Matrix gamma-carboxyglutamic acid (Gla)-containing protein (MGP) was found to be present in articular cartilage by Western-blot analysis of guanidinium chloride extracts of human and bovine cartilage and was further localized by immunohistochemical studies on human and monkey specimens. In newborn articular cartilage MGP was present diffusely throughout the matrix, whereas in growth-plate cartilage it was seen mainly in late hypertrophic and calcifying-zone chondrocytes. In adult articular cartilage MGP was present primarily in chondrocytes and the pericellular matrix. Immunoelectron microscopy studies revealed an association between MGP and vesicular structures with an appearance consistent with matrix vesicles. MGP may be an important regulator of cartilage calcification because of its localization in cartilage and the known affinity of Gla-containing proteins for Ca2+ and hydroxyapatite.
A nonhepatic vitamin K-dependent protein, matrix Gla protein, has recently been identified in cartilage where it may play an important role in the control of mineralization or matrix development. We have investigated the vitamin K cycle in chondrocytes isolated from bovine and rabbit articular cartilage and examined these cells for their ability to synthesize vitamin K-dependent proteins. Chondrocytes were found to have an active vitamin K-dependent carboxylation system. Preincubation of the cells with warfarin resulted in a significant increase in the measured carboxylase activity. Both vitamin K epoxide reductase and DT-diaphorase (EC 1.6.99.2) activity were present indicating that chondrocytes are capable of producing reduced vitamin K1H2, the cofactor for the vitamin K-dependent carboxylase. Specific 14C-labeling of microsomal vitamin K-dependent protein precursors demonstrated synthesis of several vitamin K-dependent proteins by chondrocytes. 35S-labeling of chondrocyte proteins provided evidence that matrix Gla protein is synthesized by these cells.
An osteoblast-like human osteosarcoma cell line (U2-OS) has been shown to possess a vitamin K-dependent carboxylation system which is similar to the system in human HepG2 cells and in liver and lung from the rat. In an 'in vitro' system prepared from these cells, vitamin K1 was shown to overcome warfarin inhibition of gamma-carboxylation carried out by the vitamin K-dependent carboxylase. The data suggest that osteoblasts, the cells involved in synthesis of vitamin K-dependent proteins in bone, can use vitamin K1 as an antidote to warfarin poisoning if enough vitamin K1 can accumulate in the tissue. Five precursors of vitamin K-dependent proteins were identified in osteosarcoma and HepG2 cells respectively. In microsomes (microsomal fractions) from the osteosarcoma cells these precursors revealed apparent molecular masses of 85, 78, 56, 35 and 31 kDa. When osteosarcoma cells were cultured in the presence of warfarin, vitamin K-dependent 14C-labelling of the 78 kDa precursor was enhanced. Selective 14C-labelling of one precursor was also demonstrated in microsomes from HepG2 cells and from rat lung after warfarin treatment. In HepG2 cells this precursor was identified as the precursor of (clotting) Factor X. This unique 14C-labelling pattern of precursors of vitamin K-dependent proteins in microsomes from different cells and tissues reflects a new mechanism underlying the action of warfarin.
Animal models used in vitamin K (VK) studies require VK tissue manipulation while maintaining adequate coagulation. To investigate this in a rodent model used for the study of VK on cartilage, male Sprague Dawley rats (3mo, n=20) were fed either a VK‐deficient diet [21.9±3.9 μg phylloquinone (PK)/kg, D group] or control diet (1035±131μg PK/kg, C group) following destabilization of the medial meniscus knee surgery. Bleeding (in feces) was observed in D group only when housed in wire bottomed cages for 3–5 d. The bleeding stopped once moved to conventional cages. Fecal samples (n=5 group) were obtained at 3.5 wk. In D group, PK concentrations were only 2% of that in C group. No menaquinone‐4 (MK4) was detected in feces in either group whereas MK6–13 concentrations were present in both (p=0.49). At 6 wk, no MK4 was detected in either D or C group in serum or liver; PK was detected in C group only. Kidney PK and MK4 concentrations were 3.4±2.6 and 39.1±3.7nmol/kg, respectively in D group, which were 24% and 50% of their respective values in C group. Our data suggest that conventional cages used with a VK deficient diet reduces PK and MK‐4 concentrations in tissues while avoiding abnormal bleeding. The biological availability of long chain MKs from feces is not known.
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