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Report |
Address correspondence to Gerard Karsenty, Baylor College of Medicine, One Baylor Plaza, Rm. S921, Houston, TX 77030. Tel.: (713) 798-5489. Fax: (713) 798-1530. email: karsenty{at}bcm.tmc.edu
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Abstract |
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Key Words: MGP; osteocalcin; ECM; mineralization; local regulation
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Introduction |
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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 -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, 1983; Celeste et al., 1986). Mgp 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.
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Results and discussion |
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To extend these observations, we asked whether bone mineralization could be affected by either local or systemic expression of Mgp. To that end, we studied bone mineralization at 4 wk old in WT, ApoE-Mgp, and in 1(I)Col-Mgp mice that express Mgp only in osteoblasts (Fig. 1 B). Skeletal preparations of 10-d-old skulls showed no mineralization defect in ApoE-Mgp mice, whereas a severe decrease in intramembranous bone mineralization was observed in
1(I) Col-Mgp mice (Fig. 3 E). Likewise, histological analysis of vertebrae of ApoE-Mgp mice failed to show any increase in unmineralized bone, whereas histological analysis of vertebrae of
1(I)Col-Mgp mice showed a marked, i.e., 812-fold, increase of unmineralized bone tissue (Fig. 3 F). When together, the analyses of Mgp/; SM22
-Mgp, ApoE-Mgp, and
1(I)Col-Mgp mice establish that, in animals fed a normal diet, MGP inhibits ECMM locally and not systemically.
MGP and osteocalcin do not share an antimineralization function despite structural similarities
The osteoidosis, i.e., increase in unmineralized bone ECM, observed in the 1(I)Col-Mgp mice provided us with an in vivo model to test the function of the gla residues within MGP and other proteins. In this context, we generated transgenic mice that produced two distinct mutated forms of MGP in osteoblasts. In MGP mutant1 (MGPm1), three of the four glutamic acid residues present in the mouse protein were replaced by aspartic acid residues. In MGP mutant2 (MGPm2) all four glutamic acid residues undergoing
-carboxylation were replaced by aspartic acid residues. Histological analysis performed in 4-wk-old mice failed to detect any evidence of osteoidosis in the
1(I)Col-Mgpm2 mice, whereas osteoidosis was considerably milder in
1(I)Col-Mgpm1 mice than in
1(I)Col-Mgp (Fig. 4 C). These results establish that the gla residues are required for MGP antimineralization function.
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SM22-Osteocalcin mice did not show any metabolic or histological abnormalities (Table I and not depicted) despite a six- to eightfold increase in serum osteocalcin indicating efficient transcription/translation of the transgene (Fig. 5 A). SM22
-Osteocalcin mice were then intercrossed with Mgp+/ mice to obtain Mgp/; SM22
-Osteocalcin mice. Surprisingly, these latter mutant mice did not survive past 2 mo old and possessed the phenotype of Mgp/ mice (unpublished data). Indeed, Alizarin red staining of skeletal preparations and histological analysis showed that at 1 mo old the aorta of Mgp/; SM22
-Osteocalcin was fully mineralized (Fig. 5, B and C). This result indicates that unlike MGP and despite the presence of three gla residues, osteocalcin cannot inhibit ECMM in arteries. Alternatively, it could mean that this function of osteocalcin could not be incurred outside bone, its physiological site of expression. To test this possibility, we generated transgenic mice overexpressing Osteocalcin in osteoblasts under the control of the
1(I) collagen promoter. Again, unlike what we observed in
1(I)Col-Mgp transgenic mice,
1(I)Col-Osteocalcin mice had normally mineralized bone. In particular, they had a normal osteoid volume relative to total bone volume (Fig. 5 D). Together, the results of these two experiments indicate that despite the presence of gla residues, osteocalcin is not an inhibitor of ECMM in vivo. Based on the gain of function experiments presented here and on the loss of function experiments described previously it appears that the gla residues do not affect MGP and osteocalcin functions in the same way. Our results raise the hypothesis that, as it is the case for thrombin, only decarboxylated osteocalcin may have a function (Furie and Furie, 1988). Consistent with this hypothesis, it was shown recently that serum level of decarboxylated osteocalcin is a reliable indicator of the severity of osteoporosis in postmenopausal women (Szulc et al., 1996).
