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J Sodek

Publications and source records attributed to J Sodek.

At least 37 records · Page 2Linked to original sources
PubMed2000

Osteopontin.

Osteopontin (OPN) is a highly phosphorylated sialoprotein that is a prominent component of the mineralized extracellular matrices of bones and teeth. OPN is characterized by the presence of a polyaspartic acid sequence and sites of Ser/Thr phosphorylation that mediate hydroxyapatite binding, and a highly conserved RGD motif that mediates cell attachment/signaling. Expression of OPN in a variety of tissues indicates a multiplicity of functions that involve one or more of these conserved motifs. While the lack of a clear phenotype in OPN "knockout" mice has not established a definitive role for OPN in any tissue, recent studies have provided some novel and intriguing insights into the versatility of this enigmatic protein in diverse biological events, including developmental processes, wound healing, immunological responses, tumorigenesis, bone resorption, and calcification. The ability of OPN to stimulate cell activity through multiple receptors linked to several interactive signaling pathways can account for much of the functional diversity. In this review, we discuss the structural features of OPN that relate to its function in the formation, remodeling, and maintenance of bones and teeth.

Amino Acid Sequence↗
PubMed1999

Transcription of the bone sialoprotein gene is stimulated by v-Src acting through an inverted CCAAT box.

Bone sialoprotein (BSP) is an early marker of differentiated osteoblasts that has been implicated in the nucleation of hydroxyapatite crystal formation during de novo bone formation. Although essentially specific to mineralizing connective tissues, BSP is also expressed ectopically by carcinomas that exhibit microcalcification and which metastasize to bone with high frequency. However, it is not known how BSP is regulated in transformed cells. Because the v-src oncogene induces expression of a number of genes that are involved in tumor growth and metastasis, including osteopontin, we have studied the effects of v-Src on transcription of the BSP gene. Transfection of mouse src-/- cells with a v-src expression vector increased the transcriptional activity of rat BSP promoter/luciferase chimeric constructs approximately 5-fold. Deletion analysis revealed that the v-Src activity was targeted to an inverted CCAAT box located immediately upstream from an inverted TATA box in the BSP promoter. Although mutation of the CCAAT box diminished the basal transcription activity of the BSP promoter, the Src-induced stimulation was completely abolished. Gel mobility shift analysis identified four nuclear factors that bound to this region of the BSP promoter, two of which required an intact CCAAT sequence. Monoclonal antibodies identified nuclear factor-Y (NF-Y) as the principal nuclear factor that bound to the CCAAT box; the second factor (beta) showing strong binding only in short constructs containing the CCAAT sequence. Transcription analyses with a dominant negative NF-Y expression vector confirmed that NF-Y mediated the action of v-Src. These studies indicate that BSP gene expression in transformed cells can be up-regulated by Src kinase activity through a mechanism mediated by the NF-Y transcription factor, which targets an inverted CCAAT box in the BSP gene promoter.

3T3 Cells↗
PubMed1999

Expression of core binding factor Osf2/Cbfa-1 and bone sialoprotein in tooth development.

The transcription factor Osf2/Cbfa1 is a key regulator of osteogenic differentiation while BSP, a major non-collagenous protein, is a marker of osteoblastic differentiation. To determine the relationship between Osf2/Cbfa1 and the formation of mineralized tissues in tooth development we have studied the temporal expression of Osf2/Cbfa1 and BSP mRNA using in situ hybridization. These studies show that Osf2/Cbfa1 is expressed early in mesenchymal and epithelial tissues destined to form the mineralized tissues of the tooth and periodontal tissues, whereas BSP provides a specific marker for the differentiated cells in each of these tissues. Expression of Osf2/Cbfa1, but not BSP, was observed in the periodontal ligament indicating that expression of Osf2/Cbfa1 is not restricted to mineralizing tissues.

Age Factors↗
PubMed1999

Activation of neutrophil collagenase in periodontitis.

Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate. Analysis of gingival crevicular fluid in periodontitis, gingivitis, and controls revealed sixfold (P < 0.001)-higher levels of active collagenase in periodontitis (n = 12) samples compared to gingivitis (n = 17) samples, which exhibited low levels of activity, while controls (n = 25) showed no activity. After gingival crevicular fluid was collected, no further activation of latent collagenase occurred in vitro. Although both MMP-1 and MMP-8, but not MMP-13, could be detected by immunoblots, blocking antibodies to MMP-1 showed that collagenase activity was largely contributed by MMP-8, which was localized to the matrix of diseased tissues. The MMP-8 in gingival crevicular fluid migrated primarily as a 60-kDa form with smaller amounts of a 78-kDa species, whereas MMP-8 isolated from peripheral neutrophils migrated at 70 and 89 kDa, corresponding to active and latent forms of the enzyme, respectively. Most of the MMP-8 in the 60- and 70-kDa bands selectively bound to tissue inhibitor of metalloproteinase 2 and collagen, indicating that most, but not all, of the enzyme in these bands was in an activated form. However, the amounts of the 78- and 60-kDa forms from gingival crevicular fluid in different samples did not correlate (r2 = 0.028) with the latent and active enzyme measured by collagenase assay. Collectively, these studies have identified distinct forms of latent and active MMP-8 in gingival crevicular fluid that appear to result from a unique activation mechanism that occurs in periodontitis. The complexity of MMP-8 activation is further indicated by the presence of latent, activated, and superactivated forms of MMP-8 in the 60- and 70-kDa bands obtained from gingival crevicular fluid and neutrophil samples, respectively.

Case-Control Studies↗
PubMed1999

Bone sialoprotein.

The search for a protein nucleator of hydroxyapatite crystal formation has been a focus for the isolation and characterization of the major non-collagenous proteins in bone. Of the proteins characterized to date, bone sialoprotein (BSP) has emerged as the only bona fide candidate for nucleation. BSP is a highly glycosylated and sulphated phosphoprotein that is found almost exclusively in mineralized connective tissues. Characteristically, polyglutamic acid and arginine-glycine-aspartate (RGD) motifs with the ability to bind hydroxyapatite and cell-surface integrins, respectively, have been conserved in the protein sequence. Expression of the BSP gene, which is induced in newly formed osteoblasts, is up-regulated by hormones and cytokines that promote bone formation and down-regulated by factors that suppress bone formation. Thus, BSP has the biophysical and chemical properties of a nucleator, and its temporo-spatial expression coincides with de novo mineralization in bone and cementum. Moreover, BSP has been associated with mineral crystal formation in several pathologies, including breast carcinomas. However, the ability of BSP to mediate cell attachment and to signal through the RGD motif points to alternate functions for BSP which need further investigation. In combination, the hydroxyapatite-binding polyglutamic acid sequences and the RGD provide bi-functional entities through which BSP may mediate the targeting and attachment of normal and metastasizing cells to the bone surface.

Amino Acid Sequence↗
PubMed1999

Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice.

Bone sialoprotein (BSP) and osteopontin (OPN) are two major noncollagenous matrix proteins in mineralized connective tissue that have discrete roles in bone matrix formation, mineralization, and remodeling. The osteotropic secosteroid, 1,25-dihydroxyvitamin D3, a potent regulator of bone remodeling required for normal bone development, has been shown to exert differential effects on OPN and BSP expression by bone cells in vitro. To investigate these effects in vivo, we induced vitamin D3 deficiency in a transgenic mouse line (rBSP2.7Luc) that has a 2.7 kb rat BSP promoter linked to a luciferase reporter gene in its genome. Pregnant rBSP2.7Luc mice were fed vitamin D3-deficient food and demineralized water for 6 weeks. Their offspring were weaned at 3 weeks of age and then fed vitamin D-deficient food for an additional week. The control group were fed normal rodent pellets and water during the entire experimental procedure. Bone tissues from 40, 4-week-old offspring in each group were analyzed for BSP, OPN and luciferase expression. Vitamin D3-deficient mice displayed a rachitic phenotype that included reduced size and malformation of bones. Assays of the BSP promoter transgene in calvariae, mandibles, and tibiae of the rachitic mice showed increases in luciferase activity of 3.1-, 1.9-, and 4.6-fold, respectively, when compared with control littermates. Semiquantitative reverse transcriptase polymerase chain reaction assays of BSP mRNA revealed increases of 7-, 74-, and 66-fold, respectively, in the same rachitic bones, while OPN mRNA was reduced 12.5-fold in calvariae and 2-fold in tibiae and mandibles. In situ hybridization using mouse cRNA probes revealed that the increased BSP expression and decreased OPN expression in the vitamin D3-deficient mice was primarily in osteoblastic cells on the surface of calvariae and endosteal spaces of alveolar bone, on newly formed epiphyseal bone, and in cementoblasts and in hypertrophic chondrocytes. These studies are the first to show that BSP and OPN are differentially regulated by vitamin D3 in vivo, reflecting the diverse roles of these protein in bone remodeling. Moreover, the increased expression of the BSP transgene in the rachitic mice demonstrates that vitamin D3 regulation of BSP expression is mediated, in part, by element(s) within the 2.7 kb promoter region.

