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At least 19 recordsLinked to original sources
PubMed2005

Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors.

The transcription factor Sox9 is expressed in all chondroprogenitors and has an essential role in chondrogenesis. Sox9 is also expressed in other tissues, including central nervous system, neural crest, intestine, pancreas, testis, and endocardial cushions, and plays a crucial role in cell proliferation and differentiation in several of these tissues. To determine the cell fate of Sox9-expressing cells during mouse embryogenesis, we generated mice in which a Cre recombinase gene preceded by an internal ribosome entry site was inserted into the 3' untranslated region of the Sox9 gene (Sox9-Cre knock-in). In the developing skeleton, Sox9 was expressed before Runx2, an early osteoblast marker gene. Cell fate mapping by using Sox9-Cre;ROSA26 reporter (R26R) mice revealed that Sox9-expressing limb bud mesenchymal cells gave rise to both chondrocytes and osteoblasts. Furthermore, a mutant in which the Osterix gene was inactivated in Sox9-expressing cells exhibited a lack of endochondral and intramembranous ossification and a lack of mature osteoblasts comparable with Osterix-null mutants. In addition, Sox9-expressing limb bud mesenchymal cells also contributed to tendon and synovium formation. By using Sox9-Cre;R26R mice, we also were able to systematically follow Sox9-expressing cells from embryonic day 8.0 to 17.0. Our results showed that Sox9-expressing cells contributed to the formation of all cell types of the spinal cord, epithelium of the intestine, pancreas, and mesenchyme of the testis. Thus, our results strongly suggest that all osteo-chondroprogenitor cells, as well as progenitors in a variety of tissues, are derived from Sox9-expressing precursors during mouse embryogenesis.

Animals↗
PubMed2006

An in situ hybridization study of Runx2, Osterix, and Sox9 at the onset of condylar cartilage formation in fetal mouse mandible.

Mandibular condylar cartilage is the principal secondary cartilage, differing from primary cartilage in its rapid differentiation from progenitor cells (preosteoblasts/skeletoblasts) to hypertrophic chondrocytes. The expression of three transcription factors related to bone and cartilage formation, namely Runx2, Osterix and Sox9, was investigated at the onset of mouse mandibular condylar cartilage formation by in situ hybridization. Messenger RNAs for these three molecules were expressed in the condylar anlage, consisting of preosteoblasts/skeletoblasts, at embryonic day (E)14. Hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. Runx2 mRNA was expressed in the embryonic zone at the posterior position of the newly formed cartilage, in the bone collar and in the newly formed cartilage, but expression intensity in the newly formed cartilage was slightly weaker. Osterix mRNA was also expressed in the embryonic zone and in the bone collar, but was at markedly lower levels in the newly formed cartilage. Sox9 mRNA was continuously expressed from the embryonic zone to the newly formed cartilage. At this stage, Sox5 mRNA was expressed only in the newly formed cartilage. These results suggest that reduced expression of Osterix in combination with Sox9-Sox5 expression is important for the onset of condylar (secondary) cartilage formation.

Animals↗
PubMed2006

Response to mechanical strain in an immortalized pre-osteoblast cell is dependent on ERK1/2.

Mechanical strain inhibits osteoclastogenesis by regulating osteoblast functions: We have shown that strain inhibits receptor activator of NF-kappaB ligand (RANKL) expression and increases endothelial nitric oxide synthase (eNOS) and nitric oxide levels through ERK1/2 signaling in primary bone stromal cells. The primary stromal culture system, while contributing greatly to understanding of how the microenvironment regulates bone remodeling is limited in use for biochemical assays and studies of other osteoprogenitor cell responses to mechanical strain: Stromal cells proliferate poorly and lose aspects of the strain response after a relatively short time in culture. In this study, we used the established mouse osteoblast cell line, conditionally immortalized murine calvarial (CIMC-4), harvested from mouse calvariae conditionally immortalized by insertion of the gene coding for a temperature-sensitive mutant of SV40 large T antigen (TAg) and support osteoclastogenesis. Mechanical strain (0.5-2%, 10 cycles per min, equibiaxial) caused magnitude-dependent decreases in RANKL expression to less than 50% those of unstrained cultures. Overnight strains of 2% also increased osterix (OSX) and RUNX2 expression by nearly twofold as measured by RT-PCR. Importantly, the ERK1/2 inhibitor, PD98059, completely abrogated the strain effects bringing RANKL, OSX, and RUNX2 gene expression completely back to control levels. These data indicate that the strain effects on CIMC-4 cells require activation of ERK1/2 pathway. Therefore, the CIMC-4 cell line is a useful alternative in vitro model which effectively recapitulates aspects of the primary stromal cells and adds an extended capacity to study osteoblast control of bone remodeling in a mechanically active environment.

