Matrix GLA protein (MGP) has been identified as a calcification inhibitor in cartilage and vasculature. Part of this effect may be attributed to its influence on osteoinductive activity of bone morphogenetic protein-2 (BMP-2). To detect binding between MGP and BMP-2, we performed immunoprecipitation using MGP and BMP-2 tagged with FLAG and c-Myc. The results showed coprecipitation of BMP-2 with MGP. To quantify the effect of MGP on BMP-2 activity, we assayed for alkaline phosphatase activity and showed a dose-dependent effect. Low levels of MGP relative to BMP-2 (<1-fold excess) resulted in mild enhancement of osteoinduction, whereas intermediate levels (1-15-fold excess) resulted in strong inhibition. High levels of MGP (>15-fold excess), however, resulted in pronounced enhancement of the osteoinductive effect of BMP-2. Cross-linking studies showed that inhibitory levels of MGP abolished BMP-2 receptor binding. Immunoblotting showed a corresponding decrease in activation of Smad1, part of the BMP signaling system. Enhancing levels of MGP resulted in increased Smad1 activation. To determine the cellular localization of BMP-2 in the presence of MGP, binding assays were performed on whole cells and cellsynthesized matrix. Inhibitory levels of MGP yielded increased matrix binding of BMP-2, suggesting that MGP inhibits BMP-2 in part via matrix association. These results suggest that MGP is a BMP-2 regulatory protein. MGP1 is a small matrix protein that was initially isolated from bone and characterized by Price and Williamson (1). MGP deficiency in mice results in premature calcification in bone, calcification of normally noncalcifying cartilage, such as the trachea, and severe vascular calcification leading to premature death (2). Thus, MGP functions as a calcification inhibitor; however, its molecular mechanism is incompletely understood.MGP appears to play a role in cell differentiation. In the artery wall of the MGP knockout mouse, medial smooth muscle cells are replaced by chondrocyte-like cells undergoing endochondral ossification, and in the growth plate of growing bones, hypertrophic chondrocytes are lacking (2). Further support for an effect of MGP on cell differentiation comes from the work of Yagami et al. (3), who show that overexpression of MGP in developing limbs delays chondrocyte maturation and blocks endochondral ossification. In addition, MGP inactivation triggers mineralization in cultured hypertrophic chondrocytes but not in immature chondrocytes. This is consistent with recent data from Newman et al. (4) demonstrating that overexpression of MGP in hypertrophic chondrocytes reduces mineralization. These authors also show that MGP expression is biphasic and stage-specific during chondrocyte differentiation and that MGP has an effect on chondrocyte viability. Increased expression of MGP induces apoptosis in maturing chondrocytes, whereas decreased expression induces apoptosis in proliferative and hypertrophic chondrocytes.Previous studies from our laboratory using the multipotent cell line C3H10T1/...

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Expression of matrix GLA protein (MGP), an alleged calcification inhibitor, is increased in calcified arteries. We used calcifying vascular cells (CVC) that form calcified nodules in vitro to clarify the importance of MGP in vascular cell calcification and differentiation. Unexpectedly, MGP dose-dependently increased calcification in CVC. It also increased expression of the osteogenic marker Cbfal, while decreasing expression of the smooth muscle marker alpha-actin as assessed by immunoblotting. Bone morphogenetic protein-2 (BMP-2), a known osteoinductive factor also increased calcification and osteogenic differentiation in CVC. We hypothesized that the effect of MGP was linked to that of BMP-2 since previous studies show that MGP modulates BMP-2 activity. Therefore, we compared the effect of MGP at different levels of exogenous BMP-2. Results showed that high BMP-2 levels significantly increased the stimulatory effect of low levels of MGP. A relative inhibition of calcification was observed at intermediate levels of MGP and a trend towards renewed stimulation at high levels of MGP. Thus, addition of MGP either promoted or inhibited calcification, depending on the relative amounts of BMP-2 and MGP. This was confirmed in human CVC with different relative expression of BMP-2 and MGP. Calcification in CVC with high relative expression of BMP-2 was inhibited by MGP, while calcification in CVC with low relative expression of BMP-2 was stimulated by MGP. MGP and BMP-2 both accelerated nodule formation, but had opposite effects on nodule size; MGP decreased while BMP-2 increased nodule size. The effect of BMP-2 may partly be explained by a BMP-2 induced decrease in MGP expression. Together, our results suggest that the effect of MGP on calcification and osteogenic differentiation is determined by availability of BMP-2.

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The potential role of vascular endothelium in atherosclerotic calcification is unknown. Endothelial cells (EC) express bone morphogenetic proteins (BMP), and EC-conditioned medium is osteoinductive in marrow stromal cells. To test whether EC are osteoinductive in vascular cells, we used calcifying vascular cells (CVC) that form nodules and mineralize in vitro. We established a coculture model with EC grown opposite CVC on membranes coated with collagen I or collagen IV, both of which are expressed in atherosclerotic lesions. On collagen I, EC did not alter CVC nodule formation, calcification or expression of the osteogenic marker Cbfa1, the chondrogenic marker collagen IX or smooth muscle cell α-actin. However, on collagen IV, EC abolished nodule formation and calcification, and expression of cell markers decreased, suggesting dedifferentiation. Matrix GLA protein (MGP), also expressed in atherosclerotic lesions, was added to CVC in coculture. Unexpectedly, MGP enhanced Cbfa1 expression in CVC on both collagen I and IV. The enhancement was most apparent on collagen IV, where calcification also increased. However, MGP did not restore nodule formation on collagen IV, suggesting that nodule formation and cell differentiation are separate processes. The effect of EC on CVC calcification was suppressed by noggin, an inhibitor of BMP activity, and in part mimicked by replacement of EC by BMP-2. Our results support a role for endothelium in vascular calcification, modulated by collagens and MGP.