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PubMed1992

Changes in expression of alpha 1 type 1 collagen and osteocalcin mRNA in osteoblasts and odontoblasts at different stages of maturity as shown by in situ hybridization.

We investigated whether the expressed phenotype of osteoblasts and odontoblasts is changing with increasing maturity of the cells. Thus we determined, using in situ hybridization techniques, whether the expression of mRNA's for osteocalcin and the alpha 1 type 1 collagen chain was different in newly developed and more mature cuboidal osteoblasts of the primary and secondary spongiosa of radii of 8 day old rats, in mature cuboidal and older flat osteoblasts in the metatarsals of pig embryos, and in apical and coronal odontoblasts of the developing unerupted molars of pig embryos. The results indicate that newly differentiated osteoblasts in the primary spongiosa of the 8 day rat radius contained approximately the same amount of type 1 collagen message as more mature osteoblasts in the secondary spongiosa. Osteocalcin mRNA, on the other hand, was undetectable in the newly differentiated osteoblasts but clearly detectable in the mature osteoblasts of the secondary spongiosa. When we compared expression of osteocalcin and collagen type 1 mRNA in mature cuboidal and older flat osteoblasts, we found that the amount of osteocalcin mRNA relative to collagen type 1 mRNA was higher in flat osteoblasts than in cuboidal osteoblasts. In odontoblasts, however, the steady state level of collagen type 1 mRNA was higher in the older coronal odontoblasts, and the level of osteocalcin message lower, when compared to the younger apical odontoblasts. The results indicate that relative levels of osteocalcin and collagen mRNA in osteoblasts and odontoblasts vary depending on the stage in their secretory lifetime. This heterogeneity of the osteoblast and odontoblast population suggests that the composition of the matrix produced by these cells also differs.

Animals↗

PubMed1991

Gene expression in osteoblastic cells.

Osteoblasts, the bone-forming cells, synthesize the macromolecules of the bone matrix including: type I collagen; osteocalcin; osteonectin; osteopontin; proteoglycan I and II; bone sialoprotein; matrix gla-protein; bone glycoprotein 75; several other proteins, which have not been extensively characterized; growth factors, including transforming growth factor beta and fibroblast growth factor. Osteoblasts also have high levels of the membrane-bound enzyme, alkaline phosphatase, which plays a role in matrix mineralization, and receptors for tissue-specific hormones, such as parathyroid hormone, as well as many other hormones, cytokines and growth factors, which regulate bone growth, differentiation and metabolism. The expression of these various proteins, most of which are not unique to bone but which together characterize the bone phenotype, is induced during osteoblastic differentiation in a stepwise fashion, suggestive of multiple regulatory factors. The detailed sequence of the expression of osteoblastic genes in situ has not been fully characterized. It appears that type I collagen and alkaline phosphatase are expressed early during the commitment to the osteoblastic phenotype, whereas osteopontin and osteocalcin appear late during osteoblastic differentiation. Diversity among "osteoblastic" cells is also apparent, probably not all osteoblastic cells express all the features. A large number of osteoblastic models are currently available to study the expression of osteoblast-related genes in vitro. These include primary cultures from calvaria or trabecular bone from several species, including humans, osteosarcoma-derived cell lines, and experimentally immortalized cells. Some of these in vitro models, especially the calvaria-derived cultures, undergo changes which mimic osteoblastic differentiation in vivo. The study of these and other cell models started providing insights into the regulation of gene expression in osteoblastic cells. In addition to a vast body of information on the conditions required for the expression of various proteins in culture and their regulation by hormones and growth factors, more detailed information on specific genes has recently been obtained. For example, regulation of type I collagen gene expression has been studied in osteosarcoma cell lines where 1,25(OH)2 vitamin D3 was shown to act via specific DNA segment(s) in the 5' flanking region of the gene, while parathyroid hormone affected gene expression by altering the stability of the transcripts. TGF beta 1, which stimulates osteogenesis, was shown to promote the transcription of osteopontin and type I collagen, the latter effect requiring the binding site for the transactivating protein, nuclear factor I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

[Osteoblasts during various functional states].

