administration resulted in net bone loss, because bone resorption preceded bone formation at all times. In contrast, over a wide range of model parameters, short application of PTH resulted in a net increase in bone mass, because osteoclasts were rapidly removed upon PTH withdrawal, enabling osteoblasts to rebuild the bone. In excellent agreement with experimental findings, increase in the rate of osteoclast death abolished the anabolic effect of PTH on bone. This study presents an original concept for the regulation of bone remodeling by PTH, currently the only approved anabolic treatment for osteoporosis.

Animals↗

PubMed1991

Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells.

We have reported that osteoblastic cells are required for differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts. In the present study we examined the pathogenesis of the osteoclast deficiency in osteopetrotic (op/op) mice using a coculture system of spleen cells and osteoblastic cells. When spleen cells obtained from op/op or normal (+/?) littermates of op/+ parent mice were cocultured with osteoblastic cells obtained from calvaria of normal ddy strain mice, numerous tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) were formed in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Most of the TRAP-positive MNCs bound [125I]salmon calcitonin. This suggests that there is no abnormality in the osteoclast progenitors present in the splenic tissues of op/op mice. When osteoblastic cells from +/? littermates were cocultured with normal spleen cells from ddy mice, TRAP-positive MNCs were similarly formed in response to 1 alpha,25(OH)2D3. In contrast, in cocultures of op/op osteoblastic cells with normal spleen cells, no TRAP-positive cells appeared, even in the presence of 1 alpha,25(OH)2D3. The op/op mutation was recently reported to exist in the coding region of the macrophage colony-stimulating factor (M-CSF) gene. Adding M-CSF and 1 alpha,25(OH)2D3 to the coculture with op/op osteoblastic cells induced the appearance of TRAP-positive MNCs with calcitonin receptors. These results clearly indicate that osteoclast deficiency in op/op mice is due to a defect in the local microenvironment in bone, in which M-CSF produced by osteoblastic cells plays a critical role in osteoclast development.

Acid Phosphatase↗

PubMed1992

Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor.

Recent studies have demonstrated the presence of estrogen receptor (ER) in both normal human osteoblast-like and osteoblast-like osteosarcoma cells. The number of ER in cultured osteoblastic cells is very low (200-500 sites/cell). This has complicated characterization of the biological role of estrogens in bone cells. To study the responsiveness of bone cells to estrogens, we established osteoblast-like cell lines expressing higher ER levels. ROS 17/2.8, an osteoblastic cell line, was stably transfected with the cDNA encoding for the mouse ER. After a selection period, positive clones were isolated and evaluated for the presence of ER by both Northern blot analysis and ligand binding assays. Using these techniques, we detected a significant increase in the level of both ER transcript and binding compared to that in wild-type cells. The levels of expressed ER protein were similar to those reported in normal human osteoblast-like cells in primary culture (approximately 2000 sites/cell). To test whether the exogenously inserted ER was responsive, both wild-type and ER stably transfected cells were transiently transfected with a reporter construct containing an estrogen-responsive element linked to a truncated thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Exposure of the cells to increased concentrations of estradiol induced a slight increase in CAT activity in wild-type cells (approximately 1.5-fold) at maximal stimulation; however, it provoked a clear concentration-dependent increase in CAT activity in the ER stably transfected cells, with a maximal stimulation of approximately 10-fold. This event was receptor mediated, since ICI 164,384, an ER antagonist, blocked the enhancement of estradiol-induced CAT activity, and it was specific, since other steroid hormones did not stimulate CAT activity. Finally, we evaluated the ability of ER to modulate an endogenous estrogen-responsive gene by measuring the activity of the enzyme alkaline phosphatase. In addition, diethylstilbestrol, a synthetic estrogen agonist, increased the activity of both the CAT reporter gene and the endogenous alkaline phosphatase enzyme. In summary, we have established osteoblast-like cells expressing high levels of an exogenously inserted ER, which has characteristics similar to those of the endogenous ER in terms of its Kd. Finally, the exogenous ER regulates both exogenously inserted construct (VITERECAT) and endogenous properties of the cells (enzymatic activity and proliferation).

Alkaline Phosphatase↗

PubMed1995

Inhibition of osteoblastic cell proliferation and ornithine decarboxylase activity by ethanol.

