Biosynthesis of bone proteins by fetal porcine calvariae in vitro. Rapid association of sulfated sialoproteins (secreted phosphoprotein-1 and bone sialoprotein) and chondroitin sulfate proteoglycan (CS-PGIII) with bone mineral

Abstract

To study the biosynthesis of bone proteins, fragments of fetal porcine calvariae were cultured in the presence of 50 micrograms/ml ascorbate and 10 mM beta-glycerophosphate and individual cultures labeled for either 4 h or 48 h with [35S]-methionine, Na2[35SO4], Na3[32PO4] or [14C]-glycine plus [14C]-proline. The radiolabeled proteins in tissue extracts were obtained by sequential extraction with 4 M GuHCl (G1-extract), 0.5 M EDTA (E-extract), and again with 4 M GuHCl (G2-extract) and analyzed together with the radiolabeled proteins secreted into the medium. SPP-1 (secreted phosphoprotein 1, osteopontin) was the major non-collagenous protein deposited into the bone matrix, with lesser amounts of BSP (bone sialoprotein), osteocalcin and chondroitin sulfate proteoglycans (CS-PG II and CS-PG III). SPP-1 was also the major phosphorylated protein and was recovered, together with several fragmented forms, almost entirely in the demineralizing extracts. Moreover, approximately one-half of the [35SO4] incorporated into E-extract proteins was present in SPP-1, the remainder being incorporated into PGs with smaller amounts associated with BSP. Over 65% of the [35SO4] in the proteoglycans of the demineralizing extracts was recovered in the small CS-PG III with less than 35% in CS-PG II, the bone homologue of DS-PG II (decorin). In contrast, CS-PG II was the predominant small proteoglycan in culture media and in guanidine extracts. Some sulfated BSP was also observed in guanidine extracts and small amounts appeared to bind to collagen. Radiolabeled SPARC (osteonectin), a prominent protein of fetal porcine bone, was not detected in the mineralized bone tissues but was prominent in the culture medium. These results demonstrate that following secretion, the major proteins expressed by osteoblastic cells are initially incorporated into different tissue compartments, with most of the sulfated sialoproteins and CS-PG III associating rapidly with the hydroxyapatite crystals. The initial distribution of these proteins is of importance in the evaluation of their role in bone formation and mineralization.