At the electron microscopical investigation of osteoblasts in different zones of osteogenesis (enchondral foci, metaphyses, endosteum) in the rat and rabbit femoral bone it has been revealed that their population is heteromorphic. As demonstrate cytochemical data and radioautography, using 3H-uridine, 3H-glycine, 35S-sulfate, 45Ca, results of measurements in osteoblast population, 4 morpho-functional states (or types) are defined. In areas of an intensive osteoplastic process there are young osteoblasts (I type), mature functionally active osteoblasts (II type), osteoblasts with a hypertrophic endoplasmic network (cell-depots of the secrete--III type). In preosteoblasts and osteoblasts of the I type a higher than in osteoblasts of other types intensity of 35S-sulfate incorporation and alkaline phosphatase activity is revealed. In osteoblasts of the II type processes of biosynthesis of collagenous proteins predominate. Osteoblasts of the III type are subjected to destruction during secretion process. In the areas where osteopoesis is dying away, osteoblasts of the I and II types transform into a poorly active state, concerning specific biosynthesis (osteoblasts of the IV type). Presence of osteoblasts having various functional states in the areas of intensive osteopoiesis, depends on certain asynchronity of specific processes of biosyntheses, that occur in them. The morpho-functional states described are regarded as a specific peculiarity in function of collagene-producing cells.

Alkaline Phosphatase↗

PubMed1997

In vitro cell behavior of osteoblasts on Pyrost bone substitute.

BACKGROUND: Pyrost bone substitute has been shown to be a promising orthopedic biomaterial. However, little is known about mechanisms that are responsible for the genesis and development of the bond between bone and the Pyrost bone substitute. The purpose of this study is to elucidate the in vitro cell behavior of osteoblasts on Pyrost bone substitute. METHODS: By using primary culture of rat osteoblasts, the changes in cell morphology during adhesion and flattening onto the surface of Pyrost bone substitute were studied in vitro. At 1 hour, at 3 hours, and at days 1, 3, and 7 after layering, the cell behavior was observed with scanning electron microscope. RESULTS: The processes of trypsinized osteoblast adhesion and spreading on Pyrost bone substitute consisted of 1) contact of rounded osteoblasts with the Pyrost substrate; 2) attachment of osteoblasts at point of contact; 3) centrifugal growth of filopodia; 4) flattening and spreading of the osteoblasts on the Pyrost substrate; 5) division and growth of osteoblasts; and 6) suspension of the osteoblasts across the pores by their processes. CONCLUSIONS: These results demonstrated that Pyrost can form a physico-chemical bond with osteoblasts. The Pyrost bone substitute not only supports osteoblasts attachment but also allows proliferation of the osteoblasts.

Animals↗

PubMed1999

Expression of mRNAs for type-I collagen, bone sialoprotein, osteocalcin, and osteopontin at different stages of osteoblastic differentiation and their regulation by 1,25 dihydroxyvitamin D3.

We have used in situ hybridization to evaluate the effects of 1,25 dihydroxyvitamin D3 (1,25 (OH)2 D3) on the expression of mRNA for bone-matrix proteins and to determine whether mature osteoblasts respond differently to 1,25 (OH)2 D3 than younger, newly differentiated osteoblasts. Rat calvaria cells were cultured for 7, 12, 15, and 19 days to obtain a range of nodules from very young to very mature. At each time point, some cultures were treated with 10 nM 1,25 (OH)2 D3 for 24 h prior to fixation. In control cultures, type-I collagen mRNA was detectable in osteoblastic cells in very young nodules and increased with increasing maturity of the nodules and the osteoblasts lining them. The bone sialoprotein mRNA signal was weak in young osteoblasts, increased in older osteoblasts, and decreased in mature osteoblasts. Weak osteocalcin and osteopontin signals were seen only in osteoblasts of intermediate and mature nodules. 1,25 (OH)2 D3 treatment markedly upregulated osteocalcin and osteopontin mRNAs and downregulated mRNA levels of bone sialoprotein and, to a lesser extent, type-I collagen in both young and mature osteoblasts. However, a marked diversity of signal levels for bone sialoprotein, osteocalcin, and osteopontin existed between neighboring mature osteoblasts, particularly after 1,25 (OH)2 D3 treatment, which may therefore selectively affect mature osteoblasts, depending on their differentiation status or functional stage of activity.