Low bone mass and an increased prevalence of skeletal fractures are evident in the alcoholic population. Histomorphometric analysis of skeletal tissue from alcoholic patients reveals reduced osteoblast number and suppressed bone formation activity, with relative sparing of resorptive indexes. The decreased number of osteoblasts observed in alcoholic subjects results from either impaired proliferation or accelerated senescence. Polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, are essential for cell proliferation in a variety of cell types. To determine whether the consequences of ethanol on osteoblast number involve the modulation of polyamine biosynthesis, we examined the effect of ethanol on parameters of cell growth and ODC activity in a rat osteoblast-like osteosarcoma cell line (UMR 106-01). Ethanol markedly impaired DNA synthesis and cell proliferation in a dose-dependent fashion. Difluoromethylornithine, a specific inhibitor of ODC activity, induced a similar inhibition of UMR 106-01 cell proliferation, indicating the importance of the polyamine pathway in this osteoblastic cell line. Induction of ODC activity was impaired in ethanol-exposed cell cultures in a dose-dependent fashion that paralleled the antiproliferative effects. Finally, supplemental polyamine administration substantially improved DNA synthesis in ethanol-exposed UMR 106-01 cell cultures. These data confirm a direct inhibitory effect of ethanol on osteoblast proliferation that may in part explain the reduced bone mass observed in subjects who consume excessive amounts of alcohol. These findings also suggest that altered polyamine metabolism may be an important mechanism responsible for the antiproliferative effects of ethanol on the osteoblast.

Animals↗

PubMed1996

Osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell contact.

We have established a method for obtaining an enriched preparation of functionally active osteoclast-like multinucleated cells (enriched OCLs) from co-cultures of mouse primary osteoblasts and bone marrow cells. Using these enriched OCLs, the effect of osteoblastic cells on osteoclast function was examined in two assays: a pit formation assay and an assay for actin ring formation. The enriched OCLs cultured for 24 h on dentine slices formed only a few resorption pits. When various numbers of primary osteoblasts were added to the enriched OCLs, the areas of the resorption pits increased proportionally to the number of osteoblasts added. Like primary osteoblasts, the established cell lines of osteoblastic cells (MC3T3-E1 and KS-4) and bone marrow-derived stromal cells (MC3T3-G2/PA6 and ST2) potentiated the pit formation caused by enriched OCLs. In contrast, the fibroblastic cell lines NIH3T3 and C3H10T1/2) and the myoblastic cell line (C2C12) failed to activate OCL function. When cell-to-cell contact between MC3T3-E1 cells and enriched OCLs was prevented, only a few resorption pits were formed. Pit formation by enriched rat osteoclasts placed on dentine slices was also stimulated by adding MC3T3-E1 cells. Actin ring formation and pit forming activity were well correlated in either culture of enriched mouse OCLs or authentic rat osteoclasts on dentine slices. These results indicate that osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell and/or cell-to matrix contact.

3T3 Cells↗

PubMed1996

Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption.

Current knowledge indicates that osteoblasts play an integral role in osteoclastic bone resorption through an osteoclast-stimulating activity produced by osteoblasts in response to resorption-promoting osteotropic factors. Previously, we have shown that the inhibitory action of the bisphosphonates on bone resorption in part is mediated by osteoblasts. The aim of the present study was to investigate whether the bisphosphonate-generated inhibition is due to these compounds decreasing the synthesis of the osteoclast-stimulating activity or is the result of osteoblasts synthesizing an osteoclast resorption inhibitor. Using the osteoblastic cell line CRP 10/30, which produces osteoclast- stimulating activity, constitutively and employing isolated rat osteoclasts cultured on ivory, evidence was obtained indicating that the bisphosphonates ibandronate and alendronate at a concentration of 10(-7) M induce osteoblasts to synthesize an osteoclast inhibitor that reduces pit formation by more than 50%. The inhibitor is heat and proteinase labile and has a molecular mass between 1-10 kDa. The reduction of resorption pits is paralleled by a decrease in tartrate-resistant acid phosphatase-positive mono- and multinucleated cells, whereas the mean area resorbed per pit was not changed, suggesting that the inhibitor affects osteoclast formation and/or survival and probably not the osteoclast resorption activity. Rat preosteoblastic cells and rat dermal fibroblasts were found not to produce the inhibitor. In conclusion, osteoblasts aside from their role of mediating osteoclastic resorption promoters are also involved in inhibiting bone resorption through the synthesis of an osteoclast resorption inhibitor.

Acid Phosphatase↗

PubMed1996

Osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell contact.