Animals↗
PubMed1999

Assessment of a novel screening test for neutrophil collagenase activity in the diagnosis of periodontal diseases.

BACKGROUND: Increased levels of active neutrophil collagenase (MMP-8) in the gingival crevicular fluid (GCF) are associated with progressive periodontitis. The measurement of this enzyme in GCF could facilitate diagnosis. However, assays with sufficient sensitivity to detect collagenase in whole-mouth GCF currently use radiolabeled substrates and require several days to complete. To provide more rapid analyses of collagenase activity that are better adapted to clinical studies, we developed and validated a novel assay (soluble biotinylated-collagen assay: SBA) based on chemiluminescent detection of biotinylated collagen digestion products. METHODS: The concordance of the novel SBA assay with a radioactive collagen substrate assay was assessed by parallel analyses of enzyme from 35 neutrophil preparations and from 41 samples of GCF from periodontitis patients, followed by Pearson correlation analysis. To test whether the assay appropriately measured MMP-8 activity, enzyme activity was assessed after incubation with specific collagenase blockers. We examined the diagnostic utility of the SBA in cross-sectional and longitudinal analyses of 125 patients with adult periodontitis, 5 patients with early-onset periodontitis, 1 edentulous patient, and in 32 control patients without periodontitis. RESULTS: The assay detected <56 pg collagen degraded/hour/microl sample, which is comparable to the most sensitive radioactive assay. The total assay time was 22 hours and reproducibility on replicate measurements was high (r = 0.96). In direct comparisons of MMP-8 activity in GCF with enzyme from peripheral blood neutrophils using the SBA and radioactive assays, there was a high correlation (r = 0.97). As expected, EDTA and TIMP-1 and -2, known inhibitors of MMP-8, completely blocked enzyme activity with this assay. Cross-sectional and longitudinal analyses of GCF showed that MMP-8 activity was >18-fold higher in severe periodontitis than in stable periodontitis and decreased to <25% of pretreatment levels following therapy. Based on measurements of collagenase activity in different disease groups, we estimated a value of 80 nano units as a threshold for severe periodontitis. CONCLUSIONS: These results indicate that active MMP-8 is detected in GCF by a novel assay that is specific, simple, rapid, and reproducible and which may facilitate diagnostic discrimination between stable and progressive lesions.

Adult↗
PubMed1998

Flow cytometric analysis of recombinant human osteogenic protein-1 (BMP-7) responsive subpopulations from fetal rat calvaria based on intracellular osteopontin content.

