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 289 records · Page 16Linked to original sources

PubMed2003

Hydroxyapatite accelerates differentiation and suppresses growth of MC3T3-E1 osteoblasts.

Hydroxyapatite describes both the natural mineral phase of bone as well as the widely used calcium-phosphate implant substitute. Given that hydroxyapatite is a major component of the in vivo surface with which osteoblasts interact, it is surprising that most studies examining the regulation of osteoblast growth and differentiation utilize plastic surfaces. Here we demonstrate that the phenotype of mouse MC3T3-E1 osteoblasts is significantly altered on hydroxyapatite compared with plastic surfaces. Specifically, alkaline phosphatase activity and messenger RNA levels, markers of early stages of osteoblast differentiation, are increased in osteoblasts cultured on hydroxyapatite. The precocious appearance of alkaline phosphatase activity on the hydroxyapatite surface suggests that osteoblast differentiation is activated earlier compared with plastic surfaces. Osteocalcin expression, a marker of late-stage differentiation, is also increased on hydroxyapatite and further demonstrates enhanced differentiation. Cell counts indicate that fewer osteoblasts are present on hydroxyapatite versus plastic surfaces 24 h after plating. Measurement of osteoblast attachment, apoptosis, and necrosis indicated no differences between surfaces. In contrast, the number of bromodeoxyuridine-incorporating cells was significantly decreased on hydroxyapatite compared with plastic surfaces. Taken together, our findings indicate that hydroxyapatite enhances osteoblast differentiation while also suppressing growth.

Alkaline Phosphatase↗

PubMed1996

Expression and phorbol ester-induced down-regulation of protein kinase C isozymes in osteoblasts.

The protein kinase C (PKC) enzyme family consists of at least 11 isozymes in three classes, with characteristic tissue distributions. Phorbol esters activate and ultimately down-regulate phorbol-sensitive isozymes. PKC is a signal transducer in bone, and phorbol esters influence bone resorption. Little is known about specific PKC isozymes in this tissue, however. We describe here the expression and phorbol ester-induced down-regulation of PKC isozymes in osteoblasts. Normal mouse osteoblasts and seven osteoblastic cell lines (rat UMR-106, ROS 17/2.8, ROS 24/1, and human MG-63, G-292, SaOS-2, HOS-TE85) were screened for isozyme expression by Western immunoblotting using isozyme-specific anti-PKC antibodies. The conventional alpha and beta I isozymes, but not gamma, were present in each of the osteoblasts examined; PKC-beta II was detectable in all but the ROS 24/1 line. PKC-epsilon was expressed in all osteoblasts screened, but other novel PKCs, delta, eta, and theta, were detectable only in select lines. The atypical zeta and iota/lambda PKCs were in all osteoblasts examined. To determine the sensitivity of the isozymes to prolonged phorbol ester treatment, normal osteoblasts and the UMR-106 cell line were treated with vehicle or 1 microM phorbol 12, 13-dibutyrate (PDB) for 1, 3, 6, 12, 24, or 48 h, and Western blot analysis was performed. Normal and UMR-106 cells showed similar phorbol sensitivities; conventional (alpha, beta I) and novel (delta, epsilon, eta) isozymes were down-regulated by prolonged phorbol treatment but atypical isozymes were not. Down-regulation of all sensitive PKCs was detectable within 6 h of phorbol treatment; the novel delta and epsilon isozymes, however, showed more rapid and dramatic down-regulation than conventional isozymes. The observed down-regulation was dose-dependent (0.3-3 microM) and specific; 48 h treatment with the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), failed to down-regulate PDB-sensitive isozymes. The phorbol-induced down-regulation was also reversible; 24 h after withdrawing PDB, all phorbol-sensitive isozymes, except PKC-eta, had recovered at least partially. These studies, the first to characterize thoroughly PKC isozyme expression in osteoblastic cells from several species, demonstrate that osteoblasts have a characteristic PKC isozyme profile, including both phorbol ester-sensitive and -insensitive isozymes. The time course of down-regulation and the presence of phorbol-insensitive PKCs must be considered in interpreting the effects of phorbol esters on bone remodeling.

