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PubMed2005

Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1 stimulate osteoblast-specific osteocalcin gene expression.

The role of ATF4 (activating transcription factor 4) in osteoblast differentiation and bone formation was recently described using ATF4-deficient mice (Yang, X., Matsuda, K., Bialek, P., Jacquot, S., Masuoka, H. C., Schinke, T., Li, L., Brancorsini, S., Sassone-Corsi, P., Townes, T. M., Hanauer, A., and Karsenty, G. (2004) Cell 117, 387-398). However, the mechanisms of ATF4 in bone cells are still not clear. In this study, we determined the molecular mechanisms through which ATF4 activates the mouse osteocalcin (Ocn) gene 2 (mOG2) expression and mOG2 promoter activity. ATF4 increased the levels of Ocn mRNA and mOG2 promoter activity in Runx2-containing osteoblasts but not in non-osteoblastic cells that lack detectable Runx2 protein. However, ATF4 increased Ocn mRNA and mOG2 promoter activity in non-osteoblastic cells when Runx2 was co-expressed. Mutational analysis of the OSE1 (ATF4-binding site) and the two OSE2s (Runx2-binding sites) in the 657-bp mOG2 promoter demonstrated that ATF4 and Runx2 activate Ocn via cooperative interactions with these sites. Pull-down assays using nuclear extracts from osteoblasts or COS-7 cells overexpressing ATF4 and Runx2 showed that both factors are present in either anti-ATF4 and anti-Runx2 immunoprecipitates. In contrast, pull-down assays using purified glutathione S-transferase fusion proteins were unable to demonstrate a direct physical interaction between ATF4 and Runx2. Thus, accessory factors are likely involved in stabilizing interactions between these two molecules. Regions within Runx2 required for ATF4 complex formation and activation were identified. Deletion analysis showed that the leucine zipper domain of ATF4 is critical for Runx2 activation. This study is the first demonstration that cooperative interactions between ATF4 and Runx2/Cbfa1 stimulate osteoblast-specific Ocn expression and suggests that this regulation may represent a novel intramolecular mechanism regulating Runx2 activity and, thereby, osteoblast differentiation and bone formation.

Activating Transcription Factor 4↗

PubMed2006

Up-regulation of endogenous RGS2 mediates cross-desensitization between Gs and Gq signaling in osteoblasts.

Regulator of G protein signaling (RGS) proteins limit G protein signals. In this study, we investigated the role of RGS2 in the control of G protein signaling cascades in osteoblasts, the cells responsible for bone formation. Expression of RGS2 was up-regulated in primary cultures of mouse calvarial osteoblasts by parathyroid hormone-related peptide (PTHrP)-(1-34), which stimulates G(s) signaling. RGS2 was also up-regulated by extracellular ATP, which selectively activates G(q), as well as by forskolin and phorbol myristate acetate, which activate targets downstream of G(s) and G(q), respectively. To assess the role of endogenous RGS2, we characterized G(s) and G(q) signaling in osteoblasts derived from wild type and rgs2(-/-) mice. Under control conditions, nucleotide-stimulated calcium release, endothelin-stimulated accumulation of inositol phosphates, and PTHrP-stimulated cAMP accumulation were equivalent in osteoblasts isolated from wild type and rgs2(-/-) mice. Thus, basal levels of endogenous RGS2 do not appear to regulate G(s) or G(q) signaling in osteoblasts. Interestingly, forskolin treatment of wild type but not rgs2(-/-) osteoblasts suppressed both endothelin-stimulated accumulation of inositol phosphates and nucleotide-stimulated calcium release, indicating that up-regulation of RGS2 by G(s) signaling desensitizes G(q) signals. Furthermore, pretreatment with ATP suppressed PTHrP-dependent cAMP accumulation in wild type but not rgs2(-/-) osteoblasts, implying that up-regulation of RGS2 by G(q) signaling desensitizes G(s) signals. Our findings demonstrate that endogenously expressed RGS2 can limit G(s) signaling. Moreover, up-regulation of RGS2 contributes to cross-desensitization of G(s)- and G(q)-coupled signals.

Adenosine Triphosphate↗

PubMed2002

ERK MAP Kinase is required in 1,25(OH)2D3-induced differentiation in human osteoblasts.

