Osteoblast Induction Research Articles

Downregulation of osteoblast markers and induction of the glial fibrillary acidic protein by oncostatin M in osteosarcoma cells require PKCdelta and STAT3.

The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCdelta and STAT3, offering new lines of therapeutic investigations. Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation. Proliferation was analyzed by 3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated. OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCdelta inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction. These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCdelta, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.

Pressure simulation of orthodontic force in osteoblasts: a pilot study.

To elucidate the RUNX2 gene expression induction in human osteoblasts after mechanical loading. Using a stringent pulse-chase protocol human osteoblasts were exposed to centrifugal pressure force for 30 and 90 min. Untreated control cells were processed in parallel. Before, and at defined times after centrifugation, total RNA was isolated. RUNX2 gene expression was measured using real-time quantitative reverse transcriptase polymerase chain reaction. The stress/control ratio was used to illustrate possible stimulatory or diminishing effects of force application. Immediately after 30 min of force application the RUNX2 gene expression was induced by a factor of 1.7 +/- 0.14 as compared with the negative control. This induction decreased rapidly and reached its pre-load levels within 30 min. Longer force applications (up to 90 min) did not change the RUNX2 gene expression. In mature osteoblasts centrifugal pressure force stimulates RUNX2 gene expression within a narrow time frame: loading of mature cells results in a temporary increase of RUNX2 expression and a fast downregulation back to its pre-load expression level. With this pilot study the gene expression behavior after mechanical stimuli could be determined with a simple laboratory setup.

Cot/Tpl2 is essential for RANKL induction by lipid A in osteoblasts.

Lipopolysaccharide (LPS) is a pathogenic factor that increases bone resorption in periodontal diseases. LPS treatment of osteoblasts was shown to induce the receptor activator of NF-kappa B ligand (RANKL), an essential secretory or membrane-bound factor for osteoclast function, in a manner dependent on extracellular signal-regulated kinase (ERK) activation. However, the mechanisms regulating this process remained unknown. Here, we show that RANKL mRNA induction and ERK activation, when treated with synthetic lipid A (an active center of LPS), were markedly reduced in mouse osteoblasts lacking Cot/Tpl2, which was recently recognized as an essential kinase for the induction of TNF-alpha by LPS in macrophages. In contrast, c-Jun N-terminal kinase (JNK), p38 kinase, Raf-1, and NF-kappa B were normally activated in cot/tpl2-/- osteoblasts. These findings indicate that Cot/Tpl2 is essential for LPS-induced ERK activation and RANKL induction in osteoblasts.

Effects of Phenol Red on CFU-f Differentiation and Formation

Osteoblastic induction is commonly studied using the colony-forming unit-fibroblastic (CFU-f) assay, in which bone marrow stromal cells (BMC) are grown in a tissue culture environment permissive for osteoblastic differentiation (DMEM containing dexamethasone, ascorbic acid and beta-glycerophosphate). These cells form colonies, which express alkaline phosphatase, and form a collagenous matrix that becomes calcified. However, these same cells originate in the bone marrow where under normal circumstances they do not proliferate or differentiate despite being subjected to many of the same growth factors and hormones present within the tissue culture environment. We show here that phenol red, present within tissue culture medium as a pH indicator, may itself be a factor that permits osteoblastic recruitment. BMC cultured in the presence of the bone anabolic agents PGE2, PGA2, or bFGF, but in the absence of phenol red, failed to respond to these agents in terms of total or osteoblastic colony number. This effect was dose dependent, with low (2.5 mg/l) and high (15-20 mg/l) doses of phenol red being nonpermissive for the stimulatory effects of PGE2 whereas doses of 5-10 mg/l were permissive. Furthermore, the effects observed in the absence of phenol red could not be abrogated by the addition of 17beta-estradiol indicating that these effects cannot be attributed to estrogenic impurities within the phenol red preparation. This indicates that phenol red itself can affect the differentiation of BMC by a mechanism not previously described.