Animals↗

PubMed2003

Transcriptional profiling of human osteoblast differentiation.

Osteoblast differentiation is a key aspect of bone formation and remodeling. To further our understanding of the differentiation process, we have developed a collection of conditionally immortalized adult human osteoblast cell lines representing discrete stages of differentiation. To evaluate changes in gene expression associated with differentiation, polyA((+)) RNA from pre-osteoblasts, early and late osteoblasts, and pre-osteocytes was subjected to gene chip analysis using the Affymetrix Hu6800 chip in conjunction with an Affymetrix custom chip enriched in bone and cartilage cDNAs. Overall, the expression of 47 genes was found to change threefold or more on both chips between the pre-osteoblastic and pre-osteocytic stages of differentiation. Many of the observed differences, including down-regulation of collagen type I and collagen-processing enzymes, reflect expected patterns and support the relevance of our results. Other changes have not been reported and offer new insight into the osteoblast differentiation process. Thus, we observed regulation of factors controlling cell cycle and proliferation, reflecting decreased proliferation, and increased apoptosis in pre-osteocytic cells. Elements maintaining the cytoskeleton, extracellular matrix, and cell-cell adhesion also changed with differentiation reflecting profound alterations in cell architecture associated with the differentiation process. We also saw dramatic down-regulation of several components of complement and other immune response factors that may be involved in recruitment and differentiation of osteoclasts. The decrease in this group of genes may provide a mechanism for controlling bone remodeling of newly formed bone. Our screen also identified several signaling proteins that may control osteoblast differentiation. These include an orphan nuclear receptor DAX1 and a small ras-related GTPase associated with diabetes, both of which increased with increasing differentiation, as well as a high mobility group-box transcription factor, SOX4, that was down-regulated during differentiation. In summary, our study provides a comprehensive transcriptional profile of human osteoblast differentiation and identifies several genes of potential importance in controlling differentiation of osteoblasts.

Base Sequence↗

PubMed2005

Osteoblasts lacking the vitamin D receptor display enhanced osteogenic potential in vitro.

1,25-dihydroxyvitamin D plays an important role in the regulation of osteoblast gene expression, regulating the expression of bone matrix proteins as well as that of Runx2, a key regulator of osteoblast differentiation. Studies in mice lacking the vitamin D receptor (VDR) have revealed that the actions of the VDR on the skeleton are not required in the setting of normal mineral ion homeostasis. Since paracrine and endocrine factors can compensate for gene defects in vivo, studies were performed to determine whether ablation of the VDR alters the program of osteoblast differentiation in vitro. Studies in primary calvarial cultures revealed that ablation of the VDR enhanced osteoblast differentiation. The cells from the VDR null mice exhibited an earlier onset and increased magnitude of alkaline phosphatase activity, as well as an earlier and sustained increase in mineralized matrix formation, demonstrating that this enhancement persists throughout the program of osteoblast differentiation. The expression of bone sialoprotein, which enhances mineralization, was also increased in the VDR null cultures. To determine whether the increase in osteoblast differentiation was associated with an increase in the number of osteogenic progenitors, the number of osteoblastic colony forming units (CFU-OB) was evaluated. There was a twofold increase in the number of CFU-OB in the cultures isolated from the VDR null mice. Furthermore, the VDR null CFU-OB demonstrated an earlier onset and higher magnitude of expression of alkaline phosphatase activity when compared to the CFU-OB from their wild-type control littermates. These studies demonstrate that the VDR attenuates osteoblast differentiation in vitro and suggest that other endocrine and paracrine factors modulate the effect of the VDR on osteoblast differentiation in vivo.

Animals↗

PubMed1994

Molecular to pharmacologic control of osteoblast proliferation and differentiation.