Expression of alkaline phosphatase(ALP)activity represents a key event during the differentiation processes of osteoblasts, and the level of ALP activity has been routinely used as a relative measure of differentiation stages of osteoblasts. In human osteoblasts, we showed that vitamin D3 analogue, 1,25(OH)2D3, had a stimulatory effect on ALP activity after 3 days, compared with control. The treatment of PD098059, an ERK MAP Kinase inhibitor, had a reducing effect on ALP activity, a differentiation marker in 1,25(OH)2D3-treated primary human osteoblasts. However, SB203580, a potent p38 MAP Kinase inhibitor, had no effect on the differentiation in this system. This indicates that ERK, not p38, is directly related to 1,25(OH)2D3-stimulated ALP activity in primary human osteoblasts. These results also show that the vitamin D3 analogue stimulates ERK1 activation in primary human osteoblasts. This finding provides one of signaling pathways for differentiation in primary human osteoblasts.

Alkaline Phosphatase↗

PubMed2004

N-acetyl cysteine regulates TNF-alpha-inhibited differentiation in ROS 17/2.8 osteoblasts.

Osteoblasts play a pivotal role in bone remodeling. The alkaline phosphatase (ALPase) activity was decreased in ROS 17/2.8 osteoblast treated with TNF-alpha (2, 5 or 10 ng/ml). The treatment of TNF-alpha inhibited osteoblast differentiation such as ALPase activity in ROS 17/2.8 osteoblast. TNF-gamma (10 ng/ml) increased NF-kappaB DNA binding activity in nuclear extracts of osteoblasts. The addition of NAC (N-acetyl cysteine), free radical scavenger, completely prevented TNF-alpha-induced activation of NF-kappaB. In addition, IkappaB alpha and IkappaB beta were rapidly degraded, allowing the activated NF-kappaB to enter the nucleus and promote gene transcription. To determine whether IkappaB alpha signal transduction pathway is important in the differentiation, we generated IkappaB (KD)-stably transfected ROS 17/2.8 cells. These IkappaB (KD) transfectants did not show any regulation of ALPase in osteoblasts. Here, we suggest that the degradations of IkappaB alpha and IkappaB beta and the following activation of NF-kappaB are the targets of NAC and that NF-kappaB transcription factor is a pivotal clue to regulation of differentiation in TNFalpha-exposed osteoblasts.

Acetylcysteine↗

PubMed1993

Cell proliferation, extracellular matrix mineralization, and ovotransferrin transient expression during in vitro differentiation of chick hypertrophic chondrocytes into osteoblast-like cells.

Differentiation of hypertrophic chondrocytes toward an osteoblast-like phenotype occurs in vitro when cells are transferred to anchorage-dependent culture conditions in the presence of ascorbic acid (Descalzi Cancedda, F., C. Gentili, P. Manduca, and R. Cancedda. 1992. J. Cell Biol. 117:427-435). This process is enhanced by retinoic acid addition to the culture medium. Here we compare the growth of hypertrophic chondrocytes undergoing this differentiation process to the growth of hypertrophic chondrocytes maintained in suspension culture as such. The proliferation rate is significantly higher in the adherent hypertrophic chondrocytes differentiating to osteoblast-like cells. In cultures supplemented with retinoic acid the proliferation rate is further increased. In both cases cells stop proliferating when mineralization of the extracellular matrix begins. We also report on the ultrastructural organization of the osteoblast-like cell cultures and we show virtual identity with cultures of osteoblasts grown from bone chips. Cells are embedded in a dense meshwork of type I collagen fibers and mineral is observed in the extracellular matrix associated with collagen fibrils. Differentiating hypertrophic chondrocytes secrete large amounts of an 82-kD glycoprotein. The protein has been purified from conditioned medium and identified as ovotransferrin. It is transiently expressed during the in vitro differentiation of hypertrophic chondrocytes into osteoblast-like cells. In cultured hypertrophic chondrocytes treated with 500 nM retinoic acid, ovotransferrin is maximally expressed 3 d after retinoic acid addition, when the cartilage-bone-specific collagen shift occurs, and decays between the 5th and the 10th day, when cells have fully acquired the osteoblast-like phenotype. Similar results were obtained when retinoic acid was added to the culture at the 50 nM "physiological" concentration. Cells expressing ovotransferrin also coexpress ovotransferrin receptors. This suggests an autocrine mechanism in the control of chondrocyte differentiation to osteoblast-like cells.

Alkaline Phosphatase↗

PubMed1998

Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages.