Alkaline Phosphatase↗
PubMed2006

Transcriptional regulation of the osterix (Osx, Sp7) promoter by tumor necrosis factor identifies disparate effects of mitogen-activated protein kinase and NF kappa B pathways.

Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-alpha (TNF-alpha), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFkappaB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFkappaB nuclear entry. The forced expression of high levels of NFkappaB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis.

3T3 Cells↗
PubMed2005

JunB as a downstream mediator of PTHrP actions in cementoblasts.

UNLABELLED: The role of AP-1 family members in the action of PTHrP was examined in cementoblasts. PTHrP increased mRNA and protein levels of all Fos members, but only one Jun member (JunB) was increased. Overexpression of JunB in cementoblasts mimicked actions of PTHrP to support osteoclastogenesis and inhibit cementoblast differentiation, suggesting that the actions of PTHrP on mesenchymal cells operate through JunB. INTRODUCTION: Cementoblasts are mesenchymal cells that share phenotypic features with osteoblasts in vitro; however, unlike osteoblasts, cementoblasts rarely support osteoclastogenesis in vivo. The osteoblast-mediated support of osteoclastogenesis involves PTH-induced reduction in osteoprotegerin (OPG) expression. PTH acts on osteoblastic cells through specific signaling pathways and transcription factors such as activator protein 1 (AP-1). The purpose of this study was to determine the impact of PTH-related protein (PTHrP) on AP-1 transcription factors in cementoblasts and the role of JunB in the actions of PTHrP. MATERIALS AND METHODS: Cementoblastic cells were treated with PTHrP and evaluated for mRNA and protein levels of AP-1 family members. Stable transfectants of OCCM cells overexpressing JunB were evaluated for OPG production, ability to support osteoclastogenesis, and measures of proliferation and differentiation. RESULTS: PTHrP treatment in vitro resulted in a time-dependent upregulation of mRNA and proteins for the Fos family members, but only JunB of the Jun family. OPG mRNA and protein levels were reduced by PTHrP in OCCM and were lower in JunB overexpressing cells than controls. In co-culture experiments, TRACP+ cells were increased with RANKL treatment in JunB overexpressing cells compared with controls. Cementoblast differentiation was reduced with overexpression of JunB as measured by a decrease in mineralized nodule formation and gene expression for bone sialoprotein and osterix. Measures of proliferation including cell number and cyclin D1 levels were increased in JunB overexpressing clones. In vivo, cementoblast implants exhibited a cementoblastoid nature with copious mineral-like matrix, whereas JunB-overexpressing implants were densely cellular with little mineralized matrix. CONCLUSIONS: JunB was the only Jun family member increased by PTHrP, and its overexpression showed similar patterns of gene expression and OPG production as PTHrP treatment of controls. These data suggest that JunB may be a key mediator of PTHrP actions in cementoblasts.

Animals↗
PubMed2005

Osterix, a transcription factor for osteoblast differentiation, mediates antitumor activity in murine osteosarcoma.

Osterix is a novel zinc finger-containing transcription factor that is essential for osteoblast differentiation and bone formation. We hypothesized that osterix might have a role in osteosarcoma tumor growth and metastasis. Northern blot analysis showed that the mRNA level of osterix was decreased in two mouse osteosarcoma cell lines compared with its level in normal mouse osteoblasts. Osterix expression was also decreased in three human osteosarcoma cell lines. Transfection of the osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Osterix expression was also associated with decreased osteolysis. Using an in vitro migration assay, osterix suppressed the migration of tumor cells to lung extracts. These results suggest that osterix expression may play a role in osteosarcoma tumor growth and metastasis.