Requirement of BMP-2-induced Phosphatidylinositol 3-Kinase and Akt Serine/Threonine Kinase in Osteoblast Differentiation and Smad-dependent BMP-2 Gene Transcription

The mechanism by which bone morphogenetic protein-2 (BMP-2) induces osteoblast differentiation is not precisely known. We investigated the involvement of the phosphatidylinositol (PI) 3-kinase/Akt signal transduction pathway in modulation of this process. BMP-2 stimulated PI 3-kinase activity in osteogenic cells. Inhibition of PI 3-kinase activity with the specific inhibitor Ly-294002 prevented BMP-2-induced alkaline phosphatase, an early marker of osteoblast differentiation. Expression of dominant-negative PI 3-kinase also abolished osteoblastic induction of alkaline phosphatase in response to BMP-2, confirming the involvement of this lipid kinase in this process. BMP-2 stimulated Akt serine/threonine kinase activity in a PI 3-kinase-dependent manner in osteoblast precursor cells. Inhibition of Akt activity by a dominant-negative mutant of Akt blocked BMP-2-induced osteoblastic alkaline phosphatase activity. BMP-2 stimulates its own expression during osteoblast differentiation. Expression of dominant-negative PI 3-kinase or dominant-negative Akt inhibited BMP-2-induced BMP-2 transcription. Because all the known biological activities of BMP-2 are mediated by transcription via BMP-specific Smad proteins, we investigated the involvement of PI 3-kinase in Smad-dependent BMP-2 transcription. Smad5 stimulated BMP-2 transcription independent of addition of the ligand. Dominant-negative PI 3-kinase or dominant-negative Akt inhibited Smad5-dependent transcription of BMP-2. Furthermore dominant-negative Akt inhibited translocation of BMP-specific Smads into nucleus. Together these data provide the first evidence that activation of BMP receptor serine/threonine kinase stimulates the PI 3 kinase/Akt pathway and define a role for this signal transduction pathway in BMP-specific Smad function during osteoblast differentiation.

Effects of Parathyroid Hormone on Nasopremaxillary Growth under Different Masticatory Loading Conditions in Growing Mice

It is well accepted that reduced masticatory function resulted from a soft diet with low physical consistency causes morphologic and histologic alterations in the craniofacial skeleton in growing animals. It is also assumed that these alterations are associated with reduced proliferative activity of osteoblasts on the bone surface, indicating a significant role of mechanical stimuli mediated by various local growth factors such as insulin-like growth factor-I (IGF-I) and parathyroid hormone (PTH). A previous study demonstrated that PTH accelerated the effect of IGF-I on bone formation. This study was thus conducted to examine the effect of PTH on the induction of osteoclasts and osteoblasts and the subsequent nasopremaxillary growth under different mechanical loading conditions from mastication. In animals treated with PTH, the nasal bone length increased significantly in the solid-diet group. No significant differences in these dimensions were found between the solid-diet mice injected with physiological saline and the granulated-diet group injected with PTH. It is shown that PTH induces nasal and premaxillary growth, and the effects may be exerted if mechanical loading from mastication is sufficient. It is also demonstrated that the effects of PTH are more potent in the osteogenic activity than in the osteoclastic one, in terms of more significant expression of osteoblasts than osteoclasts.

The influence of culture conditions on extracellular matrix proteins synthesized by osteoblasts derived from rabbit bone marrow.

The influence of culture conditions on the extracellular matrix (ECM) protein expressions of rabbit bone marrow stromal cells has been studied. The focus was on the effects of two kinds of sera, fetal calf serum (FCS) and Ultroser, on cells treated with dexamethasone. The induction of osteoblastic differentiation by dexamethasone addition is confirmed, particularly when cells are cultured in FCS. Bone marrow stromal cells produce alkaline phosphatase positive CFU-F and produce ECM with some mineralized nodules. Analysis by means of two-dimensional gel electrophoresis showed important changes in the composition of ECM proteins after dexamethasone treatment. Overexpression, underexpression, and new synthesized proteins were observed. The most significant modification was linked to the synthesis of four new proteins visible in the acidic area with a low molecular weight of around 17 kDa. These proteins did not correspond to those ECM proteins known to be induced by dexamethasone. Moreover, the effect of dexamethasone on osteoblastic differentiation induction appears very limited when cells are cultured in Ultroser compared to FCS. The protein pattern with Ultroser is different to that obtained with FCS. Cells cultured in Ultroser synthesized no new protein. The different behavior of cells according to the type of medium used is discussed in terms of the osteogenic factors present in the two different sera.

