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PubMed1996

Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins.

Many proteins found in mineralized tissues have been proposed to function as regulators of the mineralization process, either as nucleators or inhibitors of hydroxyapatite (HA) formation. We have studied the HA-nucleating and HA-inhibiting properties of proteins from bone [osteocalcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondrocalcin (CC)] over a wide range of concentrations. Nucleation of HA was studied with a steady-state agarose gel system at sub-threshold [Ca] x [PO4] product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 microgram/ml (9 nM) and 10 micrograms/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 micrograms/ml. Inhibition of HA formation de novo was studied with calcium phosphate solutions buffered by autotitration. OPN was found to be a potent inhibitor of HA formation [IC50 = 0.32 microgram/ml (0.01 microM)] whereas OC was of lower potency [IC50 = 6.1 micrograms/ml (1.1 microM)]; BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 micrograms/ml. The effect of OPN on HA formation de novo is mainly to inhibit crystal growth, whereas OC delays nucleation. These findings are consistent with the view that BSP and DPP may play roles in the initiation of mineralization in bone and dentine respectively. OPN seems to be the mineralized tissue protein most likely to function in the inhibition of HA formation, possibly by preventing phase separation in tissue fluids of high supersaturation.

Animals↗

PubMed2003

Studies on osteogenic differentiation of rat bone marrow stromal cells cultured in type I collagen gel by RT-PCR analysis.

The purpose of this investigation was to examine by reverse-transcriptase polymerase chain reaction analysis the osteogenic differentiation of twice-passaged Sprague-Dawley rat bone marrow stromal cells in type I collagen gel cultured for 3 weeks. Two culture media were used here, namely Dulbecco's modified Eagle (DME) medium supplemented with vitamin C [Dex (-)] and those with vitamin C, dexamethasone and beta-glycerophosphate [Dex (+)]. Culture with Dex (-) medium in collagen gel for 3 weeks brought about the well-developed cell network and middle-stage osteogenic phenotype expression characterized by mRNA for alkaline phosphatase, osteonectin and osteopontin while those for bone sialo protein and osteocalcin were not detected. On the contrary, culture with Dex (+) medium in collagen gel for 3 weeks lead to necrosis of the cells. These results indicate that culture in collagen gel with Dex (-) DME medium containing vitamin C was useful for three-dimensional culture and middle-stage osteogenic differentiation of twice-passaged bone marrow stromal cells. This study might contribute to tissue engineering therapy to fix bone and periodontal defects in the future.

Animals↗

PubMed1998

Establishment and characterization of osteoblast-like cell lines from retrovirus (RFB MuLV)-induced osteomas in mice.

Cell lines were established by a two-step method from osteomas which had been induced by infection of mice with RFB MuLV, a bone-pathogenic, replication-competent murine retrovirus. The benign tumors, consisting of mature lamellar bone and surrounded by a thin periosteum, were cultured on sponges of denatured collagen type I fibres for up to 4 weeks. At this time osteoma cells had grown into the collagenous matrix. After release and further cultivation in monolayers, the cell lines established from these cultures varied in morphology; they expressed T1, collagen type I and type III, alkaline phosphatase, osteonectin and osteopontin mRNAs at variable levels, but not osteocalcin/BGP. They also showed alkaline phosphatase activity, but lacked responsiveness to parathyroid hormone. All cell lines established from infected mice expressed retroviral and c-myc mRNA and viral protein. In contrast to cells from control mice they showed an extended life span in culture. After growth in a three-dimensional (3-D) collagen sponge culture the cells formed an extracellular matrix containing collagen type I, alkaline phosphatase and osteocalcin/BGP. These data indicate that the two-step method facilitates the establishment of osteoblast-like cell lines from osteomas and calvaria of old mice, and provides means for further analyses of retrovirus-induced skeletal pathogenesis and bone induction.

Alkaline Phosphatase↗

PubMedDate not supplied

Acceleration of wound healing in aged rats by topical application of transforming growth factor-beta(1).