Animals↗
PubMed2006

Regulation of osteoblast differentiation by transcription factors.

Runx2, osterix, and beta-catenin are essential for osteoblast differentiation. Runx2 directs multipotent mesenchymal cells to an osteoblastic lineage, and inhibits them from differentiating into the adipocytic and chondrocytic lineages. After differentiating to preosteoblasts, beta-catenin, osterix, and Runx2 direct them to immature osteoblasts, which produce bone matrix proteins, blocking their potential to differentiate into the chondrocytic lineage. Runx2 inhibits osteoblast maturation and the transition into osteocytes, keeping osteoblasts in an immature stage. Other transcription factors including Msx1, Msx2, Dlx5, Dlx6, Twist, AP1(Fos/Jun), Knox-20, Sp3, and ATF4 are also involved in osteoblast differentiation. To gain an understanding of bone development, it is important to position these transcription factors to the right places in the processes of osteoblast differentiation.

Animals↗
PubMed2005

NFAT and Osterix cooperatively regulate bone formation.

Immunosuppressants are crucial in the prevention of detrimental immune reactions associated with allogenic organ transplantation, but they often cause adverse effects in a number of biological systems, including the skeletal system. Calcineurin inhibitors FK506 and cyclosporin A inhibit nuclear factor of activated T cells (NFAT) activity and induce strong immunosuppression. Among NFAT proteins, NFATc1 is crucial for the differentiation of bone-resorbing osteoclasts. Here we show FK506 administration induces the reduction of bone mass despite a blockade of osteoclast differentiation. This reduction is caused by severe impairment of bone formation, suggesting that NFAT transcription factors also have an important role in the transcriptional program of osteoblasts. In fact, bone formation is inhibited in Nfatc1- and Nfatc2-deficient cells as well as in FK506-treated osteoblasts. Overexpression of NFATc1 stimulates Osterix-dependent activation of the Col1a1 (encoding type I collagen) promoter, but not Runx2-dependent activation of the Bglap1 (encoding osteocalcin) promoter. NFAT and Osterix form a complex that binds to DNA, and this interaction is important for the transcriptional activity of Osterix. Thus, NFAT and Osterix cooperatively control osteoblastic bone formation. These results may provide important insight into the management of post-transplantation osteoporosis as well as a new strategy for promoting bone regeneration in osteopenic disease.

Animals↗
PubMed2006

Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells.

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of alpha1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.

Alkaline Phosphatase↗
PubMed2004

Skeletal unloading alleviates the anabolic action of intermittent PTH(1-34) in mouse tibia in association with inhibition of PTH-induced increase in c-fos mRNA in bone marrow cells.

UNLABELLED: We analyzed the effect of unloading by tail suspension on the anabolic action of intermittent PTH in the tibia of growing mice. Unloading alleviated the PTH-induced increase of bone formation and accelerated bone resorption, consequently reducing bone mass. Reduction of the PTH-induced anabolic actions on bone was associated with unloading, which was apparently related to suppression of c-fos mRNA expression in bone marrow. INTRODUCTION: The effects of intermittent parathyroid hormone (PTH) administration on unloading bone have not been well elucidated at the cellular and molecular levels. We tested the effects of PTH on unloaded tibias of tail-suspended mice. MATERIALS AND METHODS: Eighty male C57BL/6J mice, 8 weeks of age, were divided into four groups with loading or unloading and administration of PTH (40 microg/kg body weight) or vehicle five times per week. Mice were killed at 8 or 15 days, and both tibias were obtained. Bone histomorphometry of the trabecular bone in the proximal tibia, development of osteogenic cells, and mRNA expression of osteogenic molecules in bone marrow cells were assessed. RESULTS AND CONCLUSIONS: At 15 days of unloading, bone volume decreased in PTH-treated mice. The increase in the bone formation rate by PTH was depressed, and the osteoclast surface was thoroughly increased. The increase in alkaline phosphatase-positive colony-forming units-fibroblastic (CFU-f) colonies induced by PTH was maintained and that of TRACP+ multinucleated cells enhanced. The PTH-induced increase in c-fos mRNA was depressed, but the increases in Osterix and RANKL mRNA were maintained. Unloading promoted the PTH-associated osteoclastogenesis and seemed to delay the progression of osteogenic differentiation in association with reduction of the PTH-dependent increase of c-fos mRNA in bone marrow cells.

