title: Biosynthesis of bone sialoprotein by a human osteoclast-like cell line (FLG 29.1).
abstract: Biosynthesis of bone sialoprotein (BSP) by a human osteoclastic cell line (FLG 29.1) during its differentiation induced by phorbol 12-myristate 13-acetate (TPA) was studied using metabolic radiolabeling experiments. The FLG 29.1 cells were metabolically radiolabeled with [3H]glucosamine and [35S]sulfate, and the labeled glycoproteins were analyzed by anion exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation experiments. One of the major glycoproteins synthesized by the TPA-treated FLG 29.1 cells was sulfated, had an identical electrophoretic mobility to purified BSP, and could be immunoprecipitated with a specific antibody against human BSP (LF 6). Thus, this glycoprotein was tentatively identified as the BSP. Furthermore, mRNA for BSP was also detected in TPA-treated FLG 29.1 cells by RNA-polymerase chain reaction. Most BSP synthesized by FLG 29.1 cells remained cell-associated, and this is in contrast with those synthesized by osteoblasts, where the protein is rapidly released into the extracellular matrix. Immunocytochemistry using an anti-BSP antibody showed a prominent paranuclear (suggestive of Golgi apparatus) localization of BSP in the TPA-treated FLG 29.1 cells after permeabilization, while untreated cells were not significantly immunostained. Localization of BSP at the plasma membrane was also demonstrated in the TPA-treated FLG 29.1 cells by the fluorescence-activated cell sorting analysis. Since TPA has been demonstrated to induce expression of various osteoclastic characteristics in FLG 29.1 cells, induction of BSP expression by TPA suggests that the protein may play a role during the differentiation process of osteoclasts or in functions of differentiated osteoclasts.
authors.affiliation: Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: L
authors.initials: L
authors.lastname: Masi
authors.forename: M L
authors.initials: ML
authors.lastname: Brandi
authors.forename: P G
authors.initials: PG
authors.lastname: Robey
authors.forename: C
authors.initials: C
authors.lastname: Crescioli
authors.forename: J C
authors.initials: JC
authors.lastname: Calvo
authors.forename: P
authors.initials: P
authors.lastname: Bernabei
authors.forename: J M
authors.initials: JM
authors.lastname: Kerr
authors.forename: M
authors.initials: M
authors.lastname: Yanagishita
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001345:Autoradiography; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D000091344:Filaggrin Proteins; D005434:Flow Cytometry; D005944:Glucosamine / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D058574:Integrin-Binding Sialoprotein; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics; D013431:Sulfates / Q000737:chemistry; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism
chemical_list: C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D006023:Glycoproteins; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D013431:Sulfates; D013917:Thrombin; D005944:Glucosamine; D013755:Tetradecanoylphorbol Acetate