Animals↗

PubMed1999

Osteoblastic cells induce fusion and activation of osteoclasts through a mechanism independent of macrophage-colony-stimulating factor production.

Fusion and activation of osteoclasts are the final two events in osteoclastic bone resorption. To investigate the regulatory mechanism of these events, mononuclear osteoclasts (preosteoclasts, pOCs) were isolated from co-cultures of mouse osteoblastic cells and bone marrow cells. Most of the pOCs cultured without any additives died within 12 h. Survival of pOCs was supported by addition of either osteoblastic cells or macrophage-colony-stimulating factor (M-CSF). pOCs began to fuse with each other after culture for 12 h in the presence of osteoblastic cells or M-CSF. However, the properties of multinucleated osteoclast-like cells (OCLs) induced by osteoblastic cells were considerably different from those induced by M-CSF. Fusion of pOCs induced by osteoblastic cells was retarded after culture for 24 h. In contrast, M-CSF-induced fusion of pOCs continued throughout the 48-h culture period, which was not inhibited by addition of calcitonin. When pOCs together with osteoblastic cells were cultured for 48 h on dentine slices, many resorption pits were formed on the slices. Calcitonin completely inhibited the fusion and pit-forming activity of pOCs treated with osteoblastic cells. Resorption pits were hardly detected on dentine slices in pOC cultures treated with M-CSF. Osteoblastic cells prepared from osteopetrotic (op/op) mice, which cannot produce functional M-CSF, stimulated the fusion and pit-forming activity of pOCs. Recombinant RANKL (receptor activator of NF-kappaB ligand), a cytokine which is produced by osteoblastic cells and is responsible for osteoclast differentiation, induced the fusion and pit-forming activity of pOCs. These results suggested that osteoblastic cells are involved in fusion and activation of osteoclasts through a mechanism independent of M-CSF production. RANKL appears to be responsible for fusion and activation of osteoclasts induced by osteoblastic cells.

Animals↗

PubMed2007

Substance P stimulates late-stage rat osteoblastic bone formation through neurokinin-1 receptors.

Substance P (SP) is a widely distributed neuropeptide that works as a neurotransmitter and neuromodulator. Recently, SP receptors, particularly neurokinin-1 receptors (NK(1)-Rs) that have a high affinity for SP, have been observed not only in neuron and immune cells, but also in other peripheral cells, including bone cells. To identify the role of SP in bone formation, we investigated the expression of NK(1)-Rs in osteoblastic cells and the effects of SP on bone formation by rat calvarial osteoblastic cells. Rat calvarial osteoblastic cells were isolated and cultured for 3 weeks in alpha-MEM containing 10% serum, ascorbic acid, dexamethasone, and beta-glycerophosphate. We then investigated NK(1)-R expression, SP effects on osteoblastic bone formation, and osteocalcin mRNA expression in osteoblastic cells. RT-PCR and immunocytochemistry showed that NK(1)-R mRNA was expressed and NK(1)-R was present in 14-day, but not 7-day, cultured calvarial osteoblasts. Bone formation by cultured osteoblastic cells significantly increased after the addition of 10(-8)-10(-6)MSP. During 3 weeks of culture, the addition of SP in the first week did not significantly increase bone formation, whereas adding SP during the first and second week or all 3 weeks significantly increased calvarial osteoblastic bone formation. Furthermore, semi-quantitative RT-PCR indicated that SP stimulated osteocalcin mRNA expression in the osteoblasts at day 14 or day 21, whereas SP did not stimulated the runX2 or type I collagen mRNA expression at day 7 but stimulated them at day 14. These results indicate that SP stimulates bone formation by osteoblastic cells via NK(1)-Rs at late-stage bone formation. These effects were dependent on the expression of NK(1)-R in osteoblastic cells. Our findings suggest that SP secreted from sensory neurons may modulate bone formation after the expression of SP receptors.

Animals↗

PubMed2003

Comparative interspecies investigation on osteoblast cultures: data on cell viability and synthetic activity.

