Key points are not available for this paper at this time.
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 μg/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 γ-carboxylation of MGP is necessary for its binding to the serum mineral complex. 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 μg/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 γ-carboxylation of MGP is necessary for its binding to the serum mineral complex. matrix γ-carboxyglutamic acid protein bone Gla protein (osteocalcin) α2-HS-glycoprotein γ-carboxyglutamic acid 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 (1Munroe P.B. Olgunturk R.O. Fryns J.P. Maldergem L.V. Ziereisen F. Yuksel B. Gardiner R.M. Chung E. Nat. Genet. 1999; 21: 142-144Crossref PubMed Scopus (338) Google Scholar), 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 (2Keutel J. Jorgensen G. Gabriel P. Birth Defects Orig. Artic. Ser. 1972; 8: 60-68Google Scholar, 3Teebi A.S. Lambert D.M. Kaye G.M. Al-Fifi S. Tewfik T.L. Azouz E.M. Am. J. Med. Genet. 1998; 77: 182-187Crossref PubMed Scopus (59) Google Scholar). 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 (4Luo G. Ducy P. McKee M.D. Pinero G.J. Loyer E. Behringer R.R. Karsenty G. Nature. 1997; 386: 78-81Crossref PubMed Scopus (1764) Google Scholar). 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 γ-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 (5Price 1998; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). Gla protein is a protein that was in of bone is known to be by a of and The with the levels of MGP mRNA cartilage, and J. PubMed Google Scholar, J. PubMed Google Scholar), and known to MGP mRNA and J. PubMed Google Scholar, J. PubMed Google Scholar, J. PubMed Google Scholar, PubMed Scopus Google Scholar, Am. J. Google Scholar, PubMed Scopus Google Scholar). MGP mRNA at a levels of the protein have been in bone and J. PubMed Google Scholar, J. PubMed Scopus Google Scholar). This that the protein may at of calcification and that of the protein by to MGP is at the MGP of the vitamin calcium binding γ-carboxyglutamic acid PubMed Scopus Google Scholar, J. PubMed Google and of at in MGP from and PubMed Scopus Google Scholar). at a in the also been for MGP from and J. PubMed Scopus Google Scholar, J. PubMed Google Scholar). The objective of the present study was to the bisphosphonate etidronate to the of MGP in bone studies by the that etidronate and MGP to bone mineral and by studies that that etidronate with bone Gla protein a related vitamin for binding to in and that etidronate injection a a in serum levels The and of B. Scholar). report that etidronate a 25-fold in serum levels of MGP 6 h and that this is by the of a novel complex of fetuin, and MGP in serum after etidronate The and of this complex have to an of how MGP inhibits calcification in vivo. rats from was a from and was from from was from of warfarin at in and in at MGP was from bone as J. PubMed Google Scholar). fetuin as Pinero G.J. PubMed Scopus Google Scholar). other a that is and calcium by was with to with and to of In the initial rats with 8 mg of etidronate/100 g of body weight at the in was with and a of was from the to for at and serum was after in a and and at The was for the with the that the etidronate was increased to g of body of a etidronate serum levels of and matrix Gla rats subcutaneous of etidronate at a of g body weight was from at the and to the levels of and MGP is the of the levels in the serum serum serum MGP, the of warfarin the of the serum protein-mineral rats subcutaneous of mg of g of body weight, and rats of rats with etidronate at a of 8 g and of from at the for biochemical also in which warfarin etidronate and with In we a in serum levels of MGP at the the of the serum mineral complex and of bone 12 rats with mg of etidronate at and of 6 rats from the rats at and 12 and the rats at 24 h. The rats with a of etidronate at 24 and at and h. rats at h. was at and at from the rats at h as as from of and in in and of by The a by the of at The serum mineral complex was characterized by with a molecular mass In a was from rats and from rats 6 h after a of 8 mg of etidronate/100 g of body weight, and was a and of the serum the and for at to the the The and to and The was and the was The of the serum mineral complex was in serum 6 h after of etidronate at doses of 8 g and g of body the of MGP was after for at the a was after for at This was in of and for and The serum mineral complex was characterized by of that in and with and was from rats 6 h after of etidronate at a of 8 and to the of and to the of phosphate and of MGP and of and serum and in previously PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). levels in serum and other and phosphate levels in and other as Scopus Google Scholar). of a to of and the in was for in a and serum was for calcium at the of was in In the shown in of etidronate serum as in was at at The was and the was with of with the that the the of serum from a of serum from the of the after a to with fetuin in and was to a of serum and matrix Gla was from rats 6 h after subcutaneous injection with etidronate at a of of body weight and was of serum was at g for at The was and the was with of and with of The the of of and MGP in of serum and in the and in a new was from rats 6 h after subcutaneous injection with etidronate at a of of body weight and was of serum was at g for at The was and the was with of and with of The the of of and MGP in of serum and in the and The initial study was to the of etidronate serum MGP levels a subcutaneous in a was to an in