Control of osteoblast growth and development can be characterized from receptor mediated events to nuclear messengers controlling gene transcription. From this analysis it is possible to formulate a model to explain the reciprocal relationship between growth and differentiation as well as differential cytokine modulation of osteoblast function. Central to this model are putative tissue specific transcriptional switches (possibly of the bHLH gene superfamily) that may repress proliferation and permit the regulation of mature osteoblast phenotypic characteristics. This model proposes that in post-mitotic differentiated osteoblasts, tissue specific transcription factors determine the capacity to express osteoblastic characteristic, whereas receptor activated signalling cascades, namely, cAMP/protein kinase A, receptor serine/threonine kinase, and vitamin D receptor-dependent pathways, regulate mature osteoblast-specific gene expression. Activated differentiation switches also may feedback to transcriptionally repress proliferation. Conversely, in preosteoblasts, in which differentiation switches are turned off, distinct signalling cascades involving tyrosine kinases, PKC, and calcium/calmodulin regulate proliferation. Proliferating preosteoblasts also exhibit negative modulation of maturation either through inactivation of putative tissue-specific transcription factors and/or through AP-1 dependent phenotype suppression of genes expressed in mature osteoblast. Thus, the final outcome of transcriptional regulation of osteoblast function results from complex interactions between signalling pathways and permissive differentiating transcription factors. Though many aspects of this model remain speculative and require confirmation, it serves as a useful conceptual framework to further investigate the differential control of osteoblast proliferation and differentiation that may lead to improved pharmacologic ways to manipulate bone formation in vivo.

Cell Differentiation↗

PubMed2003

Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo.

Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated signaling protein that regulates diverse cellular functions. In vivo, CTGF is expressed in many tissues with highest levels in the kidney and brain. The purpose of this study was twofold; first, to localize CTGF in normal bone in vivo during growth and repair, and second, to examine CTGF expression and function in primary osteoblast cultures in vitro and test its effect on bone formation in vivo. Northern and Western blot analyses confirmed that CTGF is expressed in normal long bones during the period of growth or modeling. In situ hybridization and immunohistochemical analysis demonstrated intense staining for CTGF mRNA and protein in osteoblasts lining metaphyseal trabeculae. Examination of CTGF expression in the fracture callus demonstrated that it was primarily localized in osteoblasts lining active, osteogenic surfaces. In primary osteoblast cultures, CTGF mRNA levels demonstrated a bimodal pattern of expression, being high during the peak of the proliferative period, abating as the cells became confluent, and increasing to peak levels and remaining high during mineralization. This pattern suggests that CTGF may play a role in osteoblast proliferation and differentiation as previously demonstrated for fibroblasts and chondrocytes. Treatment of primary osteoblast cultures with anti-CTGF neutralizing antibody caused a dose-dependent inhibition of nodule formation and mineralization. Treatment of primary osteoblast cultures with recombinant CTGF (rCTGF) caused an increase in cell proliferation, alkaline phosphatase activity, and calcium deposition, thereby establishing a functional connection between CTGF and osteoblast differentiation. In vivo delivery of rCTGF into the femoral marrow cavity induced osteogenesis that was associated with increased angiogenesis. This study clearly shows that CTGF is important for osteoblast development and function both in vitro and in vivo.

Animals↗

PubMed1990

Osteoblasts display receptors for and responses to leukemia-inhibitory factor.