Upregulation by retinoic acid of transforming growth factor-β-stimulated heat shock protein 27 induction in osteoblasts: involvement of mitogen-activated protein kinases

We investigated whether transforming growth factor-β (TGF-β) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. TGF-β increased the level of HSP27 but had no effect on the HSP70 level. TGF-β stimulated the accumulation of HSP27 dose-dependently, and induced an increase in the level of mRNA for HSP27. TGF-β induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TGF-β was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. PD98059 and SB203580 suppressed the TGF-β-stimulated increase in the level of mRNA for HSP27. Retinoic acid, a vitamin A (retinol) metabolite, which alone had little effect on the HSP27 level, markedly enhanced the HSP27 accumulation stimulated by TGF-β. Retinoic acid enhanced the TGF-β-induced increase of mRNA for HSP27. The amplification of TGF-β-stimulated HSP27 accumulation by retinoic acid was reduced by PD98059 or SB203580. Retinoic acid failed to affect the TGF-β-induced phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. These results strongly suggest that p44/p42 MAP kinase and p38 MAP kinase take part in the pathways of the TGF-β-stimulated HSP27 induction in osteoblasts, and that retinoic acid upregulates the TGF-β-stimulated HSP27 induction at a point downstream from p44/p42 MAP kinase and p38 MAP kinase.

Inhibition by adenylyl cyclase-cAMP system of ET-1-induced HSP27 in osteoblasts.

We have previously reported that endothelin-1 (ET-1) stimulates heat shock protein (HSP) 27 induction in osteoblast-like MC3T3-E1 cells and that p38 mitogen-activated protein (MAP) kinase acts at a point downstream from protein kinase C (PKC) in HSP27 induction. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on ET-1-stimulated induction of HSP27 in MC3T3-E1 cells. Dibutyryl-cAMP (DBcAMP) dose dependently inhibited the HSP27 accumulation stimulated by ET-1. Forskolin and cholera toxin significantly suppressed the ET-1-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the ET-1-induced HSP27 accumulation. Forskolin reduced the p38 MAP kinase phosphorylation induced by ET-1 or 12-O-tetradecanoylphorbol-13-acetate (TPA). PGE(1), an extracellular agonist that activates cAMP production, reduced the ET-1-induced HSP27 accumulation. In addition, the phosphorylation of p38 MAP kinase induced by ET-1 or TPA was suppressed by PGE(1). Forskolin, DBcAMP, and PGE(1) suppressed the ET-1-stimulated increase in the mRNA level for HSP27. These results indicate that the adenylyl cyclase-cAMP system has an inhibitory role in ET-1-stimulated HSP27 induction in osteoblasts and that the effect is exerted at the point between PKC and p38 MAP kinase in osteoblasts.

NONSTEROIDAL ANTI-INFLAMMATORY DRUGS ARREST CELL CYCLE IN G0/G1PHASE AND INDUCE CELL DEATH IN OSTEOBLAST-ENRICHED CULTURES