Cumulative evidence indicates that osteoblasts and adipocytes share a common mesenchymal precursor and that bone morphogenetic proteins (BMPs) can induce both osteoblast and adipocyte differentiation of this precursor. In the present study, we investigated the roles of BMP receptors in differentiation along these separate lineages using a well-characterized clonal cell line, 2T3, derived from the mouse calvariae. BMP-2 induced 2T3 cells to differentiate into mature osteoblasts or adipocytes depending upon culture conditions. To test the specific roles of the type IA and IB BMP receptor components, truncated and constitutively active type IA and IB BMP receptor cDNAs were stably expressed in these cells. Overexpression of truncated type IB BMP receptor (trBMPR-IB) in 2T3 cells completely blocked BMP-2-induced osteoblast differentiation and mineralized bone matrix formation. Expression of trBMPR-IB also blocked mRNA expression of the osteoblast specific transcription factor, Osf2/ Cbfa1, and the osteoblast differentiation-related genes, alkaline phosphatase (ALP) and osteocalcin (OC). BMP-2-induced ALP activity could be rescued by transfection of wild-type (wt) BMPR-IB into 2T3 clones containing trBMPR-IB. Expression of a constitutively active BMPR-IB (caBMPR-IB) induced formation of mineralized bone matrix by 2T3 cells without addition of BMP-2. In contrast, overexpression of trBMPR-IA blocked adipocyte differentiation and expression of caBMPR-IA induced adipocyte formation in 2T3 cells. Expression of the adipocyte differentiation-related genes, adipsin and PPARgamma, correlated with the distinct phenotypic changes found after overexpression of the appropriate mutant receptors. These results demonstrate that type IB and IA BMP receptors transmit different signals to bone-derived mesenchymal progenitors and play critical roles in both the specification and differentiation of osteoblasts and adipocytes.

Adipocytes↗

PubMed2000

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

Alkaline Phosphatase↗

PubMed2005

The high mobility group transcription factor Sox8 is a negative regulator of osteoblast differentiation.

Bone remodeling is an important physiologic process that is required to maintain a constant bone mass. This is achieved through a balanced activity of bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, we identify the high mobility group transcription factor Sox8 as a physiologic regulator of bone formation. Sox8-deficient mice display a low bone mass phenotype that is caused by a precocious osteoblast differentiation. Accordingly, primary osteoblasts derived from these mice show an accelerated mineralization ex vivo and a premature expression of osteoblast differentiation markers. To confirm the function of Sox8 as a negative regulator of osteoblast differentiation we generated transgenic mice that express Sox8 under the control of an osteoblast-specific Col1a1 promoter fragment. These mice display a severely impaired bone formation that can be explained by a strongly reduced expression of runt-related transcription factor 2, a gene encoding a transcription factor required for osteoblast differentiation. Together, these data demonstrate a novel function of Sox8, whose tightly controlled expression is critical for bone formation.

Animals↗

PubMed1996

The role of gp130-mediated signals in osteoclast development: regulation of interleukin 11 production by osteoblasts and distribution of its receptor in bone marrow cultures.

Interleukin (IL)-11 is a multifunctional cytokine whose role in osteoclast development has not been fully elucidated. We examined IL-11 production by primary osteoblasts and the effects of rat monoclonal anti-mouse glycoprotein 130 (gp130) antibody on osteoclast formation, using a coculture of mouse osteoblasts and bone marrow cells. IL-1, TNF alpha, PGE2, parathyroid hormone (PTH) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) similarly induced production of IL-11 by osteoblasts, but IL-6, IL-4, and TGF beta did not. Primary osteoblasts constitutively expressed mRNAs for both IL-11 receptor (IL-11R alpha) and gp130. Osteotropic factors did not modulate IL-11R alpha mRNA at 24 h, but steady-state gp130 mRNA expression in osteoblasts was upregulated by 1 alpha,25(OH)2D3, PTH, or IL-1. In cocultures, the formation of multinucleated osteoclast-like cells (OCLs) in response to IL-11, or IL-6 together with its soluble IL-6 receptor was dose-dependently inhibited by rat monoclonal anti-mouse gp130 antibody. Furthermore, adding anti-gp130 antibody abolished OCL formation induced by IL-1, and partially inhibited OCL formation induced by PGE2, PTH, or 1 alpha,25(OH)2D3. During osteoclast formation in marrow cultures, a sequential relationship existed between the expression of calcitonin receptor mRNA and IL-11R alpha mRNA. Osteoblasts as well as OCLs expressed transcripts for IL-11R alpha, as indicated by RT-PCR analysis and in situ hybridization. These results suggest a central role of gp130-coupled cytokines, especially IL-11, in osteoclast development. Since osteoblasts and mature osteoclasts expressed IL-11R alpha mRNA, both bone-forming and bone-resorbing cells are potential targets of IL-11.