The bone morphogenetic proteins (BMPs) are characterized by their ability to induce both chondrogenic and osteogenic differentiation of mesenchymal cells in vivo and in vitro. Primary cultures of fetal rat calvarial cells contain a broad spectrum of osteogenic cells at various stages of differentiation, but the responsive subpopulations are incompletely characterized. To identify responsive cells in osteogenic cell differentiation, we have treated fetal rat calvarial cells with recombinant osteogenic protein-1 and used flow cytometric analyses of intracellular osteopontin, and of cartilage and bone nodule formation, to evaluate the effects. When administered as a single dose at confluence, osteogenic protein-1 stimulated bone nodule formation in fetal rat calvarial cultures in dose-dependently way. To determine the response of osteogenic subpopulations at two discrete stages of differentiation, fetal rat calvaria cells were cultured for 2 days (proliferative stage) or 12 days (early mineralization stage) and treated with 100 ng/ml recombinant osteogenic protein-1 for 12 h before analysis by flow cytometry. Flow cytometry analyses of cell suspensions revealed that osteogenic protein-1 increased the total protein content of cells, and selectively increased the mean expression of osteopontin and the size and granularity of osteopontin expressing cells, particularly at day 12, consistent with a stimulation of osteogenic differentiation and matrix formation. Pulse administration of 100 ng/ml osteogenic protein-1 to sorted, osteopontin-negative subpopulations enriched for stem cells reduced by more than four-fold the number and size of bone nodules while promoting chondrogenesis and adipogenesis. In contrast, a pulse administration of osteogenic protein-1 to more differentiated, large osteopontin-positive cells increased bone nodule formation two-fold. Continuous administration of 100 ng/ml osteogenic protein-1 to the large osteopontin-positive and small osteopontin-negative cell populations obliterated bone nodule formation and promoted chondrogenesis. We conclude that pulse administration of osteogenic protein-1 promotes osteogenic differentiation of cells committed to the osteogenic lineage, whereas undifferentiated periosteal cells are induced to differentiate along the chondrogenic pathway. In contrast, continuous exposure to osteogenic protein-1 promotes chondrogenesis in populations of committed osteogenic cells and in undifferentiated periosteal cells.

Adipocytes↗
PubMed1998

Enamel epithelium expresses bone sialoprotein (BSP).

Bone sialoprotein (BSP) is a major non-collagenous extracellular matrix protein in bone and other mineralized connective tissues. BSP is synthesized and secreted by bone-, dentin- and cementum-forming cells. In this study we hypothesized that BSP may be also involved in enamel formation through its postulated role in matrix mineralization. In situ hybridization with cRNA probes for rat and hamster BSP, respectively, showed strong mRNA signals in ameloblasts actively synthesizing enamel matrix in developing incisors. However, no hybridization signals were observed at an earlier developmental stage when bell-shaped molar tooth germs were being formed. Immunohistochemical analysis of tooth tissues from transgenic mice harboring a 2.7 kb rat BSP promoter ligated to a luciferase reporter gene revealed strong staining for luciferase in the enamel epithelium of the developing tooth germ. Interestingly, BSP expression was also observed in epithelial cells of an ameloblastoma. The neoplastic epithelial nests and cords demonstrated strong mRNA signals to the human BSP probe while the connective tissue stroma showed only a background level of silver grains. Immunostaining also showed deposition of BSP by the odontogenic cells of the tumor. These results demonstrate that BSP is expressed by the enamel-forming epithelium of developing teeth, suggesting a possible role for BSP in enamel formation and its subsequent mineralization. Expression of the BSP gene in ameloblastomas is consistent with the expression of BSP by the enamel epithelium and also with the expression of BSP by neoplastic tissues, suggesting a possible role in tumorigenesis.

Ameloblastoma↗
PubMed1998

Analysis of intracellular osteopontin as a marker of osteoblastic cell differentiation and mesenchymal cell migration.

Formation and repair of the hard and soft connective tissues of teeth and their supporting structures require stem cells to divide, differentiate and migrate to generate specific tissues in a defined temporo-spatial sequence. We have used antibodies to osteopontin (OPN) and fluorescence-activated cell sorting (FACS) to determine the relationship between OPN expression and cell differentiation in cultures of fetal rat calvarial cells. At different stages of osteogenic differentiation, OPN was expressed by 60-98% of cells. Populations of small OPN-negative cells with low cellular granularity (S cells) were isolated and shown to be enriched in stem cells, characterised by a lack of differentiation markers, high proliferative potential, capacity for self renewal and multipotentiality. Within 24 h of plating, S cells attached, spread and started expressing OPN, CD44, and collagens types I, II and III. Confocal microscopy of OPN in differentiating cells revealed two distinct phenotypes; a perinuclear distribution, characteristic of secreted OPN, and an intracellular perimembranous distribution co-localising with the CD44 receptor, characteristic of migrating cells in which OPN was increased > 10-fold as measured by immunoblotting. These studies show that OPN is expressed early in mesenchymal cell differentiation and is related to cell migration as well as osteogenesis.

Animals↗
PubMed1998

Characteristics of vitamin D3 receptor (VDR) binding to the vitamin D response element (VDRE) in rat bone sialoprotein gene promoter.