The impaired wound healing associated with aging may reflect inadequate secretion or delivery of cytokines. Transforming growth factor-beta(1) is a mitogenic polypeptide with beneficial effects on wound healing. In the present study we questioned whether topical administration of transforming growth factor-beta(1) could improve the wound healing process in aged rats in vivo. Wound repair (from 1 to 14 days) was analyzed in full-thickness incisional wounds from 2-year-old rats with or without a single topical application of transforming growth factor-beta(1) (1 microg/wound) at the time of wounding. Identical wounds from 3-month-old, untreated rats served as controls. Histologic analysis showed a marked delay in several aspects of wound repair in the aged rats in comparison with that noted in the younger animals. Immunostaining of the wounds for proliferating cell nuclear antigen showed a reduction in the number of cycling fibroblasts in old rats. In addition, the number of capillaries per unit area of the wound as determined by a stain for Griffonin (Bandeiraea) simplicifolia lectin, and the number of inflammatory cells as identified by an antibody specific for macrophages, were also reduced in the wound area in old rats. Treatment with transforming growth factor-beta(1) resulted in marked enhancement of the following parameters: cell proliferation, inflammatory cell and fibroblast influx, wound closure, and angiogenesis. As seen with in situ hybridization, a similar temporal pattern of expression of messenger RNAs corresponding to type I procollagen and Secreted Protein, Acidic and Rich in Cysteine (osteonectin), known to be prevalent in healing wounds, was observed in both young and aged rats. However, the levels of mRNA corresponding to these secreted proteins appeared to be reduced in wound tissue from aged rats. Treatment with transforming growth factor-beta(1) subsequently resulted in an increase in the expression of both type I procollagen and Secreted Protein, Acidic and Rich in Cysteine mRNA in the wound tissue from aged rats. In summary, a single topical application of transforming growth factor-beta(1) to the wounds of aged rats at the time of wounding was associated with a healing response that, in all the parameters of wound repair examined, was similar to that of young rats. Topical transforming growth factor-beta(1) might therefore be beneficial in the treatment of dermal wounds in the aged.

Journal Article↗

PubMed2000

Gene expression profiles in thyroid carcinomas.

The gene expression profiles of human thyroid carcinomas were analysed by serial analysis of gene expression (SAGE) which allows quantitative and simultaneous analysis of a large number of transcripts. More than 29,000 transcripts derived from a normal thyroid tissue and four thyroid tumours were analysed. While extensive similarity was noted between the expression profiles of the normal thyroid tissue and three differentiated thyroid tumours, many transcripts, such as osteonectin, a-tubulin, glyceraldehyde-3-phosphate dehydrogenase, glutathione peroxidase, and thyroglobulin, were expressed at extremely different levels in differentiated and undifferentiated carcinomas. These data provide new information that might be used to identify genes useful for the diagnosis and treatment of thyroid carcinomas.

Adenoma↗

PubMed2002

The molecular biology of distraction osteogenesis.

Distraction osteogenesis has become a mainstay in bone tissue engineering and has significantly improved our armamentarium for reconstructive craniomaxillofacial procedures. However, although the biomechanical, histological, and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in the interfragmental gap of gradually distracted bone segments remain largely unclear. Recently, a rat model of mandibular distraction was described that provides an excellent environment for deciphering the molecular mechanisms that mediate distraction osteogenesis. This article presents the hypotheses and current research that have furthered knowledge of the molecular mechanisms that govern distraction osteogenesis. Recent studies have implicated a growing number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (transforming growth factor-beta1, -beta2, -beta3, bone morphogenetic proteins, insulin-like growth factor-1, fibroblast growth factor-2) and extracellular matrix proteins (osteonectin, osteopontin) during distraction osteogenesis have been best characterized and are discussed in this article. It is believed that understanding the biomolecular mechanisms that mediate membranous distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone healing.

Animals↗

PubMed2001

Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1.

Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.

3T3 Cells↗

PubMed1984

Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography.