Animals↗

PubMed2000

Evaluation of osteoblast response to porous bioactive glass (45S5) substrates by RT-PCR analysis.

Previous studies have shown that neonatal rat calvaria osteoblasts elaborate substantial amounts of extracellular material with bone-like characteristics when cultured on porous bioactive glass substrates in vitro. However, the osteoblastic response to this material has not been fully characterized. The objective of this study was to characterize osteoblast response to porous bioactive glass substrates following the expression of the classical markers for osteoblast differentiation. In this study we synthesized porous bioactive glass substrates, seeded them with osteoblast-like cells (ROS 17/2.8) and followed the temporal expression of alkaline phosphatase (AP) activity, as well as the expression of mRNA for collagen type I (Coll-1), osteonectin (OSN), osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP). The data confirm that porous bioactive glass substrates are capable of supporting the in vitro growth and maturation of osteoblast-like cells. At a porosity of 42% and an average pore size of 80 microm, the substrates promote the expression and maintenance of the osteoblastic phenotype. The results additionally suggest that there is both a solution-mediated and a surface-controlled effect on cell activity.

Alkaline Phosphatase↗

PubMed2001

Monocyte chemoattractant protein-1 expression by osteoblasts following infection with Staphylococcus aureus or Salmonella.

Two common pathogens of bone, Staphylococcus aureus and Salmonella, were investigated for their ability to induce chemokine expression in bone-forming osteoblasts. Cultured mouse or human osteoblasts could rapidly respond to bacterial infection by upregulating the mRNA encoding the chemokine, monocyte chemoattractant protein-1 (MCP-1). This rapid induction occurred on infection with either the gram-positive pathogen, S. aureus, or the gram-negative pathogen, Salmonella. Increased mRNA expression translated into MCP-1 secretion by cultured mouse or human osteoblasts in response to viable bacteria, whereas UV-killed bacteria were less effective in stimulating chemokine secretion. There was a dose-response relationship observed between the amount of input bacteria and increases in MCP-1 secretion. Immunohistochemical staining of infected osteoblasts indicated that the majority of cells could express MCP-1, with some osteoblasts having a higher intensity of staining than others. Organ cultures of mouse calvaria (skullcap) bone showed increases in MCP-1 immunostaining following bacterial infection. The immunoreactive MCP-1 in infected calvaria localized to areas containing active osteoblasts. Taken together, these studies demonstrate a conserved osteoblast-derived MCP-1 response to two very different pathogens of bone.

Animals↗

PubMed2001

Signal transduction system for interleukin-6 synthesis stimulated by lipopolysaccharide in human osteoblasts.

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions, and one of the roles is as a powerful stimulator of bone resorption. LPS stimulated bone resorption via CD14 in mouse calvaria and was reported to function as a receptor for bacterial LPS complexed with serum proteins. Interleukin-6 (IL-6) is capable of stimulating the differentiation of osteoclasts from their hematopoietic precursors, and LPS elevates IL-6 synthesis in human osteoblastic cells. However, the signaling pathway of LPS-induced IL-6 synthesis in osteoblasts is unknown. In the present study, we could detect the existence of CD14 in human osteoblastic cells by RT-PCR analysis and show that LPS increased IL-6 mRNA and synthesis via CD14 in human osteoblastic cells. In human osteoblasts (SaM-1 cells) treated with 10 microg/ml LPS, increases in IL-6 mRNA and synthesis were inhibited by anti-CD14 antibody (MEM-18), PD98059 (an inhibitor of classic mitogen-activated protein kinase [MAPK]), or SB203580 (an inhibitor of p38 MAPK) but were not inhibited by H-89 (an inhibitor of protein kinase A [PKA]) and calphostin C (an inhibitor of protein kinase C [PKC]). Furthermore, LPS-induced IL-6 synthesis was inhibited by curcumin (an inhibitor of activating protein-1 [AP-1]) but not by pyrrolidine dithiocarbamate (PDTC) (an inhibitor of nuclear factor kappa B [NF-kappaB]). The findings of the present study suggest that the LPS receptor CD14, existent in human osteoblastic cells, and IL-6 synthesis in response to LPS probably occur via CD14, p38 MAPK, and MAP kinase/extracellular-regulated kinase kinase (MEK), leading to the transcriptional activation of AP-1 in human osteoblastic cells.

