Discovery of a High Molecular Weight Complex of Calcium, Phosphate, Fetuin, and Matrix γ-Carboxyglutamic Acid Protein in the Serum of Etidronate-treated Rats

Abstract

In the present study we report the discovery of a novel protein-mineral complex in the serum of rats treated with doses of the bone-active bisphosphonate etidronate that inhibit normal bone mineralization. The composition of this high molecular mass protein-mineral complex consists of about 18% mineral, 80% fetuin, and 2% matrix Gla protein (MGP) by weight, and the presence of the complex in serum after an injection of 8 mg etidronate/100 g of body weight elevates calcium by 1.8-fold (to 4.3 mM), phosphate by 1.6-fold (to 5.6 mM), and MGP by 25-fold (to 12 g/ml). The serum mineral complex reaches maximal levels at 6 h after subcutaneous injection of etidronate and is subsequently cleared from serum by 24 h. This highly specific complex of fetuin, MGP, and mineral prevents the growth, aggregation, and precipitation of the mineral component, which indicates that the previously reported calcification inhibitory activities of fetuin and MGP may be related to their ability to form stable complexes with nascent mineral nuclei. Treatment with the vitamin K-antagonist warfarin prevents the increase in serum MGP after etidronate injection, which shows that the increase in serum MGP is due to new synthesis and that the ␥-carboxylation of MGP is necessary for its binding to the serum mineral complex.The initial objective of the present investigations was to understand how matrix Gla protein (MGP) 1 inhibits the abnormal calcification of arteries and other soft tissues. Recent genetic and biochemical studies have established MGP as the first protein known to act as a calcification inhibitor in vivo. In humans, defects in the MGP gene that predict a non-functional MGP protein have been shown to be responsible for Keutel syndrome (1), a rare inherited disease characterized by multiple peripheral pulmonary artery stenoses, by abnormal calcification of cartilages, including costal, nasal, auricle, tracheal, and growth plate cartilage, and by nasal hypoplasia and brachytelephalangia (2, 3). In mice, targeted deletion of the MGP gene causes rapid calcification of the elastic lamellae of the arterial media, which begins at birth and is sufficiently extensive by 3-6 weeks of age that the arteries become rigid tubes that fracture, causing death by exsanguination in most of the affected mice by 6 weeks of age (4). MGP-deficient mice also display abnormal calcification of growth plate and tracheal ring cartilage. Finally, treatment of rats with the vitamin K antagonist warfarin at doses that inhibit the ␥-carboxylation of MGP causes rapid calcification of elastic lamellae of arteries and of aortic heart valves and increased expression of MGP mRNA in the calcifying artery (5, 6).Matrix Gla protein is a 10-kDa secreted protein that was originally discovered in demineralization extracts of bone but is now known to be expressed by a wide variety of tissues and cell types. The rat tissues with the highest levels of MGP mRNA are cartilage, heart, kidney, and lung (7,8), and cells known to express MGP mRNA include osteoblasts, chondrocytes...

Biochemical Characterization of the Serum Fetuin-Mineral Complex

“…This hypothesis is supported by the observation that formation of the fetuin mineral complex prevents the growth, aggregation, and precipitation of the mineral component (1) and by the observation that the fetuin-mineral complex is removed from blood in the 9 -24-h interval following etidronate injection (1).…”

“…In a previous study (1), we described the discovery of a complex of calcium, phosphate, fetuin, 1 and matrix Gla protein in the serum of rats treated with the bone active bisphosphonate etidronate and showed that the appearance of this complex in serum correlates with the inhibition of bone mineralization by etidronate. The fetuin-mineral complex reaches maximal levels 6 -9 h after etidronate injection and causes a 4-fold increase in total serum calcium without causing any increase in ionic calcium levels.…”

Low vitamin K status is associated with osteoarthritis in the hand and knee

“…Second, both mice and humans deficient in MGP express phenotypes that are similar to that seen with warfarin embryopathy, with growth plate calcification abnormalities that in theory might be mimicked by the process of osteophyte formation (11,13). Further, MGP is an important inhibitor of chondrogenesis via binding to bone morphogenetic protein 2 (39,40) and of extracellular matrix calcification via circulating complexes of fetuin and mineral (41). Therefore, inadequate vitamin K may promote both cartilage loss, with attendant JSN, and osteophyte growth.…”

Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells

“…We speculate that the failure of some type I collagen structures to mineralize in vivo is due to the presence of powerful inhibitors of mineralization. Previous studies have shown that serum itself contains high levels of one potent inhibitor of hydroxyapatite formation, fetuin (a2-HS-glycoprotein) [28][29][30], and that the calcification of cartilage in vivo is normally prevented by the calcification inhibitory activity of the vitamin K-dependent matrix Gla protein [31,32]. It seems likely that type I collagen matrices that do not ordinarily calcify, such as tendon, contain other potent inhibitors of mineralization in vivo.…”