Bovine bone morphogenetic protein (bBMP) induces differentiation of mesenchymal-type cells into cartilage and bone. bBMP has an apparent Mr of 18,500 +/- 500 and represents less than 0.001% of the wet weight of bone tissue. A Mr 34,000 protein resembling osteonectin is separated by extraction with Triton X-100. A Mr 24,000 protein and about half of a Mr 22,000 protein are disassociated from bBMP by precipitation in 1.5 M guanidine hydrochloride. Aggregates of bBMP and a Mr 14,000 protein are insoluble in aqueous media; the bBMP becomes soluble when the Mr 14,000 protein is disassociated in 6 M urea and removed from the solution by ultrafiltration. Three separate molecular species with apparent Mrs 18,500, 17,500, and 17,000 are eluted at 0.10, 0.15, and 0.20 M phosphate ion concentrations, respectively, from a hydroxy-apatite column. The Mr 18,500 protein has the amino acid composition of acidic polylpeptide and includes four half-cystine residues; the pI is 4.9-5.1. The Mr 22,000 component is a chromoprotein resembling ferritin. The NH2-terminal amino acid sequence of the Mr 17,500 protein simulates histone H2B. The Mr 17,000 protein may possess calmodulin activity. Aggregates of the Mr 18,500 and other proteins induce formation of large deposits of bone; the Mr 18,500 protein alone is rapidly absorbed and induces formation of small deposits. None of the other proteins induces bone formation.

Amino Acid Sequence↗

PubMed1996

Proteolytic activity of human osteoclast cathepsin K. Expression, purification, activation, and substrate identification.

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F.H., Dodds, R., James I., Connor J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M. (1996) J. Biol., Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not for by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (kobs/[I] = 273,000 m(-1).s(-1)). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Amino Acid Sequence↗

PubMed1999

The Hematopoietic Microenvironment: Stromal Cell Types: Characterization and Function In Situ and In Vitro.

The murine hematopoietic stroma is essential for the homing and the continued replication of hematopoietic stem cells following transplantation and appears to control their lineage proliferation. In our studies it has been found to consist of 3 cell types with differing antigenic displays, growth factor responses and interactions when placed in culture. These characteristics are in keeping with their classifications as myofibroblasts, endothelial like cells and macrophages, respectively. Cell adhesion molecules (CAMs) and early acting cytokines have been found to be associated with these cell types. Only the myofibroblast type has been found to support proliferation of the descendants of long term repopulating stem cells (LTRC) plated over their confluent layer. The endothelial like cell type, and possibly the macrophage, were found to suppress not only the growth of LTRC but, also, that of the myofibroblasts obtained from the same marrow samples. Culture medium obtained from near-confluent growth of the endothelial like cells contained a growth inhibiting molecule of less than 3000 da. capable of inhibiting cells of several types, including the stromal myofibroblasts. The endothelial like cells were shown to unilaterally express VCAM-1 in culture, as well as vWf and endothelial cell specific antigens. Tissue sections of femurs from normal irradiated recipients taken 1 hour post-transplantation revealed Lac-Z marked donor stem cells lodged just external to the endothelial lining of thin walled vessels and in contact with myofibroblasts. Previous studies had shown that some stem cells enter replication at these sites within 48 hours of lodgment. It is suggested that a balance of growth stimulation versus suppression may be maintained between the myofibroblasts and the endothelial like cell types, respectively, in the functioning marrow stroma. Both the myofibroblastoid and the endothelial like stromal cells of old mice displayed a reduced replicative rate in vivo and a reduced replicative capacity in vitro. This suggests that treatments that induce damage to the marrow microenvironment may be especially destructive in aged individuals. In recent experiments it has become clear that the stromal cell population that includes cells identifiable as alkaline phosphatase positive myofibroblasts also contains cells that produce osteocalcin, osteonectin and bone sialoprotein, and deposit von Kossa positive calcium. Whether this results from a single cell lineage or two lineages of similar morphology and alkaline phosphatase expression is under investigation.

Journal Article↗

PubMedDate not supplied

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections.

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 microm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + trade mark dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.

Animals↗

PubMed1996

Identification of stromal cell products that interact with pre-B cells.