We have established a method for obtaining an enriched preparation of functionally active osteoclast-like multinucleated cells (enriched OCLs) from co-cultures of mouse primary osteoblasts and bone marrow cells. Using these enriched OCLs, the effect of osteoblastic cells on osteoclast function was examined in two assays: a pit formation assay and an assay for actin ring formation. The enriched OCLs cultured for 24 h on dentine slices formed only a few resorption pits. When various numbers of primary osteoblasts were added to the enriched OCLs, the areas of the resorption pits increased proportionally to the number of osteoblasts added. Like primary osteoblasts, the established cell lines of osteoblastic cell (MC3T3-E1 and KS-4) and bone marrow-derived stromal cells (MC3T3-G2/PA6 and ST2) potentiated the pit formation caused by enriched OCLs. In contrast, the fibroblastic cell lines (NIH3T3 and C3H10T1/2) and the myoblastic cell line (C2C12) failed to activate OCL function. When cell-to-cell contact between MC3T3-E1 cells and enriched OCLs was prevented, only a few resorption pits were formed. Pit formation by enriched rat osteoclasts placed on dentine slices was also stimulated by adding MC3T3-E1 cells. Actin ring formation and pit forming activity were well correlated in either culture of enriched mouse OCLs or authentic rat osteoclasts on dentine slices. These results indicate that osteoclast function is activated by osteoblastic cells-through a mechanism involving cell-to-cell and/or cell-to-matrix contact.

Animals↗

PubMed1997

Rat osteoblast and osteosarcoma nuclear matrix proteins bind with sequence specificity to the rat type I collagen promoter.

The nuclear matrix mediates the 3-dimensional organization of DNA and supports DNA replication and its transcription. We hypothesize that the osteoblast nuclear matrix contributes to the transcriptional control of type I collagen (COL1A1) expression. Cis-regulatory elements of the rat COL1A1 promoter that control osteoblast expression in vivo are between -2.3 and -1.67 kilobase pairs (kb) but lie within -3.5 and -2.3 kb in cultured bone cells. This may result from differences in cell architecture between osteoblasts in tissue and those in vitro. Our aim was to identify osteoblast nuclear matrix proteins (NMPs) that associated with sequence-specificity to the COL1A1 promoter. We used osteoblasts from the rat metaphyseal femur and the rat osteosarcoma cells, ROS 17/2.8. Nuclear matrix and soluble nuclear proteins were obtained as separate subfractions. Gel mobility shift analysis, using fragments of the COL1A1 promoter, was used to identify DNA-binding proteins in the nuclear subfractions. A NMP-DNA interaction, NMP3, was observed between -2149 and -2106 nucleotide in both osteoblasts and osteosarcoma cells. NMP4 was detected between -3518 to -3406 nucleotide. Therefore, osteoblast NMPs recognize sequences in regulatory regions of the COL1A1 promoter and may link cell structure and the transcriptional regulation of this protein.

Animals↗

PubMed1998

Osteocalcin production in primary osteoblast cultures derived from normal and Hyp mice.

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17-25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

Animals↗

PubMed1998

Estrogen receptor-alpha is developmentally regulated during osteoblast differentiation and contributes to selective responsiveness of gene expression.

Estrogen responsiveness of bone is a fundamental regulatory mechanism operative in skeletal homeostasis. We examined the expression of estrogen receptor-alpha (ER) messenger RNA (mRNA) in cultured rat calvarial-derived osteoblasts during progressive development of the osteoblast phenotype. Levels of ER message were compared with the expression of traditional osteoblastic markers that have been mapped throughout the differentiation process of these cells. ER transcripts, measured using semiquantitative RT-PCR analysis, were expressed at low levels in early stage proliferating osteoblasts and increased at confluence upon initial expression of bone cell phenotypic genes. A 23-fold up-regulation of ER mRNA expression coincided with the initiation of alkaline phosphatase activity (day 8). ER mRNA levels progressively increased 70-fold, reaching a maximum level on days 22-25 in fully differentiated osteoblasts when osteocalcin expression peaked, but declined precipitously by day 32 in osteocytic cells. Analysis of RNA isolated directly from rat calvaria confirmed these in vitro results and demonstrated that ER message levels become more abundant postnatally as bone becomes more mineralized. We also examined the responsiveness of osteoblasts to 17beta-estradiol (17beta-E2) at two periods of maturation: the nodule-forming stage (day 14) and the late mineralization stage (day 30). Estradiol suppressed the levels of alkaline phosphatase, osteocalcin, osteonectin, and ER mRNAs on day 14, but up-regulated these messages on day 30. In contrast, 17beta-E2 treatment regulated the steady state levels of transforming growth factor-beta1 and type I procollagen mRNAs only in the late mineralization stage, whereas histone H4 message was unaffected by the steroid at either stage of differentiation. Thus, the observed developmental expression of ER mRNA correlates with progressive osteoblast differentiation and may be a contributing factor to differential regulation of bone cell gene expression by 17beta-E2.

Animals↗