Adult↗

PubMedDate not supplied

Surface modification by complexes of vitronectin and growth factors for serum-free culture of human osteoblasts.

Cell attachment, expansion, and migration in three-dimensional biomaterials are crucial steps for effective delivery of osteogenic cells into bone defects. Complexes composed of vitronectin (VN), insulin-like growth factors (IGFs), and insulin growth factor-binding proteins (IGFBPs) have been reported to enhance cell attachment, proliferation, and migration in a variety of cell lines in vitro. The aim of this study was to examine whether prebound complexes of VN and IGFs +/- IGFBPs could facilitate human osteoblast serum-free expansion in vitro and enhance cell attachment, proliferation, and migration in three-dimensional biomaterial constructs. Human osteoblasts derived from alveolar bone chips and the established human osteoblast cell line Saos-2 were used. These cells were seeded on tissue culture plates and porous scaffolds of type I collagen sponges and polyglycolic acid (PGA), which had been coated with VN +/- IGFBP-5 +/- IGF-I. Cell attachment, proliferation, and migration were evaluated by cell counting, confocal microscopy, and scanning electron microscopy. The number of attached human osteoblasts was significantly higher in VN-coated polystyrene culture dishes. Furthermore, significant increases in cell proliferation were observed when growth factors were bound to these surfaces in the presence of VN. In the two scaffold materials examined, greater cell attachment was found in type I collagen sponges compared with PGA scaffolds. However, coating the scaffolds with complexes composed of VN + IGF-I or VN + IGFBP-5 + IGF-I enhanced cell attachment on PGA. Moreover, the presence of VN + IGFBP-5 + IGF-I resulted in significantly greater osteoblast migration into deep pore areas as compared with untreated scaffolds or scaffolds treated with fetal calf serum. These results demonstrated that complexes of VN + IGFBP-5 + IGF-I can be used to expand osteoblasts in vitro under serum-free conditions and enhance the attachment and migration of human osteoblasts in three-dimensional culture. This in turn suggests a potential application in surface modification of biomaterials for tissue reconstruction.

Bone Substitutes↗

PubMed2001

TGF-beta-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation.

Transforming growth factor-beta (TGF-beta), a secreted factor present at high levels in bone, inhibits osteoblast differentiation in culture; yet, the mechanism of this inhibition remains unclear. We studied the effects of TGF-beta and its effectors, the Smads, on the expression and function of the osteoblast transcription factor CBFA1. TGF-beta inhibited the expression of the cbfa1 and osteocalcin genes, whose expression is controlled by CBFA1 in osteoblast-like cell lines. This inhibition was mediated by Smad3, which interacts physically with CBFA1 and represses its transcriptional activity at the CBFA1-binding OSE2 promoter sequence. The repression of CBFA1 function by Smad3 contrasts with previous observations that Smads function as transcription activators. This repression occurred in mesenchymal but not epithelial cells, and depended on the promoter sequence. Smad3-mediated repression of CBFA1 provides a central regulatory mechanism for the inhibition of osteoblast differentiation by TGF-beta, since it inhibits both cbfa1 transcription and transcriptional activation of osteoblast differentiation genes by CBFA1. Altering Smad3 signaling influenced osteoblast differentiation in the presence or absence of TGF-beta, implicating Smad3/TGF-beta-mediated repression in autocrine regulation of osteoblast differentiation.

Animals↗

PubMed2001

The osteoblastic phenotype in calcium-depleted and calcium-repleted rats: a structural and histomorphometric study.