Bone sialoprotein (BSP) is a mineralised tissue-specific protein that is highly expressed during the initial formation of bone and cementum. Expression of BSP is suppressed by the osteotropic hormone, 1,25-dihydroxyvitamin D3 (vitamin D3), which regulates bone remodelling. In previous studies, we have identified a vitamin D response element (VDRE) that is integrated with a novel inverted TATA box in the rat BSP promoter which mediates the suppression of BSP transcription (1). Although the nucleotide sequences of VDREs in different genes conform to a direct (hexamer) repeat, spaced by three nucleotides, the precise sequences are unique for each VDRE. To determine whether the nucleotide differences in the VDRE influence VDR binding, we have compared interactions of VDR proteins with various VDREs using gel mobility shift analysis. Both natural and recombinant VDRs bound to rat BSP and both mouse and porcine osteopontin (OPN) VDRE oligonucleotides in a concentration-dependent manner with a strong preference for dimer formation, whereas equal amounts of dimer and monomer were bound to the human osteocalcin VDRE. However, whereas a truncated VDR comprising the DNA binding domain alone bound the mouse osteopontin VDRE, it failed to interact with the porcine OPN and rat BSP VDREs. VDR binding to the BSP was sequence specific, as shown by mutagenesis analysis, and could be abolished by heat and VDR antibody. These studies demonstrate that subtle differences in the nucleotide sequence of VDREs affect VDR binding, which mediates the vitamin D3 response.

Animals↗
PubMed1997

Characterization of stromal progenitor cells enriched by flow cytometry.

The progenitors for cells of bone, cartilage, fat, and muscle are thought to be derived from mesenchymal stem cells but despite extensive study of stromal cell differentiation, neither mesenchymal stem cells or the more committed, tissue-specific progenitors have been well-characterized. In this study we used flow cytometry to isolate from fetal rat periosteum a population of small, slowly cycling cells with low cytoplasmic granularity (S cells) that display stem cell characteristics. On plating, S cells exhibited a 90% higher labeling index with [3H]-thymidine compared to unsorted cells and when grown in culture generated cartilage, adipocyte, and smooth muscle phenotypes, in addition to bone. Only the S-cell population showed extensive self-renewal of cells with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphatase, or osteopontin. However, after attachment, S cells became immunopositive for collagens I, II, III, osteopontin, and also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. These studies show that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacity, and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric methods. These cells may be useful for regeneration of stromal tissues.

Animals↗
PubMed1997

Transforming growth factor-beta 1 regulation of bone sialoprotein gene transcription: identification of a TGF-beta activation element in the rat BSP gene promoter.

Transforming growth factor-beta (TGF-beta) increases steady-state mRNA levels of several extracellular matrix proteins in mineralized connective tissues. Bone sialoprotein (BSP) is a major constituent of the bone matrix, thought to initiate and regulate the formation of mineral crystals. To determine the molecular pathways of TGF-beta 1 regulation of bone proteins, we have analyzed the effects of the TGF-beta 1 on the expression of the BSP in the rat osteosarcoma cell line (ROS 17/2.8). TGF-beta 1 at 1 ng/ml, increased BSP mRNA levels in ROS 17/2.8 cells approximately 8-fold: the stimulation was first evident at 3 hr, reached maximal levels at 12 hr and slowly declined thereafter. Since the stability of the BSP mRNA was not significantly affected by TGF-beta 1, and nuclear "run-on" transcription analyses revealed only a approximately 2-fold increase in the transcription of the BSP gene, most of the increase in BSP mRNA appeared to involve a nuclear post-transcriptional mechanism. Moreover, the effects of TGF-beta 1 were indirect, since the increase in BSP mRNA was abrogated by cycloheximide (28 micrograms/ml). To identify the site of transcriptional regulation by TGF-beta 1, transient transfection analyses were performed using BSP gene promoter constructs linked to a luciferase reporter gene. Constructs that included nt -801 to -426 of the promoter sequence were found to enhance transcriptional activity approximately 1.8-fold in cells treated with TGF-beta 1. Within this sequence, approximately 500 nt upstream of the transcription start site, a putative TGF-beta activation element (TAE) was identified that contained the 5'-portion of the nuclear factor-1 (NF-1) canonical sequence (TTGGC) overlapping a consensus sequence for activator protein-2 (AP-2). The functionality of the TAE was shown by an increased binding of a nuclear protein from TGF-beta 1 stimulated cells in gel mobility shift assays and from the attenuation of TGF-beta 1-induced luciferase activity when cells were co-transfected with a double-stranded TAE oligonucleotide. Competition gel mobility shift analyses revealed that the nuclear protein that binds to the TAE has similar properties to, but is distinct from, NF-1 nuclear protein. These studies have therefore identified a TGF-beta activation element (TAE) in the rat BSP gene promoter that mediates the stimulatory effects of TGF-beta 1 on BSP gene transcription.

