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At least 433 records · Page 24Linked to original sources

PubMed2005

Role of macrophages in LPS-induced osteoblast and PDL cell apoptosis.

In periradicular lesions and periodontal disease, bacterial invasion leads to chronic inflammation resulting in disruption of the structural integrity of the periodontal ligament and progressive alveolar bone destruction. The pathogenesis of these conditions has been attributed not only to bacterial-induced tissue destruction but also to a defect in periodontal tissue repair. Accumulated data have also shown that lipopolysaccharide (LPS) can directly induce cell death or apoptosis in many cell types, including macrophages, osteoblasts, vascular endothelial cells, hepatocytes and myocytes. The present study hypothesized that bacterial LPS-induced apoptosis in osteoblasts and periodontal ligament fibroblasts (PDL cells) is an important contributing factor to the defect in periodontal tissue repair in periodontal and periapical disease. Macrophages have been shown to respond to bacterial LPS by increasing the production of proinflammatory cytokines. In addition, large numbers of macrophages are present in inflamed periodontal tissue. We speculated that macrophages were a potential candidate cell for mediating apoptosis in osteoblasts and PDL cells in response to bacteria-derived LPS. The macrophage-like cell line, RAW 264.7, was stimulated with LPS, and the conditioned medium was used to treat osteoblasts and PDL cells. Bacterial LPS had no direct apoptotic effect on mouse osteoblasts or PDL cells, whereas the conditioned medium from LPS-activated macrophages was able to induce apoptosis in these cells. To evaluate the contribution of tumor necrosis factor-alpha (TNF-alpha) released from macrophages on osteoblast and PDL cell apoptosis, cells were incubated with conditioned medium from LPS-treated macrophages in the presence and absence of anti-TNF-alpha neutralizing antibodies. TNF-alpha neutralizing antibody pretreatment inhibited the effect of conditioned medium from LPS-treated macrophages on osteoblast and PDL cell apoptosis in a dose-dependent manner. These results suggest that LPS could indirectly induce apoptosis in osteoblasts and PDL cells through the induction of TNF-alpha release from macrophages. These studies provide insight into a potential mechanism by which bacterial-derived LPS could contribute to defective periodontal and bone tissue repair in periodontal and periapical disease.

Animals↗

PubMed2006

Osteoblast and osteocyte apoptosis associated with androgen action in bone: requirement of increased Bax/Bcl-2 ratio.

Both the number and the activity of osteoblasts are critical for normal bone growth and maintenance. Although a potential role for estrogen in protection of bone mass through inhibition of osteoblast apoptosis has been proposed, a function for androgen is much less clear. The aim of this study was to establish a direct role for androgen to influence osteoblast apoptosis both in vitro and in vivo. AR-MC3T3-E1 cells, with androgen receptor (AR) overexpression controlled by the type I collagen promoter, were treated with the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT). Apoptosis was assessed by three different techniques including DNA fragmentation, caspase-3 activation, and changes in mitochondrial membrane potential. Transactivation of AR by DHT enhanced apoptosis while 17beta-estradiol (E(2)) treatment reduced apoptosis in both proliferating preosteoblasts and mature osteocyte-like cells. To explore mechanism, the apoptosis regulators Bcl-2 (antiapoptotic) and Bax (proapoptotic) were evaluated. Western analysis revealed that DHT decreased Bcl-2 resulting in a significantly increased Bax/Bcl-2 ratio. Regulation of Bcl-2 was post-transcriptional since bcl-2 mRNA levels were unaffected by DHT treatment. Furthermore, ubiquitination of Bcl-2 was increased and serine phosphorylation was reduced, consistent with inhibition of MAP kinase signaling by DHT. Increased Bax/Bcl-2 ratio was essential since either Bcl-2 overexpression or Bax downregulation by RNA interference (RNAi) partially abrogated or reversed DHT-enhanced osteoblastic apoptosis. In order to establish physiologic significance in vivo, AR-transgenic mice with AR overexpression in the osteoblast lineage and thus enhanced androgen sensitivity were characterized. In male AR-transgenic mice, increased osteoblast apoptosis was observed in vivo even in association with new bone formation. Thus, although estrogen can be antiapoptotic, androgen stimulates osteoblast and osteocyte apoptosis through an increased Bax/Bcl-2 ratio even in anabolic settings. These results identify a new mechanism for androgen regulation of osteoblast activity distinct from estrogen, and suggest that enhanced apoptosis can be associated with anabolic stimulation of new bone growth. Androgens thus play a distinct role in skeletal homeostasis.