Nonsteroidal anti-inflammatory drugs have been widely prescribed for orthopaedic patients to relieve pain and chronic inflammation. However, it has been demonstrated that NSAIDs suppress bone repair and remodeling in vivo. We have reported that ketorolac inhibits bone repair in vivo and its critical effective timing is at the early stage of endochondral ossification. Our previous results showed that ketorolac and indomethacin inhibit osteoblast proliferation in vitro, suggesting that this effect may be one of the mechanisms contributing to the suppressive effect of NSAIDs on bone remodeling. Cell proliferation and death of osteoblasts should be well regulated through some relative apoptotic and mitotic factors during normal bone remodeling process. Accordingly, we proposed that the induction of osteoblastic cell death of NSAIDs might be one of the mechanisms involving their suppressive effect on bone remodeling in vivo. In this study, we investigated whether NSAIDs arrest osteoblastic cell cycle and/or induce cell death. Whether the mechanism was mediated through prostaglandin (PG) pathway. We tested the effects of ketorolac, indomethacin, diclofenac, piroxicam on cell cycle kinetics, cytotoxicity, and cell death pattern in osteoblast-enriched cultures derived from fetal rat calvaria. Our results showed that ketorolac and indomethacin arrested cell cycle at G0/G1phase. All the 4 NSAIDs had cytotoxic effects and these effects were concentration dependent. The sequence of the cytotoxic effects of these four NSAIDs at 10-4M were indomethacin > diclofenac > ketorolac > piroxicam. Both PGE1and PGE2(10-10-10-8M) also significantly elevated the LDH leakage of osteoblasts, while PGF2αhad no significant effect. These results revealed that the cytotoxic effects of NSAIDs on osteoblasts might not be through inhibiting prostaglandin synthesis. They may be through PG-independent pathways. The results from flow cytometry followed by AnnexinV-FITC and propidium iodide double staining showed that 24 hours treatment of all the 4 NSAIDs (10-6and 10-4M) significantly induced both apoptosis (p<0.01) and necrosis (p<0.01, or p<0.05) in osteoblast cultures. These effects of NSAIDs on cell cycle arrest and cell death induction in osteoblasts may be one of the important mechanisms contributing to their suppressive effect on bone repair and bone remodeling in vivo.

Regulation of osteoclast differentiation by fibroblast growth factor 2: stimulation of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor expression in osteoblasts and inhibition of macrophage colony-stimulating factor function in osteoclast precursors.

This study investigated the mechanism of direct and indirect actions of fibroblast growth factor 2 (FGF-2) on osteoclast differentiation using two mouse cell culture systems. In the coculture system of osteoblasts and bone marrow cells, FGF-2 stimulated osteoclast formation. This effect was decreased markedly by osteoprotegerin (OPG) or NS-398, a selective cyclo-oxygenase 2 (COX-2) inhibitor. FGF-2 (> or = 10(-9) M) stimulated receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) messenger RNA (mRNA) expression from 2 h to 7 days in cultured osteoblasts. NS-398 did not affect the early induction but decreased the later one, indicating that the later effect is mediated by COX-2 induction in osteoblasts. To study the direct action of FGF-2 on osteoclast precursors, we used mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of soluble RANKL/ODF (sRANKL/ODF) and macrophage colony-stimulating factor (M-CSF). Although osteoblasts expressed all FGF receptors (FGFR-1 to -4), only FGFR-1 was detected in C7 cells at various differentiation stages. FGF-2 alone or in combination with sRANKL/ODF did not induce osteoclastogenesis from C7 cells; however, FGF-2 from lower concentrations (> or = 10(-11) M) significantly decreased osteoclast formation induced by M-CSF in the presence of sRANKL/ODF. FGF-2 did not alter mRNA levels of M-CSF receptor (Fms) or RANK in C7 cells. Immunoprecipitation/ immunoblotting analyses revealed that tyrosine phosphorylation of several cellular proteins including Fms in C7 cells induced by M-CSF was inhibited by FGF-2 in the presence of sRANKL/ODF. We conclude that FGF-2 regulates osteoclast differentiation through two different mechanisms: (1) an indirect stimulatory action via osteoblasts to induce RANKL/ODF partly through COX-2 induction and prostaglandin production and (2) a direct inhibitory action on osteoclast precursors by counteracting M-CSF signaling.

Mechanism of prostaglandin D 2-stimulated heat shock protein 27 induction in osteoblasts

We previously showed that prostaglandin D 2 (PGD 2) stimulates activation of protein kinase C (PKC). We investigated whether PGD 2 stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. PGD 2 increased the levels of HSP27 while having little effect on HSP70 levels. PGD 2 stimulated the accumulation of HSP27 dose dependently in the range between 10 nM and 10 μM. PGD 2 induced an increase in the levels of mRNA for HSP27. The PGD 2-stimulated accumulation of HSP27 was reduced by staurosporine or calphostin C, inhibitors of PKC. PGD 2 induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by PGD 2 was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. Calphostin C suppressed the PGD 2-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059 or SB203580 suppressed the PGD 2-increased levels of mRNA for HSP27. These results strongly suggest that PGD 2 stimulates HSP27 induction through p44/p42 MAP kinase activation and p38 MAP kinase activation in osteoblasts and that PKC acts at a point upstream from both the MAP kinases.

