DIFFERENTIAL-EFFECTS OF WARFARIN ON MESSENGER-RNA LEVELS OF DEVELOPMENTALLY-REGULATED VITAMIN-K-DEPENDENT PROTEINS, OSTEOCALCIN, AND MATRIXCLA PROTEIN IN-VITRO

The role of the vitamin K dependent proteins, osteocalcin which is bon e specific and matrix Gla protein (MGP) found in many tissues, has bee n studied by inhibition of synthesis of their characteristic amino aci d, gamma-carboxyglutamic acid (Gla) with the anticoagulant sodium warf arin. The effect of sodium warfarin on expression of these proteins, a nd other phenotypic markers of bone and cartilage during cellular diff erentiation and development of tissue extracellular matrix, was examin ed in several model systems. Parameters assayed include cell growth (r eflected by histone gene expression) and collagen types I and II, oste opontin, alkaline phosphatase, and mineralization. Studies were carrie d out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 a nd 25/1. In normal diploid cells, warfarin consistently stimulated cel l proliferation (twofold). In osteoblast cultures, MGP mRNA levels wer e generally increased (three to tenfold). Notably, MGP mRNA levels wer e not affected in chondrocyte cultures, either with chronic or acute w arfarin treatments. Osteocalcin mRNA levels and synthesis were decreas ed up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 m u g/ml sodium warfarin) rat osteoblast cultures after 22 days. Early s tages of osteoblast phenotype development from the proliferation perio d to initial tissue formation (nodules) appeared unaffected; while aft er day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warf arin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regu lated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on end oplasmic reticulum associated enzymes. (C) 1994 Wiley-Liss, Inc.