Expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro: Inductive effects of dexamethasone on the osteoblastic phenotype

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Abstract
The nature and tissue distribution of non‐collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and β‐glycerophosphate (β‐GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone sialoprotein (BSP) and osteocalcin in DEX‐treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP‐1), type I collagen, SPARC, and alkaline phos‐phatase was stirnulated 6‐, 5‐, 3‐, and 2.5‐ fold, respectively. Metabolic labeling of proteins showed that the sialoproteins (SPP‐1 and BSP) were mostly secreted into the culture medium in the non‐mineralizing (‐β‐GP) cultures, but were the predominant non‐collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+β‐GP). Extraction of the tissue matrix with 4 M GuHCI and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non‐covalently and covalently with the collagenous matrix. SPP‐1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de‐mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP‐1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the sialoproteins; the 44 kDa SPP‐1 incorporating significantly more [32PO4] than the 55 kDa SPP‐1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non‐collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.

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