ECM mineralization (ECMM) is a physiologic process in bone, teeth, and hypertrophic cartilage, whereas in other locations it must be inhibited. To date, few proteins acting as inhibitors of ECMM have been identified through mouse and human genetic studies. They include: Ank, a transmembrane protein controlling extracellular export of pyrophosphate, a small molecule that itself inhibits ECMM; NPPS, an ectoenzyme also generating pyrophosphate extracellularly; matrix gla protein (MGP), a mineral-binding protein of the ECM; and fetuin, a circulating protein that accumulates in bone ECM (Jahnen-Dechent et al., 1997
; Luo et al., 1997
; Okawa et al., 1998
; Nakamura et al., 1999
; Hagmann, 2000
; Ho et al., 2000
; Nurnberg et al., 2001
; Schafer et al., 2003
). Understanding at the molecular level how each of these proteins inhibits ECMM is a prerequisite to better understanding how ectopic ECMM develops, such as that observed in atherosclerosis or in osteoarthritis. Elucidation of the mechanisms behind protein inhibition of ECMM may lead eventually to the identification of novel therapeutic strategies for the treatment of these diseases.
With the long-term goal of understanding how ECMM is prevented in some tissues, whereas favored in others, our laboratory has embarked on a detailed study of the functions and mechanisms of action of proteins containing gla (or γ-carboxylated glutamic acid) residues (Pudota et al., 2000
; Bandyopadhyay et al., 2002
). This posttranslational modification confers to proteins a high affinity for hydroxyapatite crystals, the major mineral crystal present in mineralized ECMs (Romberg et al., 1986
; Roy and Nishimoto, 2002
; Hoang et al., 2003
). We focused our work on two gla residue-containing proteins, namely MGP and bone gla protein (BGP or osteocalcin), the latter being a protein long thought to be involved in bone ECMM (Price et al., 1976
; Celeste et al., 1986
is expressed in vascular smooth muscle cells (VSMCs) and in chondrocytes but not in osteoblasts, whereas Osteocalcin
is expressed in osteoblasts and odontoblasts only (Ducy and Karsenty, 1995
; Luo et al., 1995
). In addition, both MGP and osteocalcin are circulating proteins (Lian et al., 1987
; Ismail et al., 1988
; Price et al., 2003
). Consistent with the pattern of Mgp
expression, MGP-deficient mice develop abnormal ECMM in their arteries and growth plate cartilage establishing that MGP is an inhibitor of ECMM in the vicinity of the cells expressing it (Luo et al., 1997
). In contrast, osteocalcin-deficient mice did not have any detectable defect of bone ECMM indicating that osteocalcin is not required for bone mineralization (Ducy et al., 1996
). This latter experiment did not address however, whether osteocalcin, like MGP, could inhibit ECMM.
The striking differences between MGP and osteocalcin functions already revealed by gene deletion experiments (Ducy et al., 1996
; Luo et al., 1997
), together with the fact that these proteins are circulating systemically raised a series of questions: first, do these proteins act only after local secretion and/or do they act systemically by reaching various tissues through the circulation? This is an important question as mice deficient in fetuin, a circulating protein, develop ectopic ECMM when fed a high calcium and high phosphorus diet (Schafer et al., 2003
). Second, can we identify in vivo the residues in MGP critical for its anti-ECMM function? Lastly, because loss of function experiments failed to uncover a function for osteocalcin during ECMM, could gain of function experiments help to provide definitive information on whether osteocalcin is involved in ECMM?
To address these questions, we used MGP-deficient mice and other transgenics to assess the vascular ECMM by gla-containing proteins, and to assess the influence of these proteins on bone mineralization. Our results are consistent with the hypothesis whereby inhibitors of ECMM act locally and not systemically. They also demonstrate that osteocalcin does not carry out the anti-ECMM function of MGP in vivo.