Alkaline Phosphatase↗
PubMed2005

[Bone formation and inflammation].

Role of BMP signaling is crucial in the osteoblast differentiation and bone formation. BMP induces expression of Runx2, a master gene for osteogenesis, and cooperates with Runx2 in mature osteoblast to express target genes. These functions are suppressed by TGF-beta-induced Smad7 together with Smurf1 by various mechanisms, including degradation of type I receptor, Smadl/5, and Runx2. Bone remodeling is achieved by "coupling" of osteoblasts and osteoclasts. Although excess maturation and activation of osteoclasts is well described during inflammation, effect of inflammation to the osteoblast function is still unclear. Further exploration in this area thus might lead to discovery of novel therapeutic targets of inflammatory bone diseases.

Animals↗
PubMed2006

[Expressions of Cbfal and osterix in osteoblasts on human acellular amniotic membrane].

OBJECTIVE: To study the differentiation of the human osteoblasts during the construction of the tissue engineered periosteum with the human acellular amniotic membrane (HAAM). METHODS: To construct the tissue engineered periosteum (n=60) with HAAM, the human fetal osteoblasts were used. The fetal osteoblasts were cultured for 2, 4, 6, 8, and 10 days, and then their total RNA was extracted, which were reversely transcripted to cDNA. The real-time PCR analysis was used to reveal Cbfal and Osterix, and the cycle threshold (Ct) was also measured. The simply-cultured osteoblasts were used as the control group (n=20). RESULTS: The expression of Cbfal was higher in the experimental group on the 2nd day when compared with that on the 4th, 6th, and 8th day (P < 0.05). The same result existed on the 10th day when compared with that on the 4th and 8th day. The expression of Osterix increased and was highest on the 8th day when compared with the other results (P < 0.05). Both of the 2 gene expressions were decreased in the control group when compared with those in the experimental group, but with no significant difference (P > 0.05). CONCLUSION: Cbfal and Osterix can be normally expressed by the osteoblasts after their integration with HAAM. As a scaffold, HAAM can be used to keep the osteoblast phenotype and differentiation with an osteoconductive ability. Such a cell-scaffold complex may provide a basis for the osteogenesis.

Amnion↗
PubMed2007

HGF inhibits BMP-induced osteoblastogenesis: possible implications for the bone disease of multiple myeloma.

The bone disease in multiple myeloma is caused by an uncoupling of bone formation from bone resorption. A key difference between patients with and patients without osteolytic lesion is that the latter have fewer and less active osteoblasts. Hepatocyte growth factor (HGF) is often produced by myeloma cells and is found at high concentrations in the bone marrow of patients with multiple myeloma. Here we show that HGF inhibited bone morphogenetic protein (BMP)-induced in vitro osteoblastogenesis. Thus, HGF inhibited BMP-induced expression of alkaline phosphatase in human mesenchymal stem cells (hMSCs) and the murine myoid cell line C2C12, as well as mineralization by hMSCs. Furthermore, the expression of the osteoblast-specific transcription factors Runx2 and Osterix was reduced by HGF treatment. HGF promoted proliferation of hMSCs, and the BMP-induced halt in proliferation was overridden by HGF, keeping the cells in a proliferative, undifferentiating state. BMP-induced nuclear translocation of receptor-activated Smads was inhibited by HGF, providing a possible explanation of how HGF inhibits BMP signaling. The in vitro data were supported by the observation of a negative correlation between HGF and a marker of osteoblast activity, bone-specific alkaline phosphatase (rho = -0.45, P = .008), in sera from 34 patients with myeloma. These observations suggest that HGF inhibits bone formation in multiple myeloma.

Animals↗
PubMed2005

Effects of implant surface microtopography on osteoblast gene expression.