serum levels of The and of B. Scholar), a vitamin bone protein related in to was from rats at after the subcutaneous of etidronate at a of 8 g of body weight, and serum was for levels of MGP and by In with the study The and of B. Scholar), serum levels of by a of and the serum was at h In serum MGP levels increased by 25-fold and the of MGP was at 6 h at h. MGP is known to inhibit the calcification of arteries and other soft (5Price 1998; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar), we that the serum MGP to etidronate be with the of a calcium phosphate mineral complex in a in which we serum levels of MGP, and phosphate at after the of shown in serum calcium and phosphate levels increased after subcutaneous injection of and by 6 h the levels of calcium and phosphate and levels of MGP increased serum calcium and phosphate with of the maximal levels at h for MGP with h for calcium and the in calcium after etidronate is due to an increase in calcium to an increase in a calcium in a rats at and 6 h after injection of 8 mg of etidronate/100 g of body weight, and the of and calcium in serum calcium at at at and at 6 calcium levels at at and at 6 h. The of etidronate to a in calcium levels indicates that the increase in calcium levels be due to the of a form of calcium in is a increase in serum phosphate and calcium levels after etidronate injection that this form of serum calcium is a complex of calcium and in a subcutaneous of etidronate a in serum and The of the to the etidronate was to the with the 8 g the first with a maximal in serum and MGP at h after treatment with g levels of and MGP at 24 h after the g of that the the serum the increase in serum calcium at 6 h after treatment with g of was increase in the of calcium at 6 which the that the increase in serum calcium is due to the of a calcium complex. etidronate of g of body weight a increase in serum and MGP at 6 h the 8 g In the bisphosphonate serum and MGP levels at and 24 h after of a g of the the calcium complex that is responsible for the in calcium calcium after etidronate injection, serum from and rats was molecular mass an and the and for and be in the increase in serum and MGP levels by the 8 g of etidronate to be due to an increase in the levels of and MGP in the high molecular mass and the levels of calcium and phosphate the for and This indicates that the in and MGP in the serum of rats treated with etidronate is due to the of a high molecular mass serum complex of and of a serum and matrix Gla was from rats 6 h after subcutaneous injection with etidronate at a of 8 g of body weight and from of the serum a molecular mass a and the levels of MGP, and for the and the for The the of of and MGP in of serum and in the and after The the of the for the rats and the in a new was from rats 6 h after subcutaneous injection with etidronate at a of 8 g of body weight and from of the serum a molecular mass a and the levels of MGP, and for the and the for The the of of and MGP in of serum and in the and after The the of the for the rats and the the of the serum mineral of serum from the rats was a at for and a molecular mass the in The levels of and MGP in the and with the for serum after serum was in with calcium levels of phosphate levels of and MGP levels of and with calcium levels of phosphate levels of and MGP levels of This shows that the calcium phosphate mineral in the serum of an at the of calcium and phosphate levels in In a of serum from the was and and to and levels of and MGP levels with the shown in which that the of the serum mineral complex is affected by of the serum the of the serum calcium phosphate complex a that be for the complex the that calcium phosphate mineral have about shown in of serum from rats treated with the of etidronate in a and the was in acid and by a was at that for at 80% of the this was to and to protein was which the of fetuin J. 8: PubMed Scopus Google Scholar). The other in the an molecular mass of and for about of the this was as serum by the of fetuin in the we the weight of fetuin to mineral phosphate in the to be an of which is shown in that of the protein mineral complex increase the levels of serum fetuin and that of the serum mineral complex about of the fetuin in is that the of the protein mineral complex be affected by the for serum the and In the and levels of and MGP in from rats and from the rats of the reported in In the the of and MGP in the and by from and rats of the reported in and the of the in the was from that shown in that the of the mineral complex to be affected by the of In serum 6 h after of 8 mg of etidronate/100 we to the of MGP after of at This indicates that the complex of a calcium phosphate mineral and protein after the 8 g etidronate the complex after the g the calcium phosphate complex in the serum of rats treated with the 8 g of we to this complex by a of calcium in the to the complex. in is a of MGP and phosphate in the of the of serum from the that is in the of serum from an The MGP in this for the of MGP in the serum to the and was MGP in the of the MGP which is about In be in the high molecular mass in the and was in a in the for the This indicates that the of MGP with the serum mineral complex is highly is established that to in and in serum The and of B. Scholar, K and Scholar). the presence of other with the serum mineral the to the high molecular mass phosphate from an and the from a normal with and by shown in is a protein in the for the that is in the for the a with an molecular mass of about this protein from the high molecular mass phosphate in the of was