Specific binding of leukemia-inhibitory factor (LIF) to osteoblasts, but not multinucleated osteoclasts, was demonstrated by receptor autoradiography by using cells isolated from newborn rat long bones. The clonal rat osteogenic sarcoma cells, UMR 106-06, which have several phenotypic properties of osteoblasts, expressed 300 LIF receptors per cell, with an apparent KD of 60 pM. Treatment of calvarial osteoblasts or UMR 106-01 cells with LIF resulted in a dose-dependent inhibition of plasminogen activator (PA) activity. Both calvarial osteoblasts and osteogenic sarcoma cells were shown by Western blotting and reverse fibrin autography to produce plasminogen activator inhibitor-1 (PAI-1), the production of which was increased by LIF treatment. Northern blot analysis revealed that LIF treatment resulted in a rapid (peak 1 hour), dose-dependent increase in mRNA for PAI-1. LIF treatment of the preosteoblast cell line, UMR 201, enhanced the alkaline phosphatase response of these cells to retinoic acid. Each of the osteoblast-like cell types (calvarial osteoblasts, UMR 106-06, and UMR 201) was shown to produce LIF by bioassay and, by using the polymerase chain reaction (PCR), was shown to express low levels of mRNA for LIF. These data establish that cells of the osteoblast lineage are targets for LIF action. The reported anabolic effects of this cytokine on bone formation in vivo could be related to inhibition of protease activity. LIF may be an important paracrine modulator in bone, or perhaps an autocrine one, based on the evidence for its production by osteoblasts and osteoblast-like cells.

Alkaline Phosphatase↗

PubMed1994

TGF beta alters growth and differentiation related gene expression in proliferating osteoblasts in vitro, preventing development of the mature bone phenotype.

This study examines the mechanism by which TGF-beta 1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-beta 1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-beta 1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-beta 1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and alpha 1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-beta 1 response throughout the course of osteoblast differentiation. The initiation of a TGF-beta 1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-beta 1. These in vitro results indicate that TGF-beta 1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml.

Animals↗

PubMed2007

Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor-beta1 (TGF-beta1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF-beta1-induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF-beta1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF-beta1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF-specific siRNA was used to prevent TGF-beta1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the requirement of CTGF for their up-regulation. To examine the effects of TGF-beta1 on osteoblast cell growth, cultures were treated with TGF-beta1 during the proliferative stage. Cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression, consistent with TGF-beta1-induced cell-cycle arrest. Cultures transfected with CTGF siRNA prior to TGF-beta1 treatment showed an even greater reduction in cell number, suggesting that TGF-beta1-induced growth arrest is independent of CTGF in osteoblasts. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF-beta1-induced ECM production in osteoblasts, but these two growth factors function independently regarding their opposing effects on osteoblast proliferation.

Animals↗

PubMed2006

Alumina particles influence the interactions of cocultured osteoblasts and macrophages.

The purpose of the current study was to evaluate the effects of alumina particles on secretion of several cytokines involved in bone resorption in cocultures of macrophages and osteoblasts. To distinguish the contribution of each individual cell type, we have established a heterologous in vitro system that makes use of mouse J774 cells and primary cultured human osteoblasts. J744 cells decreased the production of TNF-alpha when they were cocultured with osteoblasts. Treatment of J744 cells with alumina particles increased TNF-alpha secretion, but the induction was lower when cells were cocultured with osteoblasts. Secretion of IL-6 by J744 cells was very low, and increased in the presence of osteoblasts. Alumina particles were only able to stimulate the release of IL-6 by J744 cells when cells were cocultured with osteoblasts. On the other hand, incubation of osteoblasts with alumina particles enhanced the release of IL-6 and GM-CSF. Coculturing osteoblasts with J744 cells induced them to release IL-6 and GM-CSF, and treatment with alumina further increased the secretion of both mediators by osteoblasts. According to these in vitro results, it seems rather plausible that alumina particles are able to initiate an inflammatory response in vivo.

Aged↗

PubMed2006

Nitric oxide protects osteoblasts from oxidative stress-induced apoptotic insults via a mitochondria-dependent mechanism.