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Materials and methods |
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Mice
Generation of Mgp/ mice was described previously (Luo et al., 1997). Transgenic founders were generated by pronuclear injection according to standard techniques. All mice were maintained in a pathogen-free standard animal facility.
Genotyping and expression analysis
Genotypes were determined by PCR using isolated tail DNA. The following sets of primers were used: for SM22-Mgp transgene 5'-AAGGAAGGGTTTCAGGGTCCTG-3' and 5'-CGGGAAAGATGAGGAAGAAGGG-3'; for
1(I)Col-Mgp,
1(I)Col-Mgpm1, and
1(I)Col-Mgpm2 transgenes 5'-CCAGGATGCCTGAAAGATTACTAGC-3' and 5'-CGGGAAAGATGAGGAAGAAGGG-3'; for ApoE-Mgp transgene 5'-TTAGAGGAAATCACAGGGGGAGGC-3' and 5'-GGATCGCAACAAGCCTGCCTACGATATCAACAGAGATGC-3'; for SM22
-Osteocalcin transgene 5'-AAGGAAGGGTTTCAGGGTCCTG-3' and 5'-GGGGATCTGGGCTGGGGACTGAGG-3' and for
1(I)Col-Osteocalcin transgene 5'-CCAGGATGCCTGAAAGATTACTAGC-3' and 5'-GGGGATCTGGGCTGGGGACTGAGG-3'. For analysis of transgene expression, RNA was isolated as previously described and analyzed by Northern blotting (Ausubel et al., 1996). Probes used were SV40 and ApoE polyadenylation signals.
Primary osteoblast culture
WT osteoblasts were cultured in Alpha-MEM (Invitrogen) containing 10% WT or ApoE-Mgp serum and supplemented by 100 µg/ml ascorbic acid (Sigma-Aldrich). After formation of osteoblastic nodules at 4 d, 5 mM ß-glycerophosphate (Sigma-Aldrich) was added to the culture medium and cells were grown for another 4 d. Fresh medium was added in every 48 h. von Kossa staining for mineral and alkaline phosphatase staining for osteoblasts were performed at the end of the culture period.
Skeletal preparation
Thoracic aorta together with vertebrae were dissected, fixed overnight in 100% ethanol, and stained in Alcian blue dye followed by Alizarin red solution as described previously (Luo et al., 1997).
Histology
Vertebrae were fixed overnight in 4% PFA/PBS, embedded in methyl methacrylate, sectioned (7 µm), and stained by von Kossa and van Gieson. Unmineralized bone was measured using Osteomeasure software (Osteometrics Inc.). Aortas were fixed in 1% glutaraldehyde overnight, washed in 0.1 M sodium cacodylate buffer, serially dehydrated in ethanol, and embedded in paraffin. 7-µm sections were stained by von Kossa and counterstained by Toluidine blue. Images were captured with a light microscope (model DMLB; Leica) using a SPOT CCD camera, acquired with SPOT software v2.1 (Diagnostic Instruments), and processed using Adobe Photoshop®.
Serum biochemistry
Serum calcium and phosphate were measured using commercially available kits (Sigma-Aldrich). PTH was measured using an ELISA kit for immunodetection (Immunotopics). Dot blot analysis to detect serum MGP was performed using a polyclonal rabbit serum raised against a COOH-terminal MGP peptide (ERYAMVYGYNAAYNRYFRQRRGAKY). A commercially available antirabbit antibody conjugated with HRP was used as a secondary antibody. HRP activity was detected by using standard protocols and signal intensity on an imaging film was measured by NIH software for densitometric analysis. Serum osteocalcin level was measured using an osteocalcin RIA kit (Biomedical Technologies Inc.).
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Acknowledgments |
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This paper was supported by National Institutes of Health grant NIH PO1 AR42919, March of Dimes Foundation grant 1-FY99-489, and Canadian Institute of Health Research grant MT11360. M. Murshed is supported by a Postdoctoral Fellowship from American Heart Association (Application ID 0325220Y).
Submitted: 9 February 2004
Accepted: 30 April 2004
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