Our understanding of lympho-hematopoietic microenvironments is incomplete, and a new cloning strategy was developed to identify molecules that bind to B lineage lymphocyte precursors. A cell sorting procedure was used for initial enrichment of cDNAs from stromal cell mRNA that contained signal sequences and were therefore likely to encode transmembrane or secreted proteins. A second step involved expression of the library as soluble Ig fusion proteins. Finally, pools representing these proteins were screened for the ability to recognize pre-B cells. This approach resulted in the cloning of biglycan, syndecan 4, collagen type I, clusterin, matrix glycoprotein sc1, osteonectin, and one unknown molecule (designated SIM). The full-length cDNA of SIM revealed that it is a type I transmembrane protein, and its intracellular domain has weak homology with myosin heavy chain and related proteins. Staining of established cell lines and freshly isolated hematopoietic cells with the Ig fusion proteins revealed distinct patterns of reactivity and differential dependence on divalent cations. Biglycan-, sc1-, and SIM-Ig fusion proteins selectively increased interleukin 7-dependent proliferation of pre-B cells. Overexpression of the entire SIM protein affected the morphology of 293T cells, while expression of just the extracellular portion was without effect. Thus, a series of stromal cell surface molecules has been identified that interact with blood cell precursors. Three of them promoted the survival and/or proliferation of pre-B cells in culture, and all merit further study in relation to lympho-hematopoiesis.

Amino Acid Sequence↗

PubMed2004

Inferred functions of "novel" genes identified in fibroblasts chondroinduced by demineralized bone.

Little is known about the cellular mechanisms that control postnatal chondrocyte differentiation. As a first step towards identifying those mechanisms, gene expression shifts were characterized in an in vitro model of chondroinduction. In previous studies, several functional classes of genes (cytoskeletal and matrix elements, cell adhesion proteins, peptide growth factors, and signal transduction proteins) were found to be altered in human dermal fibroblasts (hDFs) cultured in porous collagen sponges with chondroinductive demineralized bone powder (DBP) for 3 days. In addition, a number of "novel" sequences were identified. In this study, molecular techniques were combined with computational methods to characterize those sequences. Gene expression of all 10 novel sequences tested was found in hDFs by RT-PCR. The sequences were compared to the human genome, and their cellular functions were inferred from genes that mapped to the same chromosomal coordinates. Only one of the novel sequences contained a protein-coding region (kinesin superfamily protein 26B). The others contained 3' untranslated (osteonectin, alpha-V integrin, RAP2B) or other untranslated regions (PTPN21, GAS6) of mRNAs. The cellular functions of the DBP-regulated genes described in this study fall into similar categories as those previously identified. These results provide new details on the cellular response of hDFs exposed to DBP.

3' Untranslated Regions↗

PubMedDate not supplied

Molecular biological evaluation of bioactive glass microspheres and adjunct bone morphogenetic protein 2 gene transfer in the enhancement of new bone formation.

Bioactive glass is a promising osteoconductive silica-based biomaterial for guidance of new bone growth. On the basis of several in vitro studies, the material appears able to promote osteoblast functions. In our in vivo study, the osteopromotive effect of bioactive glass microspheres seemed to surpass the osteoinductive action of direct adenovirus-mediated human bone morphogenetic protein 2 (BMP-2) gene transfer in a noncritical size bone defect model. The current study was initiated to elucidate the molecular mechanism behind bioactive glass action with or without adjunct BMP-2 gene transfer. A standardized bone defect of the rat tibia was filled with bioactive glass microspheres and injected with adenovirus carrying the human BMP-2 gene (RAdBMP-2). Control defects were left empty or filled with bioactive glass microspheres with injection of adenovirus carrying the lacZ reporter gene or saline. Quantitative polymerase chain reaction confirmed the expression of the transferred human BMP-2 gene at the defect area at 4 days, but not in intact reference tissues. Bone matrix components (collagens I, II, and III, osteocalcin, osteonectin, and osteopontin) and resorption markers (cathepsin K and MMP-9), determined by Northern analysis, showed a completely different pattern of gene expression in defects filled with bioactive glass compared with control defects left to heal without filling. Bioactive glass induced a long-lasting production of bone matrix with concurrent upregulation of osteoclastic markers, a sign of high bone turnover. Combining RAdBMP-2 gene transfer with bioactive glass decelerated the high turnover, but did not influence the balance of synthesis and resorption. This molecular analysis confirmed not only the highly osteopromotive effect of bioactive glass microspheres, but also the accelerated rate of new bone resorption on its surface. At least in noncritical size defects this impact of bioactive glass seems to saturate new bone formation on its surface and thereby overshadow the effect of BMP-2 gene transfer.