Nitric oxide (NO) contributes to the regulation of osteoblast activities. In this study, we evaluated the protective effects of NO pretreatment on oxidative stress-induced osteoblast apoptosis and its possible mechanism using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP) at a low concentration of 0.3 mM significantly increased cellular NO levels without affecting cell viability. However, when the concentration reached a high concentration of 2 mM, SNP increased the levels of intracellular reactive oxygen species and induced osteoblast injuries. Thus, administration of 0.3 and 2 mM SNP in osteoblasts were respectively used as sources of NO and oxidative stress. Pretreatment with NO for 24 h significantly ameliorated the oxidative stress-caused morphological alterations and decreases in alkaline phosphatase activity, and reduced cell death. Oxidative stress induced osteoblast death via an apoptotic mechanism, but NO pretreatment protected osteoblasts against the toxic effects. The mitochondrial membrane potential was significantly reduced following exposure to the oxidative stress. However, pretreatment with NO significantly lowered the suppressive effects. Oxidative stress increased cellular Bax protein production and cytochrome c release from mitochondria. Pretreatment with NO significantly decreased oxidative stress-caused augmentation of Bax and cytochrome c protein levels. In parallel with cytochrome c release, oxidative stress induced caspase-3 activation and DNA fragmentation. Pretreatment with NO significantly reduced the oxidative stress-enhanced caspase-3 activation and DNA damage. Results of this study show that NO pretreatment can protect osteoblasts from oxidative stress-induced apoptotic insults. The protective action involves a mitochondria-dependent mechanism.

Alkaline Phosphatase↗

PubMed2002

FK506 enhanced osteoblastic differentiation in mesenchymal cells.

Bone morphogenetic protein (BMP) is a bone-derived growth factor capable of promoting the differentiation of mesenchymal cells into osteogenic lineage pathways. Recently, immunosuppressants were reported to cause a moderate increase in osteoblastic differentiation in a rat osteoblast-like osteosarcoma cell line. If immunosuppressants can induce osteoblastic differentiation, it will be useful for bone tissue transplantation. We assessed the effect of immunosuppressants with or without BMP-4 on inducing osteoblastic differentiation in osteoblast-like and other mesenchymal cells. FK506, an immunosuppressant often used clinically, induced a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, one of the markers of osteoblast differentiation, in cells derived from mesenchyma. In the presence of BMP-4, ALP activity, mRNA levels of ALP and osteocalcin increased. FK506 was found to not only stimulate osteoblastic differentiation, but also to enhance BMP-4 induced osteoblastic differentiation. These results suggest that FK506 promotes differentiation of osteoblastic cells.

3T3 Cells↗

PubMed2001

1,25-Dihydroxyvitamin D3 promotes vitamin K2 metabolism in human osteoblasts.

It has been reported that vitamin K2 (menaquinone-4) promoted 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced mineralization and enhanced gamma-carboxyglutamic acid (Gla)-containing osteocalcin accumulation in cultured human osteoblasts. In the present study, we investigated whether menaquinone-4 (MK-4) was metabolized in human osteoblasts to act as a cofactor of gamma-glutamyl carboxylase. Both conversions of MK-4 to MK-4 2,3-epoxide (epoxide) and epoxide to MK-4 were observed in cell extracts of cultured human osteoblasts. The effect of 1,25(OH)2D3 and warfarin on the vitamin K cycle to cultured osteoblasts were examined. With the addition of 1 nM 1,25(OH)2D3 or 25 microM warfarin in cultured osteoblasts, the yield of epoxide from MK-4 increased. However, the conversion of epoxide to MK-4 was strongly inhibited by the addition of warfarin (2.5-25 microM), whereas it was almost not inhibited by 1,25(OH)2D3 (0.1-10 nM). To clarify the mechanism for this phenomenon, a cell-free assay system was studied. Osteoblast microsomes were incubated with 10 microM epoxide in the presence or absence of warfarin and 1,25(OH)2D3. Epoxide reductase, one of the enzymes in the vitamin K cycle was strongly inhibited by warfarin (2.5-25 microM), whereas it was not affected by 1,25(OH)2D3 (0.1-1 nM). Moreover, there was no effect of pretreatment of osteoblasts with 1 nM 1,25(OH)2D3 on the activity of epoxide reductase. However, the activity of epoxidase, that is the gamma-glutamyl carboxylase was induced by the pretreatment of osteoblasts with 1 nM 1,25(OH)2D3. In the present study, it was demonstrated that the vitamin K metabolic cycle functions in human osteoblasts as well as in the liver, the post-translational mechanism, by which 1,25(OH)2D3 caused mineralization in cooperation with vitamin K2 was clarified.