The aim of the present study was to evaluate and compare the most common parameters that characterize the expression of primary osteoblast cultures from different origin (human, rat, sheep), and of the human osteosarcoma cell line MG-63 before and after stimulation with vitamin 1,25(OH)(2)D(3). Cell viability was quite similar for primary osteoblast cultures (MTT: 1.64-2.11 OD); a significant (P < 0.005) difference was found between sheep osteoblasts and MG-63 (DeltaMTT: 0.52 +/- 0.20 OD). Osteocalcin synthesis ranged from 15.18 to 27.00 pg/ml in primary osteoblast cultures, while it was significantly (P < 0.01) lower in MG-63 (OC: 6.67 +/- 0.52 pg/ml) when compared with primary human osteoblasts. Alkaline phosphatase, C-terminal procollagen type I, and interleukin-6 were significantly (P < 0.005) lower in rat osteoblasts when compared with primary human osteoblasts, and similarly transforming growth factor-beta1 was significantly (P < 0.05) lower in rat and sheep osteoblasts when compared with primary human osteoblasts and MG-63. Nitric oxide synthesis did not show any significant difference either before or after vitamin 1,25(OH)(2)D(3) stimulation. In conclusion, the current findings confirm the presence of interspecies differences between the selected osteoblast lineages before and after stimulation with vitamin 1,25(OH)(2)D(3). Above all, the culture of sheep osteoblasts was seen to behave more similarly to that of primary human cells, mainly in terms of cell viability, osteocalcin, interleukin-6 and transforming growth factor-beta1 production.

Animals↗

PubMed2000

Etidronate inhibits human osteoblast apoptosis by inhibition of pro-apoptotic factor(s) produced by activated T cells.

Humoral factors produced by activated T cells are thought to be important in the development of bone loss in patients with rheumatoid arthritis (RA). We investigated the inhibitory effect of etidronate disodium (EHDP) on apoptosis of human osteoblasts induced by supernatants from in vitro activated T cell cultures. Human osteoblastic cell line MG63 cells and human primary osteoblast-like cells were used in the present study as human osteoblasts. T cells were incubated with interleukin-2 and further activated with 1 2-o-tetradecanoyl-phorbol 13-acetate and ionomycin, either in the presence or absence of EHDP. After we carried out the cultivation, we examined the cytotoxicity of cultured T cell supernatants toward MG63 cells and human primary osteoblast-like cells. Supernatants from activated but not resting T cell cultures efficiently induced apoptosis of MG63 cells and primary osteoblast-like cells. Supernatants from activated T cell cultures, incubated with EHDP, exhibited significantly less cytotoxicity than did supernatants incubated in the absence of EHDP. In contrast, the cytotoxicity of activated T cell culture supernatants was not affected by direct treatment of human osteoblasts with EHDP. The concentration of soluble Fas ligand in activated T cell culture supernatants was actually increased by EHDP. However, EHDP did not influence soluble Fas and tumor necrosis factor-alpha concentrations in the supernatant. Furthermore, treatment of human osteoblasts with EHDP did not alter their expression of Bcl-2/Bcl-xL or their sensitivity to anti-Fas immunoglobulin M-induced apoptosis. Our results suggest that EHDP inhibits the production of soluble factor that induces apoptosis of human osteoblasts and thus exhibits a protective action toward human osteoblast apoptosis induced by activated T cell culture supernatants. Although the exact EHDP-regulated molecule that induces apoptosis of human osteoblasts is unknown at present, our study may explain part of the therapeutic action of bisphosphonates in RA complicated by bone loss.

Apoptosis↗

PubMed2002

Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.