Characterization of demineralized bone matrix-induced osteogenesis in rat calvarial bone defects: III. Gene and protein expression.

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo (3)H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3-5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage.

Radiation retards muscle differentiation but does not affect osteoblastic differentiation induced by bone morphogenetic protein-2 in C2C12 myoblasts

Purpose : Effects of radiation on the differentiation of C2C12 myoblasts were studied using cell cultures in which the cells differentiated into either muscle cells or osteoblasts. Materials and methods : A mouse skeletal myoblast cell line, C2C12, was cultured and irradiated at confluence. Muscle differentiation was estimated by counting the number of myosin heavy chain (MHC)-positive multinucleated myotubes. Osteoblast differentiation was analysed by measuring alkaline phosphatase (ALP) activity and osteocalcin production in C2C12 cells treated with recombinant human bone morphogenetic protein-2 (rhBMP-2). Results : Myotube formation in C2C12 cells was inhibited by 2 and 4 Gy irradiation, but recovered to non-irradiated control levels following further culture. In irradiated cultures, the number of MHC-positive mononucleated cells increased significantly. Irradiation did not, however, alter the induction of osteoblastic phenotypes induced by rhBMP-2 in C2C12. Irradiation also did not affect the expression of MyoD mRNA and myogenin mRNA in myogenic differentiation and induction of osteocalcin mRNA in osteoblastic differentiation. Conclusions : During differentiation of C2C12 cells, myotube formation was delayed after irradiation, while induction of osteoblasts with BMP-2 was unaffected.

Endothelin-1 stimulates heat shock protein 27 induction in osteoblasts: involvement of p38 MAP kinase

Endothelin-1 stimulates heat shock protein 27 induction in osteoblasts: involvement of p38 MAP kinase

Endothelin-1 stimulates heat shock protein 27 induction in osteoblasts: involvement of p38 MAP kinase.

We previously reported that endothelin-1 (ET-1) activates p42/p44 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells and consequently induces synthesis of interleukin-6. In the present study, we investigated the effect of ET-1 on the induction of heat shock protein 27 (HSP 27) in MC3T3-E1 cells. ET-1 time and dose dependently stimulated HSP 27 accumulation. ET-1 induced an increase in the levels of mRNA for HSP 27. Both staurosporine and calphostin C, inhibitors of protein kinase C (PKC), suppressed the ET-1-induced HSP 27 accumulation. 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, induced the HSP 27 accumulation and the expression of mRNA for HSP 27. The ET-1-stimulated HSP 27 accumulation was reduced in PKC-downregulated MC3T3-E1 cells. The HSP 27 accumulation by ET-1 was not suppressed by PD-98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase. ET-1 or TPA induced the phosphorylation of p38 MAP kinase. SB-203580, an inhibitor of p38 MAP kinase, reduced the ET-1-stimulated HSP 27 accumulation. Calphostin C and U-73122, a phospholipase C inhibitor, suppressed the ET-1-induced phosphorylation of p38 MAP kinase. U-73122 and propranolol, a phosphatidic acid phosphohydrolase inhibitor, reduced the ET-1-stimulated HSP 27 accumulation. SB-203580 suppressed the ET-1-stimulated increase in the mRNA levels for HSP 27. These results strongly suggest that ET-1 stimulates HSP 27 induction in osteoblasts and that p38 MAP kinase activation is involved in the HSP 27 induction.

Direct bone induction in the subperiosteal space of rat calvaria with demineralized bone allografts.