Adult↗

PubMed2003

Regulated expression of G protein-coupled receptor kinases (GRK's) and beta-arrestins in osteoblasts.

Desensitization of G-protein coupled receptors (GPCR's) is largely mediated by a family of enzymes and protein co-factors termed GRKs and arrestins, respectively. In the present studies, we investigated expression of GRKs and arrestins in osteoblastic cell lines concentrating on the enzymes (GRK2 and GRK3) and protein co-factors (beta-arrestint 1 and beta-arrestin 2) that play dominant roles in regulating GPCR responsiveness in most tissues and cell types. We found that osteoblastic cells express similar amounts of GRK2 with either undetectable or lesser amounts of GRK3. In contrast, expression of beta-arrestin 1 and beta-arrestin 2 by osteoblastic cells varied between cell lines. To determine if GRK2 or beta-arrestin expression is modulated during osteoblast development, we assessed expression of GRK2 and beta-arrestin proteins during differentiation of the mouse osteoblastic cell line MC3T3-E1 cells over a 21-day period. We found that expression of GRK2 and beta-arrestin 2 increased to maximal levels by day 7 and then decreased 4-fold by day 21. In contrast, expression of beta-arrestin 1 increased to maximal levels by day 14 and then decreased 2-fold by day 21. Over this same time period (days 7-21), PTH/PTHrP receptor number decreased to a greater extent than the decrease in PTH(1-34)-induced cAMP generation, suggesting that responsiveness of individual PTH/PTHrP receptors was enhanced in differentiated cells. We conclude that (1) osteoblastic cell lines differentially express the enzymes and protein co-factors that modulate GPCR responsiveness and (2) expression of both GRK2 and beta-arrestins is temporally regulated during osteoblast development. These data are consistent with the notion that GPCR responsiveness may be differentially regulated in osteoblastic cell lines and during osteoblast development.

Animals↗

PubMed2004

Triiodothyronine (T3) and 1,25-dihydroxyvitamin D3 (1,25D3) inversely regulate OPG gene expression in dependence of the osteoblastic phenotype.

Both thyroid hormones and 1,25-dihydroxyvitamin D3 (1,25D3) are essential for normal development and maintenance of the skeleton. They regulate osteoblastic differentiation by influencing expression of osteoblast specific genes like osteocalcin (OCN). Mice deficient in OCN, the most abundant noncollagenous protein of the bone matrix, develop a phenotype characterized by higher bone mass and bone of improved quality. Osteoprotegerin (OPG), another protein important for bone metabolism, protects bone and is a regulator of the osteoclast development. 1,25D3 down regulates the basal expression of these two proteins in osteoblasts and osteoblast-like cells. The involvement of these hormones and proteins in the bone metabolism guided us to compare their regulation in cell lines with different osteoblastic phenotypes. We found that T3 increased OCN mRNA levels in MC3T3-E1 osteoblastic cells independent from their phenotype albeit with different potency but not in the bone marrow-derived stromal cell line ST2. OPG mRNA expression levels were only stimulated by T3 in mature MC3T3-E1 cells, which have the capacity to mineralize, but not in pre-osteoblastic MC3T3-E1 cells or in ST2 cells. In the mineralizing osteoblastic cells 1,25D3 inhibited T3-induced expression for OPG mRNA but not OCN mRNA. In the pre-osteoblastic cell line with undetectable OPG mRNA levels, either basal or T3-stimulated, 1,25D3 inhibited OCN mRNA expression completely. Our results emphasize the importance of balanced regulation of mRNA transcript levels for OPG and OCN, by both hormones and probably other systemic factors to enable a fine-tuning of bone metabolism at specific skeletal sites.

3T3 Cells↗

PubMed2000

The effect of carboxyl-terminal propeptide of type I collagen (c-propeptide) on collagen synthesis of preosteoblasts and osteoblasts.

