Synthesis and processing of bone sialoproteins during de novo bone formation in vitro.
Bone sialoprotein (BSP) and osteopontin (OPN) are sulphated and phosphorylated sialoglycoproteins that regulate the formation of hydroxyapatite crystals during de novo bone formation. To gain insights into the relationship between the synthesis and posttranslational modification of BSP and OPN and the mineralization of bone, pulse-chase studies were conducted on cultures of newly forming bone nodules produced by fetal rat calvarial cells in vitro. Cultures were pulse labelled with 35SO4, or with either 32PO4 or [gamma-32P]ATP to study intracellular and extracellular phosphorylation, respectively, and chased in isotope-free medium for various times up to 24 h. The presence of radiolabelled BSP and OPN was determined in the cells, in culture medium, and in various tissue compartments obtained by dissociative extraction with 4 M GuHCl (G1), 0.5 M EDTA (E), and again with 4 M GuHCl (G2) and a bacterial collagenase digestion of the demineralized collagenous tissue residue. With each isotope employed, radiolabelled BSP and OPN were detected in the E extract within the 1-h chase period and increased in amount with time. Similarly, 35SO4- and 32PO4-labelled BSP increased in the G2 extract, but OPN was not detected. In the G1 extract the 35SO4-labelled BSP decreased with chase time, whereas the 32PO4-labelled BSP increased. No differences were evident in the profiles of BSP labelled with 32PO4 or [gamma-32P]ATP. In the absence of beta-glycerophosphate, which is required for optimal mineralization of the bone nodules, 35SO4-labelled BSP was increased in the medium and G1 extract and decreased in the E extract and G2 extract after 3 h. In addition to differences in the tissue compartmentalization of BSP and OPN, these studies indicate that 35SO4 is lost from BSP during mineralization and that isoforms of BSP exist with a selective affinity for the organic and mineral phases. Moreover, the additional phosphorylation of BSP and OPN catalyzed by ectokinase activity does not appear to alter the distribution of these sialoproteins.
Zhu
X L
XL
Canadian Institutes of Health Research Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, ON.
Ganss
B
B
Goldberg
H A
HA
Sodek
J
J
D000818:Animals; D001846:Bone Development / Q000502:physiology*; D002113:Calcification, Physiologic / Q000502:physiology; D002478:Cells, Cultured; D058574:Integrin-Binding Sialoprotein; D053495:Osteopontin; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051381:Rats; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D012886:Skull; D013462:Sulfur Radioisotopes / Q000378:metabolism
C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D010761:Phosphorus Radioisotopes; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D013462:Sulfur Radioisotopes; D053495:Osteopontin