by by to protein of this that its the of fetuin J. 8: PubMed Scopus Google Scholar). of the for from the of etidronate and serum a the presence of a in the for MGP in the from the in the from the other be in the of from the that was also at levels in from the the of fetuin in the high molecular mass phosphate we of of with known of of the of in fetuin a an of of fetuin in The phosphate of is of and the weight of fetuin to phosphate is The MGP of is and the of MGP to fetuin in is The in serum levels of calcium and phosphate after etidronate injection is with a in serum calcium at this rapid in serum calcium and phosphate is due to the of the fetuin mineral complex in of serum after the injection of the 8 g etidronate also to the of This that is a of phosphate in the of the serum that is about the in a serum that the of the serum fetuin at a of fetuin to phosphate of about studies that the doses of etidronate to the of the complex of fetuin, and MGP in serum also the of the normal calcification of bone and cartilage, in the of in bone and of in the growth plate PubMed Scopus Google Scholar). rats a of mg of etidronate this of is and in the of of calcification and calcification in the In the present studies we to the of the of the complex in serum with the of growth plate mineralization. in of the of rats mg of etidronate at and 24 h and at h of calcification and calcification that to reported in the study 6 in PubMed Scopus Google with of calcification from to 12 calcification from 12 to of calcification from 24 to and calcification from to h. shown in the and which calcification was the which serum levels of and MGP the and which calcification to normal the in which serum levels of and MGP also to normal that the of the serum mineral complex after etidronate injection with the of the of growth plate of doses of etidronate 24 h serum levels of phosphate and matrix was at the from the rats in the to and serum was to the levels of phosphate and matrix Gla is the of the levels in and the the serum serum MGP, have previously shown that the vitamin K antagonist warfarin inhibits the γ-carboxylation of MGP and the calcification inhibitory of the protein and causes extensive calcification of arteries and heart valves (5Price 1998; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the γ-carboxylation of MGP is necessary for the of the protein in the serum complex of and fetuin, rats with warfarin h the of etidronate to that MGP from the of etidronate is at after etidronate injection and to serum levels of MGP, and shown in warfarin the increase in serum MGP after etidronate the of the in serum calcium and phosphate and warfarin treatment serum levels of MGP in that warfarin inhibit the synthesis of MGP the of MGP in serum after etidronate the of warfarin the etidronate serum was at 6 h after etidronate treatment from rats that treated with by phosphate levels in the high molecular that with phosphate levels in vitamin rats and MGP levels in the high molecular mass that of the in vitamin rats that the γ-carboxylation of MGP is for its the serum mineral complex and also that the MGP that in this complex from new MGP serum calcium and phosphate levels affected by warfarin the of MGP in the serum mineral complex the of the serum mineral to etidronate the of the mineral complex from of the high molecular mass from the the presence of fetuin at the in which indicates that the of fetuin the serum mineral complex is of the presence of The present study is the first to report the presence of a complex of and protein in serum and the first to this complex and to its This protein mineral complex in serum after the of the bisphosphonate and 6 h of injection with a g of the presence of this complex in serum serum calcium levels by (to phosphate levels by (to and MGP levels by (to μg/ml). calcium and phosphate by etidronate treatment the protein mineral complex be in serum in a by the of serum with to calcium phosphate mineral In in serum by a vitamin treatment that elevates and serum calcium by is of the protein mineral complex in is that the protein mineral complex is of the as a of etidronate treatment and subsequently to This the that in the initial mineral complex may after its in and the of MGP in the complex that the MGP of the complex after the initial of the complex in that the serum mineral complex is as a of the of bone by etidronate as a of the of bone this as The of the serum mineral complex and the of bone an after etidronate and PubMed Scopus Google In the of bone by etidronate and other be after injection of the PubMed Google Scholar). is the of the of bone and the of the serum mineral complex after etidronate 24 h 6 and The bisphosphonate the serum mineral complex the is to inhibit bone in rats of this been shown previously that doses inhibit normal bone P. S. PubMed Scopus Google Scholar). the of the complex be established from the that the complex be to be by a molecular mass which a of and the studies that the complex be to be in the of the which is with a of The complex may in have a of of the complex by the g etidronate of the complex The of the complex may also with the etidronate the protein mineral complex in serum the 8 g is the protein of the serum mineral with an weight of fetuin to mineral of for the complex in serum at the 8 g etidronate and an of fetuin to mineral of at the g of The MGP of the serum mineral complex with after etidronate injection, a of MGP to fetuin of the molecular mass of the serum mineral complex the complex in serum 6 h after treatment with the 8 g of etidronate of fetuin