Recently we found that the carboxyl-terminal propeptide of type I collagen (c-propeptide) is a major secretory protein of osteoblasts. Mature osteoblasts secreted 64 nM c-propeptide, and it was reported that 40 nM c-propeptide inhibited collagen synthesis at 80% of the control level. In this study, we investigated the effect of c-propeptide on collagen synthesis of preosteoblasts and osteoblasts, and found that preosteoblasts downregulated collagen synthesis by 40 nM c-propeptide, but osteoblasts were not affected by the same condition. When the binding activities of c-propeptide for preosteoblasts and osteoblasts were compared, osteoblasts showed weak affinity to c-propeptide compared with preosteoblasts, and the number of receptors for c-propeptide decreased in osteoblasts. These results imply that a decrease of receptors in osteoblasts might reduce the sensitivity of osteoblasts to c-propeptide.

3T3 Cells↗

PubMed1999

Carnitine and dehydroepiandrosterone sulfate induce protein synthesis in porcine primary osteoblast-like cells.

Age-related bone loss eventually leads to osteopenia in men and women. The etiology of age-related bone loss is currently unknown; however, decreased osteoblast activity contributes to this phenomenon. In turn, osteoblast proliferation and function is dependent on energy production, thus the loss of energy production that occurs with age may account for the deficient osteoblast activity. Carnitine and dehydroepiandrosterone-sulfate (DHEAS), both of which decline with age, promote energy production through fatty acid metabolism. Thus, we hypothesized that carnitine and DHEAS would increase osteoblast activity in vitro. Accordingly, we measured the effect of carnitine and DHEAS on palmitic acid oxidation as a measure of energy production, and alkaline phosphatase (ALP) activity and collagen type I (COL) as indices of osteoblast function in primary porcine osteoblast-like cell cultures. Carnitine (10(-3) and 10(-1) M) but not DHEAS (10(-9), 10(-8), and 10(-7) M) increased carnitine levels within the cells. Carnitine alone and in combination with DHEAS increased palmitic acid oxidation. Both carnitine and DHEAS alone and in an additive fashion increased ALP activity and COL levels. These results demonstrate that in osteoblast-like cells in vitro, energy production can be increased by carnitine and osteoblast protein production can be increased by both carnitine and DHEAS. These data suggest that carnitine and DHEAS supplementation in the elderly may stimulate osteoblast activity and decrease age-related bone loss.

Alkaline Phosphatase↗

PubMed1999

Expression of stem cell factor by osteoblasts in normal and hyperparathyroid bone: relation to ectopic mast cell differentiation.

Mast cells accumulate in hyperparathyroid bone, but the reason is not clear. We compared the distribution of mast cells and related growth factors in normal and hyperparathyroid bone. Mast cell formation was strongly affected by proximity to bone-forming surfaces of hyperparathyroid bone. Hyperparathyroidism greatly increased the production by active, bone-synthesizing osteoblasts of stem cell factor (SCF) but not of IL-3. Osteoblast SCF was distributed to the basolateral cell membranes, and its cDNA sequence (GenBank AF119835) is homologous to the murine membrane-bound SCF. Quiescent osteoblasts did not produce detectable SCF. Synthetic osteoblasts in normal bone were SCF positive, but comprised a much smaller population of cells, in keeping with the slow turnover of normal bone. Major SCF isoforms on immunoblot analysis of osteoblast-fraction proteins from high-turnover bone had M(r)s of about 48 and 40 kDa. Similar SCF isoforms were produced by MG63 osteoblast-derived cells and were identified by several anti-SCF antibodies. SCF is expressed in several mesenchymal cell types in a complementary fashion with cells bearing its receptor. SCF potently facilitates differentiation of mast cells, so the increase in paratrabecular mast cells in hyperparathyroid bone is probably driven by osteoblastic SCF. However, since mast cells are not normal components of bone, osteoblastic SCF probably regulates other cells, with mast cell differentiation occurring as a side effect greatly increased osteoblastic activity.

Bone and Bones↗