Saethre-Chotzen syndrome (SCS) is a human autosomal dominant disorder characterized by premature fusion of cranial sutures caused by mutations of the Twist gene encoding a basic helix-loop-helix (bHLH) transcription factor. We previously showed that Twist haploinsufficiency caused by a Y103X nonsense mutation in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of osteoblast differentiation. Here we show that Twist haploinsufficiency alters osteoblast apoptosis in SCS. Analysis of terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) demonstrated increased osteoblast and osteocyte apoptosis in coronal sutures from two SCS patients with nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins, and one patient with a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding. To assess the mechanisms involved, we studied osteoblast apoptosis in mutant (M-Tw) calvarial cells bearing the Y103X mutation resulting in decreased Twist mRNA and protein levels. M-Tw cells cultured in low serum conditions showed enhanced DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Biochemical analysis showed increased activity of initiator caspases-2 and -8 and downstream effector caspases-3, -6 and -7 in mutant osteoblasts. Caspase-2 was upstream of caspase-8 and effector caspases-3, -6 and -7 because their activities were suppressed by a specific caspase-2 inhibitor. M-Tw osteoblasts also showed increased cytochrome c release from the mitochondria. However, the activity of the downstream effector caspase-9 was not increased due to overexpression of the antagonist protein Hsp70. Detection of differentially expressed genes using cDNA expression array revealed increased Bax and TNFalpha mRNA levels in M-Tw compared to Nl cells, a finding confirmed by RT-PCR and western blot analyses. Neutralization of TNFalpha overexpression using anti-TNFalpha or anti-TNF receptor 1 antibodies abolished the increased activity of caspase-2, caspase-8 and caspases-3, -6 and -7 in M-Tw osteoblasts. These studies provide novel evidence that Twist haploinsufficiency in SCS promotes osteoblast apoptosis by a TNFalpha-caspase-2-caspase-8-caspases-3, -6, -7 cascade, and uncover a molecular mechanism in which Twist plays an anti-apoptotic role in human calvarial osteoblasts.

Acrocephalosyndactylia↗

PubMed2001

Osteoblasts in HIV-infected patients: HIV-1 infection and cell function.

BACKGROUND: HIV-infected patients have been shown to have a severe alteration in osteoblast function that appears to be related to the infection. OBJECTIVE: To determine whether normal human osteoblasts express CD4, whether osteoblasts from patients with HIV infection are infected by HIV-1 and whether osteoblast dysfunction observed in vivo also occurs in vitro. METHODS: Osteoblast cultures from bone marrow biopsies of HIV-infected patients (n = 14) and control patients (n = 10) were used in a cross-sectional study and a case-control prospective study. Expression of CD4 was analysed using flow cytometry and reverse transcriptase polymerase chain reaction; the presence of HIV-1 particles was determined by measuring p24 antigen in the supernatants of osteoblast cultures and viral DNA or RNA in the osteoblasts using the polymerase chain reaction. Osteoblast function was assessed by measuring cell proliferation, type I collagen and osteocalcin synthesis. RESULTS: In human osteoblasts, CD4 expression could not be determined using flow cytometry, although low levels of mRNA coding for CD4 were detected. HIV infection was not observed in osteoblast cultures from HIV-infected patients nor was there any alteration in replication and synthesis of type I collagen, although osteocalcin synthesis was increased. CONCLUSIONS: It is unlikely that HIV-1 infects human osteoblasts in vivo; therefore, the hypothesis that these cells could act as local HIV-1 reservoirs should be reconsidered.

Adult↗

PubMedDate not supplied

Regulation of osteoblast levels during bone healing.

OBJECTIVE: To confirm the occurrence of programmed cell death of osteoblasts during bone healing and to evaluate the role of interleukin-1beta (IL-1beta) in regulating osteoblast concentration. STUDY DESIGN: Electron microscopic study of the response of rats to a controlled bone injury, and a randomized controlled study of the effect of IL-1beta administered continuously for three days. METHODS: A standardized defect (1.1 millimeter in diameter, 0.5 millimeter deep) was created unilaterally on the anteromedial surface of the tibia. In some animals, the injury site was recovered five days after operation and processed for ultrastructural evaluation of osteoblasts in the callus. In another group, IL-1beta was delivered to the bone defect using micro-osmotic pumps (0.5 nanograms/hour); control rats received vehicle only. The bones were recovered one to fourteen days after injury, and concentrations of proliferating cells, osteoblasts, and apoptotic bodies were determined. The amount of callus that formed in the defect was measured. RESULTS: Osteoblasts in the callus exhibited ultrastructural changes characteristic of cells undergoing apoptosis, including condensation of chromatin, membrane blebbing, formation of apoptotic bodies, and phagocytosis by nearby osteoblasts. Addition of IL-1beta significantly increased the number of osteoblasts at the injury site and significantly decreased the number of apoptotic bodies in relation to the number of osteoblasts. The amount of callus in the bone defect was not affected by IL-1beta treatment. CONCLUSION: The role of programmed cell death of osteoblasts as a normal concomitant of bone healing was confirmed. Evidence was found suggesting that IL-1beta mediated the appearance and disappearance of osteoblasts, possibly by affecting the rates of differentiation and apoptosis, respectively. Understanding these mechanisms conceivably could lead to the ability to control osteoblast levels at an injury site.

