BIOSYNTHESIS OF BONE SIALOPROTEIN BY A HUMAN OSTEOCLAST-LIKE CELL-LINE (FLG-29.1)

Biosynthesis of bone sialoprotein (BSP) by a human osteoclastic cell l ine (FLG 29.1) during its differentiation induced by phorbol 12-myrist ate 13-acetate (TPA) was studied using metabolic radiolabeling experim ents. The FLG 29.1. cells were metabolically radiolabeled with [H-3]gl ucosamine and [S-35]sulfate, and the labeled glycoproteins were analyz ed by anion exchange chromatography, sodium dodecyl sulfate-polyacryla mide gel electrophoresis (SDS-PAGE) and immunoprecipitation experiment s. One of the major glycoproteins synthesized by the TPA-treated FLG 2 9.1 cells was sulfated, had an identical electrophoretic mobility to p urified BSP, and could be immunoprecipitated with a specific antibody against human BSP (LF 6). Thus, this glycoprotein was tentatively iden tified as the BSP. Furthermore, mRNA for BSP was also detected in TPA- treated FLG 29.1 cells by RNA-polymerase chain reaction. Most BSP synt hesized by FLG 29.1 cells remained cell-associated, and this is in con trast with those synthesized by osteoblasts, where the protein is rapi dly released into the extracellular matrix. Immunocytochemistry using an anti-BSP antibody showed a prominent paranuclear (suggestive of Gol gi apparatus) localization of BSP in the TPA-treated FLG 29.1 cells af ter permeabilization, while untreated cells were not significantly imm unostained. Localization of BSP at the plasma membrane was also demons trated in the TPA-treated FLG 29.1 cells by the fluorescence-activated cell sorting analysis. Since TPA has been demonstrated to induce expr ession of various osteoclastic characteristics in FLG 29.1 cells, indu ction of BSP expression by TPA suggests that the protein may play a ro le during the differentiation process of osteoclasts or in functions o f differentiated osteoclasts.