Animals↗
PubMed1997

Evidence of ectokinase-mediated phosphorylation of osteopontin and bone sialoprotein by osteoblasts during bone formation in vitro.

Osteopontin (OPN) and bone sialoprotein (BSP) are phosphorylated glycoproteins that, together with osteonectin/secreted protein, acidic, rich in cysteine (SPARC) and osteocalcin, comprise the major non-collagen proteins of bone. Although phosphorylation of OPN and BSP, which is known to influence the biological properties of these proteins, has been shown to occur intracellularly, recent studies have demonstrated ectokinase activity in bone cell populations [Mikuni-Takagaki, Kakai, Satoyoshi, Kawano, Suzuki, Kawase and Saito (1995) J. Bone Miner. Res. 10, 231-241]. To determine whether OPN and BSP are phosphorylated by ectokinase activity we have used [gamma-32P]ATP and [gamma-32P]GTP as cell-impenetrable phosphate donors to analyse for ectokinase activity in osteoblastic UMR106.06 cells and fetal rat calvarial cells (FRCCs). By pulse-labelling confluent cells with radiolabelled nucleotides, the phosphorylation of endogenous and exogenously added OPN and BSP was demonstrated together with the labelling of a number of cell surface proteins. These phosphorylation reactions were inhibited by a cell-impermeable ectokinase inhibitor, K252b, and cell surface phosphorylation was also inhibited by exogenously added OPN and BSP substrates, indicating competition for the ectokinase enzyme. However, phosphorylation of OPN and BSP, both of which can mediate cell attachment through Arg-Gly-Asp (RGD) motifs, was not inhibited by an RGD peptide, suggesting that binding of OPN and BSP to cell surface integrins is not required. In similar experiments, ectokinase-mediated phosphorylation of OPN and BSP was demonstrated during mineralized tissue formation by FRCCs in vitro. These studies demonstrate that OPN and BSP secreted by bone cells are phosphorylated by a casein kinase II-like ectokinase present on the surface of osteoblastic cells.

Animals↗
PubMed1997

Single cell analysis of intracellular osteopontin in osteogenic cultures of fetal rat calvarial cells.

Osteopontin (OPN), a major component of the bone matrix, is expressed at different stages of bone formation. To determine possible relationships between OPN expression and stages of osteogenic cell differentiation, we have performed single cell analyses of intracellular OPN in early (proliferating), subconfluent (differentiating), and mature (mineralizing) cultures of fetal rat calvarial cells (FRCC) using a combination of flow cytometry and confocal microscopy. At each culture stage, a high proportion (60-98%) of cells were immunoreactive for OPN (OPN+ve). Each of these populations also included a small proportion of OPN-ve cells which were characterized by their small size, low granularity, high proliferative capacity, and enhanced osteogenic potential. The OPN+ve cells displayed two distinct patterns of intracellular immunostaining: a perinuclear distribution typical of secreted proteins and a perimembrane distribution in which patches of OPN were concentrated at the cell surface. Perimembranous staining predominated in migrant cells, which contained greater than tenfold higher levels of OPN than nonmigrant cells as separated in a Boyden chamber. When cell proliferation was high (day 2), most cells were OPN + ve. At all culture stages the intensity of OPN staining was increased as cells progressed through the cell cycle. As cells differentiated and started to form matrix (days 4 and 6), the mean cell expression of OPN was also increased (fourfold), independent of changes in total cell protein. However, despite the association of OPN with osteogenic cells, we were surprised to find that a high proportion (60%) of fetal skin fibroblasts were also immunoreactive for OPN. The expression of OPN by these cell populations was confirmed by RT-PCR, and a strong correlation was observed between the quantitative flow cytometry data and Western blot analysis of cell extracts in which the high and low phosphorylated isoforms of OPN were observed. These studies, therefore, have identified several phenotypes in FRCC cultures that are based on OPN expression: small OPN-ve cell populations enriched in osteogenic precursors, differentiating osteogenic cells that synthesize and secrete OPN, and migrating stromal cells characterized by a perimembranous OPN staining pattern.