Animals↗

PubMed2004

Rosiglitazone causes bone loss in mice by suppressing osteoblast differentiation and bone formation.

Because osteoblasts and marrow adipocytes are derived from a common mesenchymal progenitor, increased adipogenesis may occur at the expense of osteoblasts, leading to bone loss. Our previous in vitro studies indicated that activation of the proadipogenic transcription factor peroxisome proliferator-activated receptor isoform gamma 2 with rosiglitazone suppressed osteoblast differentiation. Here, we show that 5-month-old Swiss-Webster mice receiving rosiglitazone for 28 d exhibited bone loss associated with an increase in marrow adipocytes, a decrease in the ratio of osteoblasts to osteoclasts, a reduction in bone formation rate, and a reduction in wall width--an index of the amount of bone formed by each team of osteoblasts. Rosiglitazone had no effect on the number of early osteoblast or osteoclast progenitors, or on osteoblast life span, but decreased the expression of the key osteoblastogenic transcription factors Runx2 and Osterix in cultures of marrow-derived mesenchymal progenitors. These effects were associated with diversion of bipotential progenitors from the osteoblast to the adipocyte lineage, and suppression of the differentiation of monopotential osteoblast progenitors. However, rosiglitazone had no effect on osteoblastic cells at later stages of differentiation. Hence, rosiglitazone attenuates osteoblast differentiation and thereby reduces bone formation rate in vivo, leading to bone loss. These findings provide a mechanistic explanation for the recent evidence that peroxisome proliferator-activated receptor isoform gamma activation is a negative regulator of bone mass and suggest that the increased production of oxidized fatty acids with age may indeed be an important mechanism for age-related osteoporosis in humans.

Adipocytes↗

PubMed1998

Inhibitory effect of glucocorticoid for osteoblast apoptosis induced by activated peripheral blood mononuclear cells.

Recent studies suggest a protective effect of glucocorticoid against progression of bone erosion and periarticular osteoporosis in patients with rheumatoid arthritis (RA), although this steroid hormone itself is believed to increase bone loss. To understand the antagonistic effect of glucocorticoid for osteopenic process in RA patients, we examined the effect of dexamethasone on Fas-mediated apoptosis of cultured human osteoblasts induced by either anti-Fas IgM or activated peripheral blood mononuclear cells (PBMC). Human osteoblastic cell line MG63 and primary osteoblast-like cells obtained from biopsy specimens were used in this study. PBMC isolated from healthy donors were cultured with or without recombinant interleukin-2 (rIL-2) followed by 12-O-tetradecanoyl-phorbol 13-acetate (PMA) with ionomycin in the presence or absence of dexamethasone. Fas was functionally expressed on MG63 and primary osteoblast-like cells, and treatment of these cells with dexamethasone affected neither Fas expression nor anti-Fas IgM-induced apoptosis. Activated PBMC expressing membrane-type Fas ligand (mFasL) efficiently killed both MG63 and primary osteoblasts-like cells, and the addition of human Fas chimeric protein (hFas-Fc) significantly diminished the cytotoxicity, indicating that interactions between mFasL of activated PBMC and Fas on human osteoblasts induce apoptosis of the latter. Although dexamethasone did not affect apoptosis of MG63 and primary osteoblast-like cells induced by anti-Fas IgM, treatment of activated PBMC with dexamethasone markedly inhibited both mFasL expression and cytotoxicity of these cells against human osteoblasts, suggesting that dexamethasone preferentially acts not on osteoblasts but PBMC. Cultured supernatants from activated PBMC induced apoptosis of human osteoblasts and the addition of hFas-Fc also inhibited the cytotoxicity of the supernatants. In addition, soluble form FasL (sFasL) was detected in the supernatants of activated PBMC. Furthermore, both the cytotoxicity and sFasL concentration of cultured supernatants of activated PBMC incubated with dexamethasone was significantly lower than that in the absence of dexamethasone. Our data suggest that glucocorticoid suppresses the apoptotic process of osteoblasts by inhibiting the expression of both mFasL and sFasL derived from activated PBMC, mediating a protective effect against periarticular bone loss and bone erosion in inflammatory arthritis such as RA.