AIM: The promotion of osteoblast attachment and differentiation has been evaluated on various implant surfaces. However, the effects of different implant surface properties on gene expression of key osteogenic factors are not fully understood. OBJECTIVE: The objectives of this study were to evaluate how topographical effects on titanium surface alter the expression of bone-related genes and transcription factors. METHODS: Osteoblasts were cultured on titanium disks prepared with a titanium dioxide grit blasting (TiOBlast) or grit blasted and etched with hydrofluoric acid (Osseospeed), grit blasted and etched (SLA-1), or grit blasted, etched and rinsed with N2 protection and stored in isotonic NaCl (SLA-2) commercially pure titanium implant discs. High-density cultures of human mesenchymal pre-osteoblastic cells (HEPM 1486, ATCC) were grown for 72 h and real-time PCR used for quantitative analysis of alkaline phosphatase (ALP), core-binding factor alpha1 (Cbfa1), Osterix, Type I Collagen, Osteocalcin and bone sialoprotein II gene expression. RESULTS: Real-time PCR showed significant (P<0.001) increases in ALP gene expression in osteoblasts grown on SLA-2, relative to all other surfaces. Cbfa1/RUNX-2 gene expression was significantly (P<0.01) increased on Osseospeed and TiOBlast surface as compared with SLA-1 and SLA-2 surfaces. The expression of Osterix had a trend similar to that of Cbfa1. CONCLUSION: In conclusion, implant surface properties may contribute to the regulation of osteoblast differentiation by influencing the level of bone-related genes and transcription factors in human mesenchymal pre-osteoblastic cells.

Cell Adhesion↗
PubMed2005

p53 functions as a negative regulator of osteoblastogenesis, osteoblast-dependent osteoclastogenesis, and bone remodeling.

p53 is a well known tumor suppressor. We show that p53 also regulates osteoblast differentiation, bone formation, and osteoblast-dependent osteoclast differentiation. Indeed, p53(-/-) mice display a high bone mass phenotype, and p53(-/-) osteoblasts show accelerated differentiation, secondary to an increase in expression of the osteoblast differentiation factor osterix, as a result. Reporter assays indicate that p53 represses osterix transcription by the minimal promoter in a DNA-binding-independent manner. In addition, p53(-/-) osteoblasts have an enhanced ability to favor osteoclast differentiation, in association with an increase in expression of macrophage-colony stimulating factor, which is under the control of osterix. Furthermore, inactivating p53 is sufficient to rescue the osteoblast differentiation defects observed in mice lacking c-Abl, a p53-interacting protein. Thus, these results identify p53 as a novel regulator of osteoblast differentiation, osteoblast-dependent osteoclastogenesis, and bone remodeling.

Animals↗
PubMedDate not supplied

[The effect of growth/differentiation factor-5 (GDF-5) on genotype and phenotype in human adult mesenchymal stem cells].

AIM: To evaluate the effects of GDF-5 on genotype and phenotype of human mesenchymal stem cells (hMSC). HYPOTHESIS: GDF-5 leads to up-regulation of the Type I-collagen (Col) gene without altering bone marker genes or alkaline phosphatase activity. METHODS: To test our hypothesis hMSC were treated with rmGDF-5. Using quantitative real-time PCR we analyzed mRNA for Col, Runx2 and Osterix (Osx). Furthermore, we analyzed alkaline phosphatase activity (ALP) as a phenotypical bone marker. ANOVA and post hoc statistical analyses were used to determine differences among treatments (p < 0.05). RESULTS: HMSC showed a biphasic response in both Col and Runx2 after rmGDF-5. Initial up-regulation was followed by a significant down-regulation below controls. Interestingly, the controls presented with changes for Col and Runx2 over time. There was no Osx expression in either treated hMSC or controls. No significant differences could be detected in ALP. CONCLUSION: Increased expression of Col and Runx2 might indicate differentiation towards both osteoblast and fibroblast lineage. However, no Osx expression and no change in ALP support the assumption that rmGDF-5 does not lead to an osteoblast phenotype in hMSC. Our in vitro studies confirm a possible therapeutic benefit of GDF-5 in the treatment of tendon and ligament injuries and tissue engineering approaches. Further research is necessary to prove its clinical value.

Alkaline Phosphatase↗