Animals↗
PubMed1997

Identification of lysyl oxidase and TRAMP as the major proteins in dissociative extracts of the demineralized collagen matrix of porcine dentine.

Carbonated apatite (dahllite) is formed within and between collagen fibrils in the mineralization of connective tissues. However, the mechanism of crystal nucleation at these sites has not been resolved. To identify non-collagenous proteins that may be involved in the nucleation process we have utilized a dissociative extraction procedure to isolate proteins associated non-covalently with the de-mineralized collagen matrix of dentine isolated from tooth roots of adult porcine incisors. Following extraction of dentine fragments with 4M GuHCl (G1-extract) and 0.5M EDTA (E-extract), de-mineralized collagen matrix-associated proteins were isolated with a second series of extractions with 4M GuHCl (G2-extract). Analysis of the G2-extracts on SDS-PAGE revealed two major 32 kDa and 24 kDa protein bands, comprising > 80% of the extracted non-collagenous proteins. The 32 kDa protein was purified by FPLC on hydroxyapatite and Mono Q resins, followed by HPLC reverse-phase chromatography. Small amounts of 26 kDa and 6 kDa proteins, which appear to represent proteolytically processed, disulphide-linked fragments of the 32 kDa protein, co-eluted with the major protein. The 32 kDa protein was identified as lysyl oxidase from amino acid sequence analysis of a 13 kDa CNBr peptide obtained from protein purified by preparative electrophoresis on SDS-PAGE. Fractionation of the 24 kDa protein on FPLC Mono Q resin generated < 5 closely eluting protein peaks. The proteins from these peaks were similar in size, staining properties, amino acid composition and CNBr digestion patterns. Each protein was immunoreactive with antibodies raised against a tyrosine-rich acidic matrix protein (TRAMP), reported previously to co-purify with lysyl oxidase. These studies, therefore, show that lysyl oxidase, which is important in collagen cross-link formation, and proteins with properties of TRAMP, a protein that can modulate collagen fibrillogenesis, are the major proteins in dissociative extracts of de-mineralized porcine dentine.

Alkylation↗
PubMed1996

Identification of a vitamin D3-response element that overlaps a unique inverted TATA box in the rat bone sialoprotein gene.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation de novo. Our studies, using the osteoblastic cell line ROS 17/2.8, have revealed that rat BSP gene expression is suppressed by 1,25-dihydroxyvitamin D3[1,25(OH)2D3], which is a powerful regulator of bone formation and resorption. To determine the molecular basis of the transcriptional suppression of BSP gene transcription by 1,25(OH)2D3, we have conducted transient transfection analyses with chimaeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. 1,25(OH)2D3 suppressed expression in all constructs, including a short construct (pLUC 3; nt -116 to +60) that contained a putative vitamin D3-response element (VDRE; AGGGTTTATAGGTCA; nt -28 to -14) that overlaps a unique inverted TATA (TTTATA) box. Mobility shift assays demonstrated strong binding of recombinant human vitamin D3 receptor protein (hVDR) to the VDRE. Point mutations introduced into each half-site and analysed for 1,25(OH)2D3-mediated suppression of transcription and for hVDR binding either decreased or increased both transcriptional suppression and binding. In comparison with activating VDREs, the rat BSP VDRE bound VDR-VDR homodimers more avidly than VDR-RXR alpha heterodimers (where RXR is retinoid X receptor). These studies have therefore identified a novel 1,25(OH)2D3 suppressor element that overlaps the inverted TATA box in the rat BSP gene and indicate that transcriptional suppression of the rat BSP gene by 1,25(OH)2D3 might involve competition between the VDR and the TATA binding protein (TBP).

Animals↗