Apoptosis↗

PubMed1999

Extracellular matrix-associated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro.

Osteoblastic differentiation is an essential part of bone formation that compensates resorbed bone matrix to maintain its structural integrity. Cells in an osteoblast lineage develop differentiated phenotypes during a long-term culture in vitro. However, intrinsic mechanisms whereby these cells differentiate into mature osteoblasts are yet unclear. Bone morphogenetic proteins (BMPs) stimulate osteoblastic differentiation and bone formation. We demonstrate that mouse osteoblastic MC3T3-E 1 cells constitutively expressed messenger RNAs (mRNAs) for BMP-2 and BMP-4 and accumulated BMPs in collagen-rich extracellular matrices. BMPs associated with the extracellular matrices were involved in the induction of osteoblastic differentiation of nonosteogenic mesenchymal cells as well as cells in the osteoblast lineage. MC3T3-E1 cells constitutively expressed type IA and type II BMP receptors. When a kinase-deficient type IA BMP receptor was stably transfected to MC3T3-E 1 cells to obliterate BMP-2/4 signaling, these cells not only failed to respond to exogenous BMP-2 but lost their capability of differentiation into osteoblasts that form mineralized nodules. These observations strongly suggest that endogenous BMP-2/4 accumulated in extracellular matrices are essential for the osteoblastic differentiation of cells in the osteoblast lineage. Therefore, the regulatory mechanism of BMP-2/4 actions in osteoblastic cells is a principal issue to be elucidated for better understanding of pathogenesis of bone losing diseases such as osteoporosis.

Animals↗

PubMed1998

Developmental association of the beta-galactoside-binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts.

The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the beta -galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.

Amino Acid Sequence↗

PubMed1982

Osteoblasts release osteoclasts from calcitonin-induced quiescence.

There is an increasing body of indirect evidence that suggests that the localization and activity of osteoclasts, the major agents of bone resorption, are controlled by osteoblasts. In this paper I provide direct evidence that osteoblasts are indeed able to alter the behaviour of osteoclasts. I used calcitonin (CT) to suppress the cytoplasmic activity of osteoclasts isolated on glass or plastic substrates, and found that, while isolated osteoclasts remain quiescent in CT for as long as the hormone is present, they regain activity when osteoblasts are added to the culture. 'Escape' from CT-induced quiescence could not be transmitted by supernatants from cultures of osteoblasts, was not due to inactivation of CT by osteoblasts, and did not occur if osteoblasts were separated from osteoclasts by a Millipore filter. When osteoblasts and osteoclasts were cultured together at low cell density in CT, I found that while those osteoclasts in the culture that remained isolated also remained inactive, osteoclasts that were in contact with osteoblasts showed a return to activity. This return to activity was most marked in the portion of osteoclast periphery in direct contact with an adjacent osteoblast. Although contact with osteoblasts did not abrogate the sensitivity of osteoclasts to added calcitonin, osteoclasts were found to escape inhibition by calcitonin at a rate proportional to the number of osteoblasts with which they were in contact.

Alkaline Phosphatase↗

PubMed2004

Identification of novel regulators associated with early-phase osteoblast differentiation.

UNLABELLED: Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation. INTRODUCTION: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation. MATERIALS AND METHODS: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation. RESULTS AND CONCLUSIONS: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes.

Animals↗