Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells

Abstract

Matrix ␥-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent ␥-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-␥-carboxylated MGP and fully ␥-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptormediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.

Effects of the blood coagulation vitamin K as an inhibitor of arterial calcification

“…Indication that such transformations could take place in the aortic wall has been provided by Watson et al (11) who showed that a subpopulation of cultured VSMCs were transformed to calcifying vascular cells (CVCs) capable of forming a mineralized matrix. We have shown (12) that BMP-2 and MGP are both synthesized by VSMCs and Murshed et al (13) have shown that MGP synthesized in the vessel wall and not blood born MGP is the in vivo pool of the protein that is active as the vascular calcification inhibitor. These findings emphasize the importance to understand the MGP-BMP-2 interaction in inhibition of vascular calcification.…”

“…At the end of the incubation period, cells were harvested and washed in DPBS. MK4 treated and ethanol treated (control) cells were lysed in RIPA buffer and solubilized proteins prepared for isoelectric focusing (IEF) by acetone, TCA and ether/ethanol washes before IEF as described by our laboratory (12). The second dimension 2D-SDS-PAGE for IEF and one-dimentional SDS-PAGE were carried out as described (12) using 8-16% gradient CRITERION gels.…”

“…Medium from each culture was collected and concentrated to equal volumes in a Vivaspin concentrator with a 3 kDa cut off. Aliquots representing total protein present in each concentrate were subjected to SDS-PAGE and Western blotting (12).…”

Low serum vitamin K in PXE results in defective carboxylation of mineralization inhibitors similar to the GGCX mutations in the PXE-like syndrome

“…Phosphorylation is thought to be carried out by the Golgi casein kinase. 8,9 It has been suggested earlier that-next to being carboxylated-MGP must be fully phosphorylated at each target serine residue to have its optimal inhibitory activity on soft tissue calcification. 9 Besides the g-carboxylase, the metabolic VK cycle also involves the reductase enzyme VKORC1.…”

“…8,9 It has been suggested earlier that-next to being carboxylated-MGP must be fully phosphorylated at each target serine residue to have its optimal inhibitory activity on soft tissue calcification. 9 Besides the g-carboxylase, the metabolic VK cycle also involves the reductase enzyme VKORC1. Mutations in the GGCX or VKORC1 genes are associated with a hereditary deficiency of the VK-dependent clotting factors as well as a clinically relevant dosing dependency of anti-coagulants.…”