In a previous report we showed that young rats fed a calcium-free diet for 28 days developed severe hypocalcaemia and showed a significant increase in serum alkaline phosphatase activity. The main histological and cytochemical changes exhibited by these animals in bone of the metaphyseal primary spongiosa were: (1) hyperplasia of osteoblasts, (2) an increase in the frequency of tartrate-resistant acid phosphatase (TRAP)-positive osteoblasts apposed to osteoid, and (3) an excessive amount of osteoid tissue. In addition to typical osteoblasts, there was a subpopulation of osteoblast-like cells with coated pits, lysosome-like bodies and large cytoplasmic processes. In the present study, we investigated how the above parameters change when calcium-depleted rats are placed on a normal diet for 7 days. Such a regimen normalized calcium concentration and alkaline phosphatase activity in the serum. The osteoid thickness returned to normal and, in some areas, was fully calcified. Most osteoblasts no longer showed TRAP activity and their ultrastructure was similar to that found in controls. Despite an intense alkaline phosphatase activity, some of them still exhibited a number of macrophagic characteristics. They were TRAP-positive, and showed electron-dense bodies in the cytoplasm facing bone, an abundance of coated pits, calcified spicules impinging on the cell membrane and large processes extending into the mineralized matrix. We concluded that calcium deficiency causes hyperplasia of osteoblasts in primary spongiosa and an increase in expression of TRAP. It also induces changes in their phenotype characterized by the acquisition of macrophagic cellular features. While TRAP activity is normalized by calcium repletion, macrophagic characteristics persist. These results suggest that the osteoblast can modulate its phenotype according to its physiological status.

Acid Phosphatase↗

PubMed1999

Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity.

The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

3T3 Cells↗

PubMed2003

Modulation of cellular differentiation by N-methyl-D-aspartate receptors in osteoblasts.

N-methyl-D-aspartate (NMDA) receptors for the central neurotransmitter l-glutamate (Glu) have been shown to be present in both osteoblasts and osteoclasts. Sustained exposure to the NMDA channel antagonist dizocilpine (MK-801) significantly prevented increases in both alkaline phosphatase activity and Ca2+ accumulation in a concentration-dependent manner in osteoblasts cultured for 7-28 days in vitro (DIV), without significantly affecting cell survivability. Osteocalcin expression was markedly reduced in the presence of MK-801 in osteoblasts cultured for 28 DIV. Both an NMDA domain antagonist and a glycine domain antagonist similarly prevented Ca2+ accumulation in osteoblasts exposed for 28 consecutive DIV. MK-801 was effective in significantly inhibiting Ca2+ accumulation determined at 28 DIV in osteoblasts exposed before 7 DIV but was ineffective in cells exposed after 11-21 DIV. Sustained exposure to MK-801 significantly inhibited DNA binding activity and expression of core binding factor alpha-1 (CBFA1) in osteoblasts exposed after 7 DIV up to 28 DIV, but not in those exposed before 7 DIV. These results suggest that heteromeric NMDA receptor channels may be functionally expressed to regulate mechanisms underlying cellular differentiation rather than proliferation and/or maturation through modulation of expression of CBFA1 in cultured rat calvarial osteoblasts.

Animals↗

PubMed2002

Effects of serum from children with newly diagnosed Crohn disease on primary cultures of rat osteoblasts.

OBJECTIVES: We propose that Crohn disease (CD) decreases bone formation via circulating inflammatory mediators. We therefore examined the effects of serum from newly diagnosed, untreated children with CD on osteoblasts in culture and the role of interleukin-6 (IL-6), a cytokine present in excess in active CD that also has direct effects on bone. METHODS: Bone mineral density was measured by dual x-ray absorptiometry. Primary cultures of rat osteoblasts were treated with serum from patients with CD and healthy controls. We measured expression of osteoblast proliferation, viability, differentiation markers, and mineralized nodule formation. Neutralizing antibodies were used to inhibit the effects of IL-6 present in serum. RESULTS: We studied 24 children with CD (14 male) and 31 controls (15 male). Spine bone mineral density was lower in patients with CD (Z score, -0.8 +/- 0.9 vs. 0.0 +/- 1.0 for controls; P < 0.05). Nodule formation was markedly decreased in osteoblasts treated with CD serum. However, CD serum did not affect osteoblast proliferation or viability. Expression of proteins characteristic of mature osteoblasts-osteocalcin and alkaline phosphatase-was reduced. Unlike our results in a model of intact bone, neutralization of IL-6 did not inhibit the effects of CD serum. Addition of IL-6 to control serum to match serum concentrations in CD had no effect either. CONCLUSIONS: CD serum affects osteoblast function and probably differentiation in vitro, suggesting a mechanism by which CD may affect bone formation. IL-6 by itself is not sufficient to cause these effects and probably needs a cofactor present in intact bone.

Absorptiometry, Photon↗