MGP of and of be that the that the protein of the complex fetuin and MGP and that the shown in indicates that molecular mass in be present in the complex. studies be to and to their in the serum complex. calcium and phosphate levels at serum the protein mineral complex is at for 24 a of the protein of the complex may be to inhibit the growth of the mineral The protein may also inhibit the and precipitation of the mineral component, is of the and precipitation of a mineral after 24 h of at the serum complex is cleared from serum 6 h of its and a of the protein may be to the complex for from The most of the serum complex is fetuin, mineral MGP, and that the of the complex the presence of fetuin in is that fetuin the of the mineral and growth of the mineral and the of nuclei. that the most for the protein of fetuin is to the binding of fetuin to mineral and to with other fetuin the mineral to inhibit that the of fetuin, which for of its weight, from the mineral and the The of in fetuin be to the of the mineral complex that in serum and to of complex with studies that fetuin inhibits the of calcium from of calcium and phosphate after for at g J. PubMed Scopus Google Scholar). in for of the inhibitory in the by which fetuin inhibits calcium precipitation was in the inhibitory was shown to be by in the of present with the calcification inhibitor of fetuin in studies and that this of the protein be with its ability to form complexes with mineral that inhibit growth and The present studies that most of the fetuin in the serum mineral complex in the fetuin in serum the injection of etidronate from the fetuin in the normal of fetuin in rats is S. J. 1998; PubMed Scopus Google and the of fetuin in the serum mineral complex is of the complex from the serum fetuin be to the serum of of the of fetuin in the and after of the complex by shows that of the complex at the etidronate about of the fetuin present in of fetuin levels present in serum at after a etidronate also to a increase in the serum fetuin as have been the fetuin in the serum mineral complex in bone the mineral of the serum mineral complex from the of bone and fetuin is an of the bone is that of the fetuin the complex in the a of etidronate fetuin synthesis by This is by the that fetuin is by in bone S. J. PubMed Google Scholar, E. P. 1998; PubMed Scopus Google and by in S. J. PubMed Google Scholar). of studies to for the synthesis of fetuin for the expression of mRNA for fetuin in the of rats Pinero G.J. PubMed Scopus Google E. P. 1998; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M.D. J. 8: PubMed Scopus Google Scholar). that expression of fetuin in the is that fetuin expression by in bone be by etidronate treatment and that bone synthesis of the protein to the of the fetuin mineral complex in serum after etidronate The present studies that MGP in serum after etidronate injection by of its binding to the serum complex. the vitamin K antagonist warfarin the of MGP in the is that the MGP that in the complex from new synthesis and that of MGP in the complex the vitamin γ-carboxylation of the that the increase in the of serum MGP after etidronate is by a of MGP from by an increased of MGP the of the MGP cleared by and the of MGP in normal serum a new synthesis and with of new MGP synthesis for the of MGP by MGP to the serum mineral complex this in This for the of MGP in serum the first 6 h after etidronate injection as as the increase in serum MGP at 6 h. 6 h the of MGP that in serum by be the of of MGP in which is This is in with the of the protein in serum at this The for the increase in serum MGP after etidronate is that the presence of the fetuin mineral complex in serum a increase in the of MGP synthesis by that MGP to we this investigations of the of MGP mRNA in have to a increase at 6 h after etidronate The present studies that MGP to the fetuin mineral complex with and The of the of MGP to the presence of MGP in the of the MGP which indicates that the of serum MGP in with MGP to the complex be The binding of MGP to the fetuin mineral complex also be highly we other with the complex other fetuin and MGP The of this is by the that the related vitamin to in the complex its known high for K and Scholar). The ability of MGP to with to the mineral complex the presence of fetuin that MGP in have a for mineral fetuin and be the inhibitor of This is by the that targeted deletion of the MGP gene in the causes rapid and extensive calcification of the elastic lamellae of arteries at birth (4Luo G. Ducy P. McKee M.D. Pinero G.J. Loyer E. Behringer R.R. Karsenty G. Nature. 1997; 386: 78-81Crossref PubMed Scopus (1764) Google Scholar), mice have of soft calcification for the of in a of mice F. S. J. 1997; PubMed Scopus Google Scholar). that the of soft to in the be due in to the ability of MGP to inhibit calcification and that the of serum MGP to inhibit calcification is to soft calcification normal of this is that a high the ability to inhibit calcification in as is by a high of mice to a of mineral a that be by the inhibitory of serum of this is that warfarin treatment and the fetuin gene deletion act to rapid calcification is with we have the ability of fetuin and MGP to the growth of the mineral of the serum is to that have other growth and bone and the of in B. J. 1999; PubMed Scopus Google Scholar, B. J. PubMed Scopus Google Scholar). MGP also and the of in S. J. PubMed Scopus Google Scholar). of studies be to fetuin and MGP their ability to the of of the serum complex. and for with and for the of fetuin in the
Price et al. (Fri,) studied this question.