In order to clarify the understanding of bone induction with crude bone morphogenetic protein (BMP)-containing allografts in subperiosteal conditions, chondrogenesis and osteogenesis were histologically evaluated following the implantation of the demineralized bone (DB) in the subperiosteal space of calvaria of 30 Wistar rats. On the forehead of the rat, DB particles were placed onto the denuded calvarial bone and covered by the skin-periosteum flap without any perforations of the marrow space of the calvaria. Sintered hydroxylapatite particles (HA) were also placed as a control. In the DB group, new bone formation on the surface of calvaria was achieved between 2 and 8 weeks after the operation. However, no chondrogenesis was seen throughout the experimental period. In the HA implantation group, fibrous tissue encapsulation of HA particles was generally seen. These results suggest that DB containing crude BMP might have the capacity for direct osteoblast induction from undifferentiated mesenchymal progenitor cells in vivo in specific situations, that is, in a subperiosteal space of uninjured rat calvaria.

Role of the α2-Integrin in Osteoblast-specific Gene Expression and Activation of the Osf2 Transcription Factor

Extracellular matrix molecules such as type I collagen are required for the adhesion, migration, proliferation, and differentiation of a number of cell types including osteoblasts. Matrix components often affect cell function by interacting with members of the integrin family of cell surface receptors. Previous work showed that collagen matrix synthesis, induced by addition of ascorbic acid to cells, precedes and is essential for the expression of osteoblast markers and induction of the osteocalcin promoter in murine MC3T3-E1 cells. This later response requires OSE2, the promoter element recognized by Osf2 (also called Cbfa1/AML3/PEBP2alphaA), a recently identified osteoblast-specific transcription factor. Osteoblasts express several integrins including alpha2beta1 which is a major receptor for type I collagen. This paper examines the role of the alpha2-integrin subunit in osteocalcin promoter activation and osteoblast differentiation. Disruption of alpha2-integrin-ECM interactions with a blocking antibody or DGEA peptide containing the cell-binding domain of type I collagen blocked activation of the mouse osteocalcin gene 2 promoter by ascorbic acid as well as induction of endogenous osteocalcin mRNA and mineralization. Furthermore, anti-alpha2-integrin blocking antibody or peptide reduced ascorbic acid-dependent binding of Osf2 to OSE2 without affecting levels of transcription factor mRNA. Time course studies revealed that ascorbic acid-dependent binding of Osf2 to OSE2 preceded increases in osteocalcin and bone sialoprotein expression and this increase in Osf2 binding was not accompanied by comparable changes in levels of transcription factor mRNA or protein. Taken together, these studies demonstrate that an alpha2-integrin-collagen interaction is required for activation of Osf2 and induction of osteoblast-specific gene expression. Furthermore, matrix signals may regulate Osf2 through a post-translational pathway or via an accessory factor.

Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM).

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell- matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix-associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM-induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

Interleukin-1α Activates an NF-κB-like Factor in Osteoclast-like Cells

We investigated the NF-kappaB transcription factor in osteoclast-like cells. Osteoclast-like cells were differentiated from mouse bone marrow cells in co-culture with mouse calvaria-derived primary osteoblasts in the presence of 1alpha,25-dihydroxyvitamin D3 and prostaglandin E2 in collagen gel-coated dishes. We enriched osteoclast-like cells from the co-cultures by Pronase treatment. When the enriched osteoclast-like cells were treated with phorbol 12-myristate 13-acetate, interleukin-1 (IL-1), calcitonin, or macrophage colony-stimulating factor, only IL-1 activated an NF-kappaB-like factor, which specifically bound to a kappaB motif DNA sequence, as detected by an electrophoretic mobility shift assay. IL-1 also activated NF-kappaB induction in osteoblasts. However, the NF-kappaB-like factor induced by IL-1-stimulated osteoclast-like cells is of smaller molecular size than the factor in osteoblasts, as shown by an electrophoretic mobility shift assay. The NF-kappaB activity of osteoclast-like cells was recognized completely by antibodies against the p50 subunit, and only partially by antibodies against the p65 subunit of NF-++kappaB. Antibodies against c-Rel, Rel B, and p52 did not recognize the NF-kappaB-like factor. These results suggest that IL-1 activates an NF-kappaB-like factor in osteoclast-like cells, which contains p50 and p65-related proteins.