Animals↗

PubMed2007

Comparison of hydrogels in the in vivo formation of tissue-engineered bone using mesenchymal stem cells and beta-tricalcium phosphate.

Availability of grafts and morbidity at the donor site limit autologous transplantation in patients requiring bone reconstruction. A tissue-engineering approach can overcome these limitations by producing bone-like tissue of custom shape and size from isolated cells. Several hydrogels facilitate osteogenesis on porous scaffolds; however, the relative suitability of various hydrogels has not been rigorously assessed. Fibrin glue, alginate, and collagen I hydrogels were mixed with swine bone marrow-derived differentiated mesenchymal stem cells (MSCs), applied to 3-dimensionally printed porous beta-tricalcium phosphate (beta-TCP) scaffolds and implanted subcutaneously in nude mice. Although noninvasive assessment of osteogenesis in 3 dimensions is desirable for monitoring new bone formation in vivo, correlations with traditional histological and mechanical testing need to be established. High-resolution volumetric computed tomography (VCT) scanning, histological examination, biomechanical compression testing, and osteonectin (ON) expression were performed on excised scaffolds after 1, 2, 4, and 6 weeks of subcutaneous implantation in mice. Statistical correlation analyses were performed between radiological density, stiffness, and ON expression. Use of collagen I as a hydrogel carrier produced superior bone formation at 6 weeks, as demonstrated using VCT scanning with densities similar to native bone and the highest compression values. Continued contribution of the seeded MSCs was demonstrated using swine-specific messenger ribonucleic acid probes. Radiological density values correlated closely with the results of histological and biomechanical testing and ON expression. High-resolution VCT is a promising method for monitoring osteogenesis.

Animals↗

PubMed1997

Expression patterns of extracellular matrix components in native and cultured normal human thyroid tissue and in human toxic adenoma tissue.

The extracellular matrix (ECM) and basement membranes (BM, a specialized form of ECM) greatly influence proliferation, differentiation, and function of cells and the structure of tissues. While a considerable amount of information is available on thyroid cellular proliferation, differentiation and function, much less is known about thyroid ECM and BM. In this study the presence of the ECM/BM components fibronectin, collagen IV, alpha1, beta1, gamma1 laminin, several laminin variants, osteonectin, and perlecan was demonstrated in cryosections of nonadenomatous and toxic adenoma human thyroid tissue. Also, positive immunohistochemical staining for collagen IV, laminin, perlecan, and fibronectin was obtained in sections of human thyroid tissue cultured in a three-dimensional (alginate) culture system. The present study provides methods and data that will facilitate the investigation of the interaction between cells and ECM in thyroid tissue.

Adenoma↗

PubMed1998

Gap junctional communication modulates gene expression in osteoblastic cells.

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106-01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter-luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2. 8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106-01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

Animals↗

PubMed1992

SARG: a new human osteosarcoma cell line. Expression of bone markers and of major histocompatibility antigens.

A new cell line (SARG) was established from a human radiation-induced osteosarcoma (OSA). It showed an epithelial-like morphology with polymorphous and sometimes bizarre nuclei. SARG had an osteoblastic differentiation pattern: almost 100% of the cells were positive for alkaline phosphatase, type I and III collagens and osteonectin. The expression of class I HLA antigens was detectable even after 40 in vitro passages. The expression of MHC antigens was greatly increased after in vitro treatment with interferon gamma (IFN-gamma), whereas interferon alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) increased the expression of class I antigens, but not of class II antigens. SARG was tumorigenic after subcutaneous injection in nude mice. Experimental metastases were never detected.

Adult↗