The vitamin K-dependent bone protein is accumulated within cultured osteosarcoma cells in the presence of the vitamin K antagonist warfarin.
Osteosarcoma cells grown in normal culture medium secrete bone gamma-carboxyglutamic acid protein (BGP, osteocalcin) which is identical with BGP purified from the bone matrix. Two tests indicate that the secreted medium protein contains the full complement of three gamma-carboxyglutamate residues present on BGP purified from the bone matrix. First, the secreted protein from ROS 17/2 and bone matrix BGP have identical isoelectric points (pI = 4.0). Second, they have identical hydroxyapatite binding behavior. If warfarin is added to the culture medium, the secreted protein has a higher isoelectric point (pI = 4.6) and a lower affinity for hydroxyapatite characteristic of thermally decarboxylated or non-gamma-carboxylated BGP. The observed shift in isoelectric point of secreted BGP after warfarin treatment from pH 4.0 to 4.6 is also reflected in the presence of pI = 4.1 and pI = 4.6 species intracellularly. These isoelectric species correspond to fully carboxylated BGP and noncarboxylated BGP, which are in the process of secretion. Addition of 10 micrograms/ml of warfarin causes a specific 47% reduction in secretion rate of BGP, while at the same time, the intracellular BGP concentration increases 3-fold. These phenomena appear related to the interruption by warfarin of the normal sequence of processing of precursor BGP proteins, as a new, immunoreactive species with a higher isoelectric pH not present in control cells appears to be responsible for the increased intracellular antigen within warfarin-treated cells. Our results show that vitamin K-dependent processing is important for normal secretion of BGP from the cell.
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D007525:Isoelectric Focusing; D015675:Osteocalcin; D012516:Osteosarcoma / Q000378:metabolism*; D051381:Rats; D014812:Vitamin K / Q000037:antagonists & inhibitors*; D014859:Warfarin / Q000494:pharmacology*
D002135:Calcium-Binding Proteins; D015675:Osteocalcin; D014812:Vitamin K; D014859:Warfarin