Androgens↗

PubMed2006

Parathyroid hormone stimulates expression of the Notch ligand Jagged1 in osteoblastic cells.

We previously demonstrated that activation of the Parathyroid Hormone Receptor (PTH1R) in osteoblastic cells increases the Notch ligand Jagged1 and expands hematopoietic stem cells (HSC) through Notch signaling. However, regulation of Jagged1 by PTH in osteoblasts is poorly understood. The present study demonstrates that PTH treatment increases Jagged1 levels in a subpopulation of osteoblastic cells in vivo and in UMR106 osteoblastic cells in vitro. Since PTH(1-34) activates both Adenylate Cyclase/Protein Kinase A (AC/PKA) and Protein Kinase C (PKC) downstream of the PTH1R in osteoblastic cells, we independently determined the effect of either pathway on Jagged1. Activation of AC with Forskolin or PKA with PTH(1-31) or cell-permeable cAMP analogues increased osteoblastic Jagged1. This PTH-dependent Jagged1 increase was blocked by H89 and PKI, specific PKA inhibitors. In contrast, PKC activation with phorbol ester (PMA) or PTH(13-34) did not stimulate Jagged1 expression, and PTH-dependent Jagged1 stimulation was not blocked by Gö6976, a conventional PKC inhibitor. Therefore, PTH treatment stimulates osteoblastic Jagged1 mainly through the AC/PKA signaling pathway downstream of the PTH1R. Since Jagged1/Notch signaling has been implicated not only in stromal-HSC interactions but also in osteoblastic differentiation, Jagged1 may play a critical role in mediating the PTH-dependent expansion of HSC, as well as the anabolic effect of PTH in bone.

Adenylyl Cyclases↗

PubMed2006

Over-expression of TIMP-1 in osteoblasts increases the anabolic response to PTH.

Intermittent PTH treatment induces structural changes that affect cancellous bone mass and have led to its indication for the treatment of osteoporosis. PTH is also known to upregulate the expression of matrix metalloproteinases (MMP) in osteoblasts. We wanted to find out whether inhibiting osteoblastic MMPs can affect the anabolic action of PTH in vivo. We had shown previously that mice over-expressing TIMP-1 (tissue inhibitor of MMPs) specifically in osteoblasts display an increase in bone mineral density and bone mass combined with an overall decrease in bone turnover. In the present study, 10-week-old wild-type (WT) and transgenic (TG) mice were treated with PTH at 40 microg/kg/day for 1.5 months. DEXA analysis was performed before and after treatment, and histomorphometric and molecular analysis were carried out at the end of the experiment. Our findings indicate that the transgene boosted the anabolic action of PTH. The femurs of PTH-treated TG mice displayed a greater increase in bone mineral density and trabecular bone volume than treated WT mice. Interestingly, the positive effect of the transgene on the action of PTH resulted from both reduced bone resorption activity and an increase in the bone formation rate. Osteoclastic surfaces that were increased in PTH-treated WT mice remained unchanged in TG mice, suggesting a decrease in osteoclastic differentiation. Histomorphometric data also indicate that PTH administration increased osteoblast activity in TG mice and affected the number of osteoblasts in WT mice. In conclusion, we demonstrate that inhibiting osteoblastic MMPs can potentiate the anabolic effect of PTH by decreasing osteoclast activity and increasing osteoblast activity. Our data also suggest that osteoblastic MMPs have some role in mediating the anabolic effects of PTH in vivo and indicate that inhibitors of MMPs could constitute a new therapy for degenerative diseases.

Animals↗

PubMed2005

Cell-to-cell contact is critical for the survival of hematopoietic progenitor cells on osteoblasts.

Osteoblasts constitute part of the stromal cell support system in marrow for hematopoiesis, however little is known as to how they interact with hematopoietic stem cells (HSCs). In vitro studies have demonstrated that the survival of HSCs in co-culture with osteoblasts requires intimate cell-to-cell contact. This suggests that the osteoblast-derived factor(s) that supports stem cell activities are produced in very small quantities, are rapidly turned over, may be membrane-anchored and/or require the engagement of cell-cell adhesion molecules that are yet to be determined. In the present report we found that the survival of hematopoietic progenitor cells on osteoblasts is dependent upon the engagement of VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) receptors using function blocking antibodies. Cell-to-cell contact is required to support progenitor activity, but can be replaced if receptor-ligand engagement of the VLA-4 and LFA-1 complexes is provided through the use of recombinant ligands (fibronectin, ICAM-1, VCAM-1). Moreover, once these receptors were engaged, conditioned medium derived from HSCs grown on osteoblast ligands supported significantly greater hematopoietic progenitors in vitro than did osteoblast-conditioned or HSC-conditioned medium alone. While the molecules present in the co-cultured medium remain to be identified, the data suggest that hematopoietic cells cooperate with osteoblasts to assemble the various marrow microenvironments by directing the synthesis of osteoblast-derived cytokines to improve HSC survival.

Adult↗

PubMed2005

Reduced osteoblastic population and defective mineralization in osteopetrotic (op/op) mice.

Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colony-stimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization.

Alkaline Phosphatase↗

PubMed2006

Wdr5, a WD-40 protein, regulates osteoblast differentiation during embryonic bone development.

Wdr5 accelerates osteoblast and chondrocyte differentiation in vitro, and is developmentally expressed in osteoblasts as well as in proliferating and hypertrophic chondrocytes. To investigate the role of Wdr5 during endochondral bone development, transgenic mice overexpressing Wdr5 under the control of the 2.3-kb fragment of the mouse alpha(1) I collagen promoter were generated. The transgene was specifically expressed in the osteoblasts of transgene positive mice and was absent in the growth plate. Histological analyses at embryonic day 14.5 demonstrated that the humeri of transgene positive embryos were longer than those isolated from wild-type littermates largely due to an expansion of the hypertrophic chondrocyte layer. Acceleration of osteoblast differentiation was observed with greater and more extensive expression of type I collagen and more extensive mineral deposition in the bone collar of transgene positive embryos. Acceleration of vascular invasion was also observed in transgene positive mice. Postnatal analyses of transgenic mice confirmed persistent acceleration of osteoblast differentiation. Targeted expression of Wdr5 to osteoblasts resulted in earlier activation of the canonical Wnt signaling pathway in the bone collar as well as in primary calvarial osteoblast cultures. In addition, overexpression of Wdr5 increased the expression of OPG, a target of the canonical Wnt signaling pathway. Overall, our findings suggest that Wdr5 accelerates osteoblast differentiation in association with activation of the canonical Wnt pathway.

Animals↗

PubMed2001

Increased osteoblast apoptosis in apert craniosynostosis: role of protein kinase C and interleukin-1.

Apert syndrome is an autosomal dominant disorder characterized by premature cranial ossification resulting from fibroblast growth factor receptor-2 (FGFR-2)-activating mutations. We have studied the effects of the prominent S252W FGFR-2 Apert mutation on apoptosis and the underlying mechanisms in human mutant osteoblasts. In vivo analysis of terminal deoxynucleotidyl transferase-mediated nick-end labeling revealed premature apoptosis of mature osteoblasts and osteocytes in the Apert suture compared to normal coronal suture. In vitro, mutant osteoblasts showed increased apoptosis, as demonstrated by terminal deoxynucleotidyl transferase-mediated nick-end labeling analysis, trypan blue staining, and DNA fragmentation. Mutant osteoblasts also showed increased activity of caspase-8 and effector caspases (-3, -6, -7) constitutively. This was related to protein kinase C activation because the selective protein kinase C inhibitor calphostin C inhibited caspase-8, effector caspases, and apoptosis in mutant osteoblasts. Apert osteoblasts also showed increased expression of interleukin (IL)-1alpha, IL-1beta, Fas, and Bax, and decreased Bcl-2 levels. Specific neutralizing anti-IL-1 antibody reduced Fas levels, Bax expression, effector caspases activity, and apoptosis in mutant cells. Thus, the Apert S252W FGFR-2 mutation promotes apoptosis in human osteoblasts through activation of protein kinase C, overexpression of IL-1 and Fas, activation of caspase-8, and increased Bax/Bcl-2 levels, leading to increased effector caspases and DNA fragmentation. This identifies a complex FGFR-2 signaling pathway involved in the premature apoptosis induced by the Apert S252W FGFR-2 mutation in human calvaria osteoblasts.

Acrocephalosyndactylia↗

PubMed2002

TWIST inactivation reduces CBFA1/RUNX2 expression and DNA binding to the osteocalcin promoter in osteoblasts.

The Saethre-Chotzen (SC) syndrome is characterized by increased osteogenesis and premature fusion of cranial sutures, resulting from mutations in TWIST, a basic helix-loop-helix transcription factor. The molecular target genes for Twist in osteoblasts are however unknown. We report here that TWIST haploinsufficiency in mutant osteoblasts reduces mRNA and protein levels for CBFA1/RUNX2, a specific osteoblast transcription factor, during both osteoblast cell growth and in vitro osteogenesis. Moreover, this is associated with altered expression of major osteoblast-specific genes. Electrophoretic mobility shift assay (EMSA) showed reduced-binding ability of Cbfa1 to its target OSE2 element in the osteocalcin promoter in mutant osteoblasts. By contrast, TWIST inactivation does not hamper Cbfa1 binding on a similar upstream element present in the alpha1(I) collagen promoter in mutant osteoblasts. This provides the first evidence that TWIST inactivation alters CBFA1/RUNX2 expression and Cbfa1 binding ability to the osteocalcin promoter, indicating that CBFA1/RUNX2 is a target gene for TWIST in human osteoblasts.

Acrocephalosyndactylia↗

PubMed1997

Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.

The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation is unknown. We previously identified an osteoblast-specific cis-acting element, termed OSE2, in the Osteocalcin promoter. We have now cloned the cDNA encoding Osf2/Cbfa1, the protein that binds to OSE2. Osf2/Cbfa1 expression is initiated in the mesenchymal condensations of the developing skeleton, is strictly restricted to cells of the osteoblast lineage thereafter, and is regulated by BMP7 and vitamin D3. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts. Finally, forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. This study identifies Osf2/Cbfa1 as an osteoblast-specific transcription factor and as a regulator of osteoblast differentiation.

Amino Acid Sequence↗

PubMed2001

Effect of L-lysine and L-arginine on primary osteoblast cultures from normal and osteopenic rats.

A therapeutic role of amino acids L-lysine (Lys) and L-arginine (Arg) in osteoporosis and fracture healing was demonstrated previously by in vivo studies. In the present study, primary cultures of osteoblasts were used to investigate the effect of amino acids on gene expression (alkaline phosphatase activity, ALP; osteocalcin, OC; type I collagen), nitric oxide production (NO) and proliferation (MTT) of cells. Cells were isolated from the distal femurs of normal and osteopenic rats. Normal and osteopenic bone-derived cells were divided into four groups: control, Lys (0.587 mg/mL/d), Arg (0.625 mg/mL/d), and Lys + Arg (0.587 + 0.625 mg/mL/d). No evidence of differences between normal and osteopenic bone-derived cultures in basal conditions was observed. A significant (P = 0.002) increase of 10.4% in NO production was observed in normal bone-derived osteoblasts treated with Lys + Arg when compared to the control group at 7 days. At the same time, normal bone-derived osteoblasts treated with Arg and Lys + Arg showed significant increases in type I collagen synthesis of 25.3% and 28.4%, respectively, when compared to the control group. Osteopenic bone-derived osteoblasts showed significant (P = 0.002) increases of 27.6% in MTT and 28.7% in cell count at 48 hours when treated with Lys + Arg in comparison with the control group. At 7 days, NO production and type I collagen synthesis increased significantly (P< 0.005) both in osteopenic bone-derived osteoblasts treated with Arg (NO: 18.5%; type I collagen: 34.4%) and Lys + Arg (NO: 23.7%; type I collagen: 20.9%) compared to the control group. Finally, a significant (P = 0.025) decrease of 5.8% in OC level was observed in osteopenic bone-derived osteoblasts treated with Arg. Results suggest that the potential therapeutic effect of Lys and Arg on bone could be related, at least in part, to an improvement of NO production and type I collagen synthesis by osteoblasts both in normal and in osteopenic bone. In osteopenic bone-derived osteoblasts this synthetic phase is preceded by an initial increase of cell proliferation.

Animals↗

PubMed2001

Expression and activity of NAD(P)H:quinone oxidoreductase (NMO1) in human osteoblastic cells.

NAD(P)H:quinone oxidoreductase (NMO1; EC 1.6.99.2), also called DT-diaphorase, is involved in the reduction of coenzyme Q, an important cellular lipophilic antioxidant that can function as an intermediate electron carrier in plasma membrane-associated electron transport regulating cell growth. We examined the ability of normal human trabecular osteoblastic cells to express NAD(P)H:quinone oxidoreductase (NMO1) and studied its modulation during cell proliferation and growth arrest. We found that confluent primary human trabecular osteoblastic cells derived from healthy individuals constitutively express NMO1 activity, measured using 2-6 dichlorophenol indophenol (DCI) or menadione (vitamin K3) as substrate, and NADPH or NADH as electron donor. We also found that NMO1 activity was related to osteoblastic cell growth. NMO1 activity increased with osteoblastic cell density. Confluent growth-arrested cells expressed an eightfold higher NMO1 specific activity than proliferative human osteoblastic cells. Reverse-transcription polymerase chain reaction analysis showed that NMO1 mRNA levels did not differ in growth-arrested confluent cell and growing cells, suggesting that the increased NMO1 activity with cell density was due to posttranslational events. Harvesting and replating the cells at low density resulted in a 93.4% loss of NMO1 enzymatic activity. Removal of serum from high-density growth-arrested cells resulted in a 48.5% decrease in NMO1 activity. NMO1 activity does not appear to be related to induction of osteoblast differentiation because treatment with the differentiating agent 1,25(OH)(2) vitamin D(3) had no effect on NMO1 activity. The finding that human osteoblastic cells express NMO1 constitutively and that NMO1 activity increases with density-dependent growth inhibition suggest a role for NAD(P)H:quinone oxidoreductase in the control of growth arrest in normal human osteoblastic cells.

Aged↗

PubMed2001

Individual osteoblasts in the developing calvaria express different gene repertoires.

Several studies in vitro and a few in vivo have suggested that mature osteoblasts heterogeneously express osteoblast markers. In one recent study of the osteoblasts associated with bone nodules formed in vitro in rat calvaria cell populations, extensive diversity was documented in the overall gene repertoires expressed. To address whether comparable heterogeneity is evident in vivo, we investigated the expression of nine osteoblast lineage markers by both in situ hybridization and immunohistochemistry. At 21 days of fetal rat development, the calvaria is a rapidly growing bone with distinct maturational zones that are readily observed in coronal sections; that is, an osteogenic front emerging at sagittal and coronal sutures is adjacent to areas of growing trabeculae of bone, followed by more mature areas of remodeling bone. Based on expression patterns, markers can be divided into two categories. One category comprises markers that are globally expressed by all osteoblasts irrespective of their position in the calvaria. Of those tested, only two, alkaline phosphatase and the pth/pthrp receptor, fit into this category. All other markers analyzed, including transcription factors (c-fos and msx-2), matrix molecules (bone sialoprotein, osteopontin, and osteocalcin), and a hormone (pthrp), were differentially expressed only in subpopulations of osteoblasts, based on cell maturational status, environment (ectocranial vs. endocranial surfaces), and microenvironment (adjacent osteoblasts). Preosteoblasts and osteocytes in different regions of the calvaria also expressed different subsets of the lineage markers. Mechanisms responsible for generating differential gene expression profiles appear to be both transcriptional and posttranscriptional. These results indicate that postproliferative, morphologically indistinguishable osteoblasts are not a homogeneous class of cells, but instead are molecularly diverse. The present results also raise the possibility that lineage progression and/or maintenance of the differentiated state may be adaptable in the calvaria.

Animals↗

PubMed2002

Discovery of osteoblast-associated genes using cDNA microarrays.

Osteoblast maturation is a complex process and involves distinct genotypic changes that are accompanied by specific phenotypic alterations. To identify new bone-related genes in osteoblasts we utilized the high-density mouse GEM1 microarray gene chip from IncyteGenomics, Inc. (St. Louis, Mo). We examined the expression profiles of over 8700 genes during the proliferation (day 3) and the mineralization (day 34) phases of MC3T3-E1 development. More than 8600 genes provided measurable signals. Of these genes, 252 were found to be differentially expressed on days 3 and 34. A large number of these genes have never been previously recognized in the context of osteoblast development. Approximately, 60% of the genes with expressions that were dominant in proliferating osteoblasts consisted of growth-related genes such as TACC3 and Pr22. The expressions of TIS21/BTG2, and a novel gene EST350, were found to peak during the differentiation phase (day 12), suggesting that they may play important roles in osteoblast differentiation. The majority of the genes with expressions that were dominant during the mineralization phase consisted of signal transduction genes and extracellular matrix (ECM) proteins such as lumican and cystatin-C. It is significant that lumican expression could not be detected on day 3, which indicates that this gene may serve as an important marker of postmitotic osteoblasts. The establishment of the expression profiles of these and other genes with various phases of MC3T3-E1 osteoblast development will allow us to distinguish the molecular events at different phases of osteoblast biology.

Animals↗

PubMed2003

Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways.

This study sought to assess the role of several signaling pathways in the fluid flow shear stress-induced proliferation and differentiation of normal human osteoblasts. We evaluated the effects of an effective dose of selective inhibitors of the extracellular signal-regulated kinases (ERK) pathway (PD98059 and U0126), the nitric oxide synthase pathway (N(omega)-nitro-L-arginine methyl ester), the cyclo-oxygenase pathway (indomethacin), or the Gi/o pathway (pertussis toxin [PTX]) on the flow-mediated effects. A 30-min steady flow shear stress at 20 dynes/cm(2) increased significantly [(3)H]thymidine incorporation (an indicator of proliferation), alkaline phosphatase activity (an index of osteoblast differentiation), phosphorylation of ERK, and expression of integrin beta1. PD98059, U0126, and N(omega)-nitro-L-arginine methyl ester completely blocked the shear stress-induced increases in ERK phosphorylation, [(3)H]thymidine incorporation, and alkaline phosphatase, but without an effect on integrin beta1 expression, indicating that the ERK and nitric oxide synthase pathways are essential for the shear stress-induced proliferation and differentiation of normal human osteoblasts and that each involves ERK activation but not integrin beta1 upregulation. Indomethacin blocked the shear stress-induced osteoblast proliferation and differentiation and integrin beta1 upregulation but not ERK activation, suggesting that the cyclo-oxygenase pathway (i.e., prostacyclin and/or prostaglandin E(2)) mediates the shear stress-induced osteoblast proliferation in an ERK-independent manner. In contrast, PTX completely blocked the flow-induced increase in integrin beta1 expression but had no effect on the increase in the ERK phosphorylation or [(3)H]thymidine incorporation. PTX not only did not inhibit but also significantly enhanced the stimulatory effect of shear stress on alkaline phosphatase activity, suggesting that a PTX-sensitive signaling pathway may have an inhibitory role in osteoblast differentiation. In summary, this study shows, for the first time, that the signal transduction mechanism of shear stress in osteoblasts is complex and involves multiple ERK-dependent and independent pathways, and provides circumstantial evidence that there may be a PTX-sensitive pathway that has completing effects with an unknown pathway on the differentiation of normal human osteoblasts.

Alkaline Phosphatase↗

PubMed1996

PCR phenotyping of cytokines, growth factors and their receptors and bone matrix proteins in human osteoblast-like cell lines.

The expression of a total of 58 cytokines, growth factors, and their corresponding receptors and bone matrix proteins was assessed using reverse transcription-linked polymerase chain reaction (RT-PCR) analysis to determine the similarity in the expression profile between clonal osteosarcoma-derived human osteoblast-like cell lines and primary human osteoblast-like cell cultures derived from human trabecular bone explants. The spectrum of cytokines, growth factors, and bone-related proteins expressed by three human osteosarcoma-derived cell lines, TE-85, MG-63, SaOS-2, and primary human osteoblast-like cells was found to be highly comparable and for the first time the expression of EGF, ECGF, FGF beta, oncostatin M, TNF beta, and SCF by human osteoblast-like cells was detected. Also the expression of several receptor types including IL-4R, IL-7R, IFN alpha/beta R, and SCFR was detected that has not been previously described for human osteoblast-like cells. For the factors examined, no qualitative variations in the expression profile were observed in the six primary human osteoblast-like cell cultures used in this study. Of the 58 factors examined, only 13 showed some degree of nonuniformity of expression between all of the three cell lines and primary cell cultures. These differences were seen especially in the expression of cytokine receptor mRNA and to a lesser extent with some cytokines. Differences in receptor expression would suggest that the possible spectrum of response to exogenously added factors, or even autocrine/ paracrine networks would be determined by the repertoire of receptors expressed by each cell type. Whether the differences are related to the status of cell maturation within the osteoblast development lineage or to their abberant regulation of expression cannot be concluded at this stage. However, this PCR-phenotyping approach rapidly provides a resource of information, which can be subsequently used for further in depth studies to facilitate the analysis of the molecular mechanisms, whereby the target gene of interest is modulated in a model cell line. In addition, this study indicates that at least based on the transcript expression profile of the factors analyzed, human osteosarcoma-derived osteoblast-like cells are useful as models for their nontransformed counterparts.

Base Sequence↗

PubMed2004

Inductive effects of dexamethasone on the gene expression of Cbfa1, Osterix and bone matrix proteins during differentiation of cultured primary rat osteoblasts.

Runx2/core binding factor alpha 1 (Cbfa1) and Osterix (Osx) are osteoblast-specific transcription factors essential for the development of a mature osteoblast phenotype and are thought to activate osteoblast marker genes in vivo to produce a bone-specific matrix. Dexamethasone (Dex) is known to be a potent stimulator of osteoblastic differentiation in vitro, however, the exact role is still unclear. To investigate the mechanisms of the stimulation of osteoblastic differentiation by Dex, we evaluated the effects of Dex on proliferation and mineralization as well as on mRNA expression of Cbfa1, Osx and osteoblast marker genes, osteocalcin (OC) and bone sialoprotein (BSP) mRNAs in differentiating foetal rat calvarial cells (FRCC), which were cultured for 35 days in the presence or absence of 10(-7) M Dex. Treatment of FRCC with Dex resulted in the stimulation of cell proliferation and increased the number of cells, which are able to produce bone-like nodules with a mineralized matrix when compared to untreated controls. Northern blot analysis revealed that, in the absence of Dex, Cbfa1 mRNA expressed at day 8, while Osx mRNA expressed at day 15. Subsequently expression of these mRNAs increased up to day 21, followed by constant expression during the culture period. The expression of OC and BSP mRNAs appeared to be synchronous with that of Osx mRNA and was detectable at day 15 with an increase thereafter. The presence of Dex resulted in an induction in Cbfa1 and Osx mRNA expression. The former appeared at day 5 and the latter appeared at day 11. Subsequently expression of Cbfa1 and Osx mRNAs increased up to day 15 with a decrease thereafter. Expression of OC and BSP mRNAs appeared to be coincident with that of Osx mRNA and was detectable at day 11 and reached a maximum at day 15 followed by constant expression. These observations indicate that induction of Cbfal and Osx mRNAs by Dex may be followed by activation of osteoblast marker genes such as OC and BSP mRNAs to produce a bone-specific matrix that subsequently becomes mineralized. Thus, it is likely that Dex may promote osteoblastic differentiation and mineralization of FRCC by inducing the expression of Cbfa1 and Osx genes in vitro.

Alkaline Phosphatase↗

PubMed2005

Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation.

Human and mouse genetic and in vitro evidence has shown that canonical Wnt signaling promotes bone formation, but we found that mice lacking the canonical Wnt antagonist Dickkopf2 (Dkk2) were osteopenic. We reaffirmed the finding that canonical Wnt signaling stimulates osteogenesis, including the differentiation from preosteoblasts to osteoblasts, in cultured osteoblast differentiation models, but we also found that canonical Wnts upregulated the expression of Dkk2 in osteoblasts. Although exogenous overexpression of Dkk before the expression of endogenous canonical Wnt (Wnt7b) suppressed osteogenesis in cultures, its expression after peak Wnt7b expression induced a phenotype resembling terminal osteoblast differentiation leading to mineralization. In addition, osteoblasts from Dkk2-null mice were poorly mineralized upon osteogenic induction in cultures, and Dkk2 deficiency led to attenuation of the expression of osteogenic markers, which could be partially reversed by exogenous expression of Dkk2. Taken together with the finding that Dkk2-null mice have increased numbers of osteoids, these data indicate that Dkk2 has a role in late stages of osteoblast differentiation into mineralized matrices. Because expression of another Wnt antagonist, FRP3, differs from Dkk2 expression in rescuing Dkk2 deficiency and regulating osteoblast differentiation, the effects of Dkk2 on terminal osteoblast differentiation may not be entirely mediated by its Wnt signaling antagonistic activity.

